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.

§Institute of Biotechnology CAS v v i BIOCEV Vestec Czech Republic

¶Department of Biochemistry Charles University Faculty of Science BIOCEV Vestec Czech Republic

From the ‡Department of Parasitology

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The retromer and retriever systems are conserved and differentially expanded in parabasalids

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis

The divergent ER-mitochondria encounter structures (ERMES) are conserved in parabasalids but lost in several anaerobic lineages with hydrogenosomes

A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle

Double-Stranded RNA Viruses Are Released From Trichomonas vaginalis Inside Small Extracellular Vesicles and Modulate the Exosomal Cargo

Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol

Anaerobic peroxisomes in Mastigamoeba balamuthi

Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis