Plasmodium falciparum SERA5 plays a non‐enzymatic role in the malarial asexual blood‐stage lifecycle

Abstract

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle.

Plasmodium falciparum subtilisin‐like ookinete protein SOPT plays an important and conserved role during ookinete infection of the Anopheles stephensi midgut

“…No protein domain in the C‐terminus of SOPT was identified by computational methods or modelling. Given the divergent catalytic triad across the genus, it is likely that SOPT is a pseudoprotease rather than an active protease in Plasmodium spp., as is the case for the protein SERA5 (serine rich antigen 5) (Stallmach et al, ).…”

“…Taken together, this indicates that the catalytic triad is not critical to the function of SOPT/PIMMS2 and suggest that it has a non‐enzymatic role in Plasmodium ookinete entry into the mosquito midgut epithelium, which is the first step of the traversal process. However, additional biochemical and structural analyses are needed for confirmation that SOPT is indeed a pseudoprotease, such as those reported previously for SERA5 (Stallmach et al, ).…”

The Plasmodium falciparum rhoptry protein RhopH3 plays essential roles in host cell invasion and nutrient uptake

“…Long-term growth was also measured using flow cytometry of hydroethidine-stained trophozoite-stage parasites, as described (Stallmach et al, 2015). Cultures adjusted to a parasitaemia of 0.1% were monitored every 48 hr for up to seven intraerythrocytic cycles.…”

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

“…For the GP1 knockdown and GP2 KO analysis, tightly synchronised parasites were treated at the young ring stage with or without 2.5‐mM GlcN and parasites were imaged at the schizont stage of the same cycle. A panel of antibodies were utilised in combination with the mouse anti‐HA 1:1,000 (Cedarlane, clone HA.C5) or rabbit polyclonal anti‐HA (Abm, 1:1,000): mouse anti‐RAP1 (1:3,000); mouse anti‐RON4 (1:2,000; Richard et al, ); rabbit anti‐AMA1 (1:2,000) (Healer, Triglia, Hodder, Gemmill, & Cowman, ); mouse anti‐EBA175 (MRA711A, 1:500; Sim et al, ; 50); rabbit anti‐GAP45 (1:2,000; Jones et al, ; 51); rabbit anti‐MSP1 (1:1,000; Wilson et al, ); rabbit anti‐SERA5 (1:1,000; Stallmach et al, ); rabbit anti‐ERD2 (1:1,000). For mitochondrion analysis, Mitotracker Red (Molecular Probes, 10 nM) was used in live imaging combined with DAPI.…”