Distinct Bacterial Pathways Influence the Efficacy of Antibiotics against Mycobacterium tuberculosis

Abstract

Effective tuberculosis treatment requires at least 6 months of combination therapy. Alterations in the physiological state of the bacterium during infection are thought to reduce drug efficacy and prolong the necessary treatment period, but the nature of these adaptations remain incompletely defined. To identify specific bacterial functions that limit drug effects during infection, we employed a comprehensive genetic screening approach to identify mutants with altered susceptibility to the first-line antibiotics in the mouse model. We identified many mutations that increase the rate of bacterial clearance, suggesting new strategies for accelerating therapy. In addition, the drug-specific effects of these mutations suggested that different antibiotics are limited by distinct factors. Rifampin efficacy is inferred to be limited by cellular permeability, whereas isoniazid is preferentially affected by replication rate. Many mutations that altered bacterial clearance in the mouse model did not have an obvious effect on drug susceptibility using in vitro assays, indicating that these chemical-genetic interactions tend to be specific to the in vivo environment. This observation suggested that a wide variety of natural genetic variants could influence drug efficacy in vivo without altering behavior in standard drug-susceptibility tests. Indeed, mutations in a number of the genes identified in our study are enriched in drug-resistant clinical isolates, identifying genetic variants that may influence treatment outcome. Together, these observations suggest new avenues for improving therapy, as well as the mechanisms of genetic adaptations that limit it. IMPORTANCE Understanding how Mycobacterium tuberculosis survives during antibiotic treatment is necessary to rationally devise more effective tuberculosis (TB) chemotherapy regimens. Using genome-wide mutant fitness profiling and the mouse model of TB, we identified genes that alter antibiotic efficacy specifically in the infection environment and associated several of these genes with natural genetic variants found in drug-resistant clinical isolates. These data suggest strategies for synergistic therapies that accelerate bacterial clearance, and they identify mechanisms of adaptation to drug exposure that could influence treatment outcome.

Host-pathogen genetic interactions underlie tuberculosis susceptibility

“…The relative representation of each Mtb mutant in the input pool versus the pools recovered from mouse spleens was quantified ( Table S4). Consistent with our previous work (Bellerose et al, 2020;, we identified 234 Mtb genes that are required for growth or survival in Mtb in B6 mice, based on significant underrepresentation of the corresponding mutant after four weeks of in vivo selection. All but one of these genes were found to be required in the larger panel, increasing confidence in this Mtb gene set ( Figure 4A and 4B).…”

“…To characterize the diversity of disease states that are possible in a genetically diverse mouse population, we infected a panel of 52 CC lines and the founder strains with Mtb. To enable TnSeq studies, the animals were infected via the intravenous route with a saturated library of Mtb transposon mutants, which in sum produce an infection that is similar to the wild-type parental strain (Bellerose et al, 2020;.…”

“…A set of 136 "core" virulence functions were required in the majority of mouse genotypes, and a second larger set of 607 "adaptive" virulence genes were required in only a subset of lines ( Table S4). The core functions included a number of genes previously found to be important in B6 mice, including those necessary for the synthesis of essential cofactors, such as pyridoxine (Pdx1) (Dick et al, 2010); for the acquisition of nutrients, such as siderophore-bound iron (irtAB) (Ryndak et al, 2010), cholesterol (mce4) (Pandey and Sassetti, 2008), glutamine (glnQ and rv2563) (Bellerose et al, 2020); and for type VII secretion (ESX1 genes) (Stanley et al, 2003). Despite the importance of these core functions, a large range in the relative abundance of these mutants was observed across the panel, and in some cases specific immunological requirements could be discerned.…”

“…Defects in this same immune axis cause the human syndrome Mendelian Susceptibility to Mycobacterial Disease (MSMD) (Altare et al, 1998;Bogunovic et al, 2012;Bustamante et al, 2014;Filipe-Santos et al, 2006), demonstrating the value of knockout (KO) mice to characterize genetic variants of large effect. Similarly, the standard mouse model has been used to define Mtb genes that are specifically required for optimal bacterial fitness during infection (Bellerose et al, 2020;Zhang et al, 2013).…”

Cell Surface Biosynthesis and Remodeling Pathways in Mycobacteria Reveal New Drug Targets

“…Several Mtb mutants displayed altered susceptibility to TB drugs in the murine model of TB disease, many of which were associated with CW pathways described herein. The findings suggest that genetic variants that may be associated with drug resistance in clinical Mtb isolates do not alter in vitro drug susceptibility but can still influence drug efficacy in vivo ( Bellerose et al., 2020 ). This suggested that the genetic backgrounds of different clinical strains can impact the applicability of new anti-mycobacterial drugs in the clinical setting.…”