Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis-derived secretory products.

We previously reported that leukotriene B4 (LTB4) contained in Trichomonas vaginalis-derived secretory products (TvSP) play an essential role in interleukin-8 (IL-8) production in human mast cell line (HMC-1 cells) via LTB4 receptor (BLT)-mediated Nuclear Factor-kappa B (NF-кB) activation. Dynamin, a GTPase, has been known to be involved in endocytosis of receptors for signaling of production of cytokine or chemokines. In the present study, we investigated the role of dynamin-mediated BLT1 endocytosis in TvSP-induced IL-8 production. When HMC-1 cells were transfected with BLT1 or BLT2 siRNA, TvSP-induced IL-8 production was significantly inhibited compared with that in cells transfected with control siRNA. In addition, pretreatment of HMC-1 cells with a dynamin inhibitor (Dynasore) reduced IL-8 production induced by TvSP or LTB4. TvSP- or LTB4- induced phosphorylation of NF-кB was also attenuated by pretreatment with Dynasore. After exposing HMC-1 cells to TvSP or LTB4, BLT1 was translocated from the intracellular compartments to the plasma membrane within 30 min. At 60 min after stimulation with TvSP or LTB4, BLT1 remigrated from the cell surface to intracellular areas. Pretreatment of HMC-1 cells with dynamin-2 siRNA blocked internalization of BLT1 induced by TvSP or LTB4. Co-immunoprecipitation experiments revealed that dynamin-2 strongly interacted with BLT1 60 min after stimulation with TvSP or LTB4. These results suggest that T. vaginalis-secreted LTB4 induces IL-8 production in HMC-1 cells via dynamin 2-mediated endocytosis of BLT1 and phosphorylation of NF-кB.

CysLT receptor-mediated NOX2 activation is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis-derived secretory products.

Trichomoniasis is caused by a sexually transmitted flagellate protozoan parasite Trichomonas vaginalis. T. vaginalis-derived secretory products (TvSP) contain lipid mediators such as leukotriene B4 (LTB4) and various cysteinyl leukotrienes (CysLTs) which included LTC4, LTD4, and LTE4. However, the signaling mechanisms by which T. vaginalis-induced CysLTs stimulate interleukin (IL)-8 production in human mast cells remain unclear. In this study, we investigated these mechanisms in human mast cells (HMC-1). Stimulation with TvSP resulted in increased intracellular reactive oxygen species (ROS) generation and NADPH oxidase 2 (NOX2) activation compared to unstimulated cells. Pre-treatment with NOX2 inhibitors such as diphenyleneiodonium chloride (DPI) or apocynin significantly reduced ROS production in TvSP-stimulated HMC-1 cells. Additionally, TvSP stimulation increased NOX2 protein expression and the translocation of p47phox from the cytosol to the membrane. Pretreatment of HMC-1 cells with PI3K or PKC inhibitors reduced TvSP-induced p47phox translocation and ROS generation. Furthermore, NOX2 inhibitors or NOX2 siRNA prevented CREB phosphorylation and IL-8 gene expression or protein secretion induced by TvSP. Pretreatment with a CysLTR antagonist significantly inhibited TvSP-induced ROS production, CREB phosphorylation, and IL-8 production. These results indicate that CysLT-mediated activation of NOX2 plays a crucial role in ROS-dependent IL-8 production in human mast cells stimulated by T. vaginalis-secreted CysLTs. These findings enhance our understanding of the inflammatory response in trichomoniasis and may inform the development of targeted therapies to mitigate this response.

Sandwich enzyme-linked aptamer-based assay for the detection of Trichomonas vaginalis.

