Cholesterol bound Plasmodium falciparum co-chaperone ‘PFA0660w’ complexes with major virulence factor ‘PfEMP1’ via chaperone ‘PfHsp70-x’

Abstract

Lethality of Plasmodium falciparum caused malaria results from ‘cytoadherence’, which is mainly effected by exported Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. Several exported P . falciparum proteins (exportome) including chaperones alongside cholesterol rich microdomains are crucial for PfEMP1 translocation to infected erythrocyte surface. An exported Hsp40 (heat shock protein 40) ‘PFA0660w’ functions as a co-chaperone of ‘PfHsp70-x’, and these co-localize to specialized intracellular mobile structures termed J-dots. Our studies attempt to understand the function of PFA0660w-PfHsp70-x chaperone pair using recombinant proteins. Biochemical assays reveal that N and C-terminal domains of PFA0660w and PfHsp70-x respectively are critical for their activity. We show the novel direct interaction of PfHsp70-x with the cytoplasmic tail of PfEMP1, and binding of PFA0660w with cholesterol. PFA0660w operates both as a chaperone and lipid binding molecule via its separate substrate and cholesterol binding sites. PfHsp70-x interacts with cholesterol bound PFA0660w and PfEMP1 simultaneously in vitro to form a complex. Collectively, our results and the past literature support the hypothesis that PFA0660w-PfHsp70-x chaperone pair assists PfEMP1 transport across the host erythrocyte through cholesterol containing ‘J-dots’. These findings further the understanding of PfEMP1 export in malaria parasites, though their in vivo validation remains to be performed.

Defining the Essential Exportome of the Malaria Parasite

“…PF3D7_0113700 and the nonessential JDP PF3D7_0501100 localise to J-dots and have been shown to be in a complex with HSP70-x and PfEMP1 using proximity ligation and coimmunoprecipitation assays (Figure 1C) [39]. Further studies support the role for PF3D7_0113700 as a cochaperone of HSP70-x, where it helps to traffic PfEMP1 within the RBC via the mobile J-dot structures [44]. Since PfEMP1 is not needed for in vitro growth, it is possible that these JDPs identified as essential might help to traffic other proteins through J-dots that are pivotal for parasite survival such as those involved in nutrient acquisition.…”

Transport mechanisms at the malaria parasite-host cell interface

“…In P . falciparum , nearly half of the 49 parasite-encoded HSP40/DnaJ proteins are predicted for export, and some of these co-chaperones have been shown to interact with effector trafficking intermediates in the host cell [ 114 – 117 ]. An exported HSP70 known as PfHSP70x is also encoded by P .…”

Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells

“…Parasite structures are formed in the iRBC to traffic parasite proteins within the host cell. These include Maurer's clefts (MCs) which are membranous structures involved in protein cargo sorting, and J‐dots, which are mobile structures affiliated with heat shock proteins and trafficking of the major virulence protein PfEMP1 (Behl et al, 2019 ; Kulzer et al, 2010 , 2012 ; Lanzer et al, 2006 ). The iRBC also becomes more permeable to plasma nutrients including isoleucine and pantothenic acid to supplement rapid parasite growth (Boddey & Cowman, 2013 , Cooke et al, 2004 , Gilson et al, 2017 , Kirk et al, 1994 , Kirk & Horner, 1995 ).…”