Virulence potential of five major pathogeniCity islands (SPI-1 to SPI-5) of Salmonella enterica serovar Enteritidis for chickens

Background  

Salmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogeniCity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogeniCity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host.     

YqiC of Salmonella enterica serovar Typhimurium is a membrane fusogenic protein required for mice colonization

Background  

Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease.     

RpoE fine tunes expression of a subset of SsrB-regulated virulence factors in Salmonella enterica serovar Typhimurium

Background  

The survival of Salmonella enterica within the intracellular host niche requires highly co-ordinated expression of virulence effectors predominantly regulated by the SsrAB two-component regulatory system. S. enterica serovar Typhimurium mutants lacking the ssrAB genes are avirulent in mice, highlighting the importance of this regulatory system in vivo. Mutants lacking the gene encoding the alternative sigma factor σ^E (rpoE) are also highly attenuated for intracellular survival, pointing to a potential connection with the SsrAB regulatory system.     

Cultivation of Salmonella enterica serovar Typhimurium in a norepinephrine-containing medium alters in vivo tissue prevalence in swine

Transporting swine to slaughter is often linked with an increase in shedding of Salmonella, but little information exists to explain the role of stress. Recent research has suggested the catecholamine norepinephrine (NE) as a potential host signal during stress. The current study sought to investigate the prevalence of Salmonella enterica serovar Typhimurium in fecal samples and various tissues following inoculation with S. Typhimurium exposed to NE in vitro. The samples were collected at 3 and 24 h post-inoculation (p.i.) from pigs inoculated with S. Typhimurium cultured in either Luria–Bertani medium (LBC) or NE-infused, SAPI minimal medium (NEC). Bacterial quantification of tissue and fecal samples revealed a difference in the concentration of Salmonella between the two infections for six tissues at the two time points, five of which were greater in the NEC animals (p<0.05). Upon observing an increase in the number of Salmonella associated with the stomach wall tissues at 3 h p.i. for the NEC culture, an experiment was conducted using an ex vivo swine contents assay to determine the effect of NE exposure on the ability of the organism to survive the conditions of the porcine stomach; NE treatment enhanced the survival of S. Typhimurium more than 2 logs (p<0.007). Our results demonstrate an increase in the number of Salmonella associated with various swine tissues following experimental inoculation with NE-treated S. Typhimurium; thus, a possible scenario could be envisioned with a Salmonella-infected pig being stressed during transportation/mixing, resulting in the shedding of NE-stimulated Salmonella and exposure of naïve, stress-compromised penmates with a “primed” microorganism.     

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

Background  

Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome.     

The Salmonella PathogeniCity Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by Salmonella enterica serovar Typhimurium

Background  

Salmonella enterica serovar Typhimurium (Typhimurium) is an important pathogen that infects a broad range of hosts. In humans, Typhimurium causes a gastroenteritis characterized by vomiting, diarrhea, and abdominal pains. Typhimurium infection occurs mainly through the ingestion of contaminated food including poultry, pork, eggs, and milk. Chickens that are asymptomatic carriers of Typhimurium constitute a potential reservoir for infection. The type three secretion systems encoded by Salmonella pathogeniCity islands (SPI) 1 and 2 are major virulence factors of Salmonella. However, only a few studies have investigated their role during the infection of chickens.     

Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation enhances its intracellular growth within macrophages

Modification of lipid A is essential for bacterial adaptation to its host. Salmonella Typhimurium LpxR potentially detoxifies lipid A by 3′-O-deacylation; however, the involvement of deacylation in its adaptation remains unclear. LpxR-dependent 3′-O-deacylation was observed in the stationary phase. When macrophages were infected with stationary phase bacteria, the intracellular growth of the lpxR-null strain was lower than that of the wild-type strain. Furthermore, the expression level of inducible nitric oxide synthase was higher in the cells infected with the lpxR-null strain than in the cells infected with the wild-type strain. These results indicate that lipid A 3′-O-deacylation is beneficial for intracellular growth.     

Salmonella enterica serovar Typhimurium-induced internalization and IL-8 expression in HeLa cells does not have a direct relationship with intracellular Ca levels

The invasion-associated type III secretion system (T3SS-1) of S. Typhimurium is required to initiate and sustain an acute inflammatory response in the intestine. We investigated the relationship of S. Typhimurium T3SS-1-induced IL-8 expression and invasion with intracellular Ca²⁺ mobilization in HeLa cells. Compared to the sipAsopABDE2 mutant, strains carrying a mutation in sipA, or mutations in sopABDE2 induced higher levels of IL-8 and greater bacterial internalization despite the fact that these mutants elicited similarly low intracellular concentrations of Ca²⁺. Likewise, complemented sipAsopABDE2 mutant with sopE2 did not affect intracellular Ca²⁺ concentrations or IL-8 expression, but significantly increased bacterial internalization. Treating HeLa cells with the calcium chelator BAPTA-AM or with D-BAPTA-AM, a derivative with greatly reduced Ca²⁺ chelating activity, yielded strong evidence that BAPTA-AM does not affect invasion and inhibits IL-8 secretion by a calcium-dependent mechanism. These findings suggest that, although wild-type S. Typhimurium-induced IL-8 expression and bacterial internalization in HeLa cells coincides with increased cytosolic Ca²⁺, the differing levels of IL-8 and invasion induced by strains carrying different effector proteins are unrelated to levels of intracellular Ca²⁺.     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

Oral infection with the Salmonella enterica serovar Gallinarum 9R attenuated live vaccine as a model to characterise immunity to fowl typhoid in the chicken

Background  

Salmonella enterica serovar Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid, a severe systemic disease of chickens that results in high mortality amongst infected flocks. Due to its virulence, the immune response to S. Gallinarum is poorly characterised. In this study we have utilised infection by the live attenuated S. Gallinarum 9R vaccine strain in inbred chickens to characterise humoral, cellular and cytokine responses to systemic salmonellosis.     

