Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages

Activation of macrophages by interferon gamma (IFN- ) and the subsequent production of nitric oxide (NO) are critical for the host defence against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFN- -induced NO production in RAW264.7 macrophages infected with wild-type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN- -treated macrophages infected with a nirC mutant of Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in the nirC knockout strain compared to the wild-type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of the nirC knockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade killing in activated macrophages by downregulating IFN- -induced NO production, and they highlight the critical role of nirC as a virulence gene.

Salmonella Typhimurium: Insight into the multi-faceted role of the LysR-type transcriptional regulators in Salmonella

The LysR-type transcriptional regulators (LTTRs) are widely distributed in various genera of prokaryotes LTTRs are DNA binding proteins that can positively or negatively regulate target gene expression and can also repress their own transcription Salmonella enterica comprises a group of Gram-negative bacteria capable of causing clinical syndromes that range from self-limiting diarrhoea to severe fibrinopurulent necrotizing enteritis and life threatening systemic disease. The survival and replication of Salmonella in macrophages and in infected host is brought about by the means of various two component regulatory systems, transporters and other virulence islands In Salmonella genome the existence of 44 LTTRs has been documented These LTTRs regulate bacterial stress response. systemic virulence in mice and also many virulence determinants in vitro. Here we focus on the findings that elucidate the structure and function of the LTTRs in Salmonella and discuss the importance of these LTTRs in making Salmonella a Successful pathogen...

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2.

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.

Functional transfer of Salmonella pathogenicity island 2 to Salmonella bongori and Escherichia coli.

The type III secretion system (T3SS) encoded by the Salmonella pathogenicity island 2 (SPI2) has a central role in systemic infections by Salmonella enterica and for the intracellular phenotype. Intracellular S. enterica uses the SPI2-encoded T3SS to translocate a set of effector proteins into the host cell, which modify host cell functions, enabling intracellular survival and replication of the bacteria. We sought to determine whether specific functions of the SPI2-encoded T3SS can be transferred to heterologous hosts Salmonella bongori and Escherichia coli Mutaflor, species that lack the SPI2 locus and loci encoding effector proteins. The SPI2 virulence locus was cloned and functionally expressed in S. bongori and E. coli. Here, we demonstrate that S. bongori harboring the SPI2 locus is capable of secretion of SPI2 substrate proteins under culture conditions, as well as of translocation of effector proteins under intracellular conditions. An SPI2-mediated cellular phenotype was induced by S. bongori harboring the SPI2 if the sifA locus was cotransferred. An interference with the host cell microtubule cytoskeleton, a novel SPI2-dependent phenotype, was observed in epithelial cells infected with S. bongori harboring SPI2 without additional effector genes. S. bongori harboring SPI2 showed increased intracellular persistence in a cell culture model, but SPI2 transfer was not sufficient to confer to S. bongori systemic pathogenicity in a murine model. Transfer of SPI2 to heterologous hosts offers a new tool for the study of SPI2 functions and the phenotypes of individual effectors.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria

Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 Delta pmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-gamma, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases. (C) 2014 Elsevier Ltd. All rights reserved.

Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification

Intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) manipulate their host cells through the interplay of various virulence factors. A multitude of such virulence factors are encoded on the genome of S. Typhimurium and are usually organized in pathogenicity islands. The virulence-associated genomic stretch of STM3117-3120 has structural features of pathogenicity islands and is present exclusively in non-typhoidal serovars of Salmonella. It encodes metabolic enzymes predicted to be involved in methylglyoxal metabolism. STM3117-encoded lactoylglutathione lyase significantly impacts the proliferation of intracellular Salmonella. The deletion mutant of STM3117 (Delta lgl) fails to grow in epithelial cells but hyper-replicates in macrophages. This difference in proliferation outcome was the consequence of failure to detoxify methylglyoxal by Delta lgl, which was also reflected in the form of oxidative DNA damage and upregulation of kefB in the mutant. Within macrophages, the toxicity of methylglyoxal adducts elicits the potassium efflux channel (KefB) in the mutant which subsequently modulates the acidification of mutant-containing vacuoles (MCVs). The perturbation in the pH of the MCV milieu and bacterial cytosol enhances the Salmonella pathogenicity island 2 translocation in Delta lgl, increasing its net growth within macrophages. In epithelial cells, however, the maturation of Delta lgl-containing vacuoles were affected as these non-phagocytic cells maintain less acidic vacuoles compared to those in macrophages. Remarkably, ectopic expression of Toll-like receptors 2 and 4 on epithelial cells partially restored the survival of Delta lgl. This study identified a novel metabolic enzyme in S. Typhimurium whose activity during intracellular infection within a given host cell type differentially affected the virulence of the bacteria.

