b Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causative agents of food-borne bacterial gastroenteritis. Swift invasion through the intestinal tract and successful establishment in systemic organs are associated with the adaptability of S. Typhimurium to different stress environments. Low-pH stress serves as one of the first lines of defense in mammalian hosts, which S. Typhimurium must efficiently overcome to establish an infection. Therefore, a better understanding of the molecular mechanisms underlying the adaptability of S. Typhimurium to acid stress is highly relevant. In this study, we have performed a transcriptome analysis of S. Typhimurium under the acid tolerance response (ATR) and found a large number of genes (ϳ47%) to be differentially expressed (more than 1.5-fold or less than ؊1.5-fold; P < 0.01). Functional annotation revealed differentially expressed genes to be associated with regulation, metabolism, transport and binding, pathogenesis, and motility. Additionally, our knockout analysis of a subset of differentially regulated genes facilitated the identification of proteins that contribute to S. Typhimurium ATR and virulence. Mutants lacking genes encoding the K ؉ binding and transport protein KdpA, hypothetical protein YciG, the flagellar hook cap protein FlgD, and the nitrate reductase subunit NarZ were significantly deficient in their ATRs and displayed varied in vitro virulence characteristics. This study offers greater insight into the transcriptome changes of S. Typhimurium under the ATR and provides a framework for further research on the subject. Salmonella enterica serovar Typhimurium is a neutralophilic, Gram-negative food-and waterborne pathogen that causes diseases ranging from gastroenteritis to systemic infection in humans. The intestinal tract of wild and domestic animals serves as a vehicle by which salmonellae find their way into humans through contaminated food and water. It has been estimated that globally this species accounts for about 80.3 million cases of food-borne gastroenteritis with about 1.5 million deaths (1). A large number of outbreaks have been linked to contaminated fruits and vegetables, including apples, mangoes, lettuce, tomatoes, celery, and unpasteurized juice (2). During host-pathogen interaction, Salmonella constantly encounters various stress conditions, such as changing pH, high osmotic pressure, low oxygen availability, and the presence of bile salts and antimicrobial peptides, that constantly test the adaptability of this pathogen. One such stress condition is low pH, and Salmonella confronts this on transit through the stomach, as well as during survival within the Salmonellacontaining vacuole (SCV) of phagocytic and nonphagocytic cells. Hence, the ability of Salmonella to perceive low-pH environments and respond to such stress is crucial for its survival and pathogenicity.The mechanism by which S. Typhimurium senses acidic environments and adapts to survive under low pH is termed the acid tolerance response (ATR) (3-...

The Gram-negative, enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is exposed to various stress conditions during pathogenesis, of which acid stress serves as a major defense mechanism in the host. Such environments are encountered in the stomach and Salmonella containing vacuole of phagocytic and non-phagocytic cells. It is only recently that small RNAs (sRNAs) have come to the forefront as major regulators of stress response networks. Consequently, the sRNA DsrA which regulates acid resistance in Escherichia coli, has not been characterized in the acid tolerance response (ATR) of Salmonella. In this study, we show dsrA to be induced two and threefold under adaptation and challenge phases of the ATR, respectively. Additionally, an isogenic mutant lacking dsrA (ΔDsrA) displayed lower viability under the ATR along with reduced motility, feeble adhesion and defective invasion efficacy in vitro. Expression analysis revealed down regulation of several Salmonella pathogenicity island-1 (SPI-1) effectors in ΔDsrA compared to the wild-type, under SPI-1 inducing conditions. Additionally, our in vivo data revealed ΔDsrA to be unable to cause gut inflammation in C57BL/6 mice at 72 h post infection, although intracellular survival and systemic dissemination remained unaffected. A possible explanation may be the significantly reduced expression of flagellin structural genes fliC and fljB in ΔDsrA, which have been implicated as major proinflammatory determinants. This study serves to highlight the role of sRNAs such as DsrA in both acid tolerance and virulence of S. Typhimurium. Additionally the robust phenotype of non-invasiveness could be exploited in developing SPI-I attenuated S. Typhimurium strains without disrupting SPI-I genes.

