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.

A key to the pathogenic success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the capacity to survive within host macrophages. Although several factors required for this survival have been identified, a comprehensive knowledge of such factors and how they work together to manipulate the host environment to benefit bacterial survival are not well understood. To systematically identify Mtb factors required for intracellular growth, we screened an arrayed, non-redundant Mtb transposon mutant library by high-content imaging to characterize the mutant-macrophage interaction. Based on a combination of imaging features, we identified mutants impaired for intracellular survival. We then characterized the phenotype of infection with each mutant by profiling the induced macrophage cytokine response. Taking a systems-level approach to understanding the biology of identified mutants, we performed a multiparametric analysis combining pathogen and host phenotypes to predict functional relationships between mutants based on clustering. Strikingly, mutants defective in two well-known virulence factors, the ESX-1 protein secretion system and the virulence lipid phthiocerol dimycocerosate (PDIM), clustered together. Building upon the shared phenotype of loss of the macrophage type I interferon (IFN) response to infection, we found that PDIM production and export are required for coordinated secretion of ESX-1-substrates, for phagosomal permeabilization, and for downstream induction of the type I IFN response. Multiparametric clustering also identified two novel genes that are required for PDIM production and induction of the type I IFN response. Thus, multiparametric analysis combining host and pathogen infection phenotypes can be used to identify novel functional relationships between genes that play a role in infection.

During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a non-replicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the non-replicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel non-replicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and non-replicating M. tuberculosis as well as compounds that inhibit the transition from non-replicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of non-replicating M. tuberculosis. In order to understand the relationship among different non-replicating models, we teste 52 of these molecules in a hypoxia model and four different chemical scaffolds in a stochastic persist model and a streptomycin dependent model. We found that compounds display varying levels of activity in different models for the non-replicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different non-replicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development.

The phylogenetic interrelationships of members of the genus Listeriu were investigated by using reverse transcriptase sequencing of 16s rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeriu monocytugenes group of species (including Listeriu innocuu, Listeria ivunovii, Listeriu seeligeri, and Listeriu welshimeri) and (ii) the species Listeriu gruyi and Listeria murruyi. At the intergeneric level a specific phylogenetic relationship between the genera Listeriu and Brochothrh was evident. The sequence data clearly demonstrated that the genus Listeriu is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobucilluceue.The genus Listeria consists of gram-positive, nonsporeforming, facultatively anaerobic, regular rod-shaped bacteria. For many years a monospecific genus, this taxon currently contains seven species, Listeria monocytogenes, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria murrayi, Listeria seeligeri, and Listeria welshimeri (23,25). The species previously named Listeria denitrijicans has recently been reclassified in the new genus Jonesia, which is a member of the order Actinomycetales (23). Although the relationships among species in the genus Listeria are now clear (21,25), the higher relationships between the genus Listeria and other gram-positive taxa remain equivocal. Until the mid-1970s the genus was traditionally associated with the coryneform group of bacteria. However, the results of numerical phenetic studies (although somewhat contradictory in detail) have pointed toward a relationship between the genus Listeria and the lactic acid group of bacteria (8,12,16,28,31). In the extensive numerical study of Wilkinson and Jones (31), listeriae clearly clustered not with the coryneform group of bacteria but with lactobacilli and Brochothrix thermosphacta, and these authors stated that the genus Listeria should be included in the family Lactobacillaceae. Chemotaxonomic criteria (in particular the presence of respiratory menaquinones and predominantly methylbranched cellular fatty acids) support a close relationship between the genus Listeria and the genus Brochothrix but not between the genus Listeria and the genus Lactobacillus or other lactic acid bacteria (4, 5, 26). The results of 16s rRNA cataloging studies have also demonstrated that there is a close relationship between the genera Listeria and Brochothrix and have indicated that these taxa are only distantly related to the genera Lactobacillus and Streptococcus (18). However, in a recent numerical phenetic study (12) members of the genus Listeria were found to be more closely related to certain lactobacilli and some asporogenous rodshaped bacteria (referred to as Thornley and Sharpe groups 2 and 3), which were assigned to the new genus Carnobac-* Corresponding author.terium (3), than to the species B . thermosphacta. These findings are contrary to the result...

Summary Successful treatment of Mycobacterium tuberculosis infection typically requires a complex regimen administered over at least six months. Interestingly, many of the antibiotics used to treat M. tuberculosis are prodrugs that require intracellular activation. Here, we describe three small molecules, active against both replicating and non-replicating M. tuberculosis, that require activation by Baeyer-Villiger monooxygenases (BVMOs). Two molecules require BVMO EthA (Rv3854c) for activation and the third molecule requires the BVMO MymA (Rv3083). While EthA is known to activate the antitubercular drug ethionamide, this is the first description of MymA as an activating enzyme of a prodrug. Further, we found that MymA also plays a role in activating ethionamide, with loss of MymA function resulting in ethionamide resistant M. tuberculosis. These findings suggest overlap in function and specificity of the BVMOs in M. tuberculosis.