Open Access

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

  • Che Julius Ngwa1,
  • Matthias Scheuermayer1,
  • Gunnar Rudolf Mair2,
  • Selina Kern1,
  • Thomas Brügl1,
  • Christine Clara Wirth1, 3,
  • Makoah Nigel Aminake1, 3,
  • Jochen Wiesner4,
  • Rainer Fischer3, 4,
  • Andreas Vilcinskas4, 5 and
  • Gabriele Pradel3Email author
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.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-256/MediaObjects/12864_2012_Article_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. falciparumGC

P. bergheimale/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. falciparumgene ID

P

Protein

Predicted feature

MW

P. bergheiortholog 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.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-256/MediaObjects/12864_2012_Article_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).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-256/MediaObjects/12864_2012_Article_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)
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa
(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

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