Exploring the transcriptome of the malaria sporozoite stage

Abstract

Most studies of gene expression in Plasmodium have been concerned with asexual and/or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A BLASTX search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) < or = 10(-4)), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.

Figure 1

Quality assessment of the generated cDNA populations. cDNA blot hybridization with stage-specific probes demonstrates that stage-specific transcript representation is not altered by cDNA amplification. (A) Ethidium bromide-stained agarose gel of cDNA amplified from salivary gland sporozoites (Sg Spz) or mixed blood stages (Blood St). Note the distinct bands visible in the sporozoite preparation. (B) Hybridization to a CS probe. (C) Hybridization to an SSP2/TRAP probe. (D) Hybridization to an MSP-1 probe. Sizes are given in kb.

Figure 2

Functional classification of P. yoelii sporozoite ESTs. One hundred sixty-one unique blastx matches were classified according to their putative biological function. Refer to Table 2 for a complete list of all blastx matches.

Figure 3

Sporozoite expression of MAEBL. (A) cDNA blot showing MAEBL expression in midgut sporozoites (Mg Spz) and salivary gland sporozoites (Sg Spz). (B) Reverse transcription–PCR confirming MAEBL expression in salivary gland sporozoites. MAEBL expression is also detected in blood stages. Amplification with MSP-1-specific primers shows MSP-1 expression in blood stages. MSP-1 expression is not detected in salivary gland sporozoites. Sizes are given in base pairs (bp). (C) Localization of MAEBL by indirect immunofluorescence assay in P. yoelii salivary gland sporozoites with antisera against the carboxyl cysteine-rich region. (D) Localization of MAEBL by indirect immunofluorescence in P. yoelii midgut sporozoites with antisera against the carboxyl cysteine-rich region. Scale bar for C and D = 1 μm.

Figure 4

Alignment of SPATR and expression in sporozoites. (A) Comparison of the deduced amino acid sequences of the P. yoelii SPATR with the homologue in P. falciparum (accession no. C71611). The conserved residues of the altered TSR are underlined with a solid line. The putative signal peptides are underlined with a dashed line. Putative signal peptide cleavage sites are marked with arrowheads (▴, ▾). Conserved cysteine residues are marked with an asterisk (*). Identical residues are shaded dark gray. Conserved amino acid changes are shaded light gray, and radical changes are not shaded. (B) cDNA blot demonstrating SPATR expression in midgut sporozoites (Mg Spz) and salivary gland sporozoites (Sg Spz). Sizes are given in kb.

Figure 5

Alignment of P52 and expression in sporozoites. (A) Comparison of the deduced amino acid sequences of the P. yoelii, Py52, with the homologue in P. falciparum, Pf52. The putative signal peptides are underlined with a dashed line. Putative signal peptide cleavage sites are marked with arrowheads (▴, ▾). Conserved cysteine residues of the tandem 6-cys motifs are marked with an asterisk (*). The carboxyl-terminal hydrophobic putative membrane anchor is underlined with a solid line. Identical residues are shaded dark gray. Conserved amino acid changes are shaded light gray, and radical changes are not shaded. (B) cDNA blot demonstrating Py52 expression in midgut sporozoites (Mg Spz) and salivary gland sporozoites (Sg Spz). Sizes are given in kb.