Trichomoniasis is the most prevalent curable, non-viral sexually transmitted infection (STI), with an estimated 156 million new infections in 2020. It can potentially result in adverse birth outcomes as well as infertility in men, whilst it also increases the risk of acquiring HIV and contracting other vaginal infections. It is mostly prevalent among women in low-income countries and especially in Africa and the Americas. This STI is caused by Trichomonas vaginalis (TV) and a robust, cost-effective, sensitive, specific and rapid diagnostic test is urgently required. We report the screening of 6 full-length and 4 truncated aptamers previously selected in our group for use in a microplate-based sandwich assay. The combination of dual aptamers comprising a short 14-mer truncated capture aptamer (termed A1_14mer) and a full-length non-truncated reporter aptamer (A6) was elucidated to be the optimum pair for a sensitive sandwich enzyme-linked aptamer assay (ELAA) for the detection of TV achieving a detection limit of 3.02 × 104 TV cells/mL. The results obtained with the A1_14mer-A6 ELAA correlate excellently with wet-mount microscopy for the detection of TV in clinical specimens, cervicovaginal lavages and vaginal swabs, highlighting the potential clinical application of this assay for cost-effective population screening and subsequent prevention of the onset of complications associated with undiagnosed and untreated TV.

Vaginitis and risk of sexually transmitted infections: results of a multi-center U.S. clinical study using STI nucleic acid amplification testing.

Significant increases in rates of sexually transmitted infections (STIs) caused by Trichomonas vaginalis (TV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) are occurring in the United States. We present results of a U.S. study examining the intersection of STIs and vaginitis. Among 1,051 women with diagnoses for the presence or absence of bacterial vaginosis (BV) and/or symptomatic vulvovaginal candidiasis (VVC), 195 (18.5%) had one or more STIs, including 101 (9.6%) with TV, 24 (2.3%) with CT, 9 (0.8%) with NG, and 93 (8.8%) with MG. STI prevalence in BV-positive women was 26.3% (136/518), significantly higher than STI prevalence of 12.5% (59/474) in BV-negative women (P < 0.0002). Unlike infections with CT or NG, solo infections of MG or TV were each significantly associated with a diagnosis of BV-positive/VVC-negative (OR 3.0751; 95% CI 1.5797-5.9858, P = 0.0113, and OR 2.873; 95% CI 1.5687-5.2619, P = 0.0017, respectively) and with mixed infections containing MG and TV (OR 3.4886; 95% CI 1.8901-6.439, P = 0.0042, and OR 3.1858; 95% CI 1.809-5.6103, P = 0.0014, respectively). TV and MG infection rates were higher in all Nugent score (NS) categories than CT and NG infection rates; however, both STIs had similar comparative prevalence ratios to CT in NS 6-10 vs NS 0-5 (CT: 3.06% vs 1.4%, 2.2-fold; MG: 10.7% vs 6.1%, 1.8-fold; TV: 14.5% vs 7.0%, 2.1-fold). NG prevalence was relatively invariant by the NS category. These results highlight the complexity of associations of STIs with two major causes of vaginitis and underscore the importance of STI testing in women seeking care for abnormal vaginal discharge and inflammation. IMPORTANCE: This study reports high rates for sexually transmitted infections (STIs) in women seeking care for symptoms of vaginitis and bacterial vaginosis, revealing highly complex associations of STIs with two of the major causes of vaginal dysbiosis. These results underscore the importance of STI testing in women seeking care for abnormal vaginal discharge and inflammation.

Anal HPV prevalence in individuals with and without other concomitant sexually transmitted infections.

The association between human papillomavirus (HPV) and other sexually transmitted infections (STIs) in anal lesions still remains unclear. Aim of the study was to evaluate the prevalence of simultaneous infection of HPV and Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis in individuals screened for HPV anal infection. A total of 507 anal samples were tested for both anal HPV and STIs: 16% resulted positive for one or more non-HPV STIs. Specifically, C. trachomatis, M. genitalium, and N. gonorrhoeae were detected in 8%, 5%, and 4% of cases, respectively. Two groups were considered, including a positive STI group and a negative STI group. The prevalence of HPV was similar in patients in both groups: high risk (HR)-HPV and low risk (LR)-HPV were 67% and 53% versus 62% (p = 0.361) and 54% (p = 0.864) of patients, respectively. However, HPV 16, 18, 35, 51, 59, and 69 were significantly more frequent in patients tested positive for other STIs versus HPV infection alone (p < 0.05). No significant differences between the two groups were observed in vaccination coverage, 28% versus 32% (p = 0.463), and HIV status, 86% versus 84% (p = 0.658). The study shows that the overall HPV status is not directly correlated to other STIs in the investigated population, except for certain HPV types, including HR-HPV 16, reinforcing the urge for a greater vaccination coverage.

