Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance.

Errors are inherent in all biological systems. Errors in protein translation are particularly frequent giving rise to a collection of protein quasi-species, the diversity of which will vary according to the error rate. As mistranslation rates rise, these new proteins could produce new phenotypes, although none have been identified to date. Here, we find that mycobacteria substitute glutamate for glutamine and aspartate for asparagine at high rates under specific growth conditions. Increasing the substitution rate results in remarkable phenotypic resistance to rifampicin, whereas decreasing mistranslation produces increased susceptibility to the antibiotic. These phenotypic changes are reflected in differential susceptibility of RNA polymerase to the drug. We propose that altering translational fidelity represents a unique form of environmental adaptation.

Post-translational regulation via Clp protease is critical for survival of Mycobacterium tuberculosis.

Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.

Genetic modification of the O-polysaccharide of Francisella tularensis results in an avirulent live attenuated vaccine.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, is a highly virulent microbe. One significant virulence factor of F. tularensis is the O-polysaccharide (O-PS) portion of the organism's lipopolysaccharide. METHODS: A wzy (O-antigen polymerase) deletion mutant of Ft. live attenuated vaccine strain (Ft.LVS), designated Ft.LVS::Δwzy, was created and evaluated as a live attenuated vaccine. Specifically, the mutant's virulence potential and its protective efficacy against type A and type B strains were investigated by challenge of immunized mice. RESULTS: F. tularensis LVS::Δwzy expressed only 1 repeating unit of O-PS and yet, upon immunization, induced O-PS-specific antibodies. Compared with Ft.LVS, the mutant was highly sensitive to complement-mediated lysis, significantly attenuated in virulence, and was recovered in much lower numbers from the organs of infected mice. Intranasal immunization with Ft.LVS::Δwzy provided protection against subsequent intranasal infection with the highly virulent type A strain SchuS4 and with Ft.LVS. Immunization with Ft.LVS::Δwzy elicited both humoral and cell-mediated immunity. CONCLUSIONS: Ft.LVS::Δwzy was avirulent in mice and, despite expressing only 1 repeating unit of the O-PS, induced antibodies to the full-length O-PS. Vaccination with Ft.LVS::Δwzy protected mice against intranasal challenge with both type A and type B strains of F. tularensis and induced functional immunity through both humoral and cellular mechanisms.

Characterization of the O-antigen polymerase (Wzy) of Francisella tularensis.

The O-antigen polymerase of gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly(176), Asp(177), Gly(323), and Tyr(324)) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface.

Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis.

Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.

Cellular and humoral immunity are synergistic in protection against types A and B Francisella tularensis.

Herein we report studies with a novel combination vaccine that, when administered to mice, conferred protection against highly virulent strains of Francisella tularensis by stimulating both arms of the immune system. Our earlier studies with Ft.LVS::wbtA, an O-polysaccharide (OPS)-negative mutant derived from the available live vaccine strain of F. tularensis (Ft.LVS), elucidated the role of antibodies to the OPS - a key virulence determinant - in protection against virulent type A organisms. However, when expressed on the organism, the OPS enhances virulence. In contrast, in purified form, the OPS is completely benign. We hypothesized that a novel combination vaccine containing both a component that induces humoral immunity and a component that induces cellular immunity to this intracellular microbe would have an enhanced protective capacity over either component alone and would be much safer than the LVS vaccine. Thus we developed a combination vaccine containing both OPS (supplied in an OPS-tetanus toxoid glycoconjugate) to induce a humoral antibody response and strain Ft.LVS::wbtA (which is markedly attenuated by its lack of OPS) to induce a cell-mediated protective response. This vaccine protected mice against otherwise-lethal intranasal and intradermal challenge with wild-type F. tularensis strains Schu S4 (type A) and FSC 108 (type B). These results represent a significant advance in our understanding of immunity to F. tularensis and provide important insight into the development of a safer vaccine effective against infections caused by clinical type A and B strains of F. tularensis.