Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito

  • Che Julius Ngwa1,

    Affiliated with

    • Matthias Scheuermayer1,

      Affiliated with

      • Gunnar Rudolf Mair2,

        Affiliated with

        • Selina Kern1,

          Affiliated with

          • Thomas Brügl1,

            Affiliated with

            • Christine Clara Wirth1, 3,

              Affiliated with

              • Makoah Nigel Aminake1, 3,

                Affiliated with

                • Jochen Wiesner4,

                  Affiliated with

                  • Rainer Fischer3, 4,

                    Affiliated with

                    • Andreas Vilcinskas4, 5 and

                      Affiliated with

                      • Gabriele Pradel3Email author

                        Affiliated with

                        BMC Genomics201314:256

                        DOI: 10.1186/1471-2164-14-256

                        Received: 19 September 2012

                        Accepted: 1 April 2013

                        Published: 15 April 2013

                        Abstract

                        Background

                        The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector.

                        Results

                        To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence.

                        For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy.

                        Conclusions

                        The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector.

                        Keywords

                        Malaria Plasmodium falciparum Gametocyte Gametogenesis Transmission Mosquito Transcriptome

                        Background

                        Up to date, the tropical disease malaria is one of the most devastating infectious diseases in the world, causing 216 million new cases and approximately 655.000 casualties each year [1]. Disease treatment and control measures are undermined by the spread of drug resistance in malaria parasites, particularly in populations of Plasmodium falciparum, the agent responsible for malaria tropica (reviewed in [2]).

                        The transmission of malaria parasites from the human to the mosquito is mediated by specialized sexual precursor cells, the intraerythrocytic gametocytes. Maturation of P. falciparum gametocytes from stage I to stage V takes approximately 10 days, and during this period the gametocytes maintain a stable cell cycle arrest (reviewed in [3, 4]). The mature gametocytes circulate in the human’s blood stream, but remain dormant until they are taken up by a blood-feeding mosquito.

                        When entering the mosquito midgut together with the blood meal, the gametocytes become activated from the dormant stage by external stimuli, i.e. a drop in temperature and the contact with the mosquito-derived molecule xanthurenic acid (XA) (reviewed in [5, 6]). Gametocyte activation leads to rounding up of the cell, followed by parasite egress from the enveloping erythrocyte, which involves the rupture of two membranes, the parasitophorous vacuole membrane (PVM) and the erythrocyte membrane [7] (reviewed [8]). During gametogenesis the microgametocyte replicates its genome three times in order to produce eight motile microgametes. Following the fusion of micro- and macrogametes a zygote forms and develops into an infective ookinete within the following 24 hours. The motile ookinete possesses an apical complex which enables it to traverse the midgut epithelium before settling down and forming an oocyst between epithelium and basal lamina (reviewed in [5]).

                        During gametocytogenesis, P. falciparum expresses a new set of genes important for sexual development [914]. Ingestion by the blood-feeding mosquito again triggers molecular changes in the sexual stage parasites, which help adjusting the gametocytes to the insect and which on the one hand initiate sexual reproduction and further development of the parasite in the vector, on the other hand prepare the emerging gametes for the hostile environment of the mosquito midgut. Noteworthy, the midgut stages have to persevere outside a host cell for more than one day. During this time period, the cells are highly vulnerable to the aggressive factors of the gut, which among others include bacteria as well as human immune cells, antibodies and complement proteins present in the blood meal, and this exposure results in an approximate 1000-fold loss of parasite abundance [15] (reviewed in [5, 6]).

                        Gametocyte maturation and gametogenesis are particularly accompanied by the coordinated expression of numerous adhesive surface proteins, including the EGF domain-containing proteins Pfs25 and Pfs28, the 6-cys proteins Pfs230 and Pfs48/45, and the LCCL domain-containing PfCCp proteins. It is noteworthy that the majority of these proteins can be divided into two classes: One class of the adhesion proteins, including Pfs230, Pfs48/45 and the six PfCCp proteins, is expressed within the parasitophorous vacuole (PV) of the developing gametocyte, and the majority of these proteins assemble to multimeric protein complexes [16, 17] (reviewed in [18]). The adhesion proteins are subsequently present on the gamete surface, but expression of these proteins usually ceases during fertilization. The expression of the second class of surface proteins starts following parasite transmission to the mosquito, as was shown for Pfs25 and Pfs28, and expression often persists until the ookinete has formed (reviewed in [5]). One reason for this sudden onset of protein expression following gametocyte activation in the mosquito midgut is the translational repression of messenger RNA (mRNA) encoding for some of these proteins. This was inter alia shown in the rodent malaria parasite P. berghei for the repression of Pbs25 and Pbs28 by the RNA helicase DOZI (development of zygote inhibited) as part of a ribonucleoprotein complex [19]. DOZI and its subsequently identified interaction partner CITH (CAR-I and fly trailer hitch) are considered repressors of maternally supplied mRNA important for ookinete development [20].

                        While astonishing advances have been made in previous years in unveiling the molecular basis of parasite transmission from the mammalian host to the mosquito, the currently available data are fragmented and major key players for gametogenesis and adaptation have not yet been identified. We thus aimed at investigating the changes in transcript levels during gametocyte activation in order to gain in-depth knowledge on the molecular switch-over that takes place in the parasite during transmission. We selected the suppression subtractive hybridization (SSH) technique to experimentally screen for genes differentially expressed during gametocyte activation, because this PCR-based method amplifies induced genes while simultaneously suppressing house-keeping genes, and is thus particularly suited to discover new or unexpected genes [21]. Our data provide insights into the regulated gene expression of malaria parasites during transmission to the mosquito and enable the identification of novel proteins with essential functions for the mosquito midgut stages, which might be suitable targets for transmission blocking interventions.

                        Results and discussion

                        To determine changes in the transcriptome of malaria parasites during the initial phase of transmission to the mosquito, we conducted a SSH on mRNA isolated from mature non-activated gametocytes and from gametocytes at 30 min after in vitro activation, which have completed gametogenesis [7]. A subtracted cDNA library was constructed and the resulting cDNA was assigned to the respective plasmodial genes.

                        We identified a total number of 126 genes, for which expression levels changed in the gametocytes during activation. The majority of genes can be assigned to six major ontology groups (Figure 1, Table 1): 22 genes (17.5%) have putative functions in signaling, 18 genes (14.3%) are assigned to processes linked to cell cycle and gene expression, 11 genes (8.7%) can be linked to the cytoskeleton or the inner membrane complex (IMC), 10 genes (7.9%) have putative functions in protein trafficking, stabilization and degradation (proteostasis), eight genes (6.4%) are linked to general metabolic functions, and 16 genes (12.7%) are proteins of the cell surface or PVM. Furthermore, 15 genes (11.9%) can be assigned to other functions, including protein synthesis and processing. A total of 26 genes (20.6%) encode for proteins with unknown function, eight of which have sequences for transmembrane domains and five of which encode for a signal peptide.
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-256/MediaObjects/12864_2012_4956_Fig1_HTML.jpg
                        Figure 1

                        Ontology groups of P. falciparum genes with changes in transcript expression during gametocyte activation. The changes in transcript abundance were determined by SSH between the mRNA of mature non-activated gametocytes and gametocytes at 30 min p.a. in vitro. A total number of 126 genes were identified (the numbers of genes per group are indicated).

                        Table 1

                        Genes of the Plasmodium falciparum gametocyte transcriptome as identified by SSH (comparing mature non-activated gametocytes and gametocytes at 30 min p.a.)

                        Plasmodb ID

                        Protein

                        Predicted features

                        pred. MW

                        Signaling

                        PF3D7_0214600

                        serine/threonine protein kinase

                         

                        204.3

                        PF3D7_1356800

                        serine/threonine protein kinase ARK3

                         

                        475.7

                        PF3D7_0302100

                        serine/threonine protein kinase PfCLK-4/SRPK1

                        Phosphorylation of SR splicing factors

                        157.4

                        PF3D7_1230900

                        atypical protein kinase, RIO family

                         

                        111.5

                        PF3D7_1238900

                        calcium/calmodulin-dependent protein kinase, PK2

                         

                        58.9

                        PF3D7_0624000

                        hexokinase, HK

                         

                        55.3

                        PF3D7_0515300

                        phosphatidylinositol 3-kinase, PI3K

                         

                        255.9

                        PF3D7_1113100

                        protein tyrosine phosphatase, PRL

                         

                        25.1

                        PF3D7_1403900

                        phosphatase

                         

                        35.2

                        PF3D7_1464600

                        phosphatase

                        SP

                        170.5

                        PF3D7_0210600

                        conserved Plasmodium protein

                        SP, calcium-lipid-binding domain CaLB

                        53.7

                        PF3D7_1238100

                        calcyclin binding protein

                        Calcyclin-binding domain

                        266.4

                        PF3D7_1442200

                        GTP binding protein

                         

                        118.7

                        PF3D7_1358600

                        zinc finger protein

                        Zinc-Finger domain

                        118.3

                        PF3D7_0927200

                        zinc finger protein

                        Zinc-Finger domain

                        189.7

                        PF3D7_1004300

                        zinc finger, C3HC4 type

                         

                        131.7

                        PF3D7_1433400

                        conserved Plasmodium membrane protein

                        7 TMs, PHD C4HC3 zinc-finger-like motifs

                        682.5

                        PF3D7_1463900

                        conserved Plasmodium membrane protein

                        SP, 7 TMs, EF hand calcium-binding domain

                        127.1

                        PF3D7_1233600

                        asparagine and aspartate rich protein 1, AARP1

                        Zinc-finger domain, Ring/U-Box

                        146.3

                        PF3D7_0530200

                        triose phosphate transporter iTPT

                        SP, 7 TMs

                        59.8

                        PF3D7_0303900

                        conserved Plasmodium protein

                        SP, phosphatidyl-ethanolamine-binding protein PEBP domain

                        23.2

                        PF3D7_0818200

                        14-3-3 protein

                        Regulative protein, binds signaling enzymes like kinases or TM receptors

                        30.2

                        Cell cycle and gene expression

                        PF3D7_1445900

                        DEAD/DEAH box ATP-dependent RNA helicase

                         

                        60.0

                        PF3D7_1019000

                        eukaryotic translation initiation factor subunit eIF2A

                         

                        280.8

                        PF3D7_1417200

                        NOT family protein

                        Regulation of transcript

                        519.8

                        PF3D7_1103800

                        CCR4-NOT transcription complex subunit 1, NOT1

                        4 TMs

                        392.0

                        PF3D7_0622900

                        ApiAP2 protein

                        Transcription factor

                        237.4

                        PF3D7_0420300

                        ApiAP2 protein

                        Transcription factor

                        400.2

                        PF3D7_0615400

                        ribonuclease

                         

                        323.2

                        PF3D7_0811300

                        CCR4-associated factor 1, CAF1

                         

                        200.2

                        PF3D7_1034900

                        methionine-tRNA ligase

                         

                        101.2

                        PF3D7_1469700

                        conserved Plasmodium protein

                        Homology to the transcriptional mediator subunit Med6

                        24.6

                        PF3D7_0513600

                        deoxyribodipyrimidine photolyase

                         

                        129.2

                        PF3D7_1105500

                        centrin-4 (CEN4)

                         

                        20.1

                        PF3D7_1018500

                        PHF5-like protein

                        mRNA splicing factor RDS3 domain

                        12.5

                        PF3D7_0827800

                        SET domain protein SET3

                        Chromatin regulator, nuclear protein, in male gametocytes

                        283.6

                        PF3D7_1115200

                        SET domain protein, SET7

                        Chromatin regulator

                        94.3

                        PF3D7_1360900

                        polyadenylate-binding protein

                        RNA-binding domain

                        44.8

                        PF3D7_1011800

                        QF122 antigen

                        KH domain type 1, RNA binding

                        131.6

                        PF3D7_1347500

                        DNA/RNA-binding protein Alba 4, ALBA4

                         

                        42.2

                        Cytoskeleton, IMC, Motor complex

                        PF3D7_0422300

                        alpha-tubulin II

                        Cytoskeletal element

                        49.7

                        PF3D7_1246200

                        actin I

                        IMC component

                        41.2

                        PF3D7_0503400

                        actin-depolymerizing factor 1, ADF1

                         

