Repeatability of computed tomography liver radiomic features in a nonhuman primate model of diet-induced steatosis.

Purpose: We describe a method to identify repeatable liver computed tomography (CT) radiomic features, suitable for detection of steatosis, in nonhuman primates. Criteria used for feature selection exclude nonrepeatable features and may be useful to improve the performance and robustness of radiomics-based predictive models. Approach: Six crab-eating macaques were equally assigned to two experimental groups, fed regular chow or an atherogenic diet. High-resolution CT images were acquired over several days for each macaque. First-order and second-order radiomic features were extracted from six regions in the liver parenchyma, either with or without liver-to-spleen intensity normalization from images reconstructed using either a standard (B-filter) or a bone-enhanced (D-filter) kernel. Intrasubject repeatability of each feature was assessed using a paired t-test for all scans and the minimum p-value was identified for each macaque. Repeatable features were defined as having a minimum p-value among all macaques above the significance level after Bonferroni's correction. Features showing a significant difference with respect to diet group were identified using a two-sample t-test. Results: A list of repeatable features was generated for each type of image. The largest number of repeatable features was achieved from spleen-normalized D-filtered images, which also produced the largest number of second-order radiomic features that were repeatable and different between diet groups. Conclusions: Repeatability depends on reconstruction kernel and normalization. Features were quantified and ranked based on their repeatability. Features to be excluded for more robust models were identified. Features that were repeatable but different between diet groups were also identified.

One Size Fits All? Not in In Vivo Modeling of Tuberculosis Chemotherapeutics.

Tuberculosis (TB) remains a global health problem despite almost universal efforts to provide patients with highly effective chemotherapy, in part, because many infected individuals are not diagnosed and treated, others do not complete treatment, and a small proportion harbor Mycobacterium tuberculosis (Mtb) strains that have become resistant to drugs in the standard regimen. Development and approval of new drugs for TB have accelerated in the last 10 years, but more drugs are needed due to both Mtb's development of resistance and the desire to shorten therapy to 4 months or less. The drug development process needs predictive animal models that recapitulate the complex pathology and bacterial burden distribution of human disease. The human host response to pulmonary infection with Mtb is granulomatous inflammation usually resulting in contained lesions and limited bacterial replication. In those who develop progressive or active disease, regions of necrosis and cavitation can develop leading to lasting lung damage and possible death. This review describes the major vertebrate animal models used in evaluating compound activity against Mtb and the disease presentation that develops. Each of the models, including the zebrafish, various mice, guinea pigs, rabbits, and non-human primates provides data on number of Mtb bacteria and pathology resolution. The models where individual lesions can be dissected from the tissue or sampled can also provide data on lesion-specific bacterial loads and lesion-specific drug concentrations. With the inclusion of medical imaging, a compound's effect on resolution of pathology within individual lesions and animals can also be determined over time. Incorporation of measurement of drug exposure and drug distribution within animals and their tissues is important for choosing the best compounds to push toward the clinic and to the development of better regimens. We review the practical aspects of each model and the advantages and limitations of each in order to promote choosing a rational combination of them for a compound's development.

Development and Optimization of Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis Reporter Constructs.

Mycobacterium tuberculosis resides in the lungs in various lesion types with unique microenvironmental conditions. This diversity is in line with heterogeneous disease progression and divergent drug efficiency. Fluorescent reporter strains can be used to decipher the micromilieu and to guide future treatment regimens. Current reporters using replicating plasmids, however, are not suitable for long-term mouse infections or studies in non-human primates. Using a combination of recombinant DNA and protein optimization techniques, we have developed reporter strains based on integrative plasmids, which exhibit stimulus-response characteristics and fluorescence intensities comparable to those based on replicating plasmids. We successfully applied the concepts by constructing a multi-color reporter strain able to detect simultaneous changes in environmental pH, Mg2+ concentrations, and protein expression levels.

Plasticity of the Mycobacterium tuberculosis respiratory chain and its impact on tuberculosis drug development.

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.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

De novo pyrimidine synthesis is necessary for intestinal colonization of Salmonella Typhimurium in chicks.

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.

Contribution of Asparagine Catabolism to Salmonella Virulence.

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.

