RSpred, a set of Hidden Markov Models to detect and classify the RIFIN and STEVOR proteins of Plasmodium falciparum

Abstract

Background: Many parasites use multicopy protein families to avoid their host's immune system through a strategy called antigenic variation. RIFIN and STEVOR proteins are variable surface antigens uniquely found in the malaria parasites Plasmodium falciparum and P. reichenowi. Although these two protein families are different, they have more similarity to each other than to any other proteins described to date. As a result, they have been grouped together in one Pfam domain. However, a recent study has described the sub-division of the RIFIN protein family into several functionally distinct groups. These sub-groups require phylogenetic analysis to sort out, which is not practical for large-scale projects, such as the sequencing of patient isolates and meta-genomic analysis.

Results: We have manually curated the rif and stevor gene repertoires of two Plasmodium falciparum genomes, isolates DD2 and HB3. We have identified 25% of mis-annotated and ~30 missing rif and stevor genes. Using these data sets, as well as sequences from the well curated reference genome (isolate 3D7) and field isolate data from Uniprot, we have developed a tool named RSpred. The tool, based on a set of hidden Markov models and an evaluation program, automatically identifies STEVOR and RIFIN sequences as well as the sub-groups: A-RIFIN, B-RIFIN, B1-RIFIN and B2-RIFIN. In addition to these groups, we distinguish a small subset of STEVOR proteins that we named STEVOR-like, as they either differ remarkably from typical STEVOR proteins or are too fragmented to reach a high enough score. When compared to Pfam and TIGRFAMs, RSpred proves to be a more robust and more sensitive method. We have applied RSpred to the proteomes of several P. falciparum strains, P. reichenowi, P. vivax, P. knowlesi and the rodent malaria species. All groups were found in the P. falciparum strains, and also in the P. reichenowi parasite, whereas none were predicted in the other species.

Conclusions: We have generated a tool for the sorting of RIFIN and STEVOR proteins, large antigenic variant protein groups, into homogeneous sub-families. Assigning functions to such protein families requires their subdivision into meaningful groups such as we have shown for the RIFIN protein family. RSpred removes the need for complicated and time consuming phylogenetic analysis methods. It will benefit both research groups sequencing whole genomes as well as others working with field isolates. RSpred is freely accessible via http://www.ifm.liu.se/bioinfo/.

Figure 1

Schematic representation of RIFIN and STEVOR proteins. Schematic representation of A-RIFIN, B-RIFIN and STEVOR proteins (not to scale). Aa: amino acid; Indel: insertion/deletion; SP?: potential signal peptide; V1: first variable region; PEXEL: Plasmodium export element; C1: first conserved region; TM?: questionable transmembrane region; V2: second variable region; TM: highly probable transmembrane region; C2: second conserved region.

Figure 2

Missing genes and corrections from automatic predictions. (A) Artemis visualization of Broad Institute Annotations (BIA) and manual annotations. Manually curated genes are represented in blue, proteins in red. BIA genes and proteins are in green. (B) Artemis Comparison Tool visualization of the BLAST results between HB3 supercontig vs. HB3 automatically predicted genes. A missing gene, labelled "New gene" in this figure, is visible between two predicted genes. Blue and red represent BLAST hits between the (upper) supercontig and the (lower) predicted genes. BLAST hits are coloured according to the strand on which the hit is found (red for the Crick strand, blue for the Watson strand).

Figure 3

Phylogenetic tree of RIFIN and STEVOR proteins. The phylogenetic tree shows the segregation of low scoring STEVOR proteins, which we call STEVOR-like. RIFIN sub-groups have been collapsed to improve readability of the tree. Bootstrap support (in %), after 500 replicates, is only shown for values >60%.

Figure 4

Evaluation program. The evaluation program compares the scores of all HMMs in database searches. Depending on each sequence's scores, the program designates the sequence as "False", i.e. neither STEVOR nor RIFIN, or as belonging to one of the groups: STEVOR, STEVOR-like, RIFIN, A-RIFIN, B-RIFIN, B1-RIFIN or B2-RIFIN.