Deciphering the physiology of drug tolerant and resistant Mycobacterium tuberculosis

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Stellenbosch : Stellenbosch University
ENGLISH ABSTRACT: Poor adherence to treatment for tuberculosis (TB) disease and the rising incidents of drug resistant Mycobacterium tuberculosis strains are factors that negatively influence TB control. The current study was designed to explore some of the key knowledge gaps concerning M. tuberculosis physiology; looking at the effect of the diverse M. tuberculosis genetic backgrounds and the presence of ropB mutation on the transcriptome, looking at M. tuberculosis host response and the likelihood as to whether induced mycobacterial tolerance can provide a reservoir from which genetic resistance can arise. Exploring some of these key knowledge gaps was imperative, given the fact that, lengthy anti-TB drug treatment could be required to entirely eradicate some of M. tuberculosis strains, and non-compliance with completing treatment might lead to the emergence of multidrug (MDR)-TB. Firstly, we investigated the effect of M. tuberculosis strains with different genetic backgrounds on their total transcriptomic profiles (as a proxy for the physiological state). Secondly, we examined the influence of rpoB Ser531Leu mutation and the effect of isoniazid (INH) treatment (24h) at sub-lethal concentrations on the transcriptomic profiles of rifampicin (RIF)-resistant (K636RIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains, using RNA-sequencing (RNA-Seq) and Real-Time quantitative polymerase chain reaction (RT-qPCR) techniques. RNA-Seq analysis identified significantly differentially expressed genes in the transcriptomes of K636WT, H37RVWT and K636RIF M. tuberculosis strains. Our comparative transcriptomic data of K636WT relative to H37RvWT M. tuberculosis strains demonstrated that different genetic backgrounds influenced the total transcriptome. We demonstrated that rpoB Ser531Leu mutation has an impact on the transcriptional responses of K636WT relative to K636RIF M. tuberculosis strains. Our data did not demonstrate an effect of INH treatment on the transcriptomes of M. tuberculosis strains from different genetic backgrounds, making this one of our limitations. We then assessed the host immune response after infection with RIF-resistant (K636RIF and H37RvRIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains using the luminex x multi-analyte profiling (xMAP) technology and enzyme-linked immunosorbent assay (ELISA). Our host response data (Chapter 4) revealed no differences in host response to K636WT and H37RvWT M. tuberculosis strains from different genetic backgrounds. In contrast, there were differences in host response to K636WT and K636RIF M. tuberculosis strains in a RAW264.7 macrophage model of infection. This was confirmed by the observed varying secretion levels of cytokines and chemokines (IL-6, IL-12p40 and RANTES) required to mediate M. tuberculosis growth and survival after 24 - 48h of infection. We further investigated whether viable but non-replicating (VBNR) persisters Mycobacterium smegmatis sub-populations, when exposed to high INH concentrations, may provide a pool from which genetic resistant mutants can arise. We used a combination of a fluorescence dilution (FD) reporter system, flow cytometry and fluorescence-activated cell sorting (FACS) to detect, quantify and separate VBNR and actively replicating (AR) M. smegmatis bacterial populations following INH treatment. Subsequently, we performed PCR to amplify the katG and inhA promoter and Sanger sequencing to identify mutations in these genes that are commonly associated with INH resistance. Our flow cytometry results showed successful detection of VBNR and AR bacterial populations in M. smegmatis::pTiGc following INH pre-treatment at high concentration (30x MIC) for 72h. Mutation frequencies of different sorted populations were determined as 3.51% for M. smegmatisVBNR, 5.20% for M. smegmatisAR and 0.02% for M. smegmatis UNT. Sanger sequencing data demonstrated a high percentage of mutations in the inhA promoter (C-15T) (76% in VBNR; 64% in AR) compared to mutations in the katG gene (48% in VBNR; 44% in AR). However, the difference was not statistically significant (p > 0.1). This study addressed the following knowledge gaps: it advanced our understanding about the M. tuberculosis physiology. It confirmed that strain genetic background and the presence of rpoB Ser531Leu mutation may play a role in the physiological state of M. tuberculosis strains as reflected in their transcriptomes. It confirmed that host response in vitro is influenced by M. tuberculosis strain genotype and that infection with K636WT, H37RvWT and H37RvRIF M. tuberculosis strains will result in the secretion of pro-inflammatory cytokines and chemokines while infection with K636RIF M. tuberculosis strain (with rpoB Ser531Leu mutation) might induce secretion of anti-inflammatory cytokines (second line of host defense). This study was the first to successfully use a FACS analysis in combination with the FD reporter system to detect, isolate and quantify VBNR from AR M. smegmatis, following INH pre-treatment at high concentrations. We speculate that our results showed that the VBNR persisters‟ sub-population is likely to provide a reservoir from which genetic resistant mutants can arise, when treated with high INH concentrations as made evident by the observed INH resistant mutants in VBNR M. smegmatis. This work contributed further knowledge to finding better strategies to prevent the spread of emerging MDR, as well as extensively drug resistant M. tuberculosis.
