Culturing and drug treatment
Culturing of parasites and drug treatment experiments were conducted under aseptic conditions. P. falciparum isolate 3D7 parasites were cultured in 75 cm2 flasks (Nalgene) in human erythrocytes (blood group A+) and RPMI 1640 medium supplemented with 25 mM HEPES, 0.0088% hypoxanthine, 40 ug/ml gentamycin (all Sigma) and 0.5% Albumax (Invitrogen). Parasites were maintained in continuous culture in a 10 ml volume, and were scaled up to 50 ml for drug treatments. The parasitemia and morphological forms detected in the cultures were determined and estimated by Giemsa (Fluka) stained smears under 1000× light microscopy.
Prior to morphology studies and drug treatment, cultures were synchronized with 5% D-sorbitol  for 5 min at 37°C to select for parasites in the ring stage. Sorbitol treatment was repeated two days later. A morphological analysis of the culture was performed in the presence and absence of 2 mM cyclohexylamine (corresponding IC99 value) in DMSO over a 48 h period. Smears of the cultures were prepared every 6 h post drug treatment. Fresh cultures were scaled up to 50 ml (5% hematocrit, 10% parasitemia), synchronized twice and treated with 2 mM cyclohexylamine at invasion (K. Lüersen, personal communication). Fifteen milliliters of culture was harvested at late ring/early trophozoite (18 hpi), mid trophozoite (25 hpi) and late trophozoite (30 hpi) stages. Similar volumes were harvested from untreated P. falciparum 3D7 control cultures at the same time points. Parallel drug treatments were conducted, serving as biological replicates.
Erythrocytes from harvested cultures were pelleted, washed once with 1× PBS to remove traces of culture medium, and then frozen at -80°C until RNA extractions were performed. RNA was extracted by the method described by  and quantified using the Nanodrop 1000 spectrophotometer (Thermo Scientific). The integrity was confirmed on an Agilent Bioanalyzer.
Universal Reference RNA pool (URR)
A reference pool of RNA from mixed-stage in vitro cultures was constructed. Cultures were grown to high parasitemia, without synchronisation. Twenty-four (24) cultures were harvested and flash-frozen in liquid nitrogen. RNA was extracted as described previously. Care was taken to avoid bias towards any specific life cycle stage. The percentage of spots on the microarray to which the URR hybridized was determined in order to gauge accurate expression ratios for as many spots as possible. Equal amounts of URR were labelled with either Cy3 or Cy5 and hybridized to a microarray (as described in next section). URR coverage was defined as the percentage of spots called "present" when local background-subtracted intensity of a spot exceeded the average background intensity of all spots in that channel .
cDNA synthesis and array hybridization
A reference design was employed for array hybridisation, utilising the URR pool described previously. All solvent-control and drug-treated samples were hybridised to Operon slides, along with the URR. For each time point and each untreated/treated sample, three microarray slides were processed, such that a total of eighteen slides were processed in the study. Two independent cDNA samples (biological replicates) were prepared for each untreated and drug-treated sample at each time point. One of the biological replicate cDNA samples were additionally hybridised to a third slide (representing the technical replicate). cDNA synthesis reactions of 12 μg each were set up for each sample or reference RNA in a total of 50 μl. Prior to denaturation at 70°C for 10 min, 2 μl of oligo d(T) primer (2 μg/μl) and 8 μl of random nanomer primer (0.5 μg/μl) (both New England Biolabs) were added. cDNA synthesis proceeded overnight at 42°C after the addition of an aminoallyl-dNTP mix (Fermentas), DTT, reaction buffer and 200 U of SuperScript III Reverse Transcriptase (Invitrogen). RNA was hydrolyzed by the addition of 10 μl each of 1 M NaOH and 0.5 M EDTA solutions and incubated at 65°C for 10 min. Unincorporated aminoallyl dUTP was removed by purification using the NucleoSpin Extract II Kit (Macherey-Nagel). The eluted cDNA was quantified using a Nanodrop spectrophotometer and similar quantities of test and reference cDNA (typically 2 μg) were dried down to a volume of 2.5 μl in a vacuum drier. Five microliters