A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus.

Supplementary Fig. S3D: List of putative genes that have been reported to expressed during 24 hrs to 50 hrs in the liver stages (Reference no. 31) were downregulated in pbspeld ko EEF's. The plasmoDB ID of each of these genes with their functions are indicated.

Supplementary Fig. S4A
Supplementary Fig. S4A: Validating the expression of selected genes by qRT-PCR upregulated in microarray. cDNA was generated from HepG2 cultures infected with WT and pbspeld ko at 36h time point. The cDNA was used as template for analyzing the expression of indicated genes. **p value<0.005 compared to WT.

Supplementary Fig. S4B
Supplementary Fig. S4B: Validating the expression of selected genes by qRT-PCR downregulated in microarray. cDNA was generated from HepG2 cultures infected with WT and pbspeld ko at 36h time point. The cDNA was used as template for analyzing the expression of indicated genes. ***p value<0.0005 and ** p value 0.005 compared to WT. and Sanger gene data base (http://www.genedb.org/Homepage/Pberghei) were used to retrieve the PBANKA_091090 gene sequence and its Plasmodium homologs and PBANKA_030600 (P230p) gene sequence.

Generation of P. berghei ANKA WT GFP line
To achieve a successful DCO homologous recombination, sequences corresponding to fragment A and B within the P230p ORF were cloned on either ends of the GFP-hDHFR cassette using XhoI/ClaI and NotI/AscI sites respectively. The targeting construct was separated from vector backbone by digesting with XhoI/AscI and electroporated into the schizont stages. Genomic DNA was isolated from drug resistant GFP positive parasites and correct site-specific integration at fragment A and B was confirmed by primers designed at sites beyond recombination. A diagnostic PCR was performed to amplify product of 890 bp using primer set FP12/RP12 and product of 1140 bp using primer set FP13/RP13, that confirmed the correct integrations at region A and B within the p230p locus. Following limiting dilution, the clonal lines were confirmed for successful disruption of the gene by diagnostic PCR using primer set FP11/RP11 that amplified a PCR product of 869 bp only from WT genomic DNA.
The PCR product was sequenced and confirmed. The mCherry ORF with restriction sites XhoI and SpeI was cloned in pTZ57R/T vector. A 3' regulatory sequence of HSP70 was PCR amplified using forward primer, FP-5' TATACTAGTTTATTGTTCTGTACTTCTTTT3' (SpeI site underlined) and reverse primer, RP-5'ACTCCCGGGAAAATACCAATAATACCGTTT3' (XmaI site underlined) from pBC-GFP-hDHFR vector and following sequence confirmation, this fragment was cloned into the pTZ57R/T in tandem to mCherry ORF using restriction sites SpeI and SmaI/XmaI. The mCherry ORF along with HSP70 3' UTR was released from pTZ57R/T vector and cloned into pBC-GFP-hDHFR vector using XhoI and XmaI replacing GFP cassette. The vector was named pBC-mCherry-hDHFR.

Labeling and microarray hybridization
The samples for gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). Total RNA were reverse transcribed at 40°C using oligodT primer tagged to a T7 polymerase promoter and converted to double stranded cDNA. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP (Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up using Qiagen RNeasy columns (Qiagen).

Hybridization and Scanning
The labeled cRNA sample were fragmented at 60C and hybridized on to an Agilent Custom Plasmodium berghei Gene Expression Microarray 4x44k designed by Genotypic Technology Private Limited (AMADID No: 067226). Fragmentation of labeled cRNA and hybridization were done using the gene expression hybridization kit (Agilent Technologies, In situ Hybridization kit). Hybridization was carried out in Agilent's surehyb chambers at 65 C for 16 hrs. The hybridized slides were washed using Agilent gene expression wash buffers (Agilent Technologies) and scanned using the Agilent Microarray Scanner (Agilent Technologies) at 5 micron resolution. Data extraction from Images was done using Agilent Feature Extraction software.

Microarray data analysis
Feature extracted raw data was analyzed using Agilent GeneSpring GX software. Normalization of the data was done in GeneSpring GX using the 75th percentile shift method. Percentile shift normalization is a global normalization, where the locations of all the spot intensities in an array are adjusted. This normalization takes each column in an experiment independently, and computes the n th percentile of the expression values for this array, across all spots (where n has a range from 0-100 and n=75 is the median). It subtracts this value from the expression value of each entity and fold change values were obtained by comparing test samples with respect to specific control samples. Significant genes upregulated fold> 0.8 (logbase2) and downregulated <-0.8 (logbase2) in the test samples with respect to control sample were identified. Statistical student Ttest p-value among the replicates was calculated based on volcano Plot Algorithm. Differentially regulated genes were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category and pathways using Biological Analysis tool DAVID (http://david.abcc.ncifcrf.gov/).