Mario L. Arrieta-Ortiz

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global tuberculosis (TB) control, yet the mechanisms enabling rapid evolution of resistance in Mtb remain poorly understood. Here, we show that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INH ^R ), challenging the paradigm that resistance mutations must precede compensatory adaptation. Using Mycobacterium smegmatis mc ² 155 (Msm) as a model, we demonstrate that brief exposure to sublethal isoniazid (INH) enriches for “low-level resistance and tolerance” (LLRT) mutants in a single step. LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (>500× IC ₅₀ ) at ~6-fold higher rates than wildtype, primarily through otherwise deleterious mycothiol biosynthesis mutations that become tolerable in an oxidative stress-buffered background. Crucially, sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH ^R evolution. Analysis of 1578 clinical Mtb isolates revealed significant enrichment of oxidative stress-related loci among those associated with INH ^R . Reanalysis of genome-wide CRISPRi data further linked oxidative stress response pathways to survival under multiple antibiotics. Together, these findings suggest that host-imposed oxidative stress and suboptimal drug exposure may prime Mtb populations for rapid resistance evolution, highlighting oxidative stress defenses as potential targets to limit resistance emergence.

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global TB control, yet the mechanisms enabling rapid evolution of drug resistance in Mtb remain poorly understood. Here we reveal that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INH ^R ) without fitness costs, challenging the paradigm that resistance mutations always precede their fitness compensatory adaptations. Using M. smegmatis mc ² 155 (Msm) as a model, we show that brief exposure to sublethal INH (2× IC ₅₀ ) enriches for "low-level resistance and tolerance" (LLRT) mutants in a single step. These LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (> 500× IC ₅₀ ) at 6-fold higher rates than wildtype, primarily through otherwise-deleterious mycothiol biosynthesis mutations that become tolerable in the oxidative stress-buffered background. Crucially, we demonstrate that sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH resistance evolution in Msm. Bayesian analysis of 1,578 clinical Mtb isolates from Vietnam confirmed that mutations in oxidative stress response genes were significantly associated with the emergence of INH ^R strains ( p-value = 1.09×10 ^-7 ). Independently, reanalysis of genome-wide CRISPRi screens revealed that the OSR network and high Bayes probability genes are functionally associated with treatment escape and survival with multiple antibiotics, including isoniazid, rifampicin, ethambutol, bedaquiline, vancomycin, clarithromycin, linezolid, and streptomycin. Our findings that host-imposed oxidative stress and inadequate drug penetration may synergistically prime Mtb populations for rapid resistance evolution suggest that targeting pre-resistance mechanisms, such as oxidative stress defenses, could help slow the emergence of antibiotic resistance in tuberculosis.

Antimicrobial resistance (AMR) is projected to be the cause of >10 million deaths annually by 2050. While efforts to find new potent antibiotics are effective, they are expensive and outpaced by the rate at which new resistant strains emerge.

Mycobacterium tuberculosis (MTB) generates phenotypic diversity to persist and survive the harsh conditions encountered during infection. MTB avoids immune effectors and antibacterial killing by entering into distinct physiological states. The surviving cells, persisters, are a major barrier to the timely and relapse-free treatment of tuberculosis (TB). We present for the first time, PerSort, a method to isolate and characterize persisters in the absence of antibiotic or other pressure. We demonstrate the value of PerSort to isolate translationally dormant cells that preexisted in small numbers within Mycobacterium species cultures growing under optimal conditions but that dramatically increased in proportion under stress conditions. The translationally dormant subpopulation exhibited multidrug tolerance and regrowth properties consistent with those of persister cells. Furthermore, PerSort enabled single-cell transcriptional profiling that provided evidence that the translationally dormant persisters were generated through a variety of mechanisms, including vapC30, mazF, and relA/spoT overexpression. Finally, we demonstrate that notwithstanding the varied mechanisms by which the persister cells were generated, they converge on a similar low-oxygen metabolic state that was reversed through activation of respiration to rapidly eliminate persisters fostered under host-relevant stress conditions. We conclude that PerSort provides a new tool to study MTB persisters, enabling targeted strategies to improve and shorten the treatment of TB. IMPORTANCEMycobacterium tuberculosis (MTB) persists and survives antibiotic treatments by generating phenotypically heterogeneous drug-tolerant subpopulations. The surviving cells, persisters, are a major barrier to the relapse-free treatment of tuberculosis (TB), which is already killing >1.8 million people every year and becoming deadlier with the emergence of multidrug-resistant strains. This study describes PerSort, a cell sorting method to isolate and characterize, without antibiotic treatment, translationally dormant persisters that preexist in small numbers within Mycobacterium cultures. Characterization of this subpopulation has discovered multiple mechanisms by which mycobacterial persisters emerge and unveiled the physiological basis for their dormant and multidrug-tolerant physiological state. This analysis has discovered that activating oxygen respiratory physiology using l-cysteine eliminates preexisting persister subpopulations, potentiating rapid antibiotic killing of mycobacteria under host-relevant stress. PerSort serves as a new tool to study MTB persisters for enabling targeted strategies to improve and shorten the treatment of TB.

There is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing >700,000 deaths annually. Here, we demonstrate that tetracycline resistant (Tet ^R ) Escherichia coli undergoes global transcriptional and metabolic remodeling, including down-regulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Targeted knockout of ArcA, identified by network analysis as a master regulator among 25 transcription factors of this new compensatory physiological state, significantly increased the susceptibility of Tet ^R E. coli to tetracycline treatment. A drug, sertraline, which generated a similar metabolome profile as the arcA knockout strain also synergistically re-sensitized Tet ^R E. coli to tetracycline. The potentiating effect of sertraline was eliminated upon knocking out arcA , demonstrating that the mechanism of synergy was through action of sertraline on the tetracycline-induced ArcA network in the Tet ^R strain. Our findings demonstrate that targeting mechanistic drivers of compensatory physiological states could be a generalizable strategy to re-sensitize AMR pathogens to lost antibiotics.

The success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection. One Sentence Summary: Novel technology (Path-seq) discovers cell wall remodeling program during Mycobacterium tuberculosis infection of macrophages