Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis Graphical abstract Highlights d Titratable CRISPRi enables quantification of target vulnerability in mycobacteria d Essential genes and processes vary widely in their vulnerability d Differential vulnerability predicts differential antibacterial susceptibility d Generalizable approach allows prioritization of high-value targets for drug discovery

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified > 10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECT's ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.transport | pore | secretion

Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target.

Exposure to hypoxia causes structural changes in the endothelial cell layer that alter its permeability and its interaction with leukocytes and platelets. One of the well characterized cytoskeletal changes in response to stress involves the reorganization of the actin cytoskeleton and the formation of stress fibers. This report describes cytoskeletal changes in pulmonary microvascular endothelial cells in response to hypoxia and potential mechanisms involved in this process. The hypoxia-induced actin redistribution appears to be mediated by components downstream of MAPK p38, which is activated in pulmonary endothelial cells in response to hypoxia. Our results indicate that kinase MK2, which is a substrate of p38, becomes activated by hypoxia, leading to the phosphorylation of one of its substrates, HSP27. Because HSP27 phosphorylation is known to alter actin distribution in response to other stimuli, we postulate that it also causes the actin redistribution observed in hypoxia. This notion is supported by the observations that similar actin redistribution occurs in cells overexpressing constitutively active MK2 or phosphomimicking HSP27 mutant. Overexpressing dominant negative MK2 blocks the effects of hypoxia on the actin cytoskeleton. Taken together these results indicate that hypoxia stimulates the p38-MK2-HSP27 pathway leading to significant alteration in the actin cytoskeleton.Hypoxia causes injury in a variety of organs and has been associated with many lung diseases including the acute respiratory distress syndrome, pulmonary embolism, and ischemiareperfusion injury. Hypoxia has been shown to increase the permeability of the endothelial barrier both in vitro (1-4) and in vivo (5). Moreover, hypoxia increases endothelial adhesiveness to neutrophils (6, 7). In that respect, endothelial cells respond to hypoxia in a manner similar to their response to inflammation. However, as opposed to the response of endothelial cells to inflammatory products, which has been extensively explored, the signal transduction pathways involved in the endothelial response to hypoxia remain poorly understood. Recent reports have demonstrated activation of the stress-activated MAPK 1 p38 in response to hypoxia (8 -16). For example, we have described the activation of p38 in hypoxic pulmonary microvascular endothelial cells and implicated it as one of the mechanisms of activation of the reactive oxygen-producing enzyme, xanthine oxidase (16). The enzyme MK2, immediately downstream of p38, is known to phosphorylate the small heat shock protein HSP27 (17). Because HSP27 interacts with actin and modulates cytoskeletal organization (18, 19), we investigated whether the MK2 pathway is activated by hypoxia and whether this process can lead to cytoskeletal changes. Our findings indicate that MK2 is indeed activated by hypoxia in RPMEC, and that HSP27 phosphorylation is increased concomitantly with reorganization of the actin cytoskeleton. The effect of hypoxia on the actin cytoskeleton is mimicked by overexpressing constitutively active M...