The effects of herbal composition Gambigyeongsinhwan (4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty rats and HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatic steatosis has risen rapidly in parallel with a dramatic increase in obesity. The aim of this study was to determine whether the herbal composition Gambigyeongsinhwan (4) (GGH(4)), composed of Curcuma longa L. (Zingiberaceae), Alnus japonica (Thunb.) Steud. (Betulaceae), and the fermented traditional Korean medicine Massa Medicata Fermentata, regulates hepatic steatosis and inflammation. MATERIALS AND METHODS: The effects of GGH(4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty (OLETF) rats and HepG2 cells were examined using Oil red O, hematoxylin and eosin, and toluidine blue staining, immunohistochemistry, quantitative real-time polymerase chain reaction, and peroxisome proliferator-activated receptor α (PPARα) transactivation assay. RESULTS: Administration of GGH(4) to OLETF rats improved hepatic steatosis and lowered serum levels of alanine transaminase, total cholesterol, triglycerides, and free fatty acids. GGH(4) increased mRNA levels of fatty acid oxidation enzymes (ACOX, HD, CPT-1, and MCAD) and decreased mRNA levels of lipogenesis genes (FAS, ACC1, C/EBPα, and SREBP-1c) in the liver of OLETF rats. In addition, infiltration of inflammatory cells and expression of inflammatory cytokines (CD68, TNFα, and MCP-1) in liver tissue were reduced by GGH(4). Treatment of HepG2 cells with a mixture of oleic acid and palmitoleic acid induced significant lipid accumulation, but GGH(4) inhibited lipid accumulation by regulating the expression of hepatic fatty acid oxidation and lipogenic genes. GGH(4) also increased PPARα reporter gene expression. These effects of GGH(4) were similar to those of the PPARα activator fenofibrate, whereas the PPARα antagonist GW6471 reversed the inhibitory effects of GGH(4) on lipid accumulation in HepG2 cells. CONCLUSIONS: These results suggest that GGH(4) inhibits obesity-induced hepatic steatosis and that this process may be mediated by regulation of the expression of PPARα target genes and lipogenic genes. GGH(4) also suppressed obesity-related hepatic inflammation. Thus, GGH(4) may be a promising drug for the treatment of obesity-related liver diseases.

Intravital excitation increases detection sensitivity for pulmonary tuberculosis by whole-body imaging with ß-lactamase reporter enzyme fluorescence.

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 (ß-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.

Real-time Imaging of Mycobacterium tuberculosis, Using a Novel Near-Infrared Fluorescent Substrate.

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 (ß-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.

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks.

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.

Intravital Fluorescence Excitation in Whole-Animal Optical Imaging.

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU.

Assessment of bacterial community structure in nitrifying biofilm under inorganic carbon-sufficient and -limited conditions.

In this work, nitrification and changes in the composition of the total bacterial community under inorganic carbon (IC)-limited conditions, in a nitrifying moving bed biofilm reactor, was investigated. A culture-independent analysis of cloning and sequencing based on the 16S rRNA gene was applied to quantify the bacterial diversity and to determine bacterial taxonomic assignment. IC concentrations had significant effects on the stability of ammonia-oxidation as indicated by the reduction of the nitrogen conversion rate with high NH4(+)-N loadings. The predominance of Nitrosomonas europaea was maintained in spite of changes in the IC concentration. In contrast, heterotrophic bacterial species contributed to a high bacterial diversity, and to a dynamic shift in the bacterial community structure, under IC-limited conditions. In this study, individual functions of heterotrophic bacteria were estimated based on taxonomic information. Possible key roles of coexisting heterotrophic bacteria are the assimilation of organic compounds of extracellular polymeric substances produced by nitrifiers, and biofilm formation by providing a filamentous structure and aggregation properties.

Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

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.

Only one of the two type VI secretion systems encoded in the Salmonella enterica serotype Dublin genome is involved in colonization of the avian and murine hosts.

