Abstract
Trichomonas vaginalis is the parasitic protozoan residing in human urogenital tract causing trichomoniasis, which is the leading non-viral sexually transmitted disease. It has cosmopolitan distribution throughout the globe and affects both men and women. Lifecycle of the parasite has been traditionally described as consisting of motile and symptom-causing trophozoites. Chemical and temperature perturbations in trophozoites have been shown to aid conversion to pseudocysts, which is poorly investigated. In the current study, we show the formation of viable cyst-like structures (CLS) in stationary phase of T. vaginalis axenic culture. We used a fluorescent stain called calcofluor white, which specifically binds to chitin and cellulose-containing structures, to score for T. vaginalis CLS. Using flow cytometry, we demonstrated and quantitated the processes of encystation as well as excystation; thus, completing the parasite's lifecycle in vitro without any chemical/temperature alterations. Like cysts from other protozoan parasites such as Entamoeba histolytica and Giardia lamblia, T. vaginalis CLS appeared spherical, immotile, and resistant to osmotic lysis and detergent treatments. Ultrastructure of CLS demonstrated by Transmission Electron Microscopy showed a thick electron-dense deposition along its outer membrane. To probe the physiological role of CLS, we exposed parasites to vaginal pH and observed that trophozoites took this as a cue to convert to CLS. Further, upon co- culturing with cells of cervical origin, CLS rapidly excysted to form trophozoites which abrogated the cervical cell monolayer in a dose-dependent manner. To further corroborate the presence of two distinct forms in T. vaginalis, we performed two-dimensional gel electrophoresis and global, untargeted mass spectrometry to highlight differences in the proteome with trophozoites. Interestingly, CLS remained viable in chlorinated swimming pool water implicating the possibility of its role as environmentally resistant structures involved in non-sexual mode of parasite transmission. Finally, we showed that symptomatic human patient vaginal swabs had both T. vaginalis trophozoites and CLS; thus, highlighting its importance in clinical infections. Overall, our study highlights the plasticity of the pathogen and its rapid adaption when subjected to stressful environmental cues and suggests an important role of CLS in the parasite's life cycle, pathogenesis and transmission.
Highlights
Trichomonas vaginalis is a protozoan parasite and the causative agent of the most common non-viral, sexually transmitted disease (STD) in humans known as trichomoniasis (Schwebke and Burgess, 2004)
We observed that T. vaginalis occurs as cyst-like structures (CLS) under conditions of prolonged growth and consequent nutrition depletion
Every 12 h, cells were stained with Calcofluor White (CFW) and Fluorescein Diacetate (FDA)
Summary
Trichomonas vaginalis is a protozoan parasite and the causative agent of the most common non-viral, sexually transmitted disease (STD) in humans known as trichomoniasis (Schwebke and Burgess, 2004). A recent study reported that 56% patients attending STD clinics were infected with T. vaginalis (Johnston and Mabey, 2008). Most studies on T. vaginalis have reported only the trophozoite form as the active, motile, and infective form of the parasite which is sexually transmitted between individuals (Warton and Honigberg, 1979) and amoeboid form characterized by an increase in surface contact with epithelial cells from vagina, cervix, and prostate (Hirt, 2013). A recent comparative study of proteomes of T. vaginalis trophozoites and pseudocysts reported major differences between protein content and abundance of various sets of proteins in the two forms (DiasLopes et al, 2018). We demonstrated the presence of both trophozoite and CLS in human female clinical cases of T. vaginalis which strengthens our hypothesis of relevance of cyst-form in in vivo disease condition. Our results provide credence to the possibility that T. vaginalis may not exclusively be an STD and additional level of caution must be exercised by clinicians when diagnosing and treating patients with this disease
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