Metabolomic profiling and stable isotope labelling of Trichomonas vaginalis and Tritrichomonas foetus reveal major differences in amino acid metabolism including the production of 2-hydroxyisocaproic acid, cystathionine and S-methylcysteine

Trichomonas vaginalis and Tritrichomonas foetus are pathogens that parasitise, respectively, human and bovine urogenital tracts causing disease. Using LC-MS, reference metabolomic profiles were obtained for both species and stable isotope labelling with D-[U-13C6] glucose was used to analyse central carbon metabolism. This facilitated a comparison of the metabolic pathways of T. vaginalis and T. foetus, extending earlier targeted biochemical studies. 43 metabolites, whose identities were confirmed by comparison of their retention times with authentic standards, occurred at more than 3-fold difference in peak intensity between T. vaginalis and T. foetus. 18 metabolites that were removed from or released into the medium during growth also showed more than 3-fold difference between the species. Major differences were observed in cysteine and methionine metabolism in which homocysteine, produced as a bi-product of trans-methylation, is catabolised by methionine γ-lyase in T. vaginalis but converted to cystathionine in T. foetus. Both species synthesise methylthioadenosine by an unusual mechanism, but it is not used as a substrate for methionine recycling. T. vaginalis also produces and exports high levels of S-methylcysteine, whereas only negligible levels were found in T. foetus which maintains significantly higher intracellular levels of cysteine. 13C-labeling confirmed that both cysteine and S-methylcysteine are synthesised by T. vaginalis; S-methylcysteine can be generated by recombinant T. vaginalis cysteine synthase using phosphoserine and methanethiol. T. foetus contained higher levels of ornithine and citrulline than T. vaginalis and exported increased levels of putrescine, suggesting greater flux through the arginine dihydrolase pathway. T. vaginalis produced and exported hydroxy acid derivatives of certain amino acids, particularly 2-hydroxyisocaproic acid derived from leucine, whereas negligible levels of these metabolites occurred in T. foetus

Genomic and metabolomic polymorphism among experimentally selected paromomycin-resistant Leishmania donovani strains

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study paromomycin (PMM) resistance (PMM-R) was induced in three Nepalese clinical strains of L. donovani, with different inherent susceptibility to antimony drugs (Sb), by step-wise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites even though a single cloned population was used at the start of selection. PMM IC50 values for PMM-R parasites varied between 104-481 µM at the promastigotes stage and 32-195 µM at the intracellular amastigotes stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote but not the promastigote stage (p < 0.05). This effect was most marked in the Sb-R PMM-R clone, where PMM-R resistance was associated with a significant upregulation in glutathione compared to its WT (p < 0.05) although there was no change in trypanothione (detected in its oxidised form). Interestingly, PMM-R strains showed an increase in either the keto acid derivative of isoleucine (Sb-I PMM-R) or the 2-hydroxy acids derived from arginine and tyrosine (Sb-S PMM-R and Sb-R PMM-R). These results are consistent with the recent finding that upregulation of the branch-chain amino acid aminotransferase and the D-lactate dehydrogenase are linked to PMM-R. In addition, we found that PMM-R was associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow rapid selection of genetic changes that confer a survival advantage

Acceptability of HIV self-sampling kits (TINY vial) among people of black African ethnicity in the UK: a qualitative study

Background: Increasing routine HIV testing among key populations is a public health imperative, so improving access to acceptable testing options for those in need is a priority. Despite increasing targeted distribution and uptake of HIV self-sampling kits (SSKs) among men who have sex with men in the UK, little is known about why targeted SSK interventions for black African users are not as wide-spread or well-used. This paper addresses this key gap, offering insight into why some groups may be less likely than others to adopt certain types of SSK interventions in particular contexts. These data were collected during the development phase of a larger study to explore the feasibility and acceptability of targeted distribution of SSKs to black African people. Methods: We undertook 6 focus groups with members of the public who self-identified as black African (n = 48), 6 groups with specialists providing HIV and social services to black African people (n = 53), and interviews with HIV specialist consultants and policy-makers (n = 9). Framework analysis was undertaken, using inductive and deductive analysis to develop and check themes. Results: We found three valuable components of targeted SSK interventions for this population: the use of settings and technologies that increase choice and autonomy; targeted offers of HIV testing that preserve privacy and do not exacerbate HIV stigma; and ensuring that the specific kit being used (in this case, the TINY vial) is perceived as simple and reliable. Conclusions: This unique and rigorous research offers insights into participants’ views on SSK interventions, offering key considerations when targeting this population.. Given the plethora of HIV testing options, our work demonstrates that those commissioning and delivering SSK interventions will need to clarify (for users and providers) how each kit type and intervention design adds value. Most significantly, these findings demonstrate that without a strong locus of control over their own circumstances and personal information, black African people are less likely to feel that they can pursue an HIV test that is safe and secure. Thus, where profound social inequalities persist, so will inequalities in HIV testing uptake – by any means