Antiparasitic activity of chalcones analogue against Trichomonas vaginalis: biochemical, molecular and in silico aspects.

Trichomonas vaginalis is the etiologic agent of trichomoniasis, a worldwide distributed sexually transmitted infection (STI) that affects the genitourinary tract. Even though this disease already has a treatment in the prescription of drugs of the 5-nitroimidazole class, described low treatments adhesion, adverse side effects and cases of resistant isolates demonstrate the need for new formulations. With this in mind, chalcones emerge as a potential alternative to be tested, being compounds widely distributed in nature, easy to chemically synthesize and presenting several biological activities already reported. In this experiment, we evaluated the antiparasitic activity of 10 chalcone at a concentration of 100 µM against ATCC 30236 T. vaginalis isolates, considering negative (live trophozoites), positive (Metronidazole 100 µM) and vehicle (DMSO 0.6%) controls. Compounds 3a, 3c, 3 g and 3i showed promising results, with MICs set at 70 µM, 80 µM, 90 µM and 90 µM, respectively (p < 0,05). Cytotoxicity assays were performed on VERO and HMVII cell lines and revealed low inhibition rates at concentrations bellow 20 µM. To elucidate a possible mechanism of action for these molecules, the DPPH, ABTS and FRAP assays were performed, in which none of the four compounds presented antioxidant activity. Assays to verify ROS and lipid peroxidation in the parasite membrane were performed. None of the tested compounds identified ROS accumulation after incubation with trophozoites. 3 g molecule promoted an increase in MDA production after incubation. Results presented in this paper demonstrate the promising trichomonicidal profile, although further tests are still needed to optimize their performance and better elucidate the mechanisms of action involved.

Role of a novel uropod-like cell membrane protrusion in the pathogenesis of the parasite Trichomonas vaginalis.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease worldwide. As an extracellular parasite, adhesion to host cells is essential for the development of infection. During attachment, the parasite changes its tear ovoid shape to a flat ameboid form, expanding the contact surface and migrating through tissues. Here, we have identified a novel structure formed at the posterior pole of adherent parasite strains, resembling the previously described uropod, which appears to play a pivotal role as an anchor during the attachment process. Moreover, our research demonstrates that the overexpression of the tetraspanin T. vaginalis TSP5 protein (TvTSP5), which is localized on the cell surface of the parasite, notably enhances the formation of this posterior anchor structure in adherent strains. Finally, we demonstrate that parasites that overexpress TvTSP5 possess an increased ability to adhere to host cells, enhanced aggregation and reduced migration on agar plates. Overall, these findings unveil novel proteins and structures involved in the intricate mechanisms of T. vaginalis interactions with host cells.

Endocytosis in anaerobic parasitic protists.

The incorporation of different molecules by eukaryotic cells occurs through endocytosis, which is critical to the cell's survival and ability to reproduce. Although this process has been studied in greater detail in mammalian and yeast cells, several groups working with pathogenic protists have made relevant contributions. This review analysed the most relevant data on the endocytic process in anaerobic protists (Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, and Tritrichomonas foetus). Many protozoa can exert endocytic activity across their entire surface and do so with great intensity, as with E. histolytica. The available data on the endocytic pathway and the participation of PI-3 kinase, Rab, and Rho molecular complexes is reviewed from a historical perspective.

Investigation of the Antitrichomonal Activity of Cinnamaldehyde, Carvacrol and Thymol and Synergy with Metronidazole.

