Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes

Abstract

The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α‐ and β‐subunits of succinyl‐CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α‐ and β‐SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α‐ and β‐SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α‐ nor β‐SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain‐containing membrane‐associated protein and tetraspanin family surface protein, as well as soluble‐secreted β‐amylase‐1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α‐ and β‐SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.

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

“…To test the TVV transmission between trichomonads, we prepared two cell lines. The donor line was TV79-49c1 + that was transformed with pTagVagV5-Pur ( Rada et al, 2019 ), enabling the expression of biotin ligase (BirA) as a marker under puromycin selection (TV79-49c1 + PAC-BirA). The recipient strain was TV79-49c1 – that was transfected with pTagVag2 ( Rada et al, 2015 ) for expression of cytosolic HA-tagged phosphofructokinase under geneticin selection (TV79-49c1 – G418-PFK).…”

In-depth comparative analysis of Tritrichomonas foetus transcriptomics reveals genes linked to host-adaptation

“…In addition, we have been able to identify a gene TRFO_27838 that codi es a putative precursor of adhesin AP65-1 in cluster 66 and another putative adhesin (TRFO_07867) in cluster 619. This type of adhesins possesses a role as moonlighting proteins (a subclass of multifunctional proteins previously documented in trichomonads parasites with a role in pathogenesis and adaptation to host cells) (34). Interestingly, at cluster 140, a gene that encodes for a tetraspanin protein (TRFO_34204) was found grouped with ribosomal proteins and rubrerythrin gene, a protein related to oxidative stress protection (35).…”

Perils and Promises of Pathogenic Protozoan Extracellular Vesicles

“…Incidentally, exosomes or microvesicle in T. cruzi conceivably have their origin from the tubular network of the endoplasmic reticulum and Golgi (Lozano et al, 2017). Also, there could be plasma membrane-derived vesicles and exosomes formed through fusion of MVBs with the flagellar pocket of epimastigote and metacyclic stages of T. cruzi (Bayer-Santos et al, 2013), whereas Trichomonas vaginalis microvesicles are derived from endocytic compartments or the plasma membrane (Rada et al, 2019). Bizarre forms of T. brucei vesicles which are independent of ESCRT machinery and autophagy have been observed in addition to EV exocytosis from the flagellar pocket, parasite surface, and MVBs (Eliaz et al, 2017).…”

“…Likewise, disintegration of T. vaginalis during in vitro cultivation produced vesicles which subsequently bind to the cell surface (Rada et al, 2019 ) and add to the existing subpopulations of EVs. The use of antibiotics as component of culture media may equally trigger typical secretion of EVs, although the implication of this was not clearly mentioned with the use of gentamicin as a component of culture media in the studies of P. falciparum EVs (Mantel et al, 2016 ; Sisquella et al, 2017 ; Castelli et al, 2019 ).…”

“…The secreted exosomes of L. major promastigotes and amastigotes function as the main protein secretory pathway (Leitherer et al, 2017 ), and the involvement of T. vaginalis vesicles in the export of adhesin proteins has been confirmed by immunofluorescence analyses (Rada et al, 2019 ). Of the total proteins released by T. cruzi EVs, prediction holds that about 57% were secreted through classical and non-classical pathways.…”