Interactome analyses of Salmonella pathogenicity islands reveal SicA indispensable for virulence

Background: Serovars of Salmonella enterica, namely Typhi and Typhimurium, reportedly, are the bacterial pathogens causing systemic infections like gastroenteritis and typhoid fever. To elucidate the role and importance in such infection, the proteins of the Type III secretion system of Salmonella pathogenicity islands and two component signal transduction systems, have been mainly focused. However, the most indispensable of these virulent ones and their hierarchical role has not yet been studied extensively. Results: We have adopted a theoretical approach to build an interactome comprising the proteins from the Salmonella pathogeneicity islands (SPI) and two component signal transduction systems. This interactome was then analyzed by using network parameters like centrality and k-core measures. An initial step to capture the fingerprint of the core network resulted in a set of proteins which are involved in the process of invasion and colonization, thereby becoming more important in the process of infection. These proteins pertained to the Inv, Org, Prg, Sip, Spa, Ssa and Sse operons along with chaperone protein SicA. Amongst them, SicA was figured out to be the most indispensable protein from different network parametric analyses. Subsequently, the gene expression levels of all these theoretically identified important proteins were confirmed by microarray data analysis. Finally, we have proposed a hierarchy of the proteins involved in the total infection process. This theoretical approach is the first of its kind to figure out potential virulence determinants encoded by SPI for therapeutic targets for enteric infection. Conclusions: A set of responsible virulent proteins was identified and the expression level of their genes was validated by using independent, published microarray data. The result was a targeted set of proteins that could serve as sensitive predictors and form the foundation for a series of trials in the wet-lab setting. Understanding these regulatory and virulent proteins would provide insight into conditions which are encountered by this intracellular enteric pathogen during the course of infection. This would further contribute in identifying novel targets for antimicrobial agents. (C) 2014 Elsevier Ltd. All rights reserved.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

Infiltration of Matrix-Non-producers Weakens the Salmonella Biofilm and Impairs Its Antimicrobial Tolerance and Pathogenicity

Bacterial biofilms display a collective lifestyle, wherein the cells secrete extracellular polymeric substances (EPS) that helps in adhesion, aggregation, stability, and to protect the bacteria from antimicrobials. We asked whether the BPS could act as a public good for the biofilm and observed that infiltration of cells that do not produce matrix components weakened the biofilm of Salmonella enterica serovar Typhimurium. PS production was costly for the producing cells, as indicated by a significant reduction in the fitness of wild type (WT) cells during competitive planktonic growth relative to the non-producers. Infiltration frequency of non-producers in the biofilm showed a concomitant decrease in overall productivity. It was apparent in the confocal images that the non producing cells benefit from the BPS produced by the Wild Type (WT) to stay in the biofilm. The biofilm containing non-producing cells were more significantly susceptible to sodium hypochlorite and ciprofloxacin treatment than the WT biofilm. Biofilm infiltrated with non-producers delayed the pathogenesis, as tested in a murine model. The cell types were spatially assorted, with non producers being edged out in the biofilm. However, cellulose was found to act as a barrier to keep the non-producers away from the WT microcolony. Our results show that the infiltration of non-cooperating cell types can substantially weaken the biofilm making it vulnerable to antibacterials and delay their pathogenesis. Cellulose, a component of BPS, was shown to play a pivotal role of acting as the main public good, and to edge-out the non-producers away from the cooperating microcolony.

Role of neutrophils in murine salmonellosis

Gastrointestinal infections with Salmonella enterica serovars have different clinical outcomes that range from localized inflammation to a life-threatening systemic disease in the case of typhoid fever. Using a mouse model of systemic salmonellosis, we investigated the contribution of neutrophils to the innate immune defense against Salmonella after oral infection. Neutrophil infiltration was dependent on the bacterial burden in various infected organs (Peyer's patches, mesenteric lymph nodes, spleen, and liver). However, the massive infiltration of neutrophils did not allow clearance of an infection with wild-type Salmonella, presumably due to protection of intracellular Salmonella against neutrophil activities. A Salmonella mutant strain deficient in Salmonella pathogenicity island 2 (SPI2) was able to infect systemic sites, but its replication was highly restricted and it did not cause detectable attraction of neutrophils. Neutrophil depletion by antibody treatment of mice did not restore the virulence of SPI2 or auxotrophic mutant strains, supporting the hypothesis that attenuation of the strains is not due to greater susceptibility to neutrophil killing. Our observations reveal that neutrophils have completely different roles during systemic salmonellosis and localized gastrointestinal infections. In the latter conditions, rapid neutrophil attraction efficiently prevents the spread of the pathogen, whereas the neutrophil influx is delayed during systemic infections and cannot protect against lethal bacteremia.

sRNAscanner: A Computational Tool for Intergenic Small RNA Detection in Bacterial Genomes