                        13.7

                        PF3D7_1020200

                        kinesin

                         

                        99.1

                        PF3D7_0724900

                        kinesin-like protein

                         

                        222.6

                        PF3D7_1246400

                        myosin A tail domain interacting protein, MTIP

                        IMC component

                        23.5

                        PF3D7_1251200

                        coronin

                        Actin-binding protein

                        69.0

                        PF3D7_0708000

                        cytoskeleton-associated protein

                        Cap-Gly domain

                        103.6

                        PF3D7_0515700

                        glideosome-associated protein 40, GAP40

                        10 TMs, IMC component

                        51.8

                        PF3D7_0918000

                        glideosome-associated protein 50, GAP50

                        SP, 2 TMs, IMC component

                        44.6

                        PF3D7_1351700

                        Alveolin, ALV6

                        IMC component

                        151.2

                        Proteostasis (Protein trafficking, stabilization and degradation)

                        PF3D7_0807500

                        proteasome subunit alpha type 6

                        Component oft he proteasome core particle

                        29.5

                        PF3D7_1466300

                        proteasome subunit RPN2

                        Proteasome non-ATPase regulatory subunit 1

                        132.8

                        PF3D7_0815800

                        vacuolar sorting protein, VPS9

                        Ortholog of Saccharomyces cerevisae vacuolar sorting protein 9

                        214.7

                        PF3D7_1136400

                        conserved Plasmodium protein

                        TPR domain, mediates protein interactions and multi-protein complexes

                        107.1

                        PF3D7_0215400

                        conserved Plasmodium protein

                        WD40-domain protein, assembly and stabilization of MPCs

                        110.8

                        PF3D7_0818900

                        heat shock protein 70, HSP70-1

                         

                        73.9

                        PF3D7_0917900

                        heat shock protein 70, HSP70-2

                         

                        72.4

                        PF3D7_0306200

                        activator of Hsp90 ATPase, AHA1

                         

                        41.6

                        PF3D7_0708400

                        heat shock protein 90, HSP90

                         

                        86.2

                        PF3D7_0102200

                        ring-infected erythrocyte surface antigen, RESA

                        PHIST and DnaJ domains

                        126.5

                        Metabolism, general

                        mal_mito_2

                        cytochrome c oxidase subunit I, cox1

                        SP, 12 TMs, component of the respiratory chain in mitochondria

                        53.0

                        PF3D7_1444800

                        fructose 1,6-biphosphate aldolase

                        Glycolysis pathway

                        40.1

                        PF3D7_0806800

                        vacuolar proton translocating ATPase subunit A

                        6 TMs

                        123.0

                        PF3D7_0922200

                        S-adenosylmethionine synthetase, SAMS

                         

                        44.8

                        PF3D7_0801900

                        conserved Plasmodium protein

                        FAD/NADP-binding domain

                        552.0

                        PF3D7_0403200

                        conserved Plasmodium protein

                        Possibly acetyl-CoA-synthetase

                        69.4

                        PF3D7_0511800

                        myo-inositol 1-phosphate synthase

                         

                        69.1

                        PF3D7_0104400

                        4-hydroxy-3-methylbut-2-enyl diphosphate reductase, LytB

                        1 TM, protein export domain, non-mevalonate pathway

                        62.5

                        cell surface and PVM

                        PF3D7_1035300

                        glutamate-rich protein; GLURP

                        SP, on parasite surface

                        141.1

                        PF3D7_1335300

                        reticulocyte binding protein 2, RH2b

                        1 TM, on parasite surface, involved in erythrocyte binding

                        382.9

                        PF3D7_1228600

                        merozoite Surface Protein 9, MSP-9

                        SP, on merozoite surface, involved in erythrocyte binding

                        86.6

                        PF3D7_1028700

                        merozoite TRAP-like protein, MTRAP

                        SP, 1 TM, belongs to TRAP family, in micronemes and on merozoite surface

                        58.1

                        PF3D7_0731500

                        erythrocyte binding antigen 175, EBA-175

                        SP, 1 TM, Duffy binding domain, on merozoite surface

                        174.6

                        PF3D7_0102500

                        erythrocyte binding antigen-181, EBA-181

                        SP, 2 TMs, Duffy binding domain, on merozoite surface

                        181.2

                        PF3D7_1035700

                        duffy binding-like merozoite surface protein, DBLMSP

                        SP, Duffy binding domain, SPAM

                        80.3

                        PF3D7_0202500

                        early transcribed membrane protein 2, ETRAMP2

                        SP, 1 TM, integral PVM protein

                        11.5

                        PF3D7_0423700

                        early transcribed membrane protein 4, ETRAMP4

                        SP, 2TMs, integral PVM protein

                        10.2

                        PF3D7_1033200

                        early transcribed membrane protein 10.2, ETRAMP 10.2

                        SP, 2TMs, integral PVM protein

                        38.9

                        PF3D7_1103500

                        CPW-WPC family protein

                        SP, adhesin, cysteine-rich CPW-WPC domain

                        67.3

                        PF3D7_0406200

                        Pfs16

                        SP, 2 TMs, integral PVM protein of gametocytes

                        16.7

                        PF3D7_0930300

                        merozoite surface protein 1, MSP1

                        SP, GPI anchor, EGF domain, on merozoite surface, part of MSP complex

                        195.7

                        PF3D7_1218800

                        conserved Plasmodium protein, PSOP17

                        SP, lectin domain, laminin G2 domain

                        39.6

                        PF3D7_0707300

                        rhoptry-associated membrane antigen, RAMA

                        GPI anchor, involved in erythrocyte binding

                        103.6

                        PF3D7_1021900

                        10b antigen

                        Ankyrin repeat region, PHAX RNA-binding domain, interacts with surface proteins like PfEMP1 (Y2H)

                        267.4

                        Others

                        PF3D7_1103100

                        60S ribosomal protein P1

                         

                        13.0

                        PF3D7_1027800

                        60S ribosomal protein L3

                         

                        44.2

                        PF3D7_1408600

                        40S ribosomal protein S8e

                         

                        25.1

                        PF3D7_1358800

                        40S ribosomal protein S13

                         

                        17.3

                        PF3D7_0206200

                        metabolite/drug transporter

                        11 TMs, Major facilitator superfamily

                        62.7

                        PF3D7_1207700

                        blood stage antigen 41–3 precursor

                        SP

                        43.4

                        PF3D7_1457000

                        signal peptide peptidase, SPP

                        8 TMs

                        47.6

                        PF3D7_0320700

                        signal peptidase complex subunit 2, SPC2

                        2 TMs, SPC25 domain

                        21.0

                        PF3D7_0207700

                        serine repeat antigen 4, SERA-4

                        SP, papain family cysteine proteinase

                        108.7

                        PF3D7_1033800

                        plasmepsin VII

                        SP, 1 TM, aspartyl protease

                        52.3

                        PF3D7_0930000

                        procollagen lysine 5-dioxygenase

                        2 TMs

                        66.8

                        PF3D7_0817600

                        conserved Plasmodium protein

                        MAC/PF domain

                        112.2

                        PF3D7_0403800

                        conserved Plasmodium protein

                        Alpha/beta-hydrolase

                        83.4

                        PF3D7_0709900

                        conserved Plasmodium membrane protein

                        4 TMs, alpha/beta-hydrolase

                        274.0

                        PF3D7_0311600

                        conserved Plasmodium protein, unknown function

                        SP, 1 TM, ribophorin-I domain,putative dolichyl-diphospho-oligosaccharide-protein

                        84.0

                        Unknown

                        PF3D7_0904200

                        conserved Plasmodium protein

                        SP, 1 TM

                        34.0

                        PF3D7_1024800

                        conserved Plasmodium protein

                        SP, 2 TMs

                        171.1

                        PF3D7_1239400

                        conserved Plasmodium protein

                        SP

                        22.0

                        PF3D7_0730800.1

                        Plasmodium exported protein

                        SP

                        32.1

                        PF3D7_1316700

                        conserved Plasmodium protein

                        SP

                        73.4

                        PF3D7_0601900

                        conserved Plasmodium protein

                        1 TM

                        14.7

                        PF3D7_0704100

                        conserved Plasmodium membrane protein

                        6 TMs

                        425.3

                        PF3D7_1023000

                        conserved Plasmodium membrane protein

                        5 TMs

                        48.9

                        PF3D7_1439600

                        conserved Plasmodium protein

                        1 TM

                        22.6

                        PF3D7_1021700

                        conserved Plasmodium membrane protein

                        4 TMs

                        830.2

                        PF3D7_0417400

                        conserved Plasmodium protein

                        2 TMs, interacts with PF3D7_1021700 (Y2H)

                        822.4

                        PF3D7_1026600

                        conserved Plasmodium protein

                        Interacts with RNA-associated proteins (Y2H)

                        196.5

                        PF3D7_1362600

                        conserved Plasmodium protein

                        Interacts with splicing factor 3B subunit 2 (Y2H)

                        32.1

                        PF3D7_0202400

                        conserved Plasmodium protein

                        Pfg27 domain

                        141.9

                        PF3D7_1124200

                        conserved Plasmodium protein

                        UAS domain

                        49.3

                        PF3D7_1452400

                        conserved Plasmodium protein

                         

                        107.5

                        PF3D7_1225600

                        conserved Plasmodium protein

                         

                        93.1

                        PF3D7_1451200

                        conserved Plasmodium protein

                         

                        176.2

                        PF3D7_1438800

                        conserved Plasmodium protein

                         

                        84.0

                        PF3D7_1114600

                        conserved Plasmodium protein

                         

                        39.6

                        PF3D7_1026100

                        conserved Plasmodium protein

                         

                        23.9

                        PF3D7_1230100

                        conserved Plasmodium protein

                         

                        61.5

                        PF3D7_1321000

                        conserved Plasmodium protein

                         

                        80.6

                        PF3D7_0411000

                        conserved Plasmodium protein

                         

                        179.7

                        PF3D7_1126700

                        conserved Plasmodium protein

                         

                        110.1

                        PF3D7_0508900

                        conserved Plasmodium protein

                         

                        370.2

                        IMC, inner membrane complex; PVM, parasitophorous vacuole membrane; SP, signal peptide; TM, transmembrane; Y2H, yeast-two-hybrid.

                        Roughly 60% (76/126) of the SSH genes have been detected in the currently available P. falciparum gametocyte proteomes, while for 40% there is as yet no protein evidence (Table 2) [9, 12, 13, 22]. Almost identical numbers apply to the 113 orthologs of the rodent malaria parasite P. berghei; the 13 P. falciparum genes without P. berghei orthologs include amongst others the three etramp genes, pfs16 and resa.
                        Table 2

                        Comparison of genes identified by SSH with P. falciparum and P. berghei gametocyte proteome data

                           

                        Pf

                        P. falciparum GC

                        P. berghei male/female GC

                        Total

                         

                        PF_ID

                        Gene Ontology

                        MW

                        Troph

                        I & II

                        IV & V

                        V

                        I.II.IV.V

                        FG1

                        FG 3

                        FG (1+3)

                        MG1

                        MG3

                        MG (1+3)

                        FG +MG

                        FG.MG.PF

                        PB_ID

                        PF3D7_0210600

                        signaling

                        53.7

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_030750

                        PF3D7_0214600

                        signaling

                        204.3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_031140

                        PF3D7_0302100

                        signaling

                        157.4

                        2

                        3

                        1

                        7

                        11

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        11

                        PBANKA_040110

                        PF3D7_0303900

                        signaling

                        23.2

                        0

                        4

                        4

                        11

                        19

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        19

                        PBANKA_040250

                        PF3D7_0515300

                        signaling

                        255.9

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_111490

                        PF3D7_0530200

                        signaling

                        59.8

                        0

                        0

                        0

                        3

                        3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        3

                        PBANKA_124460

                        PF3D7_0624000

                        signaling

                        55.3

                        17

                        20

                        2

                        24

                        46

                        6

                        13

                        19

                        11

                        10

                        21

                        40

                        86

                        PBANKA_112290

                        PF3D7_0818200

                        signaling

                        30.2

                        18

                        21

                        14

                        25

                        60

                        11

                        20

                        31

                        9

                        11

                        20

                        51

                        111

                        PBANKA_071260

                        PF3D7_0927200

                        signaling

                        189.7

                        0

                        13

                        2

                        5

                        20

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        20

                        PBANKA_082800

                        PF3D7_1004300

                        signaling

                        131.7

                        3

                        5

                        1

                        0

                        6

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        6

                        PBANKA_120260

                        PF3D7_1113100

                        signaling

                        25.1

                        1

                        2

                        1

                        3

                        6

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        6

                        PBANKA_093450

                        PF3D7_1230900

                        signaling

                        111.5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_144560

                        PF3D7_1233600

                        signaling

                        146.3

                        0

                        4

                        0

                        8

                        12

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        12

                        PBANKA_144820

                        PF3D7_1238100

                        signaling

                        266.4

                        7

                        9

                        3

                        9

                        21

                        2

                        6

                        8

                        8

                        7

                        15

                        23

                        44

                        PBANKA_145260

                        PF3D7_1238900

                        signaling

                        58.9

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_145340

                        PF3D7_1356800

                        signaling

                        475.7

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_113310

                        PF3D7_1358600

                        signaling

                        118.3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_113490

                        PF3D7_1403900

                        signaling

                        35.2

                        6

                        9

                        5

                        12

                        26

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        26

                        ×

                        PF3D7_1433400

                        signaling

                        682.5

                        0

                        0

                        2

                        0

                        2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        2

                        PBANKA_101150

                        PF3D7_1442200

                        signaling

                        118.7

                        0

                        0

                        1

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_130610

                        PF3D7_1463900

                        signaling

                        127.1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_132710

                        PF3D7_1464600

                        signaling

                        170.5

                        23

                        31

                        0

                        3

                        34

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        34

                        PBANKA_132800

                        PF3D7_0420300

                        cell cycle & gene expr.