The type VI secretion system (T6SS) is a virulence factor for many Gram-negative bacteria. Salmonella genus harbors five phylogenetically distinct T6SS loci encoded in Salmonella Pathogenicity Islands (SPIs) SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22, which are differentially distributed among serotypes. The T6SSs encoded in SPI-6 and SPI-19 contribute to pathogenesis of serotypes Typhimurium and Gallinarum in mice and chickens, respectively. Salmonella Dublin is a pathogen restricted to cattle where it causes a systemic disease. Also, it can colonize other hosts such as chickens and mice, which can act as reservoirs of this serotype. Salmonella Dublin harbors the genes for both T6SS(SPI-6) and T6SS(SPI-19). This study has determined the contribution of T6SS(SPI-6) and T6SS(SPI-19) to host-colonization by Salmonella Dublin using avian and murine models of infection. Competitive index experiments showed that, a mutant strain lacking both T6SSs (∆T6SS(SPI-6)/∆T6SS(SPI-19)) presents a strong colonization defect in cecum of chickens, similar to the defect observed for the ∆T6SS(SPI-6) mutant, suggesting that this serotype requires a functional T6SS(SPI-6) for efficient colonization of the avian gastrointestinal tract. Colonization of mice was also defective, although to a lesser extent than in chickens. In contrast, the T6SS(SPI-19) was not necessary for colonization of either chickens or mice. Transfer of T6SS(SPI-6), but not T6SS(SPI-19), restored the ability of the double mutant to colonize both animal hosts. Our data indicate that Salmonella Dublin requires only the T6SS(SPI-6) for efficient colonization of mice and chickens, and that the T6SS(SPI-6) and T6SS(SPI-19) are not functionally redundant.

High-rate partial nitritation using porous poly(vinyl alcohol) sponge.

Poly(vinyl alcohol) (PVA) has been utilized as a support material for the immobilization of nitrifying bacteria without the comprehensive survey of partial nitritation. In the present study, the activities of nitrifiers and the maximum nitrogen conversion rate of partial nitritation with PVA sponge-cubes were specified according to different conditions. The selective enrichment of ammonia-oxidizing bacteria (AOB) on PVA sponge-cubes was achieved by the competition between AOB and nitrite-oxidizing bacteria for dissolved oxygen. The efficiency of ammonia oxidation was proportional to the concentration of HCO3 (-) with the molar ratio of HCO3 (-)-C/NH4 (+)-N = 1.91 and a half of the ratio was applied to the further experiments to ensure stable partial nitritation. The maximum nitrogen conversion rate of partial nitritation was dependent on the volume, not the size of sponge-cubes. The partial nitritation showed the superior rate performance of 3.09 kg N/m(3) day with the packing ratio of 32 % of 5 × 5 × 5 mm(3) PVA sponge-cubes.

Novel determinants of intestinal colonization of Salmonella enterica serotype typhimurium identified in bovine enteric infection.

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle.

The type VI secretion system encoded in SPI-6 plays a role in gastrointestinal colonization and systemic spread of Salmonella enterica serovar Typhimurium in the chicken.

The role of the Salmonella Pathogenicity Islands (SPIs) in pathogenesis of Salmonella enterica Typhimurium infection in the chicken is poorly studied, while many studies have been completed in murine models. The Type VI Secretion System (T6SS) is a recently described protein secretion system in Gram-negative bacteria. The genus Salmonella contains five phylogenetically distinct T6SS encoded in differentially distributed genomic islands. S. Typhimurium harbors a T6SS encoded in SPI-6 (T6SSSPI-6), which contributes to the ability of Salmonella to colonize mice. On the other hand, serotype Gallinarum harbors a T6SS encoded in SPI-19 (T6SSSPI-19) that is required for colonization of chicks. In this work, we investigated the role of T6SSSPI-6 in infection of chicks by S. Typhimurium. Oral infection of White Leghorn chicks showed that a ΔT6SSSPI-6 mutant had reduced colonization of the gut and internal organs, compared with the wild-type strain. Transfer of the intact T6SSSPI-6 gene cluster into the T6SS mutant restored bacterial colonization. In addition, our results showed that transfer of T6SSSPI-19 from S. Gallinarum to the ΔT6SSSPI-6 mutant of S. Typhimurium not only complemented the colonization defect but also resulted in a transient increase in the colonization of the cecum and ileum of chicks at days 1 and 3 post-infection. Our data indicates that T6SSSPI-6 contributes to chicken colonization and suggests that both T6SSSPI-6 and T6SSSPI-19 perform similar functions in vivo despite belonging to different phylogenetic families.

L-asparaginase II produced by Salmonella typhimurium inhibits T cell responses and mediates virulence.

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.

A rapid change in virulence gene expression during the transition from the intestinal lumen into tissue promotes systemic dissemination of Salmonella.

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.