Objective: Trichomonas vaginalis is a sexually transmitted protozoan parasite that usually causes infections in women. Metronidazole is used as the first choice in the treatment of this parasitic disease, but there is a need for new drugs since 1980's with increasing numbers of reported resistance. In this study, it was aimed to determine the antitrichomonal activity of the major components of Cinnamomum zeylanicum (cinnamon) and Thymus vulgaris (thyme) essential oils, cinnamaldehyde, carvacrol and thymol against metronidazole resistant and susceptible T. vaginalis strains, and to determine their interaction with metronidazole by checkerboard method. Methods: Cinnamaldehyde, carvacrol, thymol and metronidazole were obtained commercially. Two clinical isolates and one metronidazole resistant T. vaginalis reference strain were used in the study. MIC50 and MLC values of essential oil components and metronidazole were determined by broth microdilution method. The combinations of essential oil components with metronidazole were determined by the checkerboard method. Results: According to in vitro activity tests, cinnamaldehyde was determined to be most effective essential oil component. Clinical isolates were susceptible to metronidazole. In combination study, metronidazole showed synergy with cinnamaldehyde and carvacrol, and partial synergy with thymol. Conclusion: It was determined that cinnamaldehyde, carvacrol and thymol, which are known to have high antimicrobial activity, also have strong activity against T. vaginalis isolates and show a synergistic interaction with metronidazole. The use of metronidazole at lower doses in the synergistic interaction may contribute to the literature in terms of reducing drug side effects, creating a versatile antimicrobial target, and reducing the rate of resistance development.

Bacterial vaginosis (BV) and Trichomonas vaginalis (TV) co-infection, and bacterial antibiogram profile of pregnant women studied in Lagos, Nigeria.

AIM: This study was undertaken to determine the prevalence of Bacterial Vaginosis (BV), Trichomonas Vaginalis (TV) co-infection, and the antibacterial sensitivity profile of bacterial isolates. METHODS: The study was a cross-sectional study of 232 pregnant women on a routine antenatal visit between April 2019 and Sept. 2020, at Amukoko clinic in Lagos, Nigeria. The gynaecologist conducted the clinical examination on each patient looking for vaginal discharge and its consistency/homogeneity, colour and odour. Two High Vaginal Swab (HVS) samples were taken from every patient and a semi-structured questionnaire was used to gather the socio-demographic, practices/attitudes, and clinical information of each participant. One sample was employed for wet preparation to identify the TV and BV diagnosis using Amsel's criteria and Whiff's test. The second sample was used for bacterial culture and antibiogram was conducted using the disc diffusion technique. The Clinical Laboratory Standard Institutes' (CLSI) interpretative criteria were used to categorise the results. RESULTS: The mean age of the clients was 28.11 ± 7.08 years of age. The majority (88%) were aged 15-35 years. Only 81 (34.9%) had microbial organisms isolated or seen from their specimens and 19 (8.2%) of such were classified as having BV (Bacteriods or Gardnerella isolated). Of the 81 infected, 33 (40.8%) had only bacterial infection, 36 (44.4%) had TV alone and 12 (14.8%) had bacteria co-infected with TV. From the clinical records, the population that was classified as having UTI or vaginitis was only 46 (20.7%) The study observed age (15-35 years) related association between vaginosis/ TV co-infection (X2 = 7.9; P = 0.005). Participants with symptoms of vaginitis or UTI (mainly E. coli & pseudomonas spp. isolated), BV/co-infection with TV significantly associated with female traders (X2 = 8.5; P = 0.003) and were more associated with those from polygamous relationships (X2 = 18.79, P = 0.0001). Women in their 3rd and 2nd. trimester were more significantly associated with vaginal infection (X2 = 9.47, P = 0.002; X2 = 4.79, P = 0.029) respectively. The Pseudomonas showed susceptibility to ciprofloxacin (CIP) and cefuroxime (CXM). While, E. coli isolates were susceptible to cefepime, ciprofloxacin, and imipenem. CONCLUSION: There is a relatively low prevalence of BV and flagellate co-infection in the community studied. RECOMMENDATION: We recommend screening of antenatal women with underlying symptoms for BV and flagellates co-infection to avoid its progression to vaginitis.