                        400.2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_052170

                        PF3D7_0513600

                        cell cycle & gene expr.

                        129.2

                        10

                        29

                        0

                        10

                        39

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        39

                        PBANKA_111330

                        PF3D7_0615400

                        cell cycle & gene expr.

                        323.2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_123010

                        PF3D7_0622900

                        cell cycle & gene expr.

                        237.4

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_112180

                        PF3D7_0811300

                        cell cycle & gene expr.

                        200.2

                        0

                        2

                        1

                        2

                        5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        5

                        PBANKA_142620

                        PF3D7_0827800

                        cell cycle & gene expr.

                        283.6

                        0

                        0

                        1

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_070290

                        PF3D7_1011800

                        cell cycle & gene expr.

                        131.6

                        41

                        22

                        6

                        17

                        45

                        4

                        11

                        15

                        14

                        0

                        14

                        29

                        74

                        PBANKA_121020

                        PF3D7_1018500

                        cell cycle & gene expr.

                        12.5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_050270

                        PF3D7_1019000

                        cell cycle & gene expr.

                        280.8

                        7

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_050320

                        PF3D7_1034900

                        cell cycle & gene expr.

                        101.2

                        21

                        31

                        0

                        49

                        80

                        6

                        0

                        6

                        3

                        0

                        3

                        9

                        89

                        PBANKA_051870

                        PF3D7_1103800

                        cell cycle & gene expr.

                        392.0

                        7

                        12

                        0

                        23

                        35

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        35

                        PBANKA_094310

                        PF3D7_1105500

                        cell cycle & gene expr.

                        20.1

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_094140

                        PF3D7_1115200

                        cell cycle & gene expr.

                        94.3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_093250

                        PF3D7_1347500

                        cell cycle & gene expr.

                        42.2

                        18

                        15

                        30

                        22

                        67

                        11

                        0

                        11

                        7

                        10

                        17

                        28

                        95

                        PBANKA_136030

                        PF3D7_1360900

                        cell cycle & gene expr.

                        44.8

                        8

                        11

                        11

                        12

                        34

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        34

                        ×

                        PF3D7_1417200

                        cell cycle & gene expr.

                        519.8

                        5

                        6

                        0

                        6

                        12

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        12

                        PBANKA_102550

                        PF3D7_1445900

                        cell cycle & gene expr.

                        60.0

                        16

                        25

                        3

                        21

                        49

                        0

                        0

                        0

                        1

                        0

                        1

                        1

                        50

                        PBANKA_130970

                        PF3D7_1469700

                        cell cycle & gene expr.

