Salmonella enterica Serovar Typhimurium Lacking hfq Gene Confers Protective Immunity against Murine Typhoid

Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Dhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+) T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

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.

Host-specificity of Salmonella enterica serovar Gallinarum: Insights from comparative genomics

In this study, we have identified the possible genetic factors responsible for fowl-adaptation of Salmonella enterica serovar Gallinarum (S. Gallinarum). By comparing the genes related to Salmonella pathogenicity islands (SPI) of S. Gallinarum with those of Salmonella enterica serovar Enteritidis (S. Enteritidis) we have identified twenty-four positively selected genes. Our results suggest that the genes encoding the structural components of SPI-2 encoded type three secretion apparatus (TTSS) and the effector proteins that are secreted via SPI-1 encoded TTSS have evolved under positive selection pressure in these serovars. We propose that these positively selected genes play important roles in conferring different host-specificities to S. Gallinarum and S. Enteritidis.

Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

lac Repressor Is an Antivirulence Factor of Salmonella enterica: Its Role in the Evolution of Virulence in Salmonella

The genus Salmonella includes many pathogens of great medical and veterinary importance. Bacteria belonging to this genus are very closely related to those belonging to the genus Escherichia. lacZYA operon and lacI are present in Escherichia coli, but not in Salmonella enterica. It has been proposed that Salmonella has lost lacZYA operon and lacI during evolution. In this study, we have investigated the physiological and evolutionary significance of the absence of lacI in Salmonella enterica. Using murine model of typhoid fever, we show that the expression of Lacl causes a remarkable reduction in the virulence of Salmonella enterica. Lacl also suppresses the ability of Salmonella enterica to proliferate inside murine macrophages. Microarray analysis revealed that Lacl interferes with the expression of virulence genes of Salmonella pathogenicity island 2. This effect was confirmed by RT-PCR and Western blot analysis. Interestingly, we found that SBG0326 of Salmonella bongori is homologous to lacI of Escherichia coli. Salmonella bongori is the only other species of the genus Salmonella and it lacks the virulence genes of Salmonella pathogenicity island 2. Overall, our results demonstrate that Lacl is an antivirulence factor of Salmonella enterica and suggest that absence of lacI has facilitated the acquisition of virulence genes of Salmonella pathogenicity island 2 in Salmonella enterica making it a successful systemic pathogen.

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.

Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual "Salmonella translocated effector." Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS(-/-) mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style.

Curcumin Increases the Pathogenicity of Salmonella enterica Serovar Typhimurium in Murine Model

Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Contrary to this, our study reveals that it regulates the defense pathways of Salmonella enterica serovar Typhimurium ( S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in Peyer's,patches, mesenteric lymph node, spleen and liver, when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. This increased tolerance might be attributed to the up-regulation of genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that iron chelation property of curcumin have a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ regulatory system. Curcumin downregulates SPI1 genes, required for entry into epithelial cells and upregulates SPI2 genes required to intracellular survival. Since it is known that the SPI1 and SPI2 system can be regulated by the PhoPQ system, this common regulator could explain curcumin's mode of action. This data urges us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks.

Detection of IgG, IgA, IgM and IgE Antibodies in Invasive Amoebiasis in Endemic Areas

Entamoeba histolytica-specific serum IgG, IgA, IgM and IgE antibodies were assayed in cases of amoebiasis in an endemic area. Patient groups consisted of amoebic liver abscess (n=18), pre-abscess hepatic amoebiasis (n=22) and amoebic colitis (n=30). Control subjects comprised 26 asymptomatic cyst passers, 13 giardiasis cases, 20 typhoid patients and 24 non-amoebic individuals. Serum IgG was assayed by ELISA, using a monoclonal anti IgG β- galactosidase (IgG β-gal) conjugate, a polyclonal avidin biotin horse radish peroxidase (AB-HRP), and a polyclonal anti IgG horse radish peroxidase (IgG HRP) conjugate. IgA and IgM were assayed by the β-gal ELISA and IgE by AB-HRP. Diagnostically significant IgG and IgA while lower IgM and IgE antibody levels were seen in extraintestinal cases. About 40% of suspected pre-abscess hepatic amoebiasis cases were confirmed by antibody estimation. All isotype levels in most dysentery cases were in the range of the controls.

Acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella

During the course of infection, Salmonella has to face several potentially lethal environmental conditions, one such being acidic pH. The ability to sense and respond to the acidic pH is crucial for the survival and replication of Salmonella. The physiological role of one gene (STM1485) involved in this response, which is upregulated inside the host cells (by 90- to 113-fold) is functionally characterized in Salmonella pathogenesis. In vitro, the DSTM1485 neither exhibited any growth defect at pH 4.5 nor any difference in the acid tolerance response. The DSTM1485 was compromised in its capacity to proliferate inside the host cells and complementation with STM1485 gene restored its virulence. We further demonstrate that the surface translocation of Salmonella pathogenicity island-2 (SPI-2) encoded translocon proteins, SseB and SseD were reduced in the DSTM1485. The increase in co-localization of this mutant with lysosomes was also observed. In addition, the DSTM1485 displayed significantly reduced competitive indices (CI) in spleen, liver and mesenteric lymph nodes in murine typhoid model when infected by intra-gastric route. Based on these results, we conclude that the acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

Salmonella enterica serovars Typhimurium and Typhi as model organisms Revealing paradigm of host-pathogen interactions

The lifestyle of intracellular pathogens has always questioned the skill of a microbiologist in the context of finding the permanent cure to the diseases caused by them. The best tool utilized by these pathogens is their ability to reside inside the host cell, which enables them to easily bypass the humoral immunity of the host, such as the complement system. They further escape from the intracellular immunity, such as lysosome and inflammasome, mostly by forming a protective vacuole-bound niche derived from the host itself. Some of the most dreadful diseases are caused by these vacuolar pathogens, for example, tuberculosis by Mycobacterium or typhoid fever by Salmonella. To deal with such successful pathogens therapeutically, the knowledge of a host-pathogen interaction system becomes primarily essential, which further depends on the use of a model system. A well characterized pathogen, namely Salmonella, suits the role of a model for this purpose, which can infect a wide array of hosts causing a variety of diseases. This review focuses on various such aspects of research on Salmonella which are useful for studying the pathogenesis of other intracellular pathogens.

Curcumin reduces the antimicrobial activity of ciprofloxacin against Salmonella Typhimurium and Salmonella Typhi

Typhoidal and non-typhoidal infection by Salmonella is a serious threat to human health. Ciprofloxacin is the last drug of choice to clear the infection. Ciprofloxacin, a gyrase inhibitor, kills bacteria by inducing chromosome fragmentation, SOS response and reactive oxygen species (ROS) in the bacterial cell. Curcumin, an active ingredient from turmeric, is a major dietary molecule among Asians and possesses medicinal properties. Our research aimed at investigating whether curcumin modulates the action of ciprofloxacin. We investigated the role of curcumin in interfering with the antibacterial action of ciprofloxacin in vitro and in vivo. RTPCR, DNA fragmentation and confocal microscopy were used to investigate the modulation of ciprofloxacin-induced SOS response, DNA damage and subsequent filamentation by curcumin. Chemiluminescence and nitroblue tetrazolium reduction assays were performed to assess the interference of curcumin with ciprofloxacin-induced ROS. DNA binding and cleavage assays were done to understand the rescue of ciprofloxacin-mediated gyrase inhibition by curcumin. Curcumin interferes with the action of ciprofloxacin thereby increasing the proliferation of Salmonella Typhi and Salmonella Typhimurium in macrophages. In a murine model of typhoid fever, mice fed with curcumin had an increased bacterial burden in the reticuloendothelial system and succumbed to death faster. This was brought about by the inhibition of ciprofloxacin-mediated downstream signalling by curcumin. The antioxidant property of curcumin is crucial in protecting Salmonella against the oxidative burst induced by ciprofloxacin or interferon (IFN), a pro-inflammatory cytokine. However, curcumin is unable to rescue ciprofloxacin-induced gyrase inhibition. Curcumins ability to hinder the bactericidal action of ciprofloxacin and IFN might significantly augment Salmonella pathogenesis.

Computational studies on histidine kinase protein BaeS to target multidrug-resistant Salmonella

Multidrug-resistant Salmonella serovars have been a recent concern in curing infectious diseases like typhoid. Salmonella BaeS and BaeR are the two-component system (TCS) that signal transduction proteins found to play an important role in its multidrug resistance. A canonical TCS comprises a histidine kinase (HK) and its cognate partner response regulator (RR). The general approaches for therapeutic targeting are either the catalytic ATP-binding domain or the dimerization domain HisKA (DHp) of the HK, and in some cases, the receiver or the regulatory domain of the RR proteins. Earlier efforts of identifying novel drugs targeting the signal transduction protein have not been quite successful, as it shares similar ATP-binding domain with the key house keeping gene products of the mammalian GHL family. However, targeting the dimerization domain of HisKA through which the signals are received from the RR can be a better approach. In this article, we show stepwise procedure to specifically identify the key interacting residues involved in the dimerization with the RR along with effective targeting by ligands screened from the public database. We have found a few inhibitors which target effectively the important residues for the dimerization activity. Our results suggest a plausible de novo design of better DHp domain inhibitors.

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.

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.