Epidemiology of Trichomonas vaginalis infection in the Middle East and North Africa: systematic review, meta-analyses, and meta-regressions.

BACKGROUND: Trichomoniasis, caused by the parasite Trichomonas vaginalis (TV), remains an underappreciated sexually transmitted infection (STI), primarily due to inadequate understanding of its epidemiology and public health implications. This study aimed to characterize TV epidemiology in the Middle East and North Africa (MENA). METHODS: Systematic review and analysis of evidence sourced from international, regional, and national databases were conducted. Findings were reported following PRISMA guidelines. Random-effects meta-analyses and meta-regressions were performed to determine pooled mean prevalence, investigate associations with prevalence, and identify sources of between-study heterogeneity. FINDINGS: The review identified 263 relevant publications, encompassing 462 TV prevalence measures. The pooled mean TV prevalence was estimated as follows: 4.7% (95% CI: 3.9-5.6%) in the general population of women, 17.2% (95% CI: 5.4-33.6%) among intermediate-risk populations, 10.3% (95% CI: 6.2-15.3%) among female sex workers, 13.9% (95% CI: 12.3-15.6%) among symptomatic women, 7.4% (95% CI: 1.9-15.5%) among infertility clinic attendees, 2.3% (95% CI: 0.1-6.3%) among women with miscarriages or ectopic pregnancies, and 1.6% (95% CI: 0.8-2.7%) among STI clinic attendees. Limited data were found for men. Multivariable meta-regressions explained >40% of the prevalence variation, unveiling a hierarchical prevalence pattern by population type, an inverse correlation with national income, and a prevalence decline at a rate of 1% per calendar year. INTERPRETATION: Despite conservative sexual norms, MENA has a substantial TV prevalence, comparable to the global TV prevalence. The unexpectedly high prevalence of this curable infection may, in part, be attributed to limited access to and underutilization of STI screening and treatment services. FUNDING: This work was supported by the Qatar Research, Development, and Innovation Council [ARG01-0522-230273] and by the Biomedical Research Program at Weill Cornell Medicine-Qatar.

Imidazole Carbamates as a Promising Alternative for Treating Trichomoniasis: In Vitro Effects on the Growth and Gene Expression of Trichomonas vaginalis.

Metronidazole (MTZ) is the most common drug used against Trichomonas vaginalis (T. vaginalis) infections; however, treatment failures and high rates of recurrence of trichomoniasis have been reported, suggesting the presence of resistance in T. vaginalis to MTZ. Therefore, research into new therapeutic options against T. vaginalis infections has become increasingly urgent. This study investigated the trichomonacidal activity of a series of five imidazole carbamate compounds (AGR-1, AGR-2, AGR-3, AGR-4, and AGR-5) through in vitro susceptibility assays to determine the IC50 value of each compound. All five compounds demonstrated potent trichomonacidal activity, with IC50 values in the nanomolar range and AGR-2 being the most potent (IC50 400 nM). To gain insight into molecular events related to AGR-induced cell death in T. vaginalis, we analyzed the expression profiles of some metabolic genes in the trophozoites exposed to AGR compounds and MTZ. It was found that both AGR and MTZ compounds reduced the expression of the glycolytic genes (CK, PFK, TPI, and ENOL) and genes involved in metabolism (G6PD, TKT, TALDO, NADHOX, ACT, and TUB), suggesting that disturbing these key metabolic genes alters the survival of the T. vaginalis parasite and that they probably share a similar mechanism of action. Additionally, the compounds showed low cytotoxicity in the Caco-2 and HT29 cell lines, and the results of the ADMET analysis indicated that these compounds have pharmacokinetic properties similar to those of MTZ. The findings offer significant insights that can serve as a basis for future in vivo studies of the compounds as a potential new treatment against T. vaginalis.

