An Exported Heat Shock Protein 40 Associates with Pathogenesis-Related Knobs in Plasmodium falciparum Infected Erythrocytes

Abstract

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer’s cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.

A spiral scaffold underlies cytoadherent knobs in Plasmodium falciparum–infected erythrocytes

“…15,27 Use of gene knockout mutants has revealed that in addition to KAHRP, 2 genes, those encoding a PHIST domain protein (PFD1170c) and an Hsp40-like DNAJ Type IV protein (PF10_0381), are also important for knob formation. 15 Other parasite proteins that have been shown to associate with knob components, and which may thus form part of the knob structure, include knob-associated Hsp40, 28 the PfEMP1 trafficking protein PfEMP3, 16,28 the large variable surface antigen SURFIN, 29 the 2.5 MDa Pf332 antigen, 30 and the PHIST domain protein PFE1605w (LyMP). 31,32 PFE1605w is localized at the cell periphery 33 and has been shown to be important for cytoadherence but not for knob formation or for surface expression of PfEMP1.…”

Targeted Disruption of a Ring-infected Erythrocyte Surface Antigen (RESA)-like Export Protein Gene in Plasmodium falciparum Confers Stable Chondroitin 4-Sulfate Cytoadherence Capacity

“…In contrast to PFB0085c, disruption of PFB0090c, which has recently been shown to encode DnaJ domain-containing KAHsp40 (32,33), resulted in a marked reduction in expression of VAR2CSA and a negligible level of C4S-binding capacity in CS2 parasites (34). However, upon panning of PFB0090c-knock-out parasites on CSA-coated plates, C4S-adherent parasites were selected.…”

“…8). This is interesting, given that PFB0090c is a KAHsp40 chaperone, which interacts with knobassociated proteins, such as PfEMP1, KAHRP, and PfEPMP3, in addition to the chaperones, Hsp70 and Hsp101 (32,33). Thus, increased expression of PFB0090c could contribute at least partly to higher levels of VAR2CSA expression.…”

“…The roles of KAHRP and PfEMP3 in rigid knob formation and strengthening of the adhesive interactions between PfEMP1s and their receptors have been well established (16,17,19,22). Recently, PF3D7_0201800 (PFB0090c) has been shown to be an Hsp40, named KAHsp40, that interacts with KAHRP, PfEMP1, PfEMP3, Hsp70, and Hsp101 (32,33). KAHsp40 functions as a chaperone for proteins involved in knob assembly formation (32,33).…”

“…Recently, PF3D7_0201800 (PFB0090c) has been shown to be an Hsp40, named KAHsp40, that interacts with KAHRP, PfEMP1, PfEMP3, Hsp70, and Hsp101 (32,33). KAHsp40 functions as a chaperone for proteins involved in knob assembly formation (32,33). A targeted disruption of PFB0085c showed no obvious change in PfEMP1 expression and C4S adherence ability of parasites, whereas that of PFB0090c resulted in var gene switching to express different PfEMP1 (34).…”