The viability of Mycobacterium tuberculosis (Mtb) depends on energy generated by its respiratory chain. Cytochrome bc1-aa3 oxidase and type-2 NADH dehydrogenase (NDH-2) are respiratory chain components predicted to be essential, and are currently targeted for drug development. Here we demonstrate that an Mtb cytochrome bc1-aa3 oxidase deletion mutant is viable and only partially attenuated in mice. Moreover, treatment of Mtb-infected marmosets with a cytochrome bc1-aa3 oxidase inhibitor controls disease progression and reduces lesion-associated inflammation, but most lesions become cavitary. Deletion of both NDH-2 encoding genes (Δndh-2 mutant) reveals that the essentiality of NDH-2 as shown in standard growth media is due to the presence of fatty acids. The Δndh-2 mutant is only mildly attenuated in mice and not differently susceptible to clofazimine, a drug in clinical use proposed to engage NDH-2. These results demonstrate the intrinsic plasticity of Mtb’s respiratory chain, and highlight the challenges associated with targeting the pathogen’s respiratory enzymes for tuberculosis drug development.

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

SummarySalmonella enterica subspecies I serotypes are responsible for the vast majority of salmonellosis in mammals and birds, yet only a few factors specific to this group that allow them to persist in this niche have been identified. We show that STM0557, a S. enterica subspecies I-specific gene encoding an inner membrane protein, is critical for faecal shedding and intestinal persistence of S. enterica serotype Typhimurium ATCC14028 in Salmonella-resistant mice, but mutations in this gene do not diminish short-term intestinal colonization or invasion of cultured epithelial cells. STM0557 and two neighbouring genes, located on a pathogenicity island termed SPI-16, resemble genes of the gtrA,B, gtr(type) cluster in seroconverting bacteriophages. In general, the gtr genes encode proteins responsible for serotype conversion of the infected bacterium by addition glucose residues to repeating O-antigen subunits of lipopolysaccharide (LPS). In lysogenized Shigella, such modifications have been previously shown to be constitutively expressed and to facilitate invasion of host cells. We show that serotype Typhimurium gtr orthologues, STM0557-0559, are responsible for 'form variation' or glucosylation of the O12 antigen galactose (4 position) to generate the 12-2 variant. Form variation in Typhimurium is not constitutive, but occurred upon exposure and during intracellular growth of serotype Typhimurium in J774 macrophages. Our data suggest that the 12-2 antigen is a S. enterica subspecies I-specific LPS modification that enhances long-term intestinal colonization, and is in contrast to the role of O-antigen variation described for Shigella.

SUMMARY Salmonella enterica serovar Typhimurium avoids clearance by the host immune system by suppressing T cell responses; however, the mechanisms that mediate this immunosuppression remain unknown. We show that S. Typhimurium inhibit T cell responses by producing L-Asparaginase II, which catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia. L-Asparaginase II is necessary and sufficient to suppress T cell blastogenesis, cytokine production, and proliferation and to downmodulate expression of the T cell receptor. Furthermore, S. Typhimurium-induced inhibition of T cells in vitro is prevented upon addition of L-asparagine. S. Typhimurium lacking the L-Asparaginase II gene (STM3106) are unable to inhibit T cell responses and exhibit attenuated virulence in vivo. L-Asparaginases are used to treat acute lymphoblastic leukemia through mechanisms that likely involve amino acid starvation of leukemic cells, and these findings indicate that pathogens similarly use L-asparagine deprivation to limit T cell responses.

Bacterial pathogens causing systemic disease commonly evolve from organisms associated with localized infections but differ from their close relatives in their ability to overcome mucosal barriers by mechanisms that remain incompletely understood. Here we investigated whether acquisition of a regulatory gene, tviA, contributed to the ability of Salmonella enterica serotype Typhi to disseminate from the intestine to systemic sites of infection during typhoid fever. To study the consequences of acquiring a new regulator by horizontal gene transfer, tviA was introduced into the chromosome of S. enterica serotype Typhimurium, a closely related pathogen causing a localized gastrointestinal infection in immunocompetent individuals. TviA repressed expression of flagellin, a pathogen associated molecular pattern (PAMP), when bacteria were grown at osmotic conditions encountered in tissue, but not at higher osmolarity present in the intestinal lumen. TviA-mediated flagellin repression enabled bacteria to evade sentinel functions of human model epithelia and resulted in increased bacterial dissemination to the spleen in a chicken model. Collectively, our data point to PAMP repression as a novel pathogenic mechanism to overcome the mucosal barrier through innate immune evasion.