Analysis of the role of VapBC-type toxin-antitoxin modules in growth, stress tolerance and drug tolerance in mycobacteria

Forty seven of the toxin-antitoxin modules in the genome of Mycobacterium tuberculosis belong to the VapBC family in which the VapC toxin is a member of the PIN-domain protein family associated with nuclease activity. The role of VapBCs in the physiology of M. tuberculosis and the cellular function(s) served by their expansion are unknown but is the subject of intense investigation as a result of the evidence suggesting an association between TAmodule function and stress adaptation as well as phenotypic drug tolerance in certain organisms. In this study, the function of ten vapBC modules from M. tuberculosis and the single vapBC from M. smegmatis was investigated. Of the vapCs assessed, Rv0549c, Rv0595c, Rv2549c and Rv2829c were growth inhibitory when conditionally expressed under the control of a tetracycline (Tet)- regulated promoter in both M. smegmatis and M. tuberculosis, with Rv0549c being less toxic than the others. The toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that in order for toxicity to be observed, the VapC level must exceed a certain threshold. Low levels of protein expression were demonstrated for Rv2456 following induction, which may account for the lack of toxicity observed for this, and the remaining ‘non-toxic’ VapCs. In addition, given that Rv3320c was toxic in both M. smegmatis and M. tuberculosis only when expressed in the absence of the Tet repressor, protein expression levels, rather than differences in (nuclease) activity appeared to be the principal determinant of VapC toxicity in this assay system. VapC toxicity was neutralized by co-expression of the cognate vapB antitoxin from both an operon with the toxin, as well as from a different chromosomal locus. However, noncognate antitoxins could not abrogate VapC toxicity thus demonstrating a specificity of interaction between VapCs and their cognate VapBs. Deletion of selected vapBC genes did not affect mycobacterial growth in vitro or mycobacterial stress adaptation, but rendered it more susceptible to growth inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’ VapCs was not increased in deletion mutant strains, even when the mutation eliminated the cognate VapB, presumably due to insufficient levels of VapC expression in this genetic background. Interestingly, low levels of VapC appeared VapCs that are induced as a result of stochastic and/or environmentally-induced vapBC expression might have a significant effect on the physiology of mycobacteria. The above-mentioned findings suggest that the vapBC family may provide an abundant source of nuclease activity in M. tuberculosis, which can vary as a function of regulated expression of individual modules, and the rates/mechanisms of antitoxin degradation. Such activity is likely to have a profound impact on the physiology of M. tuberculosis

The effects of anti-HIV nucleoside drugs on the virulence of clinically relevant candida species

Student Number: 0420652W - MSc(Med) Dissertation - School of Pathology - Faculty of Health SciencesCandida species are opportunistic yeasts that cause infections in immunocompromised individuals such as HIV and cancer patients. Recent studies show that 5fluorouracil, a nucleoside analogue used for cancer treatment, increases Candida cell virulence. The aim of this study is to determine the effects of commonly used antiHIV nucleoside analogue drugs on the virulence of Candida albicans, the predominant species associated with oral candidiasis. Oral swabs were collected from antiretroviralnaïve HIVpositive individuals. C. albicans was characterised from 39 of these swabs using standard microbiological techniques and polymerase chain reaction. The effect of nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine, stavudine, didanosine and lamivudine, at predicted drug peak concentrations in patients, as well as half and double these concentrations on select virulence factors of C. albicans isolates were studied. In addition, antifungal susceptibility to amphotericin B was assessed. Not all 39 isolates were used in the assays because of delays in obtaining reagents from respective manufacturers. Results show no change in the adherence and biofilm formation of 29 isolates upon exposure to NRTIs. In contrast, a steady increase in the number of viable cells was observed upon exposure to double the peak concentration of lamivudine to 23 of the clinical isolates. All 31 isolates tested were susceptible to amphotericin B (MIC£1mg/ml). Although these results suggest that NRTIs may have little effect on the virulence of C. albicans it is postulated, that, in a dosedependent manner, cytidine analogues act similarly to 5FU by activating a signaltransduction pathway which stimulates proliferation

VapC Toxins from Mycobacterium tuberculosis Are Ribonucleases that Differentially Inhibit Growth and Are Neutralized by Cognate VapB Antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as ‘non-toxic’. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’ VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617 – VapC proteins with similarity to Rv0549c and Rv3320c, respectively – these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism

Current Perspective of Antiviral Strategies against COVID-19.

10.1021/acsinfecdis.0c00236ACS Infect Discomplete

Efficacy of Adjunctive Tofacitinib Therapy in Mouse Models of Tuberculosis

The global tuberculosis (TB) epidemic and the spread of multi- and extensively-drug resistant strains of Mycobacterium tuberculosis (M.tb) have been fueled by low adherence to following lengthy treatment protocols, and the rapid spread of HIV (Human Immunodeficiency Virus). Persistence of the infection in immunocompetent individuals follows from the ability of M.tb to subvert host immune responses in favor of survival within macrophages. Alternative host-directed strategies are therefore being currently sought to improve treatment efficacy and duration. In this study, we evaluated tofacitinib, a new oral Janus kinase (JAK) blocker with anti-inflammatory properties, in shortening tuberculosis treatment. BALB/c mice, which are immunocompetent, showed acceleration of M.tb clearance achieving apparent sterilization after 16 weeks of adjunctive tofacitinib therapy at average exposures higher than recommended in humans, while mice receiving standard treatment alone did not achieve clearance until 24 weeks. True sterilization with tofacitinib was not achieved until five months. C3HeB/FeJ mice, which show reduced pro-inflammatory cytokines during M.tb infection, did not show improved clearance with adjunctive tofacitinib therapy, indicating that the nature of granulomatous lesions and host immunity may influence responsiveness to tofacitinib. Our findings suggest that the JAK pathway could be explored further for host-directed therapy in immunocompetent individuals

Pirfenidone Exacerbates Granulomatous Inflammation and Disease Progression in Tuberculosis

10.1164/ajrccm-conference.2016.193.1_MeetingAbstracts.A2640American Journal of Respiratory and Critical Care Medicine193A101A2640-A2640United State

OC 8711 Early biomarkers of lung inflammation and function in trials of host-directed tuberculosis therapies (TB-HDT)

10.1136/bmjgh-2019-edc.37BMJ Global Health4Suppl 3A15.2-A1