A Putative Small Solute Transporter Is Responsible for the Secretion of G377 and TRAP-Containing Secretory Vesicles during Plasmodium Gamete Egress and Sporozoite Motility

Abstract

Regulated protein secretion is required for malaria parasite life cycle progression and transmission between the mammalian host and mosquito vector. During transmission from the host to the vector, exocytosis of highly specialised secretory vesicles, such as osmiophilic bodies, is key to the dissolution of the red blood cell and parasitophorous vacuole membranes enabling gamete egress. The positioning of adhesins from the TRAP family, from micronemes to the sporozoite surface, is essential for gliding motility of the parasite and transmission from mosquito to mammalian host. Here we identify a conserved role for the putative pantothenate transporter PAT in Plasmodium berghei in vesicle fusion of two distinct classes of vesicles in gametocytes and sporozoites. PAT is a membrane component of osmiophilic bodies in gametocytes and micronemes in sporozoites. Despite normal formation and trafficking of osmiophilic bodies to the cell surface upon activation, PAT-deficient gametes fail to discharge their contents, remain intraerythrocytic and unavailable for fertilisation and further development in the mosquito. Sporozoites lacking PAT fail to secrete TRAP, are immotile and thus unable to infect the subsequent rodent host. Thus, P. berghei PAT appears to regulate exocytosis in two distinct populations of vesicles in two different life cycle forms rather than acting as pantothenic transporter during parasite transmission.

Motility precedes egress of malaria parasites from oocysts

“…By using anti-GFP antibodies in fixed sporozoites, we were not able to detect GFP-TRP1∆N on the sporozoite surface (Figure 7—figure supplement 1). Comparison of the localizations of TRP1-GFP and GFP-TRAP (Kehrer et al, 2016b) showed that the two proteins were localized differently (Figure 8). While GFP-TRAP appears mostly localized to micronemes at the front end of the sporozoite, TRP1-GFP appears to localize in what might be a subset of micronemes or a different organelle that does not extend all the way to the front.…”

“…Parasites that lack proteins involved in gliding motility (TRAP and CPβ) do not enter into salivary glands (Sultan et al, 1997; Ganter et al, 2009) and decreased gliding motility (S6/TREP/UOS3, Coronin, CSP and PAT) often goes along with decreased salivary gland invasion (Coppi et al, 2011; Kehrer et al, 2016b; Steinbuechel and Matuschewski, 2009; Combe et al, 2009; Bane et al, 2016; Tewari et al, 2002; Mikolajczak et al, 2008). Hence parasites that lack proteins involved in both egress and motility are also impaired in salivary gland invasion and thus these proteins are essential for life cycle progression at two subsequent steps.…”

The transportome of the malaria parasite

“…A GFP-tagged version of TFP1 localised to the surface of the asexual intraerythrocytic parasite when expressed from an episomal plasmid and under the control of a non-endogenous promoter (Augagneur et al, 2013). However, when expressed from its native locus and promoter, GFP-tagged TFP1 was not evident in asexual blood-stage parasites and the recombinant transporter was instead localised to the osmiophilic body of gametocytes and the micronemes and surface of sporozoites (Kehrer et al, 2016). Moreover, tfp1 was shown to be dispensable in the asexual blood stage, but was essential for infecting mosquitos -and this knockout phenotype was rescued by mCherry-tagged TFP1 (Kehrer et al, 2016).…”

“…However, when expressed from its native locus and promoter, GFP-tagged TFP1 was not evident in asexual blood-stage parasites and the recombinant transporter was instead localised to the osmiophilic body of gametocytes and the micronemes and surface of sporozoites (Kehrer et al, 2016). Moreover, tfp1 was shown to be dispensable in the asexual blood stage, but was essential for infecting mosquitos -and this knockout phenotype was rescued by mCherry-tagged TFP1 (Kehrer et al, 2016). The latter findings also concurred with the detection of TFP1 in proteomic screens of gametocytes and sporozoites as well as its absence from a proteomic screen of asexual blood-stage parasites (Lasonder et al, 2008;Silvestrini et al, 2010;Lindner et al, 2013).…”

“…That said, a number of attempts to express Plasmodium transporters in yeast have been unsuccessful (Henry et al, 2007;Lim et al, 2010), and others have either been queried (e.g. the putative pantothenate transporter PfPAT (Augagneur et al, 2013) also known as PfTFP1 (Kehrer et al, 2016); see Section V.4b and Table S2) or retracted altogether (e.g. PfMDR1; Ruetz et al, 1999).…”