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Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.

pyrE (STM3733) encodes orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), the fifth enzyme of the de novo pyrimidine biosynthetic pathway. We identified a ΔpyrE mutant as under selection in screening of a Salmonella mutant library in 4-day old chicks. Here, we confirm that a ΔpyrE mutant colonizes 4-day old chicks poorly in competitive infection with isogenic wild type, and that the ability of this mutant to colonize chicks could be restored by providing a copy of pyrE in trans. We further show that our ΔpyrE mutant grows poorly in nutrient poor conditions in vitro, and that the ability of this mutant to grow is restored, both in vitro and in chicks, when precursors to the pyrimidine salvage pathway were provided. This finding suggests that the environment in the chick intestine during our infections lacks sufficient precursors of the pyrimidine salvage pathway to support Salmonella growth. Finally, we show that the colonization defect of a ΔpyrE mutant during infection occurs in to chicks, but not in CBA/J mice or ligated ileal loops in calves. Our data suggest that de novo pyrimidine synthesis is necessary for colonization of Salmonella Typhimurium in the chick, and that the salvage pathway is not used in this niche.

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

Tuberculosis is a pulmonary disease with an especially high mortality rate in immuno-compromised populations, specifically children and HIV positive individuals. The causative agent, Mycobacterium tuberculosis ( Mtb), is a very slow growing and difficult organism to work with, making both diagnosis and development of effective treatments cumbersome. We utilize a fiber-optic fluorescence microendoscope integrated with a whole-body imaging system for in vivo Mtb detection. The system exploits an endogenous enzyme of Mtb ( β-lactamase, or BlaC) using a BlaC-specific NIR fluorogenic substrate. In the presence of BlaC, this substrate is cleaved and becomes fluorescent. Using intravital illumination of the lung to excite this probe, sensitivity of the optical system increases over trans- and epi-illumination methods of whole-body fluorescence imaging. We demonstrate that integration of these imaging technologies with BlaC-specific fluorescent reporter probe improves the level of detection to ∼100 colony forming units, a 100× increase in sensitivity in comparison to epi-illumination and a 10× increase in sensitivity in comparison to previous work in intravital excitation of tdTomato-expressing Mtb. This lower detection threshold enables the study of early stage bacterial infections with clinical strains of Mtb and longitudinal studies of disease pathogenesis and therapeutic efficacy with multiple time points in a single animal. [ABSTRACT FROM AUTHOR]

Slow growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, hinders advancement in all areas of research toward prevention and treatment. Real-time imaging with reporter enzyme fluorescence (REF) that uses custom fluorogenic substrates for bacterial enzymes allows rapid and specific detection of M. tuberculosis in live animals. We have synthesized a novel REF substrate, CNIR800, that carries a near-infrared (NIR) fluorochrome IRDye 800CW, with a quencher connected through the lactam ring that is hydrolyzed by the enzyme BlaC (β-lactamase) that is naturally expressed by M. tuberculosis. CNIR800 produces long-wavelength emission at 795 nm upon excitation (745 nm) and exhibits significantly improved signal to noise ratios for detection of M. tuberculosis. The detection threshold with CNIR800 is approximately 100 colony-forming units (CFU) in vitro and <1000 CFU in the lungs of mice. Additionally, fluorescence signal from cleaved CNIR800 reaches maximal levels 4-6 hours after administration in live animals, allowing accurate evaluation of antituberculous drug efficacy. Thus, CNIR800 represents an excellent substrate for accurate detection of M. tuberculosis rapidly and specifically in animals, facilitating research toward understanding pathogenic mechanisms, evaluation of therapeutic outcomes, and screening new vaccines. [ABSTRACT FROM AUTHOR]

Objectives: This study was performed to investigate the relationship between community residents' infection sensitivity and their levels of preventive behaviors during the 2015 Middle East Respiratory Syndrome (MERS) outbreak in Korea.Methods: Seven thousands two hundreds eighty one participants from nine areas in Gyeonggi-do including Pyeongtaek, the origin of the outbreak in 2015 agreed to participate in the survey and the data from 6,739 participants were included in the final analysis. The data on the perceived infection sensitivity were subjected to cluster analysis. The levels of stress, reliability/practice of preventive behaviors, hand washing practice and policy credibility during the outbreak period were analyzed for each cluster.Results: Cluster analysis of infection sensitivity due to the MERS outbreak resulted in classification of participants into four groups: the non-sensitive group (14.5%), social concern group (17.4%), neutral group (29.1%), and overall sensitive group (39.0%). A logistic regression analysis found that the overall sensitive group with high sensitivity had higher stress levels (17.80; 95% confidence interval [CI], 13.77 to 23.00), higher reliability on preventive behaviors (5.81; 95% CI, 4.84 to 6.98), higher practice of preventive behaviors (4.53; 95% CI, 3.83 to 5.37) and higher practice of hand washing (2.71; 95% CI, 2.13 to 3.43) during the outbreak period, compared to the non-sensitive group.Conclusions: Infection sensitivity of community residents during the MERS outbreak correlated with gender, age, occupation, and health behaviors. When there is an outbreak in the community, there is need to maintain a certain level of sensitivity while reducing excessive stress, as well as promote the practice of preventive behaviors among local residents. In particular, target groups need to be notified and policies need to be established with a consideration of the socio-demographic characteristics of the community. [ABSTRACT FROM AUTHOR]