Transient Nature of Long-Term Nonprogression and Broad Virus-Specific Proliferative T-Cell Responses with Sustained Thymic Output in HIV-1 Controllers

HIV-1(+) individuals who, without therapy, conserve cellular anti-HIV-1 responses, present with high, stable CD4(+) T-cell numbers, and control viral replication, facilitate analysis of atypical viro-immunopathology. In the absence of universal definition, immune function in such HIV controllers remains an indication of non-progression.CD4 T-cell responses to a number of HIV-1 proteins and peptide pools were assessed by IFN-gamma ELISpot and lymphoproliferative assays in HIV controllers and chronic progressors. Thymic output was assessed by sjTRECs levels. Follow-up of 41 HIV-1(+) individuals originally identified as "Long-term non-progressors" in 1996 according to clinical criteria, and longitudinal analysis of two HIV controllers over 22 years, was also performed. HIV controllers exhibited substantial IFN-gamma producing and proliferative HIV-1-specific CD4 T-cell responses to both recombinant proteins and peptide pools of Tat, Rev, Nef, Gag and Env, demonstrating functional processing and presentation. Conversely, HIV-specific T-cell responses were limited to IFN-gamma production in chronic progressors. Additionally, thymic output was approximately 19 fold higher in HIV controllers than in age-matched chronic progressors. Follow-up of 41 HIV-1(+) patients identified as LTNP in 1996 revealed the transitory characteristics of this status. IFN-gamma production and proliferative T-cell function also declines in 2 HIV controllers over 22 years.Although increased thymic output and anti-HIV-1 T-cell responses are observed in HIV controllers compared to chronic progressors, the nature of nonprogressor/controller status appears to be transitory

Metabolomic analyses of Leishmania reveal multiple species differences and large differences in amino acid metabolism

Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts

Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

Conceived and designed the experiments: FS PC BC ES AM. Performed the experiments: FS RS ES PF SR. Analyzed the data: FS BC ES PF GEK PFC AM. Contributed reagents/materials/analysis tools: PC PB GC. Wrote the paper: FS GEK BC AM.The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.Yeshttp://www.plosone.org/static/editorial#pee

Evaluation of mobile phase characteristics on three zwitterionic columns in hydrophilic interaction liquid chromatography mode for liquid chromatography-high resolution mass spectrometry based untargeted metabolite profiling of Leishmania parasites

It has been reported that HILIC column chemistry has a great effect on the number of detected metabolites in LC-HRMS-based untargeted metabolite profiling studies. However, no systematic investigation has been carried out with regard to the optimisation of mobile phase characteristics. In this study using 223 metabolite standards, we explored the retention mechanisms on three zwitterionic columns with varied mobile phase composition, demonstrated the interference from poor chromatographic peak shapes on the output of data extraction, and assessed the quality of chromatographic signals and the separation of isomers under each LC condition. As expected, on the ZIC-cHILIC column the acidic metabolites showed improved chromatographic performance at low pH which can be attributed to the opposite arrangement of the permanently charged groups on this column in comparison with the ZIC-HILIC column. Using extracts from the protozoan parasite Leishmania, we compared the numbers of repeatedly detected LC-HRMS features under different LC conditions with putative identification of metabolites not amongst the standards being based on accurate mass (±3ppm). Besides column chemistry, the pH of the mobile phase plays a key role in not only determining the retention mechanisms of solutes but also the output of the LC-HRMS data processing. Fast evaporation of ammonium carbonate produced less ion suppression in ESI source and consequently improved the detectability of the metabolites in low abundance in comparison with other ammonium salts. Our results show that the combination of a ZIC-pHILIC column with an ammonium carbonate mobile phase, pH 9.2, at 20mM in the aqueous phase or 10mM in both aqueous and organic mobile phase components, provided the most suitable LC conditions for LC-HRMS-based untargeted metabolite profiling of Leishmania parasite extracts. The signal reliability of the mass spectrometer used in this study (Exactive Orbitrap) was also investigated