                        24.6

                        0

                        2

                        0

                        3

                        5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        5

                        PBANKA_133290

                        PF3D7_0422300

                        IMC, cytoskeleton

                        49.7

                        11

                        25

                        13

                        30

                        68

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        68

                        PBANKA_052270

                        PF3D7_0503400

                        IMC, cytoskeleton

                        13.7

                        6

                        5

                        1

                        8

                        14

                        0

                        0

                        0

                        0

                        1

                        1

                        1

                        15

                        PBANKA_110310

                        PF3D7_0515700

                        IMC, cytoskeleton

                        51.8

                        3

                        0

                        0

                        7

                        7

                        1

                        0

                        1

                        0

                        0

                        0

                        1

                        8

                        PBANKA_111530

                        PF3D7_0708000

                        IMC, cytoskeleton

                        103.6

                        0

                        0

                        2

                        12

                        14

                        0

                        0

                        0

                        1

                        0

                        1

                        1

                        15

                        PBANKA_080520

                        PF3D7_0724900

                        IMC, cytoskeleton

                        222.6

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_062240

                        PF3D7_0918000

                        IMC, cytoskeleton

                        44.6

                        14

                        12

                        1

                        23

                        36

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        36

                        PBANKA_081900

                        PF3D7_1020200

                        IMC, cytoskeleton

                        99.1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_050430

                        PF3D7_1246200

                        IMC, cytoskeleton

                        41.2

                        23

                        28

                        13

                        29

                        70

                        12

                        19

                        31

                        6

                        6

                        12

                        43

                        113

                        PBANKA_145930

                        PF3D7_1246400

                        IMC, cytoskeleton

                        23.5

                        1

                        0

                        0

                        3

                        3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        3

                        PBANKA_145950

                        PF3D7_1251200

                        IMC, cytoskeleton

                        69.0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_146410

                        PF3D7_1351700

                        IMC, cytoskeleton

                        151.2

                        0

                        0

                        1

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_136440

                        PF3D7_0102200

                        proteostasis

                        126.5

                        33

                        3

                        1

                        0

                        4

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        4

                        ×

                        PF3D7_0215400

                        proteostasis

                        110.8

                        0

                        0

                        1

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_031210

                        PF3D7_0306200

                        proteostasis

                        41.6

                        4

                        7

                        0

                        8

                        15

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        15

                        PBANKA_040460

                        PF3D7_0708400

                        proteostasis

                        86.2

                        54

                        60

                        17

                        82

                        159

                        7

                        0

                        7

                        8

                        10

                        18

                        25

                        184

                        PBANKA_080570

                        PF3D7_0807500

                        proteostasis

                        29.5

                        11

                        11

                        2

                        20

                        33

                        3

                        8

                        11

                        4

                        2

                        6

                        17

                        50

                        PBANKA_122310

                        PF3D7_0815800

                        proteostasis

                        214.7

                        0

                        3

                        0

                        0

                        3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        3

                        PBANKA_071500

                        PF3D7_0818900

                        proteostasis

                        73.9

                        45

                        46

                        33

                        57

                        136

                        34

                        40

                        74

                        31

                        28

                        59

                        133

                        269

                        PBANKA_071190

                        PF3D7_0917900

                        proteostasis

                        72.4

                        45

                        40

                        42

                        51

                        133

                        24

                        46

                        70

                        16

                        16

                        32

                        102

                        235

                        PBANKA_081890

                        PF3D7_1136400

                        proteostasis

                        107.1

                        0

                        3

                        0

                        11

                        14

                        1

                        0

                        1

                        0

                        0

                        0

                        1

                        15

                        PBANKA_091220

                        PF3D7_1466300

                        proteostasis

                        132.8

                        23

                        26

                        3

                        34

                        63

                        4

                        9

                        13

                        4

                        0

                        4

                        17

                        80

                        PBANKA_132970

                        mal_mito_2

                        metabolism general

                        53.0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_MIT0002

                        PF3D7_0104400

                        metabolism general

                        62.5

                        0

                        1

                        1

                        3

                        5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        5

                        PBANKA_020870

                        PF3D7_0403200

                        metabolism general

                        69.4

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_100080

                        PF3D7_0511800

                        metabolism general

                        69.1

                        36

                        29

                        2

                        40

                        71

                        2

                        0

                        2

                        0

                        0

                        0

                        2

                        73

                        PBANKA_111140

                        PF3D7_0801900

                        metabolism general

                        552.0

                        0

                        0

                        1

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_122830

                        PF3D7_0806800

                        metabolism general

                        123.0

                        11

                        9

                        4

                        29

                        42

                        0

                        0

                        0

                        0

                        3

                        3

                        3

                        45

                        PBANKA_122380

                        PF3D7_0922200

                        metabolism general

                        44.8

                        25

                        16

                        0

                        4

                        20

                        1

                        2

                        3

                        0

                        0

                        0

                        3

                        23

                        PBANKA_082310

                        PF3D7_1444800

                        metabolism general

                        40.1

                        21

                        23

                        11

                        35

                        69

                        17

                        23

                        40

                        22

                        22

                        44

                        84

                        153

                        PBANKA_130860

                        PF3D7_0102500

                        cell surface & PVM

                        181.2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_133270

                        PF3D7_0202500

                        cell surface & PVM

                        11.5

                        1

                        0

                        0

                        0

                        0

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        0

                        ×

                        PF3D7_0406200

                        cell surface & PVM

                        16.7

                        3

                        9

                        34

                        17

                        60

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        60

                        ×

                        PF3D7_0423700

                        cell surface & PVM

                        10.2

                        0

                        5

                        0

                        2

                        7

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        7

                        ×

                        PF3D7_0707300

                        cell surface & PVM

                        103.6

                        3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_080450

                        PF3D7_0731500

                        cell surface & PVM

                        174.6

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_133270

                        PF3D7_0930300

                        cell surface & PVM

                        195.7

                        77

                        0

                        3

                        0

                        3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        3

                        PBANKA_083100

                        PF3D7_1021900

                        cell surface & PVM

                        267.4

                        11

                        26

                        4

                        31

                        61

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        61

                        PBANKA_112590/ PBANKA_141230

                        PF3D7_1028700

                        cell surface & PVM

                        58.1

                        0

                        0

                        1

                        11

                        12

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        12

                        PBANKA_051280

                        PF3D7_1033200

                        cell surface & PVM

                        38.9

                        11

                        16

                        0

                        4

                        20

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        20

                        ×

                        PF3D7_1035300

                        cell surface & PVM

                        141.1

                        0

                        0

                        1

                        0

                        1

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        1

                        ×

                        PF3D7_1035700

                        cell surface & PVM

                        80.3

                        10

                        0

                        0

                        0

                        0

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        0

                        ×

                        PF3D7_1103500

                        cell surface & PVM

                        67.3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_094340

                        PF3D7_1218800

                        cell surface & PVM

                        39.6

                        0

                        0

                        2

                        5

                        7

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        7

                        PBANKA_143440

                        PF3D7_1228600

                        cell surface & PVM

                        86.6

                        23

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_144330

                        PF3D7_1335300

                        cell surface & PVM

                        382.9

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_000220 + 10 more

                        PF3D7_0206200

                        others

                        62.7

                        0

                        0

                        0

                        2

                        2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        2

                        PBANKA_030390

                        PF3D7_0207700

                        others

                        108.7

                        11

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_030480

                        PF3D7_0311600

                        others

                        84.0

                        0

                        0

                        0

                        5

                        5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        5

                        PBANKA_040960

                        PF3D7_0320700

                        others

                        21.0

                        0

                        0

                        0

                        8

                        8

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        8

                        PBANKA_121780

                        PF3D7_0403800

                        others

                        83.4

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_100150

                        PF3D7_0709900

                        others

                        274.0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_122050

                        PF3D7_0817600

                        others

                        112.2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_071320

                        PF3D7_0930000

                        others

                        66.8

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_083070

                        PF3D7_1027800

                        others

                        44.2

                        17

                        9

                        14

                        18

                        41

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        41

                        PBANKA_051190

                        PF3D7_1033800

                        others

                        52.3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_051760

                        PF3D7_1103100

                        others

                        13.0

                        2

                        2

                        5

                        4

                        11

                        1

                        0

                        1

                        0

                        0

                        0

                        1

                        12

                        PBANKA_094360

                        PF3D7_1207700

                        others

                        43.4

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_060620

                        PF3D7_1358800

                        others

                        17.3

                        8

                        6

                        4

                        9

                        19

                        8

                        5

                        13

                        1

                        0

                        1

                        14

                        33

                        PBANKA_113510

                        PF3D7_1408600

                        others

                        25.1

                        8

                        8

                        7

                        12

                        27

                        2

                        5

                        7

                        2

                        0

                        2

                        9

                        36

                        PBANKA_103390

                        PF3D7_1457000

                        others

                        47.6

                        0

                        0

                        0

                        0

                        0

                        2

                        0

                        2

                        0

                        1

                        1

                        3

                        3

                        PBANKA_132070

                        PF3D7_0202400

                        unknown

                        141.9

                        27

                        0

                        0

                        0

                        0

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        0

                        ×

                        PF3D7_0411000

                        unknown

                        179.7

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_061240

                        PF3D7_0417400

                        unknown

                        822.4

                        0

                        0

                        1

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_071950

                        PF3D7_0508900

                        unknown

                        370.2

                        0

                        2

                        0

                        0

                        2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        2

                        PBANKA_110850

                        PF3D7_0601900

                        unknown

                        14.7

                        0

                        0

                        0

                        1

                        1

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        1

                        ×

                        PF3D7_0704100

                        unknown

                        425.3

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_080180

                        PF3D7_0730800.1

                        unknown

                        32.1

                        5

                        0

                        0

                        0

                        0

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        0

                        ×

                        PF3D7_0904200

                        unknown

                        34.0

                        0

                        1

                        0

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_041720

                        PF3D7_1021700

                        unknown

                        830.2

                        0

                        0

                        0

                        1

                        1

                        1

                        0

                        1

                        0

                        0

                        0

                        1

                        2

                        PBANKA_050590

                        PF3D7_1023000

                        unknown

                        48.9

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_050720

                        PF3D7_1024800

                        unknown

                        171.1

                        4

                        33

                        15

                        33

                        81

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        81

                        PBANKA_050900

                        PF3D7_1026100

                        unknown

                        23.9

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_051030

                        PF3D7_1026600

                        unknown

                        196.5

                        0

                        0

                        0

                        0

                        0

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        ×

                        0

                        ×

                        PF3D7_1114600

                        unknown

                        39.6

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_093310

                        PF3D7_1124200

                        unknown

                        49.3

                        0

                        0

                        0

                        7

                        7

                        0

                        0

                        0

                        1

                        0

                        1

                        1

                        8

                        PBANKA_092410

                        PF3D7_1126700

                        unknown

                        110.1

                        0

                        1

                        0

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_092170

                        PF3D7_1225600

                        unknown

                        93.1

                        0

                        1

                        0

                        0

                        1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        1

                        PBANKA_144050

                        PF3D7_1230100

                        unknown

                        61.5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_144480

                        PF3D7_1239400

                        unknown

                        22.0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_145390

                        PF3D7_1316700

                        unknown

                        73.4

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_141520

                        PF3D7_1321000

                        unknown

                        80.6

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_141930

                        PF3D7_1362600

                        unknown

                        32.1

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_113860

                        PF3D7_1438800

                        unknown

                        84.0

                        0

                        0

                        0

                        2

                        2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        2

                        PBANKA_130270

                        PF3D7_1439600

                        unknown

                        22.6

                        0

                        6

                        0

                        10

                        16

                        1

                        0

                        1

                        0

                        0

                        0

                        1

                        17

                        PBANKA_130350

                        PF3D7_1451200

                        unknown

                        176.2

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_131490

                        PF3D7_1452400

                        unknown

                        107.5

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        0

                        PBANKA_131610

                        PF troph, peptide hits for gametocyte-less P. falciparum trophozoites [12, 13]; I & II, stage I & II P. berghei gametocyte [12, 13]; V, stage V P. falciparum gametocytes [9]; I.II.IV.V, sum of all P. falciparum gametocyte data; FG1, peptide hits from highly purified female P. berghei gametocyte sample 1 [22]; FG3, peptide hits from highly purified female P. berghei gametocytes sample 3 [22]; FG(1+3), sum of both; MG1, peptide hits from highly purified male P. berghei gametocyte sample 1 [22]; MG3, peptide hits from highly purified male P. berghei gametocyte sample 3 [22]; MG(1+3), sum of both; FG+MG, total number peptide hits from all four P. berghei gametocyte samples; total FG.MG.PF, total number of P. falciparum & P. berghei gametocyte peptide hits.

                        The P. berghei orthologs of the identified P. falciparum genes were compared with a global gametocyte transcriptome analysis of P. berghei DOZI and CITH gene deletion mutants; destabilized mRNAs of these mutants are candidates for translational repression in non-activated gametocytes [19, 20]. A total of 11 genes (8.7%) were identified as candidates regulated by DOZI and CITH (Table 3); eight of those lack any gametocyte protein evidence. One of the destabilized genes was identified as a single peptide hit while the remaining two destabilized genes had 19 and 74 peptide hits in total. A number of 16 additional genes were destabilized in the combined DOZI and CITH datasets indicating they could also be under translational control (data not shown). The data comparison indicates that the majority of genes identified via SSH, which change in transcript expression levels during gametocyte activation, are not translationally controlled in non-activated gametocytes, while many such genes have been identified to function in zygote to ookinete transformation [23]. In conclusion, gene regulation appears to be important for the early events of gametocyte activation, i.e. for gametogenesis and fertilization, while translational repression particularly plays a role for the expression of proteins important for ookinete development.
                        Table 3

                        Genes identified by SSH with P. berghei orthologs showing destabilization in the absence of DOZI and CITH

                        P. falciparum gene ID

                        P

                        Protein

                        Predicted feature

                        MW

                        P. berghei ortholog gene ID

                        DOZI

                        CITH

                        PF3D7_1011800

                        74

                        QF122 antigen

                        KH domain type 1, RNA binding

                        131.6

                        PBANKA_121020

                        1.4

                        1.1

                        PF3D7_0724900

                        0

                        kinesin-like protein

                         

                        222.6

                        PBANKA_062240

                        2.4

                        1.2

                        PF3D7_1033800

                        0

                        plasmepsin VII

                        SP, 1 TM, aspartyl protease

                        52.3

                        PBANKA_051760

                        2.1

                        1.3

                        PF3D7_1316700

                        0

                        conserved Plasmodium protein

                        SP

                        73.4

                        PBANKA_141520

                        2.1

                        1.4

                        PF3D7_1321000

                        0

                        conserved Plasmodium protein

                         

                        80.6

                        PBANKA_141930

                        1.8

                        1.6

                        PF3D7_0303900

                        19

                        conserved Plasmodium protein

                        SP, phosphatidyl-ethanolamine-binding protein PEBP domain

                        23.2

                        PBANKA_040250

                        2.2

                        1.8

                        PF3D7_1103500

                        0

                        CPW-WPC family protein

                        SP, CPW-WPC domain

                        67.3

                        PBANKA_094340

                        1.3

                        2.3

                        PF3D7_1115200

                        0

                        SET domain protein, SET7

                        Chromatin regulator

                        94.3

                        PBANKA_093250

                        2.5

                        2.3

                        PF3D7_1207700

                        0

                        blood stage antigen 41–3 precursor

                        SP

                        43.4

                        PBANKA_060620

                        2.5

                        2.8

                        PF3D7_0904200

                        1

                        conserved Plasmodium protein

                        SP, 1 TM

                        34

                        PBANKA_041720

                        1.9

                        3.2

                        PF3D7_1362600

                        0

                        conserved Plasmodium protein

                        Interacts with splicing factor 3B subunit 2

                        32.1

                        PBANKA_113860

                        3.4

                        3.5

                        P, total number of peptides identified in P. falciparum and P. berghei gametocytes [9, 12, 13, 22]; DOZI and CITH, log2 values of transcript abundance WT compared to DOZI-KO, or WT compared to CITH KO [20].

                        We used real-time RT-PCR analyses to confirm the transcript changes in gametocytes during activation for a subset of the identified genes and to determine, if these genes become up- or down-regulated in their expression during gametocyte activation. Total RNA was isolated from immature (stages III and IV) and mature (stage V) gametocytes of P. falciparum strain NF54 and from activated gametocytes at 30 min post-activation (p.a.). Furthermore, we isolated RNA from mixed asexual blood stages of the gametocyte-less P. falciparum strain F12. Initially, the synthesized cDNA of each sample was tested for its stage-specificity by diagnostic RT-PCR, using primers for the asexual blood stage gene ama-1[24, 25] and for the gametocyte-specific gene pfccp2[26]. The tests confirmed that no pfccp2 transcript was present in the F12 sample, while no ama-1 expression was detected in the purified gametocyte samples of strain NF54 (Figure 2A, left). The absence of ama-1 signals in the gametocyte samples and the absence of pfccp2 in the F12 asexual blood stage sample further showed that these were devoid of any contamination by genomic DNA (gDNA). An additional test for gDNA contamination was performed by using primers specific for the gene hdac1 (histone deacetylase 1; PF3D7_0925700). In all parasite samples, i.e. in samples of F12 asexual blood stages as well as in NF54 immature, mature and activated gametocytes, hdac1 transcript was present (Figure 2A, right), as shown by diagnostic RT-PCR. In sample preparations lacking reverse transcriptase, on the other hand, no hdac1-specific PCR bands were detected.
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-256/MediaObjects/12864_2012_4956_Fig2_HTML.jpg
                        Figure 2

                        Changes in transcript expression levels during the formation and activation of P. falciparum gametocytes. (A) Diagnostic RT-PCR demonstrated the presence of stage-specific transcript in the asexual blood stages of the gametocyte-less strain F12 (F12 ABS) as well as in immature (stage III/IV) and mature (stage V) non-activated gametocytes (GC) and in gametocytes at 30 min p.a. in vitro. ama-1, marker for ABS; pfccp2, marker for GC. Transcript of gene hdac1 was detected in all stages investigated, while no hdac1-specific PCR product was detected in samples lacking reverse transcriptase (RT). (B) Real-time RT-PCR analysis showed changes of transcript expression of 27 of the SSH-identified genes between ABS, immature and mature non-activated GCs and in GCs at 30 min p.a. in vitro. Transcript expression levels were calculated by the 2-ΔCt method; the threshold cycle number (Ct) was normalized with the Ct of the gene encoding for seryl tRNA synthetase as reference.

                        We chose 27 genes from the SSH analysis for comparison of transcript abundance, with representatives from all ontology groups. The gene pfs16 was selected as an internal control, because it is known to be highly expressed in gametocytes throughout development, while it is absent in the gametes [7, 27, 28]. Furthermore two sexual stage-specific genes, actin II and pfs25, served as external controls. Pfs25 is expressed in vesicular structures during gametocyte maturation and relocates to the surface of macrogametes following activation. Pfs25 is subsequently present on the parasite surface until the ookinete stage [16, 2932]. Actin II is a sexual stage-specific actin isomer, and in P. berghei actin II was reported to play a role during microgametogenesis [33, 34]. In addition, the gene su α5, encoding for subunit (SU) α5 of the α-ring of the proteasome core particle [35], was included in the investigations.

                        Transcript expression levels were measured via real-time RT-PCR and calculated by the 2-ΔCt method [36, 37] in which the threshold cycle number (Ct), was normalized to the Ct of the endogenous control gene encoding for P. falciparum seryl tRNA synthetase (PF3D7_0717700) as reference gene [38, 39]. Transcript levels with 2-ΔCt values below 0.5 were considered negligible. Real-time RT-PCR revealed transcription in mature gametocytes for 22 out of the 27 genes. Out of these, 15 genes showed increased transcript expression in gametocytes compared to asexual blood stage parasites (Figure 2B).