of a 0.2 M Na2CO3 buffer, pH 9.0, was added to the cDNA and mixed well. Cy3 (sample) or Cy5 (reference) (2.5 μl) (GE Healthcare) ester was added and the coupling allowed to proceed for 2 h in the dark at room temperature. Uncoupled dyes were removed by purification with the RNeasy Mini Kit (Qiagen). Probe labelling was estimated on a Nanodrop. Similar amounts of labelled probe (typically 100 picomoles) were hybridized to the microarray slides. Slides were prehybridized with a solution containing 5× SSC (from 20× SSC: 3 M NaCl, 1.5 M sodium citrate, pH 7.0), 0.1% SDS and 0.1 mg/mL BSA, prewarmed to 42°C. Prehybridization proceeded in 50 ml Falcon tubes, incubated at 42°C for 45-60 min. Following prehybridization, the slides were washed in three washes of 0.1× SSC before centrifugation for 5 min at 200 × g. The combined Cy-labelled probes (4 μl) were mixed with 36 μl of hybridization buffer (Operon) for a total volume of 40 μl. The probes were denatured at 95°C for 5 min, and subsequently applied to the slide under Lifterslips (Erie Scientific Company). The microarray slides were enclosed in ArrayIt (Telechem International) hybridization chambers and submersed in a heated water bath and incubated for 16-20 h at 42°C. The slides were successively washed in low stringency (2× SSC, 0.5% SDS; heated to 42°C), medium stringency (1× SSC) and high stringency (0.1× SSC) wash buffers for 5 min each. Slides were then centrifuged (5 min, 200 × g). Scanning was performed with an Axon GenePix 4000B scanner (Molecular Devices).
Microarray data analysis
GenePix results (gpr) files were generated using GenePix 6.0 (Molecular Devices) software, without normalization. For clustering analyses, results files were normalized with DNMAD (Diagnosis and Normalization for MicroArray Data)  using print-tip loess. The normalized values were subsequently downloaded and analyzed with the Multiexperiment Viewer (MeV) in the TM4 software suite . Hierarchical Clustering (HCL, average linkage)  was performed to estimate technical and biological variation between samples and at which point cytostasis most likely occurred for comparative purposes in downstream analyses. Intensity data for individual slides were imported into LIMMA (linear models for microarray data) in the R computing environment . Pre- and post-normalization diagnostic plots were performed using MARRAY. Data from each microarray slide was normalized using print-tip loess. Data between microarrays was normalized using rquantile normalisation. Pearson correlations were computed in ExCel to estimate variation between technical and biological replicates. Spots excluded from slide correlations and normalisation were those weighted by the limma script or flagged in the Genepix results file (gpr). Additionally, spots termed Alien, Empty, Null and Operon Use Only were excluded from the correlation analyses. These spots were similarly excluded for correlations between untreated and treated samples at each time point following normalisation. Results from biological and slide replicates within each of the time points were collated, and linear models were computed to contrast gene expression between time points. A two-fold change in gene expression was used as cut-off, in conjunction with correction for false discovery (false discovery rate (FDR) = 5%). Normalised data was deposited in the Gene Expression Omnibus (GEO) database, number GSE18075. Analysis of differentially expressed genes was performed in MADIBA ( Micro Array Data Interface for Biological Annotation ).
Quantitative Real-Time PCR
cDNA samples prepared for microarray analyses were utilized for quantitative real-time PCR (RT-qPCR) validation of global expression analysis. Briefly, cDNA (with 500 nM of each primer and 1× Power SYBR Green PCR Master Mix (Applied Biosystems)) was amplified in 96 well plates in an Applied Biosystems 7500 Real-Time PCR cycler. Primers were designed using Primer3 Plus  with the qPCR module activated. Primer sequences used for polyamine as well as housekeeping genes are available on request. Following enzyme activation at 95°C for 10 min, the cDNA was amplified for 40 cycles (95°C for 15 s, 60°C for 60 s). A melt curve was performed to ascertain primer specificity. Relative expression was calculated using Applied Biosystems Sequence Detection Software.