Zipime-Weka-Schista study protocol: a longitudinal cohort study and economic evaluation of an integrated home-based approach for genital multipathogen screening in women, including female genital schistosomiasis, human papillomavirus, Trichomonas and HIV in Zambia.

INTRODUCTION: Multiplathogen home-based self-sampling offers an opportunity to increase access to screening and treatment in endemic settings with high coinfection prevalence of sexually transmitted (HIV, Trichomonas vaginalis (Tv), human papillomavirus (HPV)) and non-sexually transmitted pathogens (Schistosoma haematobium (Sh)). Chronic coinfections may lead to disability (female genital schistosomiasis) and death (cervical cancer). The Zipime-Weka-Schista (Do self-testing sister!) study aims to evaluate the validity, acceptability, uptake, impact and cost-effectiveness of multipathogen self-sampling for genital infections among women in Zambia. METHODS AND ANALYSIS: This is a longitudinal cohort study aiming to enrol 2500 non-pregnant, sexually active and non-menstruating women aged 15-50 years from two districts in Zambia with 2-year follow-up. During home visits, community health workers offer HIV and Tv self-testing and cervicovaginal self-swabs for (1) HPV by GeneXpert and, (2) Sh DNA detection by conventional (PCR)and isothermal (recombinase polymerase assay) molecular methods. Schistosoma ova and circulating anodic antigen are detected in urine. At a clinic follow-up, midwives perform the same procedures and obtain hand-held colposcopic images. High-risk HPV positive women are referred for a two-quadrant cervical biopsy according to age and HIV status. A cost-effectiveness analysis is conducted in parallel. ETHICS AND DISSEMINATION: The University of Zambia Biomedical Research Ethics Committee (UNZABREC) (reference: 1858-2021), the London School of Hygiene and Tropical Medicine (reference: 25258), Ministry of Health and local superintendents approved the study in September 2021.Written informed consent was obtained from all participants prior to enrolment. Identifiable data collected are stored securely and their confidentiality is protected in accordance with the Data Protection Act 1998.

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

BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

[Analysis of enzyme activity and substrate specificity of dolichyl-phosphate ß-glucosyltransferase].

Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated N-glycan moieties on peptide. It is of great significance to study the N-glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues in vitro. In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate ß-glucosyltransferase E (Alg5E) derived from Trichomonas vaginalis. The results demonstrated that the recombinant Alg5E, expressed in Escherichia coli, exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme's catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme's catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).

Trichomonas vaginalis extracellular vesicles up-regulate and directly transfer adherence factors promoting host cell colonization.

Trichomonas vaginalis, a common sexually transmitted parasite that colonizes the human urogenital tract, secretes extracellular vesicles (TvEVs) that are taken up by human cells and are speculated to be taken up by parasites as well. While the crosstalk between TvEVs and human cells has led to insight into host:parasite interactions, roles for TvEVs in infection have largely been one-sided, with little known about the effect of TvEV uptake by T. vaginalis. Approximately 11% of infections are found to be coinfections of multiple T. vaginalis strains. Clinical isolates often differ in their adherence to and cytolysis of host cells, underscoring the importance of understanding the effects of TvEV uptake within the parasite population. To address this question, our lab tested the ability of a less adherent strain of T. vaginalis, G3, to take up fluorescently labeled TvEVs derived from both itself (G3-EVs) and TvEVs from a more adherent strain of the parasite (B7RC2-EVs). Here, we showed that TvEVs generated from the more adherent strain are internalized more efficiently compared to the less adherent strain. Additionally, preincubation of G3 parasites with B7RC2-EVs increases parasite aggregation and adherence to host cells. Transcriptomics revealed that TvEVs up-regulate expression of predicted parasite membrane proteins and identified an adherence factor, heteropolysaccharide binding protein (HPB2). Finally, using comparative proteomics and superresolution microscopy, we demonstrated direct transfer of an adherence factor, cadherin-like protein, from TvEVs to the recipient parasite's surface. This work identifies TvEVs as a mediator of parasite:parasite communication that may impact pathogenesis during mixed infections.

