Anti-Trichomonas vaginalis activity of 1,10-phenanthroline-5,6-dione-based metallodrugs and synergistic effect with metronidazole.

Trichomonas vaginalis is responsible for the most common non-viral, sexually transmitted infection, human trichomoniasis, and is associated with an increased susceptibility to HIV. An escalation in resistance (2.5-10%) to the clinical drug, metronidazole (MTZ), has been detected and this compound also has adverse side-effects. Therefore, new treatment options are urgently required. Herein, we investigate the possible anti-T. vaginalis activity of 1,10-phenanthroline-5,6-dione (phendione) and its metal complexes, [Ag(phendione)2]ClO4 and [Cu(phendione)3](ClO4)2·4H2O. Minimum inhibitory concentration (MIC) against T. vaginalis ATCC 30236 and three fresh clinical isolates and mammalian cells were performed using serial dilution generating IC50 and CC50 values. Drugs combinations with MTZ were evaluated by chequerboard assay. A strong anti-T. vaginalis activity was found for all test compounds. IC50 values obtained for [Cu(phendione)3](ClO4)2·4H2O were similar or lower than those obtained for MTZ. In vitro assays with normal cells showed low cytotoxicity and [Cu(phendione)3](ClO4)2·4H2O presented a high selectivity index (SI) for fibroblasts (SI = 11.39) and erythrocytes (SI > 57.47). Chequerboard assay demonstrated that the combination of [Cu(phendione)3](ClO4)2·4H2O with MTZ leads to synergistic interaction, which suggests distinct mechanisms of action of the copper-phendione complex and avoiding the MTZ resistance pathways. Our results highlight the importance of phendione-based drugs as potential molecules of pharmaceutical interest.

Oxidative damage by 1,10-phenanthroline-5,6-dione and its silver and copper complexes lead to apoptotic-like death in Trichomonas vaginalis.

Trichomoniasis is a neglected, parasitic, sexually transmitted infection. Resistance to the only approved drugs is increasing worldwide, leaving millions of people without alternative medications. Thus, the search for new therapeutic options against this infection is necessary. Previously, our group reported that 1,10-phenanthroline-5,6-dione (phendione) and its silver(I) and copper (II) complexes (abbreviated as Ag-phendione and Cu-phendione, respectively) presented activity against the amitochondriate parasite T. vaginalis, with Cu-phendione being the most effective (IC50 = 0.84 µM). Methods: qRT-PCR, SEM, flow cytometry. The current study on the effects of Cu-phendione on the antioxidant metabolism of T. vaginalis by qRT-PCR revealed that the complex causes a decrease in the relative expression of mRNA of NADH oxidase, flavin reductase, superoxide dismutase, peroxiredoxin, iron-sulfur flavoprotein, rubrerythrin and osmotically inducible proteins. In contrast, the mRNA expression of flavodiiron protein was increased. Detoxification-related enzymes were downregulated, impairing oxygen metabolism in trophozoites and triggering a subsequent accumulation of the superoxide anion. Although no DNA fragmentation was observed, the treatment of parasites with Cu-phendione led to a significant reduction in cell size and a concomitant increase in granularity. The complex promoted phosphatidylserine exposure at the plasma membrane (as judged by Annexin V binding) and propidium iodide was unable to passively permeate the parasites. All of these outcomes are classical hallmarks of cell death by apoptosis. In essence, the trichomonacidal effect of Cu-phendione operates through redox homeostasis imbalance, which is a mode of action that is quite distinct from that caused by metronidazole.

Metallopeptidases as Key Virulence Attributes of Clinically Relevant Protozoa: New Discoveries, Perspectives, and Frontiers of Knowledge.

This article provides a comprehensive review of several subclasses of metallo-type peptidases expressed by the main clinically relevant protozoa, including Plasmodium spp., Toxoplasma gondii, Cryptosporidium spp., Leishmania spp., Trypanosoma spp., Entamoeba histolytica, Giardia duodenalis, and Trichomonas vaginalis. These species comprise a diverse group of unicellular eukaryotic microorganisms responsible for widespread and severe human infections. Metallopeptidases, defined as hydrolases with activity mediated by divalent metal cation, play important roles in the induction and maintenance of parasitic infections. In this context, metallopeptidases can be considered veritable virulence factors in protozoa with direct/indirect participation in several key pathophysiological processes, including adherence, invasion, evasion, excystation, central metabolism, nutrition, growth, proliferation, and differentiation. Indeed, metallopeptidases have become an important and valid target to search for new compounds with chemotherapeutic purposes. The present review aims to gather updates regarding metallopeptidase subclasses, exploring their participation in protozoa virulence as well as investigating the similarity of peptidase sequences through bioinformatic techniques in order to discover clusters of great relevance for the development of new broad antiparasitic molecules.

Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and ß-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

High rates of double-stranded RNA viruses and Mycoplasma hominis in Trichomonas vaginalis clinical isolates in South Brazil.

Trichomonas vaginalis is the etiological agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in world, with 276.4 million new cases each year. T. vaginalis can be naturally infected with Mycoplasma hominis and Trichomonasvirus species. This study aimed to evaluate the prevalence of T. vaginalis infected with four distinct T. vaginalis viruses (TVVs) and M. hominis among isolates from patients in Porto Alegre city, South Brazil. An additional goal of this study was to investigate whether there is association between metronidazole resistance and the presence of M. hominis during TVV infection. The RNA expression level of the pyruvate ferredoxin oxidoreductase (PFOR) gene was also evaluated among metronidazole-resistant and metronidazole-sensitive T. vaginalis isolates. A total of 530 urine samples were evaluated, and 5.7% samples were positive for T. vaginalis infection. Among them, 4.51% were isolated from female patients and 1.12% were from male patients. Remarkably, the prevalence rates of M. hominis and TVV-positive T. vaginalis isolates were 56.7% and 90%, respectively. Most of the T. vaginalis isolates were metronidazole-sensitive (86.7%), and only four isolates (13.3%) were resistant. There is no statistically significant association between infection by M. hominis and infection by TVVs. Our results refute the hypothesis that the presence of the M. hominis and TVVs is enough to confer metronidazole resistance to T. vaginalis isolates. Additionally, the role of PFOR RNA expression levels in metronidazole resistance as the main mechanism of resistance to metronidazole could not be established. This study is the first report of the T. vaginalis infection by M. hominis and TVVs in a large collection of isolates from South Brazil.