                        Seven genes were identified, i.e. PF3D7_0827800 (set3), Pf3D7_1246200 (actin I), PF3D7_1218800 (psop17), PF3D7_1239400, PF3D7_0704100, PF3D7_0417400, and PF3D7_1321000, for which transcript levels were increased in activated gametocytes as compared to asexual blood stages, immature gametocytes and non-activated gametocytes (Figure 2B), indicating that these genes may play an important role downstream of gametocyte activation in the mosquito midgut. SET3 was previously described to accumulate in male gametocytes, where it contributes to a prompt entry and execution of S/M phases during microgametogenesis [40]. SET domains are assigned to chromatin dynamics and are often found in histone methyltransferases, thus they play a role in the epigenetic control of gene regulation. The P. falciparum genome encodes for at least nine SET-domain-containing proteins which exhibit five different types of substrate specificities [41].

                        Actin I was described as part of the plasmodial motor complex (reviewed in [42]) and was recently also reported to be present in gametocytes [33, 43]. Furthermore, PSOP17 (putative secreted ookinete protein 17) was previously reported to be expressed in mature gametocytes of P. falciparum[9, 12] and in ookinetes of P. berghei[44]. In addition, transcript expression of the two control genes, PF3D7_1031000 (pfs25) and PF3D7_1412500 (actin II) was strong in gametocytes compared to asexual blood stage parasites and increased during gametocyte activation (Figure 2B).

                        Eight genes had an increased transcript expression in gametocytes compared to asexual blood stage parasites, and transcript levels either remained constant or decreased following activation. These include PF3D7_0302100 (clk4), PF3D7_1115200 (set7), PF3D7_0422300 (α-tubulin II), PF3D7_1103500 (encoding for a CPW-WPC protein), PF3D7_406200 (pfs16), PF3D7_1457000 (spp), PF3D7_0817600 (encoding for a MAC/PF domain, here termed PPLP6) and PF3D7_1225600 (Figure 2B). The mRNA splicing kinase CLK4 (also termed SRPK1) is expressed in the asexual blood stages and gametocytes of P. falciparum[45, 46] and a gene knock-out of the orthologous protein in P. berghei failed to exflagellate upon gametocyte activation [47]. The gene set7 encodes for one of the nine SET-domain-containing proteins (see above). Transcript of set7 is destabilized in the absence of DOZI and CITH (compare with Table 1), pointing at a translational repression of set7 in gametocytes and a role in epigenetic control mechanisms of the ookinete.

                        Pfs16 is a transmembrane protein of the gametocyte PVM [12, 27, 48, 49]. After the PVM has ruptured during the egress of the activated gametocyte from the host cell, Pfs16 is not detectable any longer in the sexual stage parasites [7] (see below), explaining the decrease of transcript following activation. Furthermore, α-tubulin II represents a tubulin isoform that is expressed in asexual blood stage parasites, in gametocytes and in microgametes [5052]. Targeted gene modification studies in P. berghei indicated that α-tubulin II plays an important role for microgametogenesis [53]. The fact that α-tubulin II transcript levels decrease during gametogenesis lets us conclude that the α-tubulin II filaments needed for the formation of the microgametes are already present in the mature gametocytes prior to activation.

                        The presenilin-like signal peptide peptidase (SPP) was previously reported to be involved in merozoite invasion and cleavage of the erythrocyte cytoskeletal protein band 3 [54]. A recent study, however, disagreed with these findings, and reported that SPP is an ER-resident protease required for growth of the erythrocytic stages [55]. SPP is considered a potential drug target of liver and blood stage parasites [5557]. The combined data indicate that SPP is present in the liver, blood and gametocyte stages, pointing to a role of SPP in multiple life-cycle stages of Plasmodium.

                        The gene product of PF3D7_0817600 comprises a MAC/PF domain, similar to the MAC/PF domains of the previously described plasmodial perforin-like proteins PPLP1-5 [58] (reviewed in [8]). We therefore termed the protein PPLP6 in this study. Furthermore, the gene product of PF3D7_1103500 is a member of a family of nine secreted proteins with cysteine-rich CPW-WPC domains [59] (reviewed in [60]). The CPW-WPC domain is a conserved domain of about 61 residues in length that exhibits six well-conserved cysteine residues and six well-conserved aromatic sites. The functions and life cycle expression patterns of the plasmodial CPW-WPC proteins are hitherto not known.

                        Three genes were predominantly expressed in the asexual blood stages and show a resurgence in transcript expression in gametocytes during maturation, i.e. PF3D7_1238900 (pk2), PF3D7_1136400 (encoding for a tetratrico peptide repeat region; TPR), and PF3D7_1021700. The calcium/calmodulin-dependent protein kinase PK2 was hitherto only investigated in the asexual blood stages of P. falciparum[61, 62] and protein expression in gametocytes has not yet been investigated. The gene product of PF3D7_1136400 comprises a TPR domain, which is known to mediate protein-protein interactions and the assembly of multiprotein complexes (reviewed in [63]), but the function of the plasmodial TPR domain protein is not yet known. While transcript levels of PF3D7_1136400 increase during gametocyte development, a drop following activation was observed.

                        For four genes, PF3D7_0818900 (hsp70-1), PF3D7_080700 (su α6), PF3D7_0807500 (gap50), and PF3D7_1444800 (encoding for fructose 1,6 bisphosphate aldolase, FBPA) expression was high in asexual blood stages and decreased during gametocyte development and following activation (Figure 2B). SU α6 is a component of the plasmodial proteasome, a proteolytic complex composed of more than 33 SUs that is responsible for the degradation and recycling of ubiquitinated proteins. As an external control we thus investigated the transcript levels of another α-ring component, su α5, and revealed similar changes in the transcript levels of su α5 in asexual blood stage parasites and gametocytes before and after activation (Figure 2B).

                        The genome of P. falciparum encodes for a variety of chaperones, including heat shock proteins (HSPs) of the HSP70, HSP90 and DnaJ/HSP40 families (reviewed in [64]). HSP70-1 was previously described to be located in the cytoplasm and nucleus of the parasite blood stages [65] as well as in the PV, pointing to a role in protein transport to the erythrocyte [66]. Another proposed function of HSP70-1 includes the protein trafficking to the apicoplast [67, 68], indicating that the chaperone has several essential functions and is important for multiple life-cycle stages. It remains to be elucidated, if other of the identified HSPs have more specific functions during parasite transmission from human to mosquito. Interestingly in this context, HSP90 belongs to a family of evolutionarily conserved chaperones which has been suggested as a capacitor for morphological evolution because reduction of its function results in phenotypic variation in Drosophila[69]. In addition, HSP90 displays multiple roles in stress adaptation and development, including spermatogenesis, oogenesis and embryogenesis in insects [70].

                        The transmembrane protein GAP50 is part of the IMC of the parasite invasive stages, including the ookinete [42, 71]. Here, it links myosin with the outer membrane of the IMC and thus contributes to gliding motility of the parasite. Recently, GAP50 was also described as a component of the gametocyte IMC [43, 72, 73], which appears to be important for the stability of the crescent-shaped cell. Noteworthy, transcript of the P. berghei orthologous protein was shown to co-precipitate with DOZI and CITH (G.R. Mair, unpublished observations), indicating that gap50 is translationally repressed in gametocytes. Thus, the release of the translational repression and onset of protein synthesis during gametocyte activation might cause the detected decrease in the gap50 transcript level.

                        FBPA is an enzyme of glycolysis, and in Plasmodium is also reported to be part of the motor complex, here linking TRAP to actin I [71, 74]. Noteworthy in this context, during gametocytogenesis, malaria parasites shift from glycolysis towards mitochondrial respiration [75], which might explain the decrease in FBPA expression in gametocytes compared to asexual blood stage parasites.

                        Transcript expression for five genes, PF3D7_0825800 (vsp9), PF3D7_0215400 (encoding for a WD40 motif), PF3D7_1351700 (alv6), PF3D7_1033200 (etramp10.2), and PF3D7_0207700 (sera4), is negligible in gametocytes. The gene PF3D7_0815800 encodes for a homolog to the yeast vacuolar sorting protein VSP9 with a predicted function in protein-protein interactions, while PF3D7_0215400 encodes for a protein with a WD40 motif. WD-repeat proteins are a large family found in all eukaryotes, implicated in a variety of functions ranging from signal transduction and transcription regulation to cell cycle control and apoptosis. The WD40 motifs act as a site for protein-protein interaction, and proteins containing WD40 repeats are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins (reviewed in [76]). WD-repeat proteins of P. falciparum have hitherto been described as receptors for protein kinase C, and as components of the myosin-driven motor complex [77, 78]. Furthermore, ALV6 is a member of the alveolin family, which comprises seven proteins in P. falciparum, associated with the membranous sacs of the IMC [79]. The fact that ALV6 expression decreases during gametocytogenesis is fairly surprising, considering that gametocytes possess an IMC (see above). It has to be elucidated, if the expression of any other alveolin is up-regulated in gametocytes.

                        The early transcribed membrane proteins (ETRAMPs) are proteins of the plasmodial PVM [80, 81]. In P. falciparum the proteins were shown to form complexes with the PVM protein exported protein 1 (EXP-1) [82]. To date, ETRAMP10.2 transcripts were found in P. falciparum trophozoites and the mixed asexual stages and the liver stages of P. yoelii[80, 81]. Because EXP-1 is also present in the gametocyte PVM [7], the expression of some of the ETRAMPs in these stages can be expected. It thus remains to be elucidated, if the other two SSH-identified ETRAMPs, ETRAMP2 and ETRAMP4, might play a specific role for gametocytes. Expression of serine repeat antigen SERA4 in the asexual blood stages has previously been described [83]. The SERA family comprises nine proteins with functions in asexual blood stage growth and host cell egress (reviewed in [8, 84]).

                        For selected genes we subsequently investigated the expression changes on the protein level. Antibodies against the proteins PK2, CLK-4, actin I and actin II, GAP50, proteasome SU α5, Pfs16, Pfs25 and PPLP6 were used to immunolabel the respective proteins in samples of P. falciparum F12 asexual blood stages, non-activated NF54 gametocytes and gametocytes at 30 min p.a. via indirect immunofluorescence assay (Figure 3A). Asexual blood stage parasites were highlighted by MSP-1 labeling; gametocytes were highlighted by labeling of Pfs25 or Pfs230, respectively. The abundance of the respective proteins in gametocytes before and after activation was quantified by measuring the average fluorescence signal intensity of a total number of 20 plotted cells per setting (Figure 3B). A significant up-regulation in the expression of PK2, actin II and Pfs25 following gametocyte activation was confirmed (Figure 3A, B). While PK2 is also expressed in asexual blood stage parasites, actin II and Pfs25 cannot be detected in these stages. On the other hand, CLK4, proteasome SU α5, and PPLP6 were detected in asexual blood stage parasites and gametocytes during maturation, but the proteins were down-regulated in gametocytes at 30 min p.a. (Figure 3B). While labeling of CLK4 and SU α5 revealed a homogenous expression of these proteins in the parasite cytoplasm and nucleus, PPLP6-labeling exhibited a punctuated expression (Figure 3A). Noteworthy, the PPLP6 labeling disappeared after the gametes have fully egressed from the enveloping erythrocyte. A role of the perforin PPLP1 was assigned to rupturing the PVM during host cell egress by the Apicomplexan parasite Toxoplasma gondii[85], and a recent study reported the involvement of PPLP2 in the lysis of the erythrocyte membrane during the exflagellation of male P. berghei gametocytes [86]. We thus hypothesize that the plasmodial PPLP6 might also play a role in the rupture of the enveloping membranes of the activated gametocytes, explaining its disappearance during gametogenesis. Similarly, the PVM-based protein Pfs16 was not present in gametocytes at 30 min p.a., since at this time point the parasites have egressed from the enveloping erythrocyte and the PVM was destroyed [7]. Furthermore, in agreement with the real-time RT-PCR results, actin I is present in both, asexual blood stages and gametocytes, and a minor up-regulation in the activated gametocytes was detected (Figure 3A, B).
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-256/MediaObjects/12864_2012_4956_Fig3_HTML.jpg
                        Figure 3

                        Changes in protein expression during formation and activation of P. falciparum gametocytes. (A) Immunofluorescence assays, using specific antibodies, detected the proteins of interest (in green) in asexual blood stage (ABS) parasites, in gametocytes (GC) and in gametocytes at 30 min p.a. (aGC). The parasite stages were highlighted with antibodies against stage-specific markers (in red; MSP1 for ABS; Pfs230 or Pfs25 for GCs and aGCs). Nuclei were highlighted by Hoechst nuclear stain (in blue). Bar, 5 μm. (B) Diagram showing the average signal intensity of the immunolabeled proteins in gametocytes before and at 30 min p.a. in vitro. Measurements were performed on 20 plotted cells per setting via quantitative confocal microscopy. Mean ± SD. *p < 0.05;**p < 0.01; ***p < 0.001 (student’s t-test).

