Evidence for a common role for the serine-type Plasmodium falciparum serine repeat antigen proteases: implications for vaccine and drug design

Abstract

Serine repeat antigens (SERAs) are a family of secreted "cysteine-like" proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six "serine-type" (SERA1 to SERA5 and SERA9) and three "cysteine-type" (SERA6 to SERA8) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1, SERA4, and SERA9 knockout lines were generated, while only SERA5, the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4-null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

FIG. 1.

Targeted disruption of four serine-type SERA genes. (A) Schematic representation detailing the outcome of expected integration events in SERA knockout parasite lines. Solid lines under the exons represent the location of the gene targeting regions used to clone fragments into pHH1 (SERA1), pTKΔSERA4, pTKΔSERA5, and pTKΔSERA9. The targeting regions of the coding sequence are indicated by the gray shaded regions. (B) Southern blots summarizing the result of the attempted targeted disruption of each SERA gene using restriction-digested genomic DNA from each transfected line following drug cycling and parental parasites. Genomic DNA was digested BstBI (B)/SphI (S) for SERA1, AflIII (A) for SERA4, XmnI (X) for SERA5, and BclI (Bc)/NotI/SwaI (Sw) for SERA9. The membranes were probed with each respective targeting sequence shown above (A). The positions of endogenous (E), plasmid (P), and integration-specific fragment (In) events that result from digestion with the respective enzymes and the probing of blots with the specific targeting region (gray) are indicated, and their molecular weights (in thousands) are given below each relevant blot. RE, restriction enzyme.

FIG. 2.

Western blot analysis of SERA4-null parasites. (A) Western blots of total material from synchronized late-blood-stage parasites comparing the parental D10 wild type with two clones of D10-ΔSERA4 were probed with either rabbit anti-Hsp70 polyclonal or rabbit anti-SERA4 polyclonal antibody. The antisera used to probe each membrane are indicated at the bottom of the gel. Molecular mass markers (kDa) are shown to the left of the membrane. (B and C) Western blots of total material from synchronized late-blood-stage parasites from wild-type D10 (B) or D10-ΔSERA4 (clone 1) (C) probed with rabbit anti-SERA1 to anti-SERA9 polyclonal antibodies specific to the N-terminal region of each SERA protein (lanes 1 to 9, respectively). The antiserum used to probe each membrane strip is indicated at the top of the lane. The strip probed with the prebleed pool is indicated by P. Molecular mass markers (kDa) are shown to the left of each membrane.

FIG. 3.

Disruption of SERA4 does not affect in vitro parasite growth. Shown are data from an extended growth rate assay comparing D10 wild-type parasites with the D10-ΔSERA4 (D10-ΔS4) parasite line over a 28-day period. Parasites were maintained below 5%. Error bars represent the standard deviations of mean parasitemias from three individual counts.

FIG. 4.

Higher levels of SERA5 mRNA are present in SERA4-null parasites. (A and B) SERA gene transcript expression levels in late-blood-stage D10 wild-type (A) andD10-ΔSERA4 (B) parasites measured by quantitative RT-PCR. Parasites in each parasite line were collected at 30 h postsynchronization and subsequent 4-h intervals for 20 h. All expression levels (mRNA) were normalized to the coregulated gene MSP-1. The peak mRNA expression level observed for SERA5 is indicated on the panels to illustrate the difference between D10 and D10ΔS4. (C) Change (n-fold) in gene expression levels between D10 and D10-ΔSERA4 parasite lines. This was calculated by dividing the mean expression levels of the individual values shown in B by those shown in A.

FIG. 5.

SERA loci across the Plasmodium and Theileria genera. HMM hits for the protease and C-terminal SERA domains are indicated schematically on the contigs on which they were found. Breaks in species tracks indicate discontinuities at contig ends. Where they exist, gene annotations have been indicated. When HMM matches existed on multiple short contigs in a species, the contigs were ordered according to orthology relationships with related species inferred from the phylogeny. SERA genes that have been successfully knocked out in P. falciparum are marked in red, while those refractory to deletion are in black. Where orthology is clearly discernible from the phylogeny, it is marked by vertical blocks that join species. Orthologies of P. gallinaceum and T. annulata genes are ambiguous due to a postspecies duplication. This ambiguity is represented by a bifurcation in the orthology block. The phylogeny of Cys SERA genes indicates a duplication of SERA8 present only in P. gallinaceum as well as a duplication resulting in SERA6 and SERA7 in Plasmodium spp. (excluding P. gallinaceum). Ambiguities arising from these duplications are represented by bifurcations in the orthology blocks. Where it is clearly present, the orthology of flanking sequence is also noted. Protease domains marked in green do not contain active-site mutations, whereas protease domains marked in blue contain the cysteine-to-serine active-site mutation. The presence of secondary-site mutations has been marked below the corresponding protease domain. RT-PCR expression levels (this study) and protein abundance as a percentages of SERA5 are shown above the P. falciparum (3D7) track (16, 17). Similarly, relative copy number expression measurements for P. vivax as a percentage of P. vivax SERA4 are shown above the P. vivax track (22). KO, knockout.

FIG. 6.

SERA sequences belong to one of two major phyletic groups that separate according to their catalytic residues. Colored segments that make up the inner disc delineate active-site and secondary-site mutations in SERA sequences. Colored segments that are part of the outer ring mark the clade to which sequences belong. Thick lines in the phylogeny indicate branches shared by MCMC, maximum-likelihood, and neighbor-joining topologies. Thin lines indicate branches supported by MCMC and one other method. Dashed lines indicate branches existing only in the MCMC topology. MCMC branch probabilities are given on the appropriate branches where they are less than 1. Members of the genus Theileria appear to have a single-copy SERA, the only observation to date of a SERA ortholog outside the genus Plasmodium. Radiation in SERA sequences containing the serine active-site mutation tends to be species specific, whereas radiation of cysteine SERA sequences has occurred prior to speciation. Branch lengths for SERA6 and SERA7 tend to be shorter than those of serine-type SERAs and those of SERA8.