Protein extraction and two-dimensional electrophoresis
Parasites from harvested cultures were released from erythrocytes by saponin lysis  and the resulting pellet washed five times in ice-cold wash medium (RPMI 1640 medium supplemented with 25 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 20 mM sodium bicarbonate and 40 μg/ml gentamycin; all Sigma). A pellet from a 15 ml culture was resuspended in 500 μl lysis buffer (8 M urea, 2 M thiourea, 2% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid), 65 mM dithiothreitol (DTT) (all Sigma) and 1% IPG buffer pH 3-10 (Amersham)). The sample was freeze-thawed five times in liquid nitrogen and then sonicated on ice (Bandelin Sonopuls sonicator, at power level 65%) using five 10 sec bursts with 10 sec of cooling in between. Care was taken to prevent warming of the sample, which introduces artifacts resulting from urea breakdown and protein carbamylation. The supernatant was clarified by ultracentrifugation at 30 000 × g for 30 min at 4°C . Protein concentration was assayed in duplicate by the RCDC protein assay kit (BioRad) against a standard curve of bovine serum albumin (BSA, BioRad). Isoelectric focusing (IEF) of total protein, 300 μg (analytical gels) and 450 μg (preparative gels), was performed as described by . After IEF, gel strips were equilibrated and cysteine residues reduced and subsequently alkylated by treatment with 1% DTT and 2.5% iodoacetamide (Sigma) in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS and bromophenol blue) for 10 min each prior to second dimension analysis. Two-dimensional electrophoresis (2DE) was subsequently performed on 12.5% polyacrylamide gels at constant current of 40 mA per gel for three hours on the Protean II Cell system (BioRad). After 2DE, analytical gels were stained with SyproRuby fluorescent stain (BioRad) and preparative gels with PageBlue Protein stain (Fermentas) according to the suppliers' protocol. Gels were imaged with Pharos FX imager (BioRad) and differential analysis performed using PDQuest™ (V 8.0.1; BioRad) software on a set of three independent gels per experimental condition. Data comparison was carried out against the untreated protein profile at 18 hpi. Spot intensity was normalized using the "local regression" model to compensate for non-expression-related variations in spot intensity. Images were analyzed for quantitative differences, using two-fold difference in expression with a t-test significance level of 95% applied.
Protein spots of interest were manually excised from PageBlue stained gels. Low abundant proteins were excised from more than one gel and gel plugs pooled to increase protein yield for mass spectrometry (MS) analysis. The number of gels that spots were excised from depended on the intensity of the protein spot. Protein spots were subjected to in-gel trypsin digestion and the resulting peptide mixture was purified using Stage tips® (Proxeon) prior to MS analysis.
MS data was acquired using a Q-STAR Elite Q-TOF mass spectrometer (Applied Biosystems) with either a MALDI (matrix-assisted laser desorption ionization) or ESI (electron spray ionization) source depending on which source was installed at the time of sample submission for MS analysis.
Samples were co-crystallized with 5 mg/ml α-cyano-4-hydroxy cinnamic acid (CHCA, Brüker Daltonics) matrix in 50% acetonitrile (ACN, Sigma), 0.1% trifluoro-acetic acid (TFA, Sigma) and spectra acquired with a 337 nm nitrogen laser operated at 20 Hz. Protein identification was based on peptide mass fingerprint (PMF) and sequencing data. The instrument was calibrated using Brüker's peptide calibration standard II using peaks at 757.39 and 2093.08 m/z. PMF spectra were acquired in positive ion mode using a range of 600-2500 m/z. MS/MS data was obtained by collision induced dissociation (CID), using argon as collision gas, from the 50 highest parent ions in the corresponding PMF spectra.
Samples were loaded in Proxeon NanoES capillaries and ionized using IonSpray voltage of 900-1200 V. The instrument was calibrated using Glu-Fibrinopeptide B (Sigma-Aldrich) using fragment ions 246.15 and 1285.54 m/z. PMF spectra were acquired in positive ion mode using a range of 450 - 1500 m/z. MS/MS data obtained via Information Dependent Acquisition (IDA) method where doubly and triply charged parent ions were selected for fragmentation by collision induced dissociation (CID), using nitrogen as collision gas.
