Heterogeneous Host-Pathogen Encounters Coordinate Antibiotic Resilience in Mycobacterium tuberculosis

Abstract

Successful treatment of tuberculosis (TB) depends on the eradication of its causative agent Mycobacterium tuberculosis (Mtb) in the host. However, the emergence of phenotypically drug-resistant Mtb in the host environment tempers the ability of antibiotics to cure disease. Host immunity produces diverse microenvironmental niches that are exploited by Mtb to mobilize adaptation programs. Such differential interactions amplify pre-existing heterogeneity in the host-pathogen milieu to influence disease pathology and therapy outcome. Therefore, comprehending the intricacies of phenotypic heterogeneity can be an empirical step forward in potentiating drug action. With this goal, we review the interconnectedness of the lesional, cellular, and bacterial heterogeneity underlying phenotypic drug resistance. Based on this information, we anticipate the development of new therapeutic strategies targeting host-pathogen heterogeneity to cure TB.

Keywords: drug tolerance; phagosomal acidification; phenotypic heterogeneity; redox metabolism.

Figure 1. Lesional Heterogeneity Modulates Mycobacterium tuberculosis (Mtb) Survival, Tissue Homeostasis, and Drug Penetration.

(A) Diverse granuloma types – cellular (red), necrotic (yellow), and cavitary (green) – coexist in the lungs of an infected host. (B) As few as two to five Mtb bacilli in aerosol droplets are adequate to infect a susceptible host. Aggregation of differentially activated macrophages and neutrophils, followed by necroptosis, leads to granuloma formation. Cellular granulomas can then morph into a closed lesion, infiltrated by epithelioid and foamy macrophages and mesenchymal stem cells (MSCs) and contained by a peripheral cuff of lymphocytes. Closed granulomas undergo macrophage necrosis and caseation in their core leading to the release of intracellular Mtb. Matrix metalloproteinases (MMPs) induce the degradation of connective tissue, which allows the expulsion of the liquefied contents into adjoining airways to form an aerobically exposed open cavity. By contrast, repopulation with fibroblasts and collagenous material may form a fibrotic granuloma primarily containing latent Mtb. Inter- and intralesional diversity arises through differences in vasculature, oxygen availability (pO₂), iron (Fe), and inflammation. These can influence Mtb physiology and drug distribution in distinct structural regions of a granuloma leading to phenotypic antimicrobial resistance (AMR). Mox, moxifloxacin; Bdq, bedaquiline; Rif, rifampicin.

Figure 2. Tissue-Specific Immune Cell Lineages Compound Phenotypic Variations in Mycobacterium tuberculosis (Mtb) during Infection.

Other than classically activated macrophages, ontologically distinct lineages of alveolar macrophages (AMs) and interstitial macrophages (IMs) as well as mesenchymal stem cells (MSCs) are known to be infection permissive in hosts. Fetal-origin AMs exhibit an M2-like phenotype with a growth-permissive environment for Mtb, whereas bone-marrow-derived IMs exhibit M1-like behavior and restrict Mtb growth. Self-renewing MSCs harbor Mtb in a drug-tolerant state and accumulate lipid droplets, which are metabolized by the bacteria for long-term persistence. FAO, fatty acid oxidation; PGE2, prostaglandin E (2); AMR, antimicrobial resistance; SRC, spare respiratory capacity.

Figure 3. Mitochondrial Activity, pH, and Cytokines Influence Survival and Phenotypic Antimicrobial Resistance (AMR) during HIV–Tuberculosis (TB) Co-infection.

Infection of macrophages harboring the latently integrated genome of HIV-1 with Mycobacterium tuberculosis (Mtb) leads to viral reactivation and bacterial proliferation. Phagosomal acidification in co-infected macrophages contributes to redox heterogeneity and phenotypic tolerance towards isoniazid (Inh) in Mtb. Co-infected macrophages experience proton leak from stressed mitochondria and non-mitochondrial reactive oxygen species (ROS), which lead to the reactivation of HIV. Neutralizing phagosomal pH using chloroquine (CQ) subverts redox heterogeneity and Inh tolerance in Mtb inside co-infected macrophages. Phagosomal maturation is controlled heterogeneously within a macrophage population, through active granulocyte-macrophage colony stimulating factor (GM-CSF) signaling and interleukin-1β (IL-1β) induction, to limit Mtb growth. HIV-TB co-infected macrophages can suppress GM-CSF signaling to prevent cytokine-mediated control of Mtb and HIV, whereas exogenous GM-CSF supplementation can reverse this effect.

Figure 4. Phagosomal Acidification and Immune Activation Induce Phenotypic Antimicrobial Resistance (AMR).

Both bacterial phenotypic variations and host immune pressures can modulate Mycobacterium tuberculosis (Mtb) proliferation in macrophages. Isogenic Mtb populations demonstrate stochastic heterogeneity in essential cellular processes such as growth, gene expression, and the management of irreversibly oxidized proteins (IOPs). Aggregation into biofilms alters the bacterial redox state with respiration driven by polyketide quinones (PkQs) serving as alternative electron acceptors in a hypoxic niche. Inside naïve macrophages, Mtb faces variable acidification in phagosomes and recalibrates metabolism and efflux activity to cope with metal and redox stress. The consequent generation of redox heterogeneity selects for an actively replicating, drug-tolerant Mtb population. Interferon-gamma (IFNγ) signaling in activated macrophages allows Mtb to be trafficked to highly acidic phagolysosomes with abundant exposure to reactive oxygen species (ROS) and nitric oxide. Enhanced adaptive responses to an increasingly hostile host environment ultimately confer metabolic quiescence on Mtb and further strengthen phenotypic AMR.

Figure 5. Redox-Altered Mycobacterium tuberculosis (Mtb) Inside Naïve Macrophages Acquires Drug Tolerance through Dissipation of Metal and Reductive Stress.

Exposure to acidic pH in macrophages leads to perturbation of redox homeostasis and the appearance of redox-altered populations of Mtb. Acidity in phagosomes induces the accumulation of soluble forms of metal ions (Fe/Cu) as well as the redox-active amino acid cysteine (CySH). The former can catalyze the generation of reactive oxygen species (ROS) by either driving the Fenton reaction or oxidizing CySH to cystine. Transcriptional profiling of intramacrophage Mtb fractions reveals that drug-tolerant E_MSH-reduced Mtb are particularly efficient in dissipating reductive and metal stress during infection. This Mtb fraction channels the flux of CySH into the reverse transsulfuration pathway leading to H_2S generation (MetB), Fe-S cluster biogenesis (SufR), and mycothiol (MSH) production. Induction of these pathways affords protection from oxidative stress by increased expression of redox- and acid-sensitive transcription factors (WhiB family and Suf system) that regulate Mtb’s antioxidant response, consume excess CySH and Fe, and induce the activity of metal and drug efflux pumps. Additionally, S-adenosylmethionine (SAM)-dependent methyltransferases are found to be highly expressed in reduced Mtb, where they can inactivate antibiotics by N-methylation. Blocking phagosomal acidification with lysosomotropic agents such as chloroquine (CQ) reverses intramacrophage redox heterogeneity and reduces isoniazid (Inh) or rifampicin (Rif) tolerance in Mtb-infected macrophages as well as in chronically infected animals. From [53], reprinted with permission from AAAS.