AFRIKAANSE OPSOMMING: Verkeerdelike gebruik van behandeling van tuberkulose (TB) en die toename in die voorkoms van middel weerstandige Mycobacterium tuberculosis is faktore wat die beheer van TB negatief beinvloed. Die huidige studie is ontwerp om van die belangrikste gapings in die kennis rondom die fisiologie van M. tuberculosis te oorbrug. Die studie het gekyk na die effek van diverse M. tuberculosis genetiese agtergronde en die voorkoms van die rpoB mutasie op die transkriptoom van M. tuberculosis, die response van die gasheer op M. tuberculosis en die waarskynlikheid dat mikobakteriële toleransie „n poel verskaf waarvan genetiese weerstandigheid kan ontstaan. Die verkenning van verskeie van hierdie belangrike gapings in kennis was noodsaaklik gegewe die feit that langdurige anti-TB behandeling benodig kan word om totaal onstlae te raak van sekere M. tuberculosis stamme. Verkeerdelike gebruik en voltooing van behandeling mag lei tot die onstaan van multiweerstandige (MDR)-TB. Eerstens het ons die effek van die M. tuberculosis stamme met verskillende genetiese agtergronde op die totale transkriptoom ondersoek (as indikasie van die fisiologiese toestand). Tweedens het ons die invloed van die rpoB Ser531Leu mutasie en die effek van isoniazid (INH) behandeling (24 uur) teen sub-dodelike konsentrasies op die transkriptoom van rifampisien (RIF) weerstandige (K636RIF) en die sensitiewe (K636WT en H37RVWT) M. tuberculosis stamme ondersoek deur gebruik te maak van RNA-volgordebepaling (RNAseq) en werklike tyd, kwantitatiewe polimerase ketting reaksie (RT-qPCR). RNAseq analise het beduidende verskille in die uitdrukking van gene in die transkriptoom van K636WT, H37RVWT en K636RIF M. tuberculosis stamme getoon. Ons vergelykende transkriptoom data van K636WT en H37RVWT M. tuberculosis stamme het gedui dat verskillede genetiese agtergronde die totale transkriptoom beinvloed. Ons het gedemonstreer dat die rpoB Ser53Leu mutasie „n impak op die transkripsionele respons van K636WT het, relatief tot K636RIF M. tuberculosis stamme. „n Beperking van die studie was dat ons data nie die effek van INH behandeling op die transkriptoom van M. tuberculosis stamme van verskillende genetiese agtergronde gedemonstreer het nie. Met die assessering van die gasheer se immuun reaksie na infeksie met die RIF-weerstanding (K636RIF en H37RVRIF) en die sensitiewe (K636WTen H37RVWT) M. tuberculosis stamme deur die gebruik van luminex x multi-analiet profiel (xMAP) tegnologie en ensiem geassosieerde immunosorbens analises (ELISA) analises. Ons gasheer respons data (Hoofstuk 4) het geen verskille in gasheer reaksies tot K636WT en H37RvWT M. tuberculosis stamme van verskillende genetiese agtergronde getoon nie. In teenstelling, daar was verskillle in die gasheer respons tot K636WT en K636RIF M. tuberculosis stamme in a RAW264.7 makrofaag infeksie model. Dit is bevestig deur die waarneming van variërende sekresie vlakke van sitokienes en chemokiene (IL-6, IL-12p40 en RANTES) wat benodig word om