The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Abstract

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Fig. 1

The rodent malaria active-site serine SERA subfamily. A. Primary structure of Plasmodium SERA proteins with active site serines (SERAser). The central papain-like cysteine protease domains are boxed in grey. Overall amino acid sequence identities of the P. berghei SERA1 (EU917224) and SERA2 (EU917225) sequences are indicated as percentages of identical residues compared with the P. falciparum SERA5 (PFB0340c). Fragments used for protein expression, purification and antibody production are indicated by bars. B. Partial conservation of the catalytic residues of the papain family within the central serine protease domain. P. berghei SERA1 and SERA2 and P. falciparum SERA5 are shown together with papain and a P. berghei cysteine family SERA protein, PbECP1/SERA5. The strictly conserved amino acid residues are boxed in grey, and the putative active-site serine or cysteine is shown in bold and boxed in black. Additional residues of the active site are marked with an ‘o’. Note that the catalytic histidine is replaced by a methionine. In contrast, the carboxy-terminal asparagine and the amino-terminal glutamine, which form the oxyanione hole, are conserved.

Fig. 2

Expression profiling of P. berghei SERAser. A. RT-PCR analysis of SERA1 and SERA2 expression in mosquito midgut (mg) and salivary gland (sg) sporozoites, mid (24 h) and late (48 h, 72 h) liver stages, liver stage merosomes (mer) and blood stage merozoites (BS mer). The merozoite and sporozoite-specific transcripts, MSP1 and TRAP, and the constitutive GAPDH transcript were added as controls. cDNAs were synthesized from mRNA in the presence (+) or absence (−) of reverse transcriptase. Genomic DNA (gDNA) was added as an amplification control. B. Quantitative RT-PCR analysis of P. berghei SERA gene expression in WT mixed blood stages (BS), purified schizonts (late BS) or infected HuH7 cell cultures 65 h post-infection (late LS). Relative gene expression was normalized to MSP1 expression level, and is shown as the mean of two independent experiments (±SD).

Fig. 3

Fluorescent tagging of P. berghei SERA1 and SERA2. A. Insertion strategy to generate the SERA1/mCherry and SERA2/mCherry parasites. The PbSERA1 and PbSERA2 genomic loci were targeted with integration plasmids containing the 3′SERA1 and SERA2 terminal fragments (dark grey box) that is fused in frame to the mCherry coding sequence (red box), the 3′ UTR of PbDHFR/TS (light grey box) and the TgDHFR/TS selectable marker (white box). Upon a single cross-over event, the region of homology is duplicated, resulting in a functional, endogenous PbSERA1 or PbSERA2 copy tagged with mCherry, followed by a truncated and non-expressed copy. B. Expression of the mCherry fusion proteins (red) was analysed by confocal fluorescence microscopy of SERA1/mCherry and SERA2/mCherry P. berghei blood stage parasites constitutively expressing GFP (green). Parasite stages are indicated by arrow heads. T, ring/trophozoite; S, schizont. Nuclei were stained with Hoechst 33342. Bars, 5 µm. C and D. Liver stage expression of the mCherry fusion proteins (red) was analysed by confocal fluorescence microscopy of SERA1/mCherry (C) and SERA2/mCherry (D) P. berghei parasites constitutively expressing GFP (green), at 24, 48 and 65 h after infection of HuH7 cells with sporozoites. Bars, 10 µm. E. Detached infected cells (merosomes) were recovered from the supernatant of infected HuH7 cultures 65 h post infection, and analysed by confocal fluorescence microscopy. Size bars, 10 µm.

Fig. 4

Immunofluorescence analysis of PbSERA1 and PbSERA2 in late liver stages. P. berghei infected HepG2 cells were fixed at different time points (45–58 h) post infection and analysed on single cell level using IFA. Developmental stages are indicated on the left. Localization of the C-terminal region of PbSERA1 (A) and the central domain of PbSERA2 (B) were determined using specific rat antibodies (red). As a marker for the PVM, we used chicken anti-EXP1 antibodies (green), and DNA was labelled with DAPI (blue). Scale bar: 5 µm. Note the distribution of SERA1C to the PV compartment in all stages, whereas SERA2M localizes both to the PV and internal parasite structures in schizonts and cytomeres, but only to the PV compartment after merozoite differentiation.

Fig. 6

Western blot analysis of PbSERA1 and PbSERA2 protein expression in P. berghei blood stages. Triton X-100 extracts of enriched schizont preparations from WT, sera1(−), sera2(−) and sera1(−)2(−) parasites were analysed by Western blot using antibodies specific for PbSERA1 and PbSERA2. Anti-HSP70 antibodies were used as a loading control. Note the absence of specific bands (arrows) in the knockout parasite lines, confirming complete gene disruption. Non-specific bands are indicated with an asterisk.

Fig. 5

Targeted gene disruption of the P. berghei SERAser genes. A and B. Replacement strategy to generate the sera1(−) and sera2(−) parasites (A), and the sera1(−)/2(−) parasites (B). The wild-type (WT) SERAser genomic loci are targeted with KpnI/SacII-linearized replacement plasmids (pREP) containing 5′ and 3′ untranslated regions adjacent to the SERAser open reading frames and the dhfr/ts-positive selectable marker. Upon a double cross-over event the open reading frame is replaced by the selectable marker. Replacement-specific test and WT primer combinations are indicated by arrows and expected fragments as lines. C. Replacement-specific PCR analysis. The successful replacement event is verified by a primer combination (test) that can only amplify a signal from the REP locus. Absence of the WT signal from sera1(−), sera2(−) and sera1(−)/2(−) parasites confirms the purity of the clonal populations. D. RT-PCR analysis of SERA1, SERA2 and MSP1 transcripts in WT, sera1(−), sera2(−) and sera1(−)/2(−) blood stage parasites. RT-PCR was performed in the presence (+) or absence (−) of reverse transcriptase (RT). Parasite genomic DNA (gDNA) was included as an amplification control.

Fig. 7

Blood stage growth of sera1(−)/2(−) double knockout P. berghei parasites. Naïve NMRI mice (n = 5) were injected intravenously with 1000 wild-type or 1000 sera1(−)/2(−) P. berghei infected erythrocytes. Infection was then monitored by daily examination of Giemsa-stained blood smears to determine the parasitemia.

Fig. 8

Upregulation of PbSERA3 expression in SERAser knockout lines. A. SERA gene expression was analysed by quantitative RT-PCR in purified schizonts from WT, sera1(−), sera2(−) and sera1(−)/2(−) parasites. Relative gene expression was normalized to MSP1 expression level, and is shown as the Log2 of the ratio knockout/WT (mean of two experiments ± SD). n.a., no amplification. B. Saponin (S) or Triton X-100 (T) extracts of purified schizonts from WT, sera1(−), sera2(−) and sera1(−)/2(−) parasites were analysed by Western blot using antibodies specific for PbSERA3. As a loading control an anti-HSP70 antibody was used.