Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.     

Catecholamines and sympathomimetic drugs decrease early Salmonella Typhimurium uptake into porcine Peyer's patches

Peyer's patches of the small intestine serve as inductive sites for mucosal immunity as well as targets for invasive enteropathogens, including Salmonella. Because they are innervated by catecholamine-containing enteric nerves, the hypothesis that the endogenous catecholamines dopamine and norepinephrine or sympathomimetic drugs alter Salmonella Typhimurium uptake into Peyer's patches was tested. Porcine jejunal Peyer's patch explants were mounted in Ussing chambers and inoculated with a porcine field isolate of Salmonella Typhimurium DT104. Salmonella recovery from gentamicin-treated tissues increased significantly between 30 and 90 min of bacterial exposure to the mucosal surface. Addition of the neuronal conduction blocker saxitoxin (0.1 micromol L(-1)) or dopamine (30 micromol L(-1)) to the contraluminal aspect of explants decreased bacterial recovery after 60 min of Salmonella exposure. The effects of dopamine were mimicked by cocaine and methamphetamine (30 micromol L(-1)), which act on catecholaminergic nerve terminals to increase synaptic neurotransmitter concentrations. These results suggest that enteric catecholaminergic nerves modulate Salmonella colonization of Peyer's patches at the earliest stages of infection, in part by altering epithelial uptake of bacteria.     

The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage

Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.     

Differences in gene expression levels and in enzymatic qualities account for the uneven contribution of superoxide dismutases SodCI and SodCII to pathogenicity in Salmonella enterica

Most Salmonella enterica serovars produce two periplasmic [Cu,Zn] superoxide dismutases, SodCI, which is prophage encoded, and SodCII, encoded by a conserved chromosomal gene. Both enzymes were proposed to enhance Salmonella virulence by protecting bacteria against products of macrophage oxidative burst. However, we previously found SodCI, but not SodCII, to play a role during mouse infection by S. enterica serovar Typhimurium. Here we have extended these findings to another serovar of epidemiological relevance: sv Enteritidis. In both serovars, the dominant role of SodCI in virulence correlates with its higher levels in bacteria proliferating in mouse tissues, relative to SodCII. To analyze the basis of these differences, the coding sequences of sodCI and sodCII genes were exchanged with the reciprocal 5'-regions (in serovar Typhimurium). The accumulation patterns of the two proteins in vivo were reversed as a result, indicating that the regulatory determinants lie entirely within the regions upstream from the initiation codon. In the construct with the sodCI gene fused to the sodCII 5'-region, SodCI contribution to virulence was reduced but remained significant. Thus, both, high-level expression and some unidentified qualities of the enzyme participate in the phenotypic dominance of SodCI over SodCII in Salmonella pathogenicity.     

Repression of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-associated proteins

The Hha/YmoA family of nucleoid-associated proteins is involved in gene regulation in enterobacteria. In Salmonella enterica serovar Typhimurium, virulence genes required for intracellular growth are induced following host cell invasion but the proteins responsible for repressing these genes prior to host cell entry have not been fully identified. We demonstrate here that Hha is the major repressor responsible for silencing virulence genes carried in Salmonella pathogenicity island 2 prior to bacteria sensing an intracellular environmental cue.     

Gene expression profiling of jejunal Peyer’s patches in juvenile and adult pigs

Peyer's patches are organized lymphoid tissues of the small intestine that play a critical role in disease resistance and oral tolerance. Peyer's patches in the jejunum contain lymphocytes, dendritic cells, macrophages, villous epithelium, and specialized follicle-associated epithelium. Little is known about the mechanisms and processes by which cells of the Peyer's patches discriminate food nutrients and commensal microflora from pathogenic microbiota. We hypothesize that the jejunal Peyer's patches express genes that mediate and regulate its essential functions. Expression patterns of approximately 2600 cDNAs from a porcine Peyer's patch subtracted library were examined by microarray profiling. Individual mRNAs of interest were further examined by quantitative RT-PCR. Innate immunity-associated genes, including complement 3 and lysozyme, and the genes for epithelial chloride channel and trappin 1 were highly expressed by jejunal Peyer's patch in both juvenile and adult pigs. The growth- and apoptosis-associated genes CIDE-B, GW112, and PSP/Reg I (pancreatic stone protein or regenerating gene) were differentially expressed in juvenile pig Peyer's patches. Many sequences which were highly expressed in jejunal Peyer's patches have previously been described with functions in epithelial cells. Animal-to-animal variation in basal jejunal Peyer's patch gene expression was considerable and reflects the dynamic physiological environment of the gut in addition to genetic, epigenetic, and microbiological variation in the small intestine.     

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.     

Long polar fimbriae and tissue tropism in Escherichia coli O157:H7

In vitro organ culture has demonstrated the human intestinal tropism of enterohaemorrhagic Escherichia coli O157:H7 for follicle associated epithelium overlying Peyer's patches of the terminal ileum. Long polar (LP) fimbriae are considered to mediate the attachment of Salmonella enterica serovar Typhimurium to Peyer's patch epithelium and, as homologous genes have been identified in O157:H7, we hypothesised that LP fimbriae in O157:H7 may perform the same function. However, mutation of LP fimbriae in O157:H7 strain 85/170 resulted in the novel phenotype of proximal and distal small intestinal colonisation with attaching/effacing lesion formation, while retaining adhesion to follicle associated epithelium. Application of whole genome DNA array technology did not identify changes in known fimbrial genes that could explain the change in tropism, but highlighted several genes that require further investigation. LP fimbrial genes are the first genes to be identified outside the locus of enterocyte effacement pathogenicity island that influence O157:H7 human intestinal tissue tropism.     

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.