Trichomonas vaginalis is responsible for the most prevalent non-viral sexually transmitted disease worldwide, and yet the mechanisms used by this parasite to establish and maintain infection are poorly understood. We previously identified a T. vaginalis homologue (TvMIF) of a human cytokine, human macrophage migration inhibitory factor (huMIF). TvMIF mimics huMIF's role in increasing cell growth and inhibiting apoptosis in human host cells. To interrogate a role of TvMIF in parasite survival during infection, we asked whether overexpression of TvMIF (TvMIF-OE) confers an advantage to the parasite under nutrient stress conditions by comparing the survival of TvMIF-OE parasites to that of empty vector (EV) parasites. We found that under conditions of serum starvation, overexpression of TvMIF resulted in increased parasite survival. Serum-starved parasites secrete 2.5-fold more intrinsic TvMIF than unstarved parasites, stimulating autocrine and paracrine signaling. Similarly, we observed that addition of recombinant TvMIF increased the survival of the parasites in the absence of serum. Recombinant huMIF likewise increased the parasite survival in the absence of serum, indicating that the parasite may use this host survival factor to resist its own death. Moreover, TvMIF-OE parasites were found to undergo significantly less apoptosis and reactive oxygen species (ROS) generation under conditions of serum starvation, consistent with increased survival being the result of blocking ROS-induced apoptosis. These studies demonstrated that a parasitic MIF enhances survival under adverse conditions and defined TvMIF and huMIF as conserved survival factors that exhibit cross talk in host-pathogen interactions.IMPORTANCE Macrophage migration inhibitory factor (MIF) is a conserved protein found in most eukaryotes which has been well characterized in mammals but poorly studied in other eukaryotes. The limited analyses of MIF proteins found in unicellular eukaryotes have focused exclusively on the effect of parasitic MIF on the mammalian host. This was the first study to assess the function of a parasite MIF in parasite biology. We demonstrate that the Trichomonas vaginalis MIF functions to suppress cell death induced by apoptosis, thereby enhancing parasite survival under adverse conditions. Our research reveals a conserved survival mechanism, shared by a parasite and its host, and indicates a role for a conserved protein in mediating cross talk in host-pathogen interactions.

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin-dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus-end tracking proteins (+TIPs) in the co-ordination of T. cruzi trypomastigote internalization and post-entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium-regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1-depleted cells. These post-entry phenotypes correlate with a generalized impairment of minus-end directed transport of lysosomes in CLASP1 knock-down cells and mimic the effects of dynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1-dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite-elicited signalling, host microtubule plus-end tracking proteins and dynein-based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells.

Background

TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties.

Methods/principle findings

Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the 'adhesive' TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to trypomastigote internalization. These motifs are not conserved in the 'non-adhesive' TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation.

Conclusion/significance

In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by parasite strains.

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.