A Cleavable Propeptide InfluencesToxoplasmaInfection by Facilitating the Trafficking and Secretion of the TgMIC2–M2AP Invasion Complex

Abstract

Propeptides regulate protein function and trafficking in many eukaryotic systems and have emerged as important features of regulated secretory proteins in parasites of the phylum Apicomplexa. Regulated protein secretion from micronemes and host cell invasion are inextricably linked and essential processes for the apicomplexan parasite Toxoplasma gondii. TgM2AP is a propeptide-containing microneme protein found in a heterohexameric complex with the microneme protein TgMIC2, a protein that has a demonstrated fundamental role in gliding motility and invasion. TgM2AP function is also central to these processes, because disruption of TgM2AP (m2apKO) results in secretory retention of TgMIC2, leading to reduced TgMIC2 secretion from the micronemes and impaired invasion. Because the TgM2AP propeptide is predicted to be processed in an intracellular site near where TgMIC2 is retained in m2apKO parasites, we hypothesized that the propeptide and its proteolytic removal influence trafficking and secretion of the complex. We found that proTgM2AP traffics through endosomal compartments and that deletion of the propeptide leads to defective trafficking of the complex within or near this site, resulting in aberrant processing and decreased secretion of TgMIC2, impaired invasion, and reduced virulence in vivo, mirroring the phenotypes observed in m2apKO parasites. In contrast, mutation of several cleavage site residues resulted in normal localization, but it affected the stability and secretion of the complex from the micronemes. Therefore, the propeptide and its cleavage site influence distinct aspects of TgMIC2-M2AP function, with both impacting the outcome of infection. INTRODUCTIONEukaryotic secretory proteins use an assortment of luminal or cytoplasmic forward targeting signals to navigate the secretory system for eventual delivery to the extracellular environment. Among the least well understood of the luminal signals are cleavable elements known as propeptides, which are typically positioned either internally or, more commonly, at the N terminus of a protein. Propeptides have been shown to serve in several capacities. They can facilitate the folding of their cognate protein, regulate its activity or oligomeric assembly, or direct it to a particular intracellular compartment within the endolysosomal or secretory system. For proproteins destined for the regulated secretory pathway, proteolytic processing (also termed proteolytic maturation) typically accompanies the condensation of immature secretory granule contents (Orci et al., 1987;Kuiper and Martens, 2000). Yet, the precise role of proteolytic maturation in the biogenesis and discharge of regulated secretory organelles remains contentious (Arvan and Halban, 2004).Toxoplasma gondii, the etiological agent of toxoplasmosis, is a genetically tractable protozoan parasite that causes widespread infection in humans resulting in significant economic losses worldwide (Jones et al., 2001). As a member of the phylum Apicomplexa, T. gondii features a distinct apical complex consis...

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

“…Several other unidentified proteins also seem to be degraded upon secretion from TgMIC5-deficient parasites. Additionally, whereas TgSUB1 processes TgMIC4 into TgMIC4 50 and TgMIC4 20 in WT parasites, it hyperprocesses TgMIC4 into a new species, TgMIC4 18 , in the absence of TgMIC5 (16). That TgMIC5 parasites do not appear to have an invasion or virulence phenotype might be the result of offsetting effects: higher efficiency trimming of the major adhesin TgMIC2 promotes parasite attachment and invasion whereas hyperprocessing or degradation of other substrates diminishes their functionality.…”

“…Parasite tachyzoites (RH, RH⌬mic5, and RH⌬sub1) were grown in human foreskin fibroblast cells and harvested as described previously (18).…”

Microneme protein 8 – a new essential invasion factor inToxoplasma gondii

“…2D). When we performed co-localisation analysis of MIC8 with an antibody against the propeptide of M2AP, which localises to early endosomes (Harper et al, 2006), we detected retained MIC8 within the same compartment (Fig. 2D).…”

“…However, no difference in initial host-cell attachment was observed in the case Upper panels: co-localisation study of MIC8Ty and GRASP-RFP (Pfluger et al, 2005) in the parasite strain T7S4-5 transiently transfected with GRASP-RFP demonstrates accumulation of MIC8 in a postGolgi compartment. Middle panels: co-localisation of MIC8Ty and the propeptide of M2AP (Harper et al, 2006) in early endosomes. Lower panels: co-localisation study of MIC8Ty and MIC3 shows that MIC3 is not localised in early endosomes because of overexpression of MIC8Ty.…”

Sialic acids: Key determinants for invasion by the Apicomplexa

“…Only after processing is TgMIC3 able to function as an adhesin through its chitin-binding-like (CBL) domain, but this processing event is dispensable for trafficking (Cerede et al, 2002;El Hajj et al, 2008). Removal of the TgM2AP propeptide is critical for stable assembly and efficient secretion of the TgMIC2-M2AP complex from the micronemes onto the parasite surface (Harper et al, 2006). The cysteine protease cathepsin L (TgCPL) that resides in compartments of the late secretory pathway is a candidate for taking part in proteolytic maturation of some MICs (Larson et al, 2009).…”