Background:Bacterial non-coding small RNAs (sRNAs) have attracted considerable attention due to their ubiquitous nature and contribution to numerous cellular processes including survival, adaptation and pathogenesis. Existing computational approaches for identifying bacterial sRNAs demonstrate varying levels of success and there remains considerable room for improvement. Methodology/Principal Findings: Here we have proposed a transcriptional signal-based computational method to identify intergenic sRNA transcriptional units (TUs) in completely sequenced bacterial genomes. Our sRNAscanner tool uses position weight matrices derived from experimentally defined E. coli K-12 MG1655 sRNA promoter and rho-independent terminator signals to identify intergenic sRNA TUs through sliding window based genome scans. Analysis of genomes representative of twelve species suggested that sRNAscanner demonstrated equivalent sensitivity to sRNAPredict2, the best performing bioinformatics tool available presently. However, each algorithm yielded substantial numbers of known and uncharacterized hits that were unique to one or the other tool only. sRNAscanner identified 118 novel putative intergenic sRNA genes in Salmonella enterica Typhimurium LT2, none of which were flagged by sRNAPredict2. Candidate sRNA locations were compared with available deep sequencing libraries derived from Hfq-co-immunoprecipitated RNA purified from a second Typhimurium strain (Sittka et al. (2008) PLoS Genetics 4: e1000163). Sixteen potential novel sRNAs computationally predicted and detected in deep sequencing libraries were selected for experimental validation by Northern analysis using total RNA isolated from bacteria grown under eleven different growth conditions. RNA bands of expected sizes were detected in Northern blots for six of the examined candidates. Furthermore, the 5'-ends of these six Northern-supported sRNA candidates were successfully mapped using 5'-RACE analysis. Conclusions/Significance: We have developed, computationally examined and experimentally validated the sRNAscanner algorithm. Data derived from this study has successfully identified six novel S. Typhimurium sRNA genes. In addition, the computational specificity analysis we have undertaken suggests that similar to 40% of sRNAscanner hits with high cumulative sum of scores represent genuine, undiscovered sRNA genes. Collectively, these data strongly support the utility of sRNAscanner and offer a glimpse of its potential to reveal large numbers of sRNA genes that have to date defied identification. sRNAscanner is available from: http://bicmku.in:8081/sRNAscanner or http://cluster.physics.iisc.ernet.in/sRNAscanner/.

Needleless Vaccine Delivery Using Micro-Shock Waves

Shock waves are one of the most efficient mechanisms of energy dissipation observed in nature. In this study, utilizing the instantaneous mechanical impulse generated behind a micro-shock wave during a controlled explosion, a novel nonintrusive needleless vaccine delivery system has been developed. It is well-known that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle-free devices for epidermal delivery have inherent problems from the perspective of the safety and comfort of the patient. The penetration depth of less than 100 mu m in the skin can elicit higher immune response without any pain. Here we show the efficient utilization of our needleless device (that uses micro-shock waves) for vaccination. The production of liquid jet was confirmed by high-speed microscopy, and the penetration in acrylamide gel and mouse skin was observed by confocal microscopy. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model, and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. Vaccination using our device elicits better protection and an IgG response even at a lower vaccine dose (10-fold less) compared to other routes of vaccination. We anticipate that our novel method can be utilized for effective, cheap, and safe vaccination in the near future.

Prediction of protein-protein interactions between human host and a pathogen and its application to three pathogenic bacteria

Molecular understanding of disease processes can be accelerated if all interactions between the host and pathogen are known. The unavailability of experimental methods for large-scale detection of interactions across host and pathogen organisms hinders this process. Here we apply a simple method to predict protein-protein interactions across a host and pathogen organisms. We use homology detection approaches against the protein-protein interaction databases. DIP and iPfam in order to predict interacting proteins in a host-pathogen pair. In the present work, we first applied this approach to the test cases involving the pairs phage T4 - Escherichia coli and phage lambda - E. coli and show that previously known interactions could be recognized using our approach. We further apply this approach to predict interactions between human and three pathogens E. coli, Salmonella enterica typhimurium and Yersinia pestis. We identified several novel interactions involving proteins of host or pathogen that could be thought of as highly relevant to the disease process. Serendipitously, many interactions involve hypothetical proteins of yet unknown function. Hypothetical proteins are predicted from computational analysis of genome sequences with no laboratory analysis on their functions yet available. The predicted interactions involving such proteins could provide hints to their functions. (C) 2011 Elsevier B.V. All rights reserved.

Typhoid fever & vaccine development: a partially answered question

Typhoid fever is a systemic disease caused by the human specific Gram-negative pathogen Salmonella enterica serovar Typhi (S Typhi). The extra-intestinal infections caused by Salmonella are very fatal. The incidence of typhoid fever remains very high in impoverished areas and the emergence of multidrug resistance has made the situation worse. To combat and to reduce the morbidity and mortality caused by typhoid fever, many preventive measures and strategies have been employed, the most important being vaccination. In recent years, many Salmonella vaccines have been developed including live attenuated as well as DNA vaccines and their clinical trials have shown encouraging results. But with the increasing antibiotic resistance, the development of potent vaccine candidate for typhoid fever is a need of the hour. This review discusses the latest trends in the typhoid vaccine development and the clinical trials which are underway.