Protein identification was performed by searching the NCBI database using the Mascot search engine. The following parameters were used for database searches with MALDI-QTOF PMF and sequencing data: monoisotopic mass, peptide charge +1, 50 ppm mass accuracy, trypsin as digesting enzyme with 1 missed cleavage allowed, carbamidomethylation of cysteine as a fixed modification, oxidation of methionine as allowable variable modification. For PMF data positive identifications equaled or exceeded the minimum significant score of 70. By MS/MS analysis, positive identifications required a minimum of two unique peptides, with at least one peptide having a significant ion score. Similar parameters were used to search ESI-QTOF PMF and sequencing data with only peptide charge set to +2 and +3. Exploratory analysis of the differentially expressed proteins was performed in MADIBA .
Polyamine metabolite analysis
Synchronized P. falciparum 3D7 cultures (two biological replicates) were treated with 2 mM cyclohexylamine as described for the transcriptome analyses. Parasites in mid-trophozoite (25 hpi) stages were selected for polyamine extraction. Triplicate samples of 20 ml parasite culture (~20% parasitemia) were harvested, pelleted by centrifugation at 2000 × g for 5 min (Boeco) and re-suspended in 4× equivalent pellet volumes of PBS. These wash steps were repeated four times in total. Subsequent cell counting was performed accurately in quadruplicate with a Neubauer Cell counting chamber (Weber).
PBS-washed cell pellets (1 ml) were subjected to protein precipitation using 5% (v/v) perchloric acid (PCA), followed by vigorous vortexing, incubation at 4°C (overnight) and subsequently precipitated by centrifugation at 16000 × g for 10 min at 4°C (Eppendorf). Polyamines were detected using benzoylation as previously reported with slight modification [50, 51]. Five hundred μl of the 5% v/v PCA extract or 500 μl polyamine standard was added to glass tubes with pure 5% PCA as blank. An internal standard (1 nmol of 1,7-diaminoheptane (Sigma-Aldrich)) was included in the P. falciparum extracts to normalize for benzoylation efficiency. One ml 2.0 M NaOH was added to each reaction, vortexed after which 5 μl of benzoyl chloride (Sigma-Aldrich) was added. The reactions were incubated at 37°C for 30 min and stopped by adding 1 ml chloroform (<99%) to allow the separation of organic derivatized molecules. The tubes were centrifuged at 1500 × g (Medifuge) for 10 min, the aqueous phases were removed to a new glass tube containing additional 1 ml chloroform for a second separation step as above. The chloroform layers were pooled and evaporated under N2 gas. One ml 0.1 M NaOH was added to the white precipitate containing tubes and these were incubated overnight at room temperature. Subsequently, 1 ml chloroform was added to the aqueous phases, centrifuged at 1500 × g for 10 min and the chloroform phases evaporated under N2. Five hundred μl of 60% MeOH was used to redissolve benzoylated products. The methanol solutions were passed through 0.2 μm Minisart RC4 (Sartorius) HPLC-certified filters.
HPLC separation was performed using a WATERS System (Waters Corporation) with a 250 mm × 4.0 mm Luna C18(2) 5 μm reverse-phase (RP) column (Phenomenex). A Guard-Pak™ Precolumn steel housing (Waters Corporation) with μBondapak C18 HPLC pre-column inserts (Waters Corporation) was connected in-line. Isocratic solvent conditions were maintained using 60% MeOH:ddH2O at flow rate of 1 ml/min. All solvents were filtered using 0.2 μm cellulose acetate filters (Sartorius) in Millipore™ housing system (Millipore) and degassed prior to use. The HPLC pump system consisted of a WATERS 600 Controller (Waters Corporation) with a 600 Series WATERS Pump (Waters Corporation) and a WATERS 712 WISP Autosampler. UV absorbance was measured at 229 nm using a WATERS 996 Photodiode array (Waters Corporation). Peak integration was performed using the Empower 2 Software Edition (2006, Waters Corporation) with Apex™ Trac functionality.