Toxin-antitoxin (TA) modules are two component “addictive” genetic elements found on either plasmid or bacterial chromosome, sometimes on both. TA systems perform a wide range of functions like biofilm formation, persistence, programmed cell death, phage abortive infection etc. Salmonella has been reported to contain several such TA systems. However, the hemolysin expression modulating protein (Hha) and its adjacent uncharacterized hypothetical protein TomB (previously known as YbaJ), have not been listed as a TA module in Salmonella. In this study we established that Hha and TomB form a bonafide TA system where Hha serves as a toxin while TomB functions as an antitoxin. Interestingly, the toxicity of Hha was conditional causing cell death under acid stress. The antitoxin attenuated the toxicity of Hha by forming a TA complex through stable interactions. The Hha-TomB TA system was found to increase persistence and inhibit programmed cell death under antibiotic stress where a phenotypically diverse population expressing differential level of TA components was observed. Therefore we propose that Hha and TomB prevent cells from committing suicide thereby promoting persister cell formation.

Salmonella Enteritidis causes food-borne gastroenteritis by the two type three secretion systems (TTSS). TTSS-1 mediates invasion through intestinal lining, and TTSS-2 facilitates phagocytic survival. The pathogens' ability to infect effectively under TTSS-1-deficient background in host's phagocytes is poorly understood. Therefore, pathobiological understanding of TTSS-1-defective nontyphoidal Salmonellosis is highly important. We performed a comparative global proteomic analysis of the isogenic TTSS-1 mutant of Salmonella Enteritidis (M1511) and its wild-type isolate P125109. Our results showed 43 proteins were differentially expressed. Functional annotation further revealed that differentially expressed proteins belong to pathogenesis, tRNA and ncRNA metabolic processes. Three proteins, tryptophan subunit alpha chain, citrate lyase subunit alpha, and hypothetical protein 3202, were selected for in vitro analysis based on their functional annotations. Deletion mutants generated for the above proteins in the M1511 strain showed reduced intracellular survival inside macrophages in vitro. In sum, this study provides mass spectrometry-based evidence for seven hypothetical proteins, which will be subject of future investigations. Our study identifies proteins influencing virulence of Salmonella in the host. The study complements and further strengthens previously published research on proteins involved in enteropathogenesis of Salmonella and extends their role in noninvasive Salmonellosis.

BackgroundSalmonella enterica serovar Enteritidis, the most common cause of human gastroenteritis, employs several virulence factors including lipopolysaccharide (LPS) for infection and establishment of disease inside the host. The LPS of S. enterica serovar Enteritidis consists of lipid A, core oligosaccharide and O-antigen (OAg). The OAg consists of repeating units containing different sugars. The sugars of OAg are synthesized and assembled by a set of enzymes encoded by genes organized into clusters. Present study focuses on the effect of deletion of genes involved in biosynthesis of OAg repeating units on resistance to antimicrobial peptides and virulence in mice.MethodsIn the present study, the OAg biosynthesis was impaired by deleting tyv, prt and wbaV genes involved in tyvelose biosynthesis and its transfer to OAg. The virulence phenotype of resulting mutants was evaluated by assessing resistance to antimicrobial peptides, serum complement, adhesion, invasion and in vivo colonization.ResultsDeletion of the above three genes resulted in the production of OAg-negative LPS. All the OAg-negative mutants showed phenotype reported for rough strains. Interestingly, ΔwbaV mutant showed increased resistance against antimicrobial peptides and normal human serum. In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt. In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant.ConclusionOAg-negative mutants are known to be avirulent; however, this study demonstrates that certain OAg negative mutants e.g. ∆wbaV may also show resistance to antimicrobial peptides and cause colitis in Streptomyces pretreated mouse model.