Abstract
The secretion of virulence factors by parasitic protists into the host environment plays a fundamental role in multifactorial host-parasite interactions. Several effector proteins are known to be secreted by Trichomonas vaginalis, a human parasite of the urogenital tract. However, a comprehensive profiling of the T. vaginalis secretome remains elusive, as do the mechanisms of protein secretion. In this study, we used high-resolution label-free quantitative MS to analyze the T. vaginalis secretome, considering that secretion is a time- and temperature-dependent process, to define the cutoff for secreted proteins. In total, we identified 2 072 extracellular proteins, 89 of which displayed significant quantitative increases over time at 37 °C. These 89 bona fide secreted proteins were sorted into 13 functional categories. Approximately half of the secreted proteins were predicted to possess transmembrane helixes. These proteins mainly include putative adhesins and leishmaniolysin-like metallopeptidases. The other half of the soluble proteins include several novel potential virulence factors, such as DNaseII, pore-forming proteins, and β-amylases. Interestingly, current bioinformatic tools predicted the secretory signal in only 18% of the identified T. vaginalis-secreted proteins. Therefore, we used β-amylases as a model to investigate the T. vaginalis secretory pathway. We demonstrated that two β-amylases (BA1 and BA2) are transported via the classical endoplasmic reticulum-to-Golgi pathways, and in the case of BA1, we showed that the protein is glycosylated with multiple N-linked glycans of Hex5HexNAc2 structure. The secretion was inhibited by brefeldin A but not by FLI-06. Another two β-amylases (BA3 and BA4), which are encoded in the T. vaginalis genome but absent from the secretome, were targeted to the lysosomal compartment. Collectively, under defined in vitro conditions, our analysis provides a comprehensive set of constitutively secreted proteins that can serve as a reference for future comparative studies, and it provides the first information about the classical secretory pathway in this parasite.
Highlights
Ture comparative studies, and it provides the first information about the classical secretory pathway in this parasite
Analysis of Secreted Proteins Using Quantitative Mass Spectrometry—To study the secretome of T. vaginalis, we developed a protocol for analyzing the time-dependent accumulation of extracellular proteins via high-resolution nano LC coupled with ESI-linear-ion trap and MS/MS label-free quantification (LFQ)
Most experimental designs for studying the secretome of parasitic protists are based on a single time period of incubation under optimal conditions to minimize cell lysis, followed by MS analysis of the proteins released to the medium [31, 34, 61,62,63,64]
Summary
Cell Cultivation—T. vaginalis strain Tv17– 48 was isolated from a symptomatic patient, and the axenic culture was immediately stored in liquid nitrogen [35]. The -amylase-coding genes BA1– 4 were amplified by PCR and subcloned into the modified pTagVag, into which we introduced a sequence encoding the biotin acceptor peptide (AP, GLNDIFEAQKIEWHE), which allowed for the production of recombinant proteins with a C-terminal AP tag. Effect of Inhibitors on -Amylase Glycosylation and Secretion— Trichomonads episomally expressing HA-tagged BA1– 4 were inoculated into TYM medium (1 ϫ 105 cells/ml) supplemented with 5 g/ml tunicamycin (Sigma-Aldrich) and grown for 24 h. The cells were removed by centrifugation, and the proteins in the supernatant were precipitated by TCA as described above and examined by Western blot analysis with mouse monoclonal ␣-HA Ab. Western blots were quantified using ImageJ v1.47 (National Institute of Health, Bethesda, MD, USA), and statisti-
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