                        Interestingly, we demonstrated a slight up-regulation in the protein expression of GAP50 at 30 min p.a., although a decrease in transcript levels was described during this process. This phenomenon can be explained by the fact that translationally repressed gap50 transcript is present in the non-activated gametocytes (see above), and that the release of repression during activation leads to rapid translation and in consequence to a loss in transcript abundance and an increase in protein abundance. It is worth mentioning in this context, that we recently showed a relocation of GAP50 from the gametocyte IMC to the plasma membrane during gametogenesis. Once on the gamete surface, GAP50 binds the human complement regulator protein factor H from the blood meal, which enables the extracellular parasite to prevent lysis by the human complement [43].

                        In summary we demonstrate that the majority of genes, which are regulated in their expression levels during gametocyte activation, have functions assigned to signaling, cell cycle and gene regulation, and proteostasis, or that they are cytoskeletal or cell surface proteins. The regulated expression of proteins involved in signaling and cell cycle control is on the one hand important for the signaling pathways that result in the induction of gametogenesis, once temperature drop and XA are perceived by the parasite [47] (reviewed in [6, 87]; on the other hand gametocyte activation releases the parasites from cell cycle arrest, subsequently leading to three rounds of rapid DNA replication during microgametogenesis [88].

                        Furthermore, induction of gametogenesis following gametocyte activation results on the one hand in the destruction of the gametocyte IMC and relocation of cytoskeletal proteins [7, 33, 34, 72, 73], on the other hand in a drastic turn-over of surface proteins (reviewed in [6]). The molecular changes in the composition of cytoskeletal and surface proteins are important for stage conversion during gametogenesis and for adaptation of the parasite to the insect host and also promote the development of the parasite midgut stages, like the ookinete. The changes in protein expression during parasite transmission from human to insect further explain the importance of chaperones and assembly proteins. The proteasome on the other hand appears to play only a minor role during gametogenesis. We hypothesize that proteins which lost their function, once the parasite has entered the mosquito vector, are only degraded after the gametes have developed and after fertilization has occurred, thus after the phase of rapid stage conversions has finished.

                        Noteworthy, the SSH analysis identified a number of unknown proteins with transmembrane domains and/or signal peptides, and for six of them, high transcript levels were shown in mature and activated gametocytes. For three of the genes, expression in the gametocytes was higher as compared to asexual blood stage parasites, and these genes might encode for proteins important for gametocyte development or gametogenesis.

                        Conclusions

                        The here presented data on the changes in the P. falciparum transcriptome during gametogenesis provide first insights into the regulated gene expression occurring while the malaria parasite passes the initial phase of transmission to the mosquito. Our findings form the basis for further studies on genes important for malaria transmission and support the identification of proteins involved in the development of the mosquito midgut stages, which might lead to the discovery of new transmission blocking targets.

                        Methods

                        Antibodies

                        The following antibodies were used in this study: mouse antisera against Pfs16 and Pfs230 (kindly provided by Kim Williamson, Loyola University Chicago), proteasome SU α5 [35] and actin II [34], as well as rabbit antisera against GAP50 (kindly provided by Julian Rayner, Sanger Institute England; [89]), Pfs230 (against the immunogenic region C) [90], MSP-1 and Pfs25 (BEI Resources, Manassas). Mouse antisera against actin I, PPLP6, PK2, and CLK4 were generated for this study (see below).

                        Parasite culture

                        Gametocyte-less F12 strain and gametocyte-forming NF54 isolate of P. falciparum were cultivated in vitro in RPMI 1640 medium complemented with 10% heat-inactivated human serum as described [91]. As soon as stage II gametocytes started to emerge in the NF54 culture, the culture medium was supplemented with 50 mM N-acetyl glucosamine (GlcNac) for approximately 5 days to kill the asexual blood stages [92]. The gametocyte culture was then maintained in normal culture medium without GlcNac until immature (stage III and IV) or mature stage V gametocytes were harvested and enriched by Percoll gradient purification [93]. The mature stage V gametocytes were divided into two aliquots, and one aliquot was subsequently incubated in gametogenesis activating solution (1.67 mg/ml glucose; 8 mg/ml NaCl; 1 mg/ml Tris–HCl (pH 8.2) [94], containing 100 μM XA for 30 min at RT.

                        Construction of a subtracted cDNA library using the SSH method

                        Total RNA was isolated from Percoll-enriched stage V gametocytes before and at 30 min p.a., using Trizol reagent (Invitrogen, Karlsruhe, Germany). Subsequently, mRNA was isolated from total RNA using the Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions, and the SSH analysis was conducted as previously described [21, 95] using the PCR-Select cDNA Subtraction Kit from Clontech (Mountain View, CA, USA), according to the manufacturer’s protocol. Briefly, 220 ng of purified mRNA were reverse-transcribed into cDNA using a cDNA synthesis primer; subsequently double-stranded cDNA was generated and digested with RsaI. The double-stranded cDNA from activated gametocytes was ligated in separate aliquots to adaptor 1 or adaptor 2R and were denaturated at 98°C for 90 s and then hybridized at 68°C for 8 h with a 30-fold excess of double-stranded cDNA of non-activated gametocytes. Subsequently, both samples were mixed together with a 10-fold excess of freshly denatured double-stranded cDNA from non-activated gametocytes and hybridized at 68°C for 16 h. The sample was then subjected to two rounds of suppression PCR with PCR-primer 1 and nested primers supplied with the kit. PCR amplifications were performed in a total volume of 25 μl using a PCR cycler (Biometra, Göttingen, Germany) with a heated lid and the 5Prime PCR Extender System (5Prime, Hamburg, Germany). An initial adapter extension at 75°C for 5 min was followed by a denaturation step at 93°C for 30 s and by 27 cycles of denaturation at 93°C for 15 s, annealing at 66°C for 30 s, and extension at 72°C for 90 s. A final 7-min 72°C step was added to allow complete extension of the products. The secondary PCR was performed with nested primer 1 and 2R on 1 μl of the primary PCR products for 15 cycles with an initial denaturation step at 93°C for 1 min, followed by denaturation at 93°C for 15 s, annealing at 68°C for 30 s and extension at 72°C for 90 s, plus a final extension step at 72°C for 7 min. Resulting PCR products of the secondary subtractive PCR were purified using the NucleoSpin Extract II kit (Macherey Nagel, Düren, Germany), ligated into the pGEM-T easy vector (Promega, Mannheim, Germany) and transformed into NEB 5-alpha competent E. coli (New England BioLabs, Frankfurt, Germany). The library was plated on 2×YT agar plates containing 100 μg/ml ampicillin and incubated at 37°C for 16 h.

                        Colony PCR, sequencing and computer analysis of cDNA sequence data

                        A preliminary colony PCR screen on 20 colonies was performed with vector-specific primers, i.e. T7-promotor: 5- TAATACGACTCACTATAGGG-3 and SP6: 5-ATTTAGGTGACACTATAG- 3 (purchased from Thermo electron, Waltham, MA, USA), using the following conditions: denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 15 s, annealing at 43°C for 20 s, and extension at 72°C for 60 s. A final 7-min 72°C step was added to allow complete extension of the products. The colony PCR showed that 75% of clones contained an insert in the vector. Subsequently, 288 randomly picked clones were screened for the presence of the vector. Plasmid isolation of 141 positively screened colonies was performed with the Fast Plasmid Mini kit (Eppendorf, Hamburg, Germany) and the inserts were sequenced (GATC Biotech, Konstanz, Germany; sequences listed in the Additional file 1: Table S1). Sequences were used to identify similar sequences of the National Center for Biotechnology Information databases using BLASTX program (BLASTX 2.2.13; http://​www.​ncbi.​nlm.​nih.​gov/​BLAST/​) and to predict signal sequences, transmembrane regions and features, using the PlasmoDB program (http://​www.​plasmodb.​org) [96].

                        RNA isolation and real-time RT-PCR

                        Total RNA was isolated from the mixed asexual F12 cultures, enriched immature NF54 gametocytes (stage III and IV), non- activated mature gametocytes and gametocytes at 30 min p.a. using the Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RNA preparations were treated with RNAse-free DNAse I (Qiagen) to remove gDNA contamination, followed by phenol/chloroform extraction and ethanol precipitation. All RNA samples had A260/A280 ratios higher than 2.1. Two μg of each total RNA sample were used for cDNA synthesis using the SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturer’s instructions. Controls without reverse transcriptase were used to investigate potential gDNA contamination by diagnostic PCR using hdac1-specific primers. RNA quality was further verified for contamination by monitoring transcripts of stage-specific genes, i.e. ama1 and pfccp2, by diagnostic RT-PCR (for primer sequences, see Additional file 2: Table S2).

                        Primers for real-time RT-PCR were designed using the Primer 3 software (http://​frodo.​wi.​mit.​edu/​primer3/​) and were initially tested on gDNA in conventional PCR with the same conditions subsequently used for real-time RT-PCR (for primer sequences, see Additional file 2: Table S2). Primers for the reference gene encoding for seryl tRNA synthetase were obtained from [38]. The PCR products were subsequently separated by agarose gel electrophoresis. Primers with high specificity were further validated by testing amplification efficiency on 10-fold dilutions of gDNA using real-time RT-PCR followed by melt curve analysis. Primers with low specificity, efficiency and poor melt curves were redesigned.

                        Real-time RT-PCR measurements were performed using the Bio-Rad CFX96 Real-Time Detection System. Reactions were prepared in triplicate in a total volume of 20 μl using the maxima SyBR green qPCR master mix (Thermo Scientific, Bonn, Germany) with 20 ng of cDNA and primer concentrations of 0.3 μM. The following PCR cycling conditions were used: an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 60 s, followed by a final extension at 95°C for 10 s and a final melt-curve analysis of 55-95°C. Controls without template and without reverse transcriptase were included in all real-time RT-PCR experiments. Transcript expression levels were calculated by the 2-ΔCt method [36, 37], using the endogenous control gene encoding for P. falciparum seryl tRNA synthetase (PF3D7_0717700) as reference [38, 39]. Control samples lacking reverse transcriptase during cDNA synthesis were routinely included during each run and exhibited an average threshold cycle number of 39.

                        Recombinant protein expression and production of mouse antisera

                        Recombinant proteins for CLK4, PK2, and actin I were expressed as fusion proteins with a GST-tag using the pGEX 4 T-1 vector (Amersham Biosciences, Freiburg, Germany), PPLP6 recombinant proteins were expressed as maltose binding protein-tagged fusion proteins using vector pIH902 (kindly provided by K. Williamson, Chicago; [90]). DNA was amplified by PCR using gene-specific primers (for primer sequences, see Additional file 2: Table S2). Recombinant proteins were expressed in BL21 (DE3) RIL cells according to the manufacturer’s protocol (Invitrogen). Recombinant proteins were isolated as either inclusion bodies (PK2, actin I) as described [16] or affinity-purified (CLK4 and PPLP6) using glutathione sepharose (GE Healthcare, Munich, Germany) and amylose resin (New England Biolabs), respectively, according to the manusfacturers´protocols. Specific immune sera were generated by immunizing 6-week-old female NMRI mice (Charles River Laboratories, Sulzfeld) with 100 μg recombinant protein emulsified in Freund’s incomplete adjuvant (Sigma-Aldrich), followed by a boost after 4 weeks. Sera were collected 10 days after the boost. The housing and handling of the animals followed the guidelines of the animal welfare committee of Lower Franconia.

