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, 9 (3)

Trichomonas vaginalis Macrophage Migration Inhibitory Factor Mediates Parasite Survival During Nutrient Stress

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Trichomonas vaginalis Macrophage Migration Inhibitory Factor Mediates Parasite Survival During Nutrient Stress

Yi-Pei Chen et al. mBio.

Abstract

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.

Keywords: Trichomonas vaginalis; apoptosis; macrophage migration inhibitory factor; nutrient starvation.

Figures

FIG 1
FIG 1
TvMIF increases parasite survival under conditions of nutrient stress. (A) Overexpression of TvMIF in the parasite enhances the survival of the parasite under conditions of serum starvation significantly after 8 h. All time points are normalized to time point 0 h for each parasite. Data are means ± standard errors of results from triplicates, and data from 1 of 3 independent experiments are shown. (B) Overexpression of TvMIF enhances parasite resistance to density stress. The cultures started at a 1 × e7/ml or 2 × e7/ml concentration and were incubated for 4 and 8 h. Data shown are means ± standard errors of results from triplicates, and data representative of 1 of 3 independent experiments are shown. *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001.
FIG 2
FIG 2
TvMIF is present in the exosome and the non-exosomal secreted fractions. (A) A secretion assay used to determine proportions of TvMIF in the exosome (Exo) and non-exosomal soluble fraction (NESF) is illustrated. Parasites were incubated at either 16°C or 37°C. Incubation at 16°C is a negative control for secretion. After the incubation, the cells were pelleted and lysed to become whole-cell lysates (Wcl) and the secreted supernatant fraction was passed through a Vivaflow crossflow cassette to separate Exo from NESF. Then, the Exo was collected by ultracentrifugation. (B) At 16°C, no detectable TvMIF was present in Exo and NESF. At 37°C, 1.4-fold more TvMIF was detected in the NESF than in the Exo fraction using immunoblotting and a TvMIF antibody. Neomycin phosphotransferase (Neo) was used to detect cellular lysis.
FIG 3
FIG 3
Endogenous TvMIF protein is induced under conditions of serum starvation. (A) Anti-TvMIF immunoblot showing that the TvMIF level was induced during serum starvation. Detection of GAPDH served as the loading control. One representative immunoblot of three independent experiments is shown. (B) Quantification of TvMIF was determined by normalizing the TvMIF signal to GAPDH for each sample; the data were compared to the 0-h time point value (set at 1) in each experiment, so no error bar was made for 0 h. Data are means ± standard errors. (C) TvMIF secretion was induced in the absence of serum. +Serum, cells grown in complete media; −Serum, cells grown without serum. Secreted TvMIF was induced ~2-fold at 3 h and ~2.5-fold at 6 h after serum starvation comparing −serum and +serum signals. Neo was used as a negative control for cell lysis.
FIG 4
FIG 4
Both recombinant TvMIF and recombinant huMIF increase the survival of the parasite under conditions of serum starvation. (A) rTvMIF (50 ng/ml) or PBS (indicated as 0 ng/ml) was added to parasites grown without serum at time point 0 h. rTvMIF at 50 ng/ml increased the parasite survival at both 4 h and 8 h after serum starvation. (B) rhuMIF (50 ng/ml) or PBS (indicated as 0 ng/ml) was added to parasites grown without serum at time point 0 h. rhuMIF at 50 ng/ml increased the parasite survival at 8 h after starvation. Results of 3 independent experiments, each done in quadruplet, are represented by the data shown here. Error bars represent means ± standard errors. *, P value ≤ 0.05; **, P value ≤ 0.01.
FIG 5
FIG 5
Increased secretion of TvMIF by TvMIF-OE parasites increases survival of EV parasites after co-culture with TvMIF-OE parasites. (A) Scheme of co-culture transwell assay. EV parasites in the wells indicated at the bottom were separated from EV or TvMIF-OE in the wells indicated at the top by a membrane with 0.4-µm pores. (B) Co-culturing EV parasites with TvMIF-OE parasites confers a survival advantage. EV parasites (bottom well in Fig. 5A) were co-cultured with TvMIF-OE parasites (black bars) or EV parasites (white bars) for the number of days indicated on the x axis. The EV parasites in the bottom well were then transferred to serum-free media for 24 h, and survival was measured. Error bars represent means ± standard errors. *, P value ≤ 0.05; **, P value ≤ 0.01. The data represent results from 3 independent experiments, each done in triplicate.
FIG 6
FIG 6
TvMIF inhibits parasite apoptosis. (A) Double staining with annexin V and Zombie Red was used to assess apoptosis. TvMIF-OE parasites (black bars) underwent less apoptosis at 16 h and 24 h after serum starvation than EV parasites (white bars). MFI, mean fluorescence intensity; Healthy, DMSO-treated control; STS, staurosporine (apoptosis inducer positive control). Error bars represent standard errors. *, P value ≤ 0.05. (B) At 0 h, both EV and TvMIF-OE nuclei were intact with little damage. (C) Nuclei were stained with DAPI. At 16 h after serum starvation, DNA damage in EV parasites was clearly visible. White arrows indicate examples of DNA fragmentation. (D) DNA damage in EV parasites was significantly greater than that observed in TvMIF-OE parasites after 16 h of serum starvation. The imaging data shown in panels B and C were quantified by counting the number of fragmented DNA in a total of 300 nuclei from both EV and TvMIF-OE parasites. *, P value ≤ 0.05. Error bars represent means ± standard errors. All data represent results from 3 independent triplicated experiments.
FIG 7
FIG 7
TvMIF inhibits parasite apoptosis under conditions of serum starvation via ROS suppression. (A) The superoxide level detected by using dihydroethidium (DHE) was significantly higher at both 16 h and 24 h in EV parasites than in TvMIF-OE parasites after serum starvation. MFI, mean fluorescence intensity. (B) Superoxide dismutase (SOD) mimetic treatment at time point 0 h decreased the superoxide level in EV at both 250 µM and 500 µM and in TvMIF-OE at 500 µM. (C) SOD mimetic treatment increased EV parasite survival at both 250 µM and 500 µM and TvMIF-OE parasite survival at 500 µM. Ns, non-significant; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001. All data represent results from 3 independent experiments, performed in triplicate.
FIG 8
FIG 8
Parasites with the TvMIF gene knocked out have significantly less survival than the wild-type (WT) parasites. (A) Immunoblot using anti-TvMIF antibody to confirm the loss of TvMIF in knockout (KO) parasites and its presence in addback parasites. GAPDH is the loading control. (B) Knockout of TvMIF in the parasite severely reduces the survival of the parasite under conditions of serum starvation. Adding back of TvMIF in knockout parasites that exogenously overexpress the protein restores and enhances the survival phenotype, reminiscent of the increased survival seen with TvMIF-OE. All time points are normalized to time point 0 h for each parasite. Data are means ± standard errors of results from triplicates, and data from 1 of 3 independent experiments are shown. *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001.

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