Standardised protocol for a prospective international multicentre clinical-based evaluation of point-of-care tests for the screening of genital and extragenital chlamydial and gonococcal infections in men who have sex with men and for the screening of genital chlamydial, gonococcal and Trichomonas vaginalis infections in at risk women.

INTRODUCTION: In 2016, WHO estimated there were roughly 374 million new infections among adults of the following four curable sexually transmitted infections (STIs): chlamydia (caused by Chlamydia trachomatis (CT)), gonorrhoea (Neisseria gonorrhoeae (NG)), syphilis (Treponema pallidum) and trichomoniasis (Trichomonas vaginalis (TV)). Accurate point-of-care tests (POCTs) for screening of genital and extragenital CT, NG and TV infections are of great value and have been developed during recent decade. Several tests are commercially available and have shown encouraging performance compared with 'gold-standard' reference tests in laboratory-based studies. However, there is limited data on their clinical performance, including at the POC. Key populations, such as men who have sex with men (MSM), are at higher risk of these STIs at genital and extragenital sites and these STIs are often asymptomatic, especially in extragenital sites and in women. We will conduct a clinical-based evaluation to assess the performance characteristics and acceptability to end-users of molecular-based diagnostic technology for POC/near patient use of the Xpert CT/NG (Cepheid, Sunnyvale, California, USA) test for screening of genital, anorectal and pharyngeal CT and NG infections in MSM and the Xpert CT/NG and Xpert TV (Cepheid, Sunnyvale, California, USA) for screening of genital CT, NG and TV among women at risk for these STIs compared with gold-standard reference nucleic acid amplification tests. This master protocol outlines the overall research approach that will be used in seven countries. METHOD AND ANALYSES: Consecutive MSM and women at risk presenting at the clinical sites in high, and low- and middle-income countries will be enrolled. The POCTs to be evaluated are Xpert CT/NG and Xpert TV. All procedures will be carried out by trained healthcare staff and tests performed in strict accordance with the manufacturer's instructions. The sensitivity, specificity, positive and negative predictive values for each POCT will be calculated. The study is ongoing with recruitment expected to be completed in all countries by mid-2022 to late-2022. ETHICS AND DISSEMINATION: Prior to enrolment, this core protocol was independently peer-reviewed and approved by the research project review panel (RP2) of the WHO Department of Sexual and Reproductive Health and Research and by the WHO Ethics Review Committee (ERC). The core protocol has been slightly adapted accordingly to individual countries and adaptations approved by both RP2 and ERC, as well as all relevant institutional review boards at each participating site. Results will be disseminated through peer-reviewed journals and presented at relevant national/international conferences.

BD Vaginal Panel assay results on the high-throughput BD COR system compared to the BD MAX system.