                        Indirect immunofluorescence assay and signal strength quantification

                        Asexual F12 blood stage parasites, non-activated NF54 gametocytes and gametocytes at 30 min p.a. were air dried on glass slides and fixed for 10 min in −80°C methanol. For membrane permeabilization and blocking of non-specific binding, fixed cells were incubated for 30 min in 0.01% saponin/0.5% BSA/PBS and 1% neutral goat serum (Sigma-Aldrich) in PBS. Preparations were then successively incubated for 1.5 h each at 37°C with the primary antibody diluted in 0.01% saponin/0.5% BSA/PBS. Binding of primary antibody was visualized using fluorescence-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Alexa Fluor 488 or Fluor 596; Molecular Probes, Karlsruhe, Germany) diluted in 0.01% saponin/0.5% BSA/PBS. Nuclei were highlighted by incubating the specimens with Hoechst nuclear stain 33342 (Molecular Probes) for 1 min. Cells were mounted with anti-fading solution AF2 (Citifluor Ltd., Leicester, UK) and sealed with nail varnish. Digital images were taken using a Leica TCS SP5 confocal laser scanning microscope and processed using Adobe Photoshop CS software. For quantitative evaluation of the average signal intensity, digital images of 20 randomly selected mature and activated gametocytes were taken using a Zeiss LSM 510 confocal laser scanning microscope under the same optimal laser scanning microscopy settings for each antibody. The average fluorescence signal intensity of each cell, as indicated by a line edging the respective cell, was measured by LSM 510 image software and recorded.

                        Declarations

                        Acknowledgements

                        We thank Meike Lietzow and Ludmilla Sologub for technical assistance. This study was supported by the Emmy Noether program of the Deutsche Forschungsgemeinschaft and the MALSIG consortium of the EU 7th framework program (GP), by an FCT grant PTDC/BIA-BCM/105610/2008 (GRM), as well as by a grant from the Hessian Ministry of Science and Art via the LOEWE-research focus “Insect Biotechnology” (AV). CJN received a fellowship by the Graduate School of Life Sciences of the University of Würzburg.

                        Authors’ Affiliations

                        (1)
                        Research Center for Infectious Diseases, University of Würzburg
                        (2)
                        Faculdade de Medicina da Universidade de Lisboa, Instituto de Medicina Molecular
                        (3)
                        Institute of Molecular Biotechnology, RWTH Aachen University
                        (4)
                        Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Bioresources Project Group
                        (5)
                        Institute of Phytopathology and Applied Zoology, University of Gießen

