Genome-wide detection of serpentine receptor-like proteins in malaria parasites

Abstract

Serpentine receptors comprise a large family of membrane receptors distributed over diverse organisms, such as bacteria, fungi, plants and all metazoans. However, the presence of serpentine receptors in protozoan parasites is largely unknown so far. In the present study we performed a genome-wide search for proteins containing seven transmembrane domains (7-TM) in the human malaria parasite Plasmodium falciparum and identified four serpentine receptor-like proteins. These proteins, denoted PfSR1, PfSR10, PfSR12 and PfSR25, show membrane topologies that resemble those exhibited by members belonging to different families of serpentine receptors. Expression of the pfsrs genes was detected by Real Time PCR in P. falciparum intraerythrocytic stages, indicating that they potentially code for functional proteins. We also found corresponding homologues for the PfSRs in five other Plasmodium species, two primate and three rodent parasites. PfSR10 and 25 are the most conserved receptors among the different species, while PfSR1 and 12 are more divergent. Interestingly, we found that PfSR10 and PfSR12 possess similarity to orphan serpentine receptors of other organisms. The identification of potential parasite membrane receptors raises a new perspective for essential aspects of malaria parasite host cell infection.

Figure 1. Summary of the strategy used to search the P. falciparum genome for serpentine receptor-like genes.

The genomic sequences available from PlasmoDB (release 4.3) were analyzed in three steps. In the first step, we used a local ORF-finder program to predict intronless ORFs longer than 500 nucleotides (green box) and searched these ORFs for seven transmembrane 7-TM proteins by using the TMHMM 2.0 transmembrane prediction program. In the second step, P. falciparum manually annotated genes (pink box) available in PlasmoDB were analyzed using TMHMM2.0 to select 7- and 8-TM proteins. In the third step, we show the automatically predicted sequences used in the work of Inoue et al. , which were retrieved by the authors from PlasmoDB (blue boxes – Glimmer, Gene finder and FullPhat are gene-finding programs) followed by their analysis with HMMTOP transmembrane prediction program to select 7-TM proteins (gray box). As our third step we reanalyzed the 69 proteins described by Inoue et al. , now using TMHMM2.0. The redundant 7- and 8-TM proteins retrieved by the three steps were eliminated and the sequences were subsequently classified according to the criteria set listed on the bottom of the figure. The graphic on the left shows the absolute number of sequences for each criteria class during the analysis, emphasizing the four P. falciparum serpentine receptor-like proteins. SP, signal peptide; AS, anchor signal; AP, apicoplast signal.

Figure 2. Characteristics of serpentine receptor-like proteins (SRs) of diverse malaria parasites species.

Each one of the four P. falciparum serpentine receptors sequences are grouped with its orthologs and differently colored: yellow, PF11_0321 (PfSR1); blue, PFL0765w (PfSR10); green, PFD1075w (PfSR12); pink, MAL7P1.64 (PfSR25). Identification numbers for most of the non-falciparum sequences are not available since these genes are not annotated in the databases; therefore, we point out the contig sequences from where they were retrieved with the nucleotides that comprise the selected gene. Scale-sized diagrams of genomic structure of serpentine receptor genes (Gene Model) and of predicted protein features are shown. Transmembrane domains predicted by TMHMM2.0 are shown in dark-green boxes, with the number of amino acids in the beginning and in the end of the 7-TM core. Potential signal peptides predicted by SignalP3.0 are shown as blue boxes with the potential cleavage sites. In the N-terminal domains of some of these receptors there are repetitive stretches of negatively charged residues (Glu/Asp-rich domains) or positively charged residues (Lys-rich domains). The position of potential N-glycosylation sites is pointed by red-arrows.

Figure 3. Phylogenetic tree showing the comparison of the 7-TM core of the Plasmodium serpentine receptors amino acid sequences.

Bootstrap values are shown at nodes with >50% support. (Scale bar: 0.2 amino acid substitutions per site). The bar indicates the number of amino acids substitution per site.

Figure 4. Phylogenetic tree of the evolutionary conserved “lung 7TM receptor” family.

The tree was compiled using the aligned amino acid sequences of the 7TM conserved core region from several eukaryote organisms. Branches supporting bootstrap values >90% are in orange, >70% in green and >50% in blue (1000 replicates). Scale bar: 0.2 amino acid substitutions per site. Accession numbers shown correspond to the following databases: GenBank for non-Plasmodium members of lung 7TM receptor family; ToxoDB for T. gondii; PlasmoDB for Plasmodium species. Identification number for non-falciparum Plasmodium species are shown as contig numbers, and complete protein sequences are available in Data S2.

Figure 5. Expression of serpentine receptors in the P. falciparum intraerythrocytic cycle.

Parasites cultures were synchronized with sorbitol and total RNA extracted from six time points during the 48 h intraerythrocytic cycle. Each RNA sample, converted to cDNA, was used as a template in a Real-time PCR reaction done in triplicates. The fluorescence values for each transcript were normalized by the ones of the 18S transcript and the expression is related to the one of the late trophozoite stage (LT). Error bars represent standard errors of two independent experiments. ER – early ring, LR – late ring, ET – early trophozoite, LT – late trophozoite, ES – early schizont, LS – late schizont.