Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in , we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.

Mycobacterium bovis, the agent of bovine tuberculosis, causes an estimated $3 billion annual losses to global agriculture due, in part, to the limitations of current diagnostics. Development of next-generation diagnostics requires a greater understanding of the interaction between the pathogen and the bovine host. Therefore, to explore the early response of the alveolar macrophage to infection, we report the first application of RNA-sequencing to define, in exquisite detail, the transcriptomes of M. bovis-infected and non-infected alveolar macrophages from ten calves at 2, 6, 24 and 48 hours post-infection. Differentially expressed sense genes were detected at these time points that revealed enrichment of innate immune signalling functions, and transcriptional suppression of host defence mechanisms (e.g., lysosome maturation). We also detected differentially expressed natural antisense transcripts, which may play a role in subverting innate immune mechanisms following infection. Furthermore, we report differential expression of novel bovine genes, some of which have immune-related functions based on orthology with human proteins. This is the first in-depth transcriptomics investigation of the alveolar macrophage response to the early stages of M. bovis infection and reveals complex patterns of gene expression and regulation that underlie the immunomodulatory mechanisms used by M. bovis to evade host defence mechanisms.

Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-L-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium's ability to persist during chronic infection in mice. Furthermore, the D,L-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host. IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PGsynthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the D,L-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

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