                        References

                        1. World Health Organization (WHO): World Malaria Report. Geneva, Switzerland: World Health Organization; 2011. http://​www.​who.​int/​malaria/​world_​malaria_​report_​2011/​en/​
                        2. Greenwood BM, Fidock DA, Kyle DE, Kappe SHI, Alonso PL, Collins FH, Duffy PE: Malaria: progress, perils, and prospects for eradication. J Clin Invest 2008, 118:1266–1276.PubMedView Article
                        3. Hammarton TC, Mottram JC, Doerig C: The cell cycle of parasitic protozoa: potential for chemotherapeutic exploitation. In Prog Cell Cycle Res 3rd edition. Edited by: Meijer L, Jézéquel A, Roberge M. 2003, 91–101.
                        4. Alano P: Plasmodium falciparum gametocytes: still many secrets of a hidden life. Mol Microbiol 2007, 66:291–302.PubMedView Article
                        5. Pradel G: Proteins of the malaria parasite sexual stages: expression, function and potential for transmission blocking strategies. Parasitology 2007, 134:1911–1929.PubMedView Article
                        6. Kuehn A, Pradel G: The coming-out of malaria gametocytes. J Biomed Biotechnol 2010, 2010:976827.PubMedView Article
                        7. Sologub L, Kuehn A, Kern S, Przyborski J, Schillig R, Pradel G: Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito. Cell Microbiol 2011, 13:897–912.PubMedView Article
                        8. Wirth CC, Pradel G: Molecular mechanisms of host cell egress by malaria parasites. Int J Med Microbiol 2012, 302:172–178.PubMedView Article
                        9. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ: A proteomic view of the Plasmodium falciparum life cycle. Nature 2002, 419:520–526.PubMedView Article
                        10. Lasonder E, Ishihama Y, Andersen JS, Vermunt AMW, Pain A, Sauerwein RW, Eling WMC, Eling WMC, Hall N, Waters AP, Stunnenberg HG, Mann M: Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 2002, 419:537–542.PubMedView Article
                        11. Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De la Vega P, Holder AA, Batalov S, Carucci DJ, Winzeler EA: Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 2003, 301:1503–1508.PubMedView Article
                        12. Silvestrini F, Bozdech Z, Lanfrancotti A, Di Giulio E, Bultrini E, Picci L, deRisi JL, Pizzi E, Alano P: Genomewide identification of genes upregulated at the onset of gametocytogenesis in Plasmodium falciparum . Mol Biochem Parasitol 2005, 143:100–110.PubMedView Article
                        13. Silvestrini F, Lasonder E, Olivieri A, Camarda G, van Schaijk B, Sanchez M, Younis Younis S, Sauerwein R, Alano P: Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum . Mol Cell Proteomics 2010, 9:1437–1448.PubMedView Article
                        14. Wass MN, Stanway R, Blagborough AM, Lal K, Prieto JH, Raine D, Sternberg MJ, Talman AM, Tomley F, Yates J, Sinden RE: Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls. Parasitology 2012, 139:1131–1145.PubMedView Article
                        15. Vaughan JA, Noden BH, Beier JC: Sporogonic development of cultured Plasmodium falciparum in six species of laboratory-reared Anopheles . AmJTrop Med Hyg 1994, 51:233–243.
                        16. Scholz SM, Simon N, Lavazec C, Dude MA, Templeton TJ, Pradel G: PfCCp proteins of Plasmodium falciparum : gametocyte-specific expression and role in complement-mediated inhibition of exflagellation. Int J Parasitol 2008, 38:327–340.PubMedView Article
                        17. Simon N, Scholz SM, Moreira CK, Templeton TJ, Kuehn A, Dude MA, Pradel G: Sexual stage adhesion proteins form multi-protein complexes in the malaria parasite Plasmodium falciparum . J Biol Chem 2009, 284:14537–14546.PubMedView Article
                        18. Kuehn A, Simon N, Pradel G: Family members stick together: multi-protein complexes of malaria parasites. Med Microbiol Immunol 2010, 199:209–226.PubMedView Article
                        19. Mair GR, Braks JAM, Garver LS, Wiegant JCAG, Hall N, Dirks RW, Khan SM, Dimopoulos G, Janse CJ, Waters AP: Regulation of sexual development of Plasmodium by translational repression. Science 2006, 313:667–669.PubMedView Article
                        20. Mair GR, Lasonder E, Garver LS, Franke-Fayard BMD, Carret CK, Wiegant JCAG, Dirks RW, Dimopoulos G, Janse CJ, Waters AP: Universal features of post-transcriptional gene regulation are critical for Plasmodium zygote development. PLoS Pathog 2010, 6:e1000767.PubMedView Article
                        21. Altincicek B, Vilcinskas A: Comparative analysis of septic injury-inducible genes in phylogenetically distant model organisms of regeneration and stem cell research, the planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris . Front Zool 2008, 5:6.PubMedView Article
                        22. Khan SM, Franke-Fayard B, Mair GR, Lasonder E, Janse CJ, Mann M, Waters AP: Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell 2005, 121:675–687.PubMedView Article
                        23. Sebastian S, Brochet M, Collins MO, Schwach F, Jones ML, Goulding D, Rayner JC, Choudhary JS, Billker O: A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs. Cell Host Microbe 2012, 12:9–19.PubMedView Article
                        24. Peterson MG, Marshall VM, Smythe JA, Crewther PE, Lew A, Silva A, Anders RF, Kemp DJ: Integral membrane protein located in the apical complex of Plasmodium falciparum . Mol Cell Biol 1989, 9:3151–3154.PubMed
                        25. Narum DL, Thomas AW: Differential localization of full-length and processed forms of PF83/AMA-1 an apical membrane antigen of Plasmodium falciparum merozoites. Mol Biochem Parasitol 1994, 67:59–68.PubMedView Article
                        26. Pradel G, Hayton K, Aravind L, Iyer LM, Abrahamsen MS, Bonawitz A, Mejia C, Templeton TJ: A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito. J Exp Med 2004, 199:1533–1544.PubMedView Article
                        27. Eksi S, Williamson KC: Protein targeting to the parasitophorous vacuole membrane of Plasmodium falciparum . Eukaryot Cell 2011, 10:744–752.PubMedView Article
                        28. Eksi S, Morahan BJ, Haile Y, Furuya T, Jiang H, Ali O, Xu H, Kiattibutr K, Suri A, Czesny B, Adeyemo A, Myers TG, Sattabongkot J, Su XZ, Williamson KC: Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development. PLoS Pathog 2012, 8:e1002964.PubMedView Article
                        29. Rener J, Graves PM, Carter R, Williams JL, Burkot TR: Target antigens of transmission blocking immunity on gametes of Plasmodium falciparum . J Exp Med 1983, 158:976–981.PubMedView Article
                        30. Vermeulen AN, Ponnudurai T, Beckers PJA, Verhave JP, Smits MA, Meuwissen JH: Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission blocking antibodies in the mosquito. J Exp Med 1985, 162:1460–1476.PubMedView Article
                        31. Vermeulen AN, Van Deursen J, Brakenhoff RH, Lensen THW, Ponnudurai T, Meuwissen JH: Characterisation of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures. Mol Biochem Parasitol 1986, 20:155–163.PubMedView Article
                        32. Quakyi IA, Carter R, Rener J, Kumar N, Good MF, Miller LH: The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies. J Immunol 1987, 139:4213–4217.PubMed
                        33. Deligianni E, Morgan RN, Bertuccini L, Kooij TW, Laforge A, Nahar C, Poulakakis N, Schüler H, Louis C, Matuschewski K, Siden-Kiamos I: Critical role for a stage-specific actin in male exflagellation of the malaria parasite. Cell Microbiol 2011, 13:1714–1730.PubMedView Article
                        34. Rupp I, Sologub L, Williamson KC, Scheuermayer M, Reininger L, Doerig C, Eksi S, Kombila DU, Frank M, Pradel G: Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut. Cell Res 2011, 21:683–696.PubMedView Article
                        35. Aminake MN, Arndt HD, Pradel G: The proteasome of malaria parasites: a multi-stage drug target for chemotherapeutic intervention? Int J Parasitol Drugs Drug Resistance 2012, 2:1–10.View Article
                        36. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001, 25:402–408.PubMedView Article
                        37. Sugden K, Tichopad A, Khan N, Craig IW, D’Souza UM: Genes within the serotonergic system are differentially expressed in human brain. BMC Neurosci 2009, 10:50.PubMedView Article
                        38. Salanti A, Staalsoe T, Lavstsen T, Jensen ATR, Sowa MP, Arnot DE, Hviid L, Theander TG: Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy associated malaria. Mol Microbiol 2003, 49:179–191.PubMedView Article
                        39. Wang CW, Mwakalinga SB, Sutherland CJ, Schwank S, Sharp S, Hermsen CC, Sauerwein RW, Theander TG, Lavstsen T: Identification of a major rif transcript common to gametocytes and sporozoites of Plasmodium falciparum . Malar J 2010, 9:147.PubMedView Article
                        40. Pace T, Olivieri A, Sanchez M, Albanesi V, Picci L, Siden Kiamos I, Janse CJ, Waters AP, Pizzi E, Ponzi M: Set regulation in asexual and sexual Plasmodium parasites reveals a novel mechanism of stage-specific expression. Mol Microbiol 2006, 60:870–882.PubMedView Article
                        41. Cui L, Fan Q, Miao J, Cui L: Histone lysine methyltransferases and demethylases in Plasmodium falciparum . Int J Parasitol 2008, 38:1083–1097.PubMedView Article
                        42. Baum J, Gilberger T, Frischknecht F, Meissner M: Host-cell invasion by malaria parasites: insights from Plasmodium and Toxoplasma . Trends Parasitol 2008, 24:557–563.PubMedView Article
                        43. Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G: Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 2013, 13:29–41.PubMedView Article
                        44. Ecker A, Bushell ES, Tewari R, Sinden RE: Reverse genetics screen identifies six proteins important for malaria development in the mosquito. Mol Microbiol 2008, 70:209–220.PubMedView Article
                        45. Dixit A, Singh PK, Sharma GP, Malhotra P, Sharma P: PfSRPK1, a novel splicing-related kinase from Plasmodium falciparum . J Biol Chem 2010, 285:38315–38323.PubMedView Article
                        46. Agarwal S, Kern S, Halbert J, Przyborski JM, Baumeister S, Dandekar T, Doerig C, Pradel G: Two nucleus-localized CDK-like kinases with crucial roles for malaria parasite erythrocytic replication are involved in phosphorylation of splicing factor. J Cell Biochem 2011, 112:1295–1310.PubMedView Article
                        47. Tewari R, Straschil U, Bateman A, Böhme U, Cherevach I, Gong P, Pain A, Billker O: The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe 2010, 8:377–387.PubMedView Article
                        48. Baker DA, Daramola O, McCrossan MV, Harmer J, Targett GA: Subcellular localisation of Pfs16, a Plasmodium falciparum gametocyte antigen. Parasitology 1994, 108:129–137.PubMedView Article
                        49. Bruce MC, Carter RN, Nakamura K, Aikawa M, Carter R: Cellular location and temporal expression of the Plasmodium falciparum sexual stage antigen Pfs16. Mol Biochem Parasitol 1994, 65:11–22.PubMedView Article
                        50. Rawlings DJ, Fujioka H, Fried M, Keister DB, Aikawa M, Kaslow DC: Alpha-tubulin II is a male-specific protein in Plasmodium falciparum . Mol Biochem Parasitol 1992, 56:239–250.PubMedView Article
                        51. Fennell BJ, Al-shatr ZA, Bell A: Isotype expression, post-translational modification and stage-dependent production of tubulins in erythrocytic Plasmodium falciparum . Int J Parasitol 2008, 38:527–539.PubMedView Article
                        52. Schwank S, Sutherland CJ, Drakeley CJ: Promiscuous expression of α-tubulin II in maturing male and female Plasmodium falciparum gametocytes. PLoS One 2010, 5:e14470.PubMedView Article
                        53. Kooij TWA, Franke-Fayard B, Renz J, Kroeze H, van Dooren MW, Ramesar J, Augustijn KD, Janse CJ, Waters AP: Plasmodium berghei α-tubulin II: A role in both male gamete formation and asexual blood stages. Mol Biochem Parasitol 2005, 141:16–26.View Article
                        54. Li X, Chen H, Oh SS, Chishti AH: A Presenilin-like protease associated with Plasmodium falcipa rum micronemes is involved in erythrocyte invasion. Mol Biochem Parasitol 2008, 158:22–31.PubMedView Article
                        55. Marapana DS, Wilson DW, Zuccala ES, Dekiwadia CD, Beeson JG, Ralph SA, Baum J: Malaria parasite signal peptide peptidase is an ER-resident protease required for growth but not for invasion. Traffic 2012, 13:1457–1465.PubMedView Article
                        56. Li X, Chen H, Bahamontes-Rosa N, Kun JF, Traore B, Crompton PD, Chishti AH: Plasmodium falciparum signal peptide peptidase is a promising drug target against blood stage malaria. Biochem Biophys Res Commun 2009, 380:454–459.PubMedView Article
                        57. Parvanova I, Epiphanio S, Fauq A, Golde TE, Prudêncio M, Mota MM: A small molecule inhibitor of signal peptide peptidase inhibits Plasmodium development in the liver and decreases malaria severity. PLoS One 2009, 4:e5078.PubMedView Article
                        58. Kaiser K, Camargo N, Coppens I, Morrisey JM, Vaidya AB, Kappe SH: A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites. Mol Biochem Parasitol 2004, 133:15–26.PubMedView Article
                        59. Saeed M, Roeffen W, Alexander N, Drakeley CJ, Targett GA, Sutherland CJ: Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte. PLoS One 2008, 3:e2280.PubMedView Article
                        60. Sutherland CJ: Surface antigens of Plasmodium falciparum gametocytes–a new class of transmission-blocking vaccine targets? Mol Biochem Parasitol 2009, 166:93–98.PubMedView Article
                        61. Ward P, Equinet L, Packer J, Doerig C: Protein kinases of the human malaria parasite Plasmodium falciparum: The kinome of a divergent eukaryote. BMC Genomics 2004, 5:79.PubMedView Article
                        62. Kato K, Sudo A, Kobayashi K, Tohya Y, Akashi H: Characterization of Plasmodium falciparum protein kinase 2. Mol Biochem Parasitol 2008, 162:87–95.PubMedView Article
                        63. Allan R, Ratajczak T: Versatile TPR domains accommodate different modes of target protein recognition and function. Cell Stress Chaperones 2011, 16:353–367.PubMedView Article
                        64. Pavithra SR, Kumar R, Tatu U: Systems analysis of chaperone networks in the malaria parasite Plasmodium falciparum . PLoS Comp Biol 2007, 2:1701–1715.
                        65. Kappes B, Suetterlin BW, Hofer-Warbinek R, Humar R, Franklin RM: Two major phosphoproteins of Plasmodium falciparum are heat shock proteins. Mol Biochem Parasitol 1993, 59:83–94.PubMedView Article
                        66. Nyalwidhe J, Lingelbach K: Proteases and chaperones are the most abundant proteins in the parasitophorous vacuole of Plasmodium falciparum -infected erythrocytes. Proteomics 2006, 6:1563–1573.PubMedView Article
                        67. Foth BJ, Ralph SA, Tonkin CJ, Struck NS, Fraunholz M, Roos DS, Cowman AF, McFadden GI: Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum . Science 2003, 299:705–708.PubMedView Article
                        68. Ramya TN, Karmodiya K, Suolia A, Surolia N: 15-deoxyspergualin primarily targets the trafficking of apicoplast proteins in Plasmodium falciparum . J Biol Chem 2007, 282:6388–6397.PubMedView Article
                        69. Rutherford SL, Lindquist S: Hsp90 as a capacitor formorphological evolution. Nature 1998, 396:336–342.PubMedView Article
                        70. Knorr E, Vilcinskas A: Post-embryonic functions of HSP90 in Tribolium castaneum include the regulation of compound eye development. Dev Genes Evol 2011, 221:357–362.PubMedView Article
                        71. Baum J, Richard D, Healer J, Rug M, Krnajski Z, Gilberger TW, Green JL, Holder AA, Cowman AF: A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. J Biol Chem 2006, 281:5197–5208.PubMedView Article
                        72. Dearnley MK, Yeoman JA, Hanssen E, Kenny S, Turnbull L, Whitchurch CB, Tilley L, Dixon MWA: Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes. J Cell Sci 2012, 125:2053–2063.PubMedView Article
                        73. Kono M, Herrmann S, Loughran NB, Cabrera A, Engelberg K, Lehmann C, Sinha D, Prinz B, Ruch U, Heussler V, Spielmann T, Parkinson J, Gilberger TW: Evolution and architecture of the inner membrane complex in asexual and sexual stages of the malaria parasite. Mol Biol Evol 2012, 29:2113–2132.PubMedView Article
                        74. Buscaglia CA, Penesetti D, Tao M, Nussenzweig V: Characterization of an aldolase-binding site in the Wiskott-Aldrich syndrome protein. J Biol Chem 2006, 281:1324–1331.PubMedView Article
                        75. van Dooren GG, Stimmler LM, McFadden GI: Metabolic maps and functions of the Plasmodium mitochondrion. FEMS Microbiol Rev 2006, 30:596–630.PubMedView Article
                        76. Stirnimann CU, Petsalaki E, Russell RB, Müller CW: WD40 proteins propel cellular networks. Trends Biochem Sci 2010, 35:565–574.PubMedView Article
                        77. Madeira L, DeMarco R, Gazarini ML, Verjovski-Almeida S, Garcia CR: Human malaria parasites display a receptor for activated C kinase ortholog. Biochem Biophys Res Commun 2003, 306:995–1001.PubMedView Article
                        78. Foth BJ, Goedecke MC, Soldati D: New insights into myosin evolution and classification. Proc Natl Acad Sci U S A 2006, 103:3681–3686.PubMedView Article
                        79. Gould SB, Tham WH, Cowman AF, McFadden GI, Waller RF: Alveolins, a new family of cortical proteins that define the protist infrakingdom Alveolata. Mol Biol Evol 2008, 25:1219–1230.PubMedView Article
                        80. Spielmann T, Fergusen DJ, Beck HP: Etramps, a new Plasmodium falciparum gene family coding for developmentally regulated and highly charged membrane proteins located at the parasite-host cell interface. Mol Biol Cell 2003, 14:1529–1544.PubMedView Article
                        81. MacKellar DC, Vaughan AM, Aly AS, DeLeon S, Kappe SH: A systematic analysis of the early transcribed membrane protein family throughout the life cycle of Plasmodium yoelii . Cell Microbiol 2011, 13:1755–1767.PubMedView Article
                        82. Spielmann T, Gardiner DL, Beck HP, Trenholme KR, Kemp DJ: Organization of ETRAMPs and EXP-1 at the parasite-host cell interface of malaria parasites. Mol Microbiol 2006, 59:779–794.PubMedView Article
                        83. Miller SK, Good RT, Drew DR, Delorenzi M, Sanders PR, Hodder AN, Speed TP, Cowman AF, de Koning-Ward TF, Crabb BS: A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle. J Biol Chem 2002, 277:47524–47532.PubMedView Article
                        84. Rosenthal PJ: Falcipains and other cysteine proteases of malaria parasites. Adv Exp Med Biol 2011, 712:30–48.PubMedView Article
                        85. Kafsack BF, Pena JD, Coppens I, Ravindran S, Boothroyd JC, Carruthers VB: Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 2009, 323:530–533.PubMedView Article
                        86. Deligianni E, Morgan RN, Bertuccini L, Wirth CC, Silmon de Monerri NC, Spanos L, Blackman MJ, Louis C, Pradel G, Siden-Kiamos I: A perforin-like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes. Cell Microbiol 2013. in press
                        87. Doerig C, Billker O, Haystead T, Sharma P, Tobin AB, Waters NC: Protein kinases of malaria parasites: an update. Trends Parasitol 2008, 24:570–577.PubMedView Article
                        88. Janse CJ, van der Klooster PF, van der Kaay HJ, van der Ploeg M, Overdulve JP: DNA synthesis in Plasmodium berghei during asexual and sexual development. Mol Biochem Parasitol 1986, 20:173–182.PubMedView Article
                        89. Jones ML, Kitson EL, Rayner JC: Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex. Mol Biochem Parasitol 2006, 147:74–84.PubMedView Article
                        90. Williamson KC, Keister DB, Muratova O, Kaslow DC: Recombinant Pfs230, a Plasmodium falciparum gametocyte protein, induces antisera that reduce the infectivity of Plasmodium falciparum to mosquitoes. Mol Biochem Parasitol 1995, 75:33–42.PubMedView Article
                        91. Ifediba T, Vanderberg JP: Complete in vitro maturation of Plasmodium falciparum gametocytes. Nature 1981, 294:364–366.PubMedView Article
                        92. Fivelman QL, McRobert L, Sharp S, Taylor CJ, Saeed M, Swales CA, Sutherland CJ, Baker DA: Improved synchronous production of Plasmodium falciparum gametocytes in vitro. Mol Biochem Parasitol 2007, 154:119–123.PubMedView Article
                        93. Kariuki MM, Kiaira JK, Mulaa FK, Mwangi JK, Wasunna MK, Martin SK: Plasmodium falciparum : purification of the various gametocyte developmental stages from in vitro-cultivated parasites. AmJTrop Med Hyg 1998, 59:505–508.
                        94. Eksi S, Stump A, Fanning SL, Shenouda MI, Fujioka H, Williamson KC: Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis. Mol Microbiol 2002, 44:1507–1516.PubMedView Article
                        95. Altincicek B, Vilcinskas A: Analysis of the immune-inducible transcriptome from microbial stress resistant, rat-tailed maggots of the drone fly Eristalis tenax . BMC Genomics 2007, 8:326.PubMedView Article
                        96. Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Miller JA, Nayak V, Pennington C, Pinney DF, Roos DS, Ross C, Stoeckert CJ: PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res 2009, 37:539–543.View Article

                        Copyright

                        © Ngwa et al.; licensee BioMed Central Ltd. 2013

                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                        Advertisement