SulA-induced filamentation in Salmonella enterica serovar Typhimurium: effects on SPI-1 expression and epithelial infection

Aims: Salmonella enterica serovar Typhimurium is capable of adopting a filamentous phenotype in response to damage. How this adaptive response affects bacterial virulence is unclear. We have examined the hypothesis that filamentation affects the ability of Salmonella to infect host cells. Methods and Results: Expression of the cell division inhibitor SulA in Salm. Typhimurium SL1344 from an arabinose‐inducible plasmid resulted in filamentation. We examined expression of the type 3 secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI‐1) using SL1344 expressing a chromosomal PprgH‐gfp reporter. Single cell analysis of SulA‐induced SL1344 PprgH‐gfp revealed a relationship between increasing cell length and decreasing propensity for prgH expression, but there was no evidence of a significant change in prgH expression evident at the whole population level. Filamentous Salm. Typhimurium were capable of initiating membrane ruffling on MDCK epithelial cells, but only nonfilamentous bacteria (<6 μm) invade. Conclusions: Induction of SulA expression in Salmonella inhibits septation. Increasing filament length is associated with down‐regulation of SPI‐1 gene expression, but a significant proportion of filaments retain the ability to produce SPI‐1 T3SS and induce membrane ruffles on epithelia. Despite an active SPI‐1 T3SS, filamentous Salmonella are unable to invade epithelial cells. Significance and Impact of the Study: Our findings that filamentous Salmonella can express an invasive phenotype but fail to invade cells suggest that their presence in food does not constitute an immediate risk of infection until septation occurs. The described SulA expression model provides a convenient model for studying the impact of filamentation in the absence of additional stresses.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

Regulation of L-arabinose transport in Salmonella typhimurium LT2

Summary The inducible L-arabinose transport system was characterized in Salmonella typhimurium LT2. Only one L-arabinose transport system with a Km of 2x10^-4 M was identified. The results suggested that araE may be the only gene which codes for L-arabinose transport activity under the conditions tested. An araE-lac fusion strain was used to study the induction of the araE gene. No araE expression was detected when the L-arabinose concentration was lower than 1 mM. The expression of araE reached a maximum in the presence of 50 mM L-arabinose, and was significantly reduced in the presence of D-glucose. Expression of the araBAD and araE genes was coordinately regulated. The concentration of L-arabinose that allowed maximum araBAD gene expression was 50-fold lower in an araE⁺ strain compared to an araE strain.     

The mkaC virulence gene of the Salmonella serovar Typhimurium 96 kb plasmid encodes a transcriptional activator

The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four MKa proteins indicated that mkaC enhances the expression of beta-galactosidase from the mkaA-, mkaB- and mkaC-lacZ gene fusions in trans. An mkaD-lacZ fusion or mkaA-lacZ fusion that did not contain DNA proximal to mkaB was not inducible with MkaC, indicating that at least mkaB and mkaA are induced together as an operon. MkaC is thus the first virulence protein whose function has been resolved.     

Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles

Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species. Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection. We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures. The analysis involved screening for mutators among revertants of S. typhimurium histidine auxotrophs selected for the His⁺ phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn. The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts. Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%). the his⁺ reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds. The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype. We calculate that the frequency of mutators in the unselected population of S. typhimurium is 1–4×10⁻⁶, an incidence of 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli.     

Microbial products from probiotic bacteria inhibit Salmonella enteritidis 857-induced IL-8 synthesis in Caco-2 cells

Oral administration of Lactobacillus spp. as probiotics is gaining importance in the treatment of intestinal inflammations. However, their mechanism of action is unknown. We investigated whether nonspecific binding Lactobacillus casei Shirota (LcS) and mannose-specific Lactobacillus plantarum 299v (Lp) and their spent culture supernatant (SCS) affect Salmonella enteritidis 857 (Se) growth, IL-8 and Hsp70 syntheses. In one set of experiments human enterocyte-like Caco-2 cells were infected with LcS, Lp or Se at 1–500 bacteria per cell for 1 h. In another set, cells were exposed to Se (0–200 per cell, 1 h) after exposure to lactobacilli (LB) (500 per cell, 30 min) or by co-incubation of Se and LB (1 h). The third set of experiments involved exposure of cells for 1 h to SCS or Se (100 per cell) pretreated (1 h) in SCS. The effect of LB SCS on Se growth was evaluated by agar plate diffusion test. IL-8 and Hsp70 were assessed over 2–24 h using ELISA and Western blotting, respectively. Neither LcS nor Lp affected the Se growth and IL-8 production. In addition, they did not induce Hsp70 expression by Caco-2 cells. Instead, their SCS inhibited the Se growth and IL-8 production and induced the expression of Hsp70 by both crypt- and villus-like cells. The beneficial effect of Lactobacillus spp. to the intestinal inflammations might be associated with a decrease in IL-8 levels. This effect could be mediated, at least in part, via a secreted antimicrobial product(s) either directly against the pathogens or indirectly through the synthesis of Hsp70.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium