| dc.description.abstract | Mycobacterium tuberculosis (Mtb) is an obligate intra-cellular pathogen that causes the disease tuberculosis (TB) in its human hosts. An estimated 1% of the world population is reported to get infected with the disease every year. The capacity of Mtb to tolerate multiple antibiotics, particularly within its host, represents a major problem in TB management.
Moreover, patients co-infected with Mtb and another global pathogen human immunodeficiency virus (HIV) showed high rates of anti-TB therapy failure as compared to
patients infected with only Mtb. The protracted therapy time for a cocktail of antibiotics often leads to non-adherence among patients resulting in ineffectiveness of the regimen. Also,
prolonged exposure to antibiotics paves the way for the acquisition of mutations that generate genetically drug-resistant Mtb strains.
Heterogeneity within Mtb populations has long been associated with refractoriness to antibiotic therapy during growth in vitro and inside host cells/tissues [Chapter 1]. It has been
reported that the micro-environment faced by Mtb inside host phagocytes promotes tolerance towards clinically-relevant anti-TB drugs, possibly contributing to reduced clearance of Mtb
from patient lesions. During chronic phase of infection, exposure to host immune pressures induces metabolic quiescence, which contributes to a drug-tolerant phenotype. Additionally,
tolerance to antibiotics has recently also been attributed to replicating Mtb in unstimulated macrophages. Expansion of this drug-tolerant Mtb population within lesions could lead to
dissemination of tolerant bacteria to new sites, ultimately reducing the efficacy of antibiotics in eradicating of Mtb. Association of host immune pressures in mobilizing drug tolerance indicates an active crosstalk between host and pathogen. In this regard, it is crucial that we identify host-specific cues and Mtb’s adaptation program in response to environmental signals
for mechanistic dissection of phenotypic drug tolerance in replicating Mtb during infection.
The major aim of this study was to characterize the cross-talk between host immune pressures and bacterial genetic determinants that sense these pressures to mediate realignment
of bacterial metabolism for survival. Upon uptake by naïve macrophages, Mtb is exposed to host stresses such as limited acidification of the phagosome (~ pH 6.2) and superoxide (O2.) stress by recruitment of vacuolar ATPases (V-ATPases) and phagocyte oxidases (NOX2) on the phagosomal membrane, respectively. Since these stresses are known to induce redox
imbalance, Mtb induces several protective mechanisms to maintain redox homeostasis for survival. A major technological advance in the redox field emerged by the development of a
fluorescent biosensor (Mrx1-roGFP2) that facilitates real-time quantification of the redox potential of the major cytosolic antioxidant in Mtb (mycothiol/MSH; EMSH) during infection.
Using this tool, it was reported that Mtb population localized within macrophages exhibits heterogeneity in EMSH as compared to uniform EMSH displayed by broth-grown bacteria.
Further, redox-diverse bacterial sub-populations demonstrated variable susceptibility towards anti-TB drugs, suggesting a link between redox physiology and drug tolerance in Mtb. On this
basis, we sought to comprehensively characterize redox-diverse fractions of intra-phagosomal Mtb to understand the underlying mechanism of phenotypic drug tolerance. Our RNAsequencing
(RNA-seq) of redox-altered Mtb provided distinct transcriptional signatures, which aided in identification of bacterial determinants of antibiotic tolerance [Chapter 2].
Phagosomal acidification is possibly one of the earliest host stresses that Mtb faces upon internalization by macrophages. Previous reports suggest that maintenance of intrabacterial
pH homeostasis, upon exposure to an acidified environment, could affect Mtb’s redox physiology. In this regard, we sought to dissect the link between phagosomal acidification,
heterogeneity in EMSH and multi-class drug tolerance in Mtb during infection [Chapter 3]. We identified that redox-diverse Mtb sub-populations within macrophages faced different degrees
of phagosomal acidification. Blocking phagosomal acidification using lysosomotropic agents subverted heterogeneity in EMSH and reversed drug tolerance to anti-TB drugs isoniazid (Inh)
and rifampicin (Rif). We also show that the pH and redox-dependent drug tolerance of Mtb is significantly higher when the pathogen infects macrophages with actively replicating HIV-1,
suggesting that it could contribute to high rates of TB therapy failure during HIV-TB coinfection.
Our data emphasizes upon the crucial role played by host acidification in generating redox-based heterogeneity and drug tolerance in intra-macrophage Mtb.
The role of the lysosomotropic agent chloroquine (CQ), in blocking acidification of sub-cellular compartments, is well-established in anti-malarial therapy as well as in autophagyrelated
studies. Based on our results in Mtb-infected macrophages, where CQ consistently decreased tolerance to Inh and Rif, we attempted to assess the effectiveness of CQ in reversing
Mtb drug tolerance in vivo [Chapter 4]. In chronically-infected BALB/c mice, coadministration of CQ with Inh or Rif dramatically reduced the fraction of drug-tolerant Mtb,
ameliorated lung pathology, and reduced post-chemotherapeutic relapse. Similar decrease inbacterial lung burden and infection-mediated tissue damage was observed in Mtb-infected
guinea pigs treated with a combination of CQ and Inh. The pharmacokinetic profile of CQ exhibited no significant drug-drug interaction with first line anti-TB drugs, making it a robust
candidate to be considered for host-directed therapy in TB management.
In summation, our data delineate a functional link between phagosomal pH, redox metabolism and multi-drug tolerance in replicating Mtb [Chapter 5]. We have attempted to
investigate the mechanistic underpinnings of how intra-phagosomal Mtb senses acidification as a cue to generate phenotypic variants of redox state which consequently exhibit differential sensitivity to anti-TB drugs. We also propose the repositioning of CQ as host-directed therapy based on its role in blocking phagosomal acidification and redox heterogeneity to potentiate antibiotic efficacy. Our findings, along with the high oral bioavailability and long half-life in patient sera, makes CQ a promising candidate to be added to current treatment regimens for
shortening TB therapy and achieving a relapse-free cure. | en_US |