M. tuberculosis groei en oorlewing te bemiddel 24 – 48 ure na infeksie. Ons het verder ondersoek ingestel of lewendige nie-repliserende (VBNR) persister Mycobacterium smegmatis sub-populasies, wanneer blootgestel aan hoë dosisse INH konsenstrasies, „n poel kan verskaf waarvan geneties weerstandige mutasies kan ontstaan. Ons het „n kombinasie van „n fluoresensie verdunning (FD) sisteem, vloeisitometrie en fluoreserensie-geaktiveerde sel sortering (FACS) gebruik om VBNR en aktief repliserende (AR) M. smegmatis populasies in respons tot INH behandeling te identifiseer, kwatifiseer en isoleer. Gevolglik het ons PKR gebruik om die katG en inhA promoter te amplifiseer en Sanger volgorde bepaling is gebruik om mutasies in hierdie gene te identifiseer, wat algemeen geassosieer is met INH weerstandigheid. Ons vloesitometrie resultate het gewys dat die identifisering van VBNR en AR bakeriële populasies in M. tuberculosis::pTiGc na voorafbehandeling met hoë konsentrasies INH (30x MIC) vir 72 uur. Mutasie frekwensies van verskillende gesorteerde populasies is bepaal as 3.51% vir M. smegmatis VBNR, 5.20% vir M. smegmatis AR en 0.02% vir M. smegmatis UNT. Sanger volgordebepaling het gedemonstreer dat „n hoë persentasie van die mutasies in die inhA protomoter (C-15T) (76% in VBNR; 64% in AR) geleë is, in vergelyking met mutasies in die katG geen (48% in VBNR, 44% in AR), maar die verskille was nie statisties beduidend nie (p>0.1). Hierdie studie adresseer die volgende gapings in kennis: dit bevorder ons begrip van M. tuberculosis fisiologie. Dit bevestig dat genetiese agtergrond van stamme en die voorkoms van die rpoB Ser153Leu mutasie „n rol mag speel in die fisiologiese toestand van M. tuberculosis stamme soos gereflekteer in hul transkriptoom. Dit bevestig dat die gasheer respons in vitro beinvloed word deur M. tuberculosis stam genotipes en dat die infeksie met K636WT, H37RVWT en H37RVRIF M. tuberculosis stamme sal lei to die sekresie van pro- inflammatoriese sitokienes en chemokiene, terwyl infeksie met K636RIF M. tuberculosis stamme (met die rpoB Ser531Leu mutasie) sekresie van anti-inflammatoriese sitokiene (2e linie gasheer beskerming) kan induseer. Hierdie is die eerste studie wat FACS analises in kominasie met die FD sisteem suskesvol gebruik om VBNR van AR M. smegmatis na die vooraf toediening van hoë konsentrasies INH te identifiseer, kwantifiseer en isoleer. Ons spekuleer dat ons resultate toon dat die VBNR persister sub-populasie waarskynlik „n poel verskaf waarvan geneties weerstandige mutasies kan ontstaan wanneer dit met hoë konsenstrasies INH behandel word, soos bewys deur die waarneming van die INH weerstandige mutasies in die VBNR M. smegmatis populasie. Hierdie werk dra by tot verdere kennis om verbeterede strategieë te identifiseer vir die verkoming van die verspreiding van MDR-TB en uitgebreide middel weerstandige M. tuberculosis.
Thesis (PhD)--Stellenbosch University, 2021.
Mycobacterium tuberculosis, UCTD, Multidrug-resistant tuberculosis, Physiology, Pathological