Exploitation of a newly-identified entry pathway into the malaria parasite-infected erythrocyte to inhibit parasite egress

Abstract

While many parasites develop within host cells to avoid antibody responses and to utilize host cytoplasmic resources, elaborate egress processes have evolved to minimize the time between escaping and invading the next cell. In human erythrocytes, malaria parasites perforate their enclosing erythrocyte membrane shortly before egress. Here, we show that these pores clearly function as an entry pathway into infected erythrocytes for compounds that inhibit parasite egress. The natural glycosaminoglycan heparin surprisingly inhibited malaria parasite egress, trapping merozoites within infected erythrocytes. Labeled heparin neither bound to nor translocated through the intact erythrocyte membrane during parasite development, but fluxed into erythrocytes at the last minute of the parasite lifecycle. This short encounter was sufficient to significantly inhibit parasite egress and dispersion. Heparin blocks egress by interacting with both the surface of intra-erythrocytic merozoites and the inner aspect of erythrocyte membranes, preventing the rupture of infected erythrocytes but not parasitophorous vacuoles, and independently interfering with merozoite disaggregation. Since this action of heparin recapitulates that of neutralizing antibodies, membrane perforation presents a brief opportunity for a new strategy to inhibit parasite egress and replication.

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

“…However, repeated attempts to generate an EXP2–BioID fusion with a verified CRISPR/Cas9 strategy for editing the 3′ end of exp2 were unsuccessful. Endogenous EXP2 can tolerate a monomeric NeonGreen (mNG, 27 kDa) fusion without an obvious fitness cost (Glushakova et al, ); thus, the bulkier size (35 kDa) or enzymatic activity of BioID may interfere with EXP2 trafficking or essential functions. To test the former possibility, we next attempted fusion with the second generation BioID2 derived from Aquifex aeolicus (Figure S1a).…”

“…To monitor EXP2 distribution in unfixed EXP1 apt parasites, we introduced a C ‐terminal mNG fusion on the endogenous copy of EXP2 and imaged parasites at a late stage corresponding with developmental arrest. Live fluorescent analysis of a parental EXP2–mNG control line with an unmodified exp1 locus (Glushakova et al, ) showed a punctate distribution at early stages that often resolved into several larger patches in trophozoites and schizonts (Figure 6a). In contrast, EXP2 distribution was substantially altered in EXP1 apt ::EXP2‐mNG parasites, often concentrating into one or two discrete points along the PVM (Figure 6a).…”

“…Primer and synthetic gene sequences are given in Table S2. To generate a BioID2 fusion to the endogenous EXP2 C ‐terminus, the coding sequence of the A. aeolicus biotin ligase with an R40G mutation (BioID2) bearing a 3' 3xHA epitope tag was amplified with primers P1/P2 from plasmid MCS‐BioID2‐HA (Kim, Jensen, et al, ; Addgene #74224) and inserted between AvrII and EagI in plasmid pyPM2GT‐EXP2‐mNG (Glushakova et al, ), replacing the AvrII site with an NheI site and resulting in the plasmid pyPM2GT‐EXP2‐BioID2‐3xHA. This plasmid was linearized at the AflII site between the 3' and 5' homology flanks and cotransfected with pUF‐Cas9‐EXP2‐CT‐gRNA (Garten et al, ) into NF54 attB .…”

“…The exp1 codon 70 in this plasmid was then changed from AGA to ACA using a QuikChange Lightning Multi Site Directed Mutagenesis kit and the primer P71, resulting in the plasmid pyEOE‐attP‐EXP1‐R70T‐3xMYC. Finally, the mNG coding sequence was amplified from pyPM2GT‐EXP2‐mNG (Glushakova et al, ) with primers P72/P73 and inserted at NheI in pyEOE‐attP‐EXP1‐WT‐3xMYC, resulting in the plasmid pyEOE‐attP‐EXP1‐mNG‐3xMYC. Complementing plasmids were cotransfected with pINT (Nkrumah et al, ) into EXP1 apt parasites to facilitate integration into the attB site on chromosome 6 and selection with 2 μM DSM1 was applied 24 hours posttransfection (in addition to 2.5 μg/mL Blasticidin‐S and 1 μM aTc for maintenance of endogenous exp1 control by the TDA system).…”

“…To monitor EXP2 by live fluorescence in EXP1 apt , an endogenous mNG fusion to EXP2 was generated by cotransfecting EXP1 apt with plasmids pyPM2GT‐EXP2‐mNG (Glushakova et al, ; linearized at AflII) and pUF‐Cas9‐EXP2‐CT‐gRNA (Garten et al, ). Selection was applied 24 hours post‐transfection with 2 μM DSM1 and parasites were cloned upon returning from selection.…”