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 β-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.
Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and ...pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.
The parasitism by
Trichomonas vaginalis
is complex and in part is mediated by cytoadherence accomplished via five surface proteins named adhesins and a glycoconjugate called lipophosphoglycan ...(TvLPG). In this study, we evaluated the ability of
T
.
vaginalis
isolates to adhere to cells, plastic (polystyrene microplates), intrauterine device (IUD), and vaginal ring. Of 32
T
.
vaginalis
isolates, 4 (12.5%) were strong adherent. The
T
.
vaginalis
isolates TV-LACM6 and TV-LACM14 (strong polystyrene-adherent) were also able to adhere to IUD and vaginal ring. Following chemical treatments, results demonstrated that the
T
.
vaginalis
components, lipophosphoglycan, cytoskeletal proteins, and surface molecules, were involved in both adherence to polystyrene and cytoadherence. The gene expression level from four adhesion proteins was highest in trophozoites adhered to cells than trophozoites adhered to the abiotic surface (polystyrene microplate). Our data indicate the major involvement of TvLPG in adherence to polystyrene, and that adhesins are important for cytoadherence. Furthermore, to our knowledge, this is the first report showing the
T
.
vaginalis
adherence to contraceptive devices, reaffirming its importance as pathogen among women in reproductive age.
Trichomonosis, caused by the flagellate protozoan
Trichomonas vaginalis
, is the most common non-viral sexually transmitted disease worldwide. Actually, the infection treatment is based on ...5-nitroimidazole drugs. However, an emergent number of resistant isolates makes important the search for new therapeutic arsenal. In this sense, the investigation of plants and their metabolites is an interesting approach. In the present study, the anti-
T. vaginalis
activity of 44 aqueous extracts from 23 Caatinga plants used in folk medicine was evaluated. After screening 44 aqueous extracts from 23 distinct plants against two isolates from ATCC and four fresh clinical isolates, only the
Polygala decumbens
root extract was effective in reducing significantly the trophozoite viability. The MIC value against all isolates tested, including the metronidazole resistant, was 1.56 mg/mL. The kinetic growth assays showed that the extract was able to completely abolish the parasite density in the first hours of incubation, confirmed by microscopy. In summary, this study describes the first report on the activity of
P. decumbens
from Caatinga against
T. vaginalis
, being directly related to the popular knowledge and use.
Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic ...characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1–5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis.
Trichomonas vaginalis is the causative agent of trichomoniasis, the most common non-viral STD in the world, and it expresses five nucleoside triphosphate diphosphohydrolase (NTPDase) genes related to parasite infection.
Dos Santos O, De Resende MCC, De Mello AL, Frazzon APG, D’Azevedo PA. The use of whole‐cell protein profile analysis by SDS‐PAGE as an accurate tool to identify species and subspecies of ...coagulase‐negative staphylococci. APMIS 2012; 120: 39–46.
We used sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as a tool to characterize coagulase‐negative staphylococci (CoNS). Of 253 clinical isolates and 10 control strains, five species and four subspecies were analyzed. All the isolates were identified using conventional phenotypic tests and SDS‐PAGE. Discrepant results between these methods, as well as less common species and subspecies, were confirmed by sodA and 16S rDNA gene sequencing. Intraspecies similarities, calculated by the Dice coefficient, were significantly higher when compared to interspecies similarities. The conventional method failed to identify eight (3.2%) molecularly defined and SDS‐PAGE‐determined isolates. Therefore, SDS‐PAGE was able to discriminate between all unidentified or misidentified isolates using a phenotypic method. In addition, SDS‐PAGE identified all atypical isolates using biochemistry and CoNS at the subspecies level.
Background and Objectives: the association between the biofilm formations an antifungal resistance has been suggested to be an important factor in the pathogenesis of several Candida species. ...Besides, studies have included invasive candidiasis from hospitalized patients; however there are few studies that evaluated the species distribution, antifungal susceptibility and biofilm formation of Candida species isolated from ambulatory patients. Thus, the aim of this study was to evaluate whether biofilm producing contributes to antifungal resistance in Candida isolates from urine sample obtained from ambulatory patients. Methods: During one year, 25 urine samples positive for yeast were collected, stored and plated on agar supplemented with chloramphenicol and Sabouread left at room temperature for 5 days for subsequent: 52% (13/25) were C. albicans, 36% (9/25) C. tropicalis, 8% (2/25) C. krusei and 4% (1/25) C. parapsilosis. Results: The ability to form biofilm was detected in 23 (92%) of the yeast studied and 15.4% (2/13) of C. albicans were fluconazole (FLU) and ketoconazole (KET) resistant, while 11.1% (1/9) of C. tropicalis were ketoconazole resistant and were anidulafungin (ANI) non-susceptible. Conclusion: our results showed the high capacity for biofilm formation among Candida isolates from ambulatory patients.
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•Guanine nucleotides were substrates for T. vaginalis ectonucleotidases.•HPLC showed consumption of extracellular GTP, with final accumulation of guanosine.•The highest gene ...expressions were found for TvNTPDase 2 and 4 by qRT-PCR.•Extracellular guanosine uptake was observed as 13CGTP nucleotide into parasite DNA.
Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5′-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5′-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5′-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4. The extracellular guanosine uptake was observed as 13CGTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as 13CATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction.
•High prevalence rates of M. hominis- and TVVs-positive T. vaginalis isolates.•No association between bacteria and viruses infection and metronidazole resistance.•First report employing sequencing ...methodology to certify the analysis of M. hominis.•Expression levels of PFOR are not the main mechanism of resistance to metronidazole.•First study of T. vaginalis infection by M. hominis and TVVs 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.
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► 42 marine-associated fungi species were selected to be screened against T. vaginalis. ► Hypocrea lixii and Penicillium citrinum were active against all isolates. ► The active ...filtrates also inhibited the metronidazole-resistant isolate growth. ► The P. citrinum F40 filtrate sample showed low cytotoxicity against Vero cells. ► Fungi from South Brazilian Coast are a potential source of antiparasitic molecules.
Trichomonas vaginalis is the causative agent of trichomonosis, the most common non-viral sexually transmitted disease. Infection with this protozoan may have serious consequences, especially for women. Currently, 5-nitroimidazole drugs are the treatment of choice for trichomonosis, but the emergence of resistance has limited the effectiveness of this therapy. In this context, this study aimed to evaluate the anti-T. vaginalis activity of marine-associated fungi found in the South Brazilian Coast. A total of 42 marine-associated fungal species (126 filtrate samples) isolated from 39 different marine organisms, mainly sponges, were selected to be screened against T. vaginalis. Of these, two filtrate samples from Hypocrea lixii F02 and Penicillium citrinum F40 showed significant growth-inhibitory activity (up to 100%) against ATCC 30236 and fresh clinical isolates, including a metronidazole-resistant isolate. Minimum inhibitory concentration (MIC) values of H. lixii F02 and P. citrinum F40 samples for all isolates tested, including the metronidazole-resistant isolate, were 2.5mg/mL. The kinetic growth curve showed that the filtrate samples were able to reduce the density of parasites to zero within 24h of incubation, which was confirmed by microscopy. Both fungal filtrate samples exhibited no hemolytic activity, and the P. citrinum F40 filtrate sample showed low cytotoxicity against Vero cells. These data suggest that marine-associated fungi from the South Brazilian Coast may produce potential candidates for further investigation and possible use in the treatment of metronidazole-resistant trichomonosis.
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