Molecular-based assays demonstrate excellent sensitivity for the detection of vaginitis causes. Here, the high-throughput BD Vaginal Panel for BD COR System (VP-COR) performance was compared to that of the predicate, BD MAX Vaginal Panel for BD MAX System (VP-MAX). Clinical or contrived samples were used to determine the agreement between VP-COR and VP-MAX. Acceptance criteria for VP-COR agreement were as follows: bacterial vaginosis (BV) required a positive percent agreement (PPA) point estimate of ≥95% and a negative percent agreement (NPA) point estimate of ≥98%; Candida group, Candida glabrata, Candida krusei, and Trichomonas vaginalis (TV) required a PPA and NPA point estimate of ≥95% [with lower bound of 95% confidence interval (95% CI) ≥90%]. PPA was 99.5% (95% CI: 97.5-100) and 97.9% (95% CI: 96.5-98.8) for BV contrived (n = 516) and BV clinical (n = 1,050) specimens, respectively. For the Candida group (clinical; n = 724), C. glabrata (contrived; n = 544), C. krusei (contrived; n = 522), and TV (clinical; n = 702), PPA was 99.4% (95% CI: 98.0-99.9), 100% (95% CI: 97.9-100), 100% (95% CI: 97.6-100), and 99.7% (95% CI: 98.3-100), respectively; the lowest lower bound CI value was 97.6%. NPA was >95% for BV contrived and BV clinical specimens. For the Candida group, C. glabrata, C. krusei, and TV, NPA was ≥98.9%; the lowest lower bound CI value was 97.3%. These results demonstrate the equivalent performance of the VP-COR assay when compared to VP-MAX.IMPORTANCEVaginitis is common among women of reproductive age, resulting in around 10 million office visits a year. Diagnosis is often difficult due to its multiple causes-including bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis-as well as variation in symptom presentation. Typically, cases are identified with a combination of symptomology, medical history, physical examination, and office- or laboratory-based testing. These traditional techniques involve subjective elements and demonstrate varying sensitivity and specificity. Inaccurate or delayed diagnosis leads to continued symptoms, repeat visits, inappropriate treatment, and unnecessary costs. Alternatively, the use of molecular-based assays increases sensitivity for the detection of vaginitis causes. With the validation of the vaginal panel molecular assay on COR (a high-throughput platform), a workflow can be streamlined in high-demand laboratories while providing high sensitivity for vaginitis detection.

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

Early endosomes sort transmembrane cargo either for lysosomal degradation or retrieval to the plasma membrane or the Golgi complex. Endosomal retrieval in eukaryotes is governed by the anciently homologous retromer or retriever complexes. Each comprises a core tri-protein subcomplex, membrane-deformation proteins and interacting partner complexes, together retrieving a variety of known cargo proteins. Trichomonas vaginalis, a sexually transmitted human parasite, uses the endomembrane system for pathogenesis. It has massively and selectively expanded its endomembrane protein complement, the evolutionary path of which has been largely unexplored. Our molecular evolutionary study of retromer, retriever and associated machinery in parabasalids and its free-living sister lineage of Anaeramoeba demonstrates specific expansion of the retromer machinery, contrasting with the retriever components. We also observed partial loss of the Commander complex and sorting nexins in Parabasalia but complete retention in Anaeramoeba. Notably, we identified putative parabasalid sorting nexin analogs. Finally, we report the first retriever protein localization in a non-metazoan group along with retromer protein localization in T. vaginalis.

Characterization of Actin-based Genotypes and Mycoplasma Endosymbionts of Trichomonas vaginalis Isolated in Sapporo, Japan.

PURPOSE: Trichomonas vaginalis is a causative agent of common non-viral sexually transmitted infections worldwide. However, the biological features, such as genotypes and endosymbionts, of T. vaginalis isolated in Japan remain unclear. The aim of this study was to characterize the actin-based genotypes and the endosymbionts of T. vaginalis isolated in Sapporo, Japan. METHODS: Three T. vaginalis clinical strains were isolated in Sapporo, Japan between 2019 and 2022. Actin-based genotyping was conducted by sequencing and phylogenetic analyses. The endosymbionts, such as Mycoplasma sp. and Trichomonasvirus, were detected using PCR and RT-PCR, respectively. Furthermore, the detected Mycoplasma spp. were identified using 16S rRNA gene sequencing. RESULTS: Of the three T. vaginalis strains, two belonged to genotype E, whereas one was genotype G as determined by actin-based genotyping. Two of the T. vaginalis strains harbored Mycoplasma spp. Using nearly full-length 16S rRNA gene sequencing, both were identified as Candidatus Mycoplasma girerdii. In contrast, the Trichomonasvirus was not found in the T. vaginalis strains. CONCLUSION: To our knowledge, this is the first report on the characterization of actin-based genotypes and the presence of endosymbiotic Ca. M. girerdii in T. vaginalis strains in Japan. Thus, this study will provide an important impetus for future research.