Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 ؋ 10 ؊8 ml/min and 1.91 ؋ 10 ؊8 ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 ؋ 10 ؊2 CFU/ml for S. Typhimurium and 4.58 ؋ 10 ؊5 CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.

Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a tradeoff between acute virulence and transmission.biofilm | magnesium | ATP

Cronobacter sakazakii is an opportunistic pathogen that actively invades host eukaryotic cells. To identify invasion factors responsible for the intestinal translocation of C. sakazakii, we constructed for the first time outer membrane protein X (OmpX) and A (OmpA) deletion mutants using the lambda Red recombination system. The ompX and ompA deletion mutants showed significantly reduced invasion of human enterocyte-like epithelial Caco-2 and human intestinal epithelial INT-407 cells, and significantly fewer mutant cells were recovered from the livers and spleens of rat pups. Furthermore, compared with intact target cells, the invasion and initial association potentials of the mutants increased at a rate similar to that of the wild type in tight-junction-disrupted target cells, suggesting that OmpX and OmpA are involved in basolateral invasion by C. sakazakii. This is the first report of C. sakazakii virulence determinants that are essential for basolateral invasion and that may be critical for the virulence of C. sakazakii.

Summary pH regulates gene expression, biochemical activities and cellular behaviors. A mildly acidic pH activates the master virulence regulatory system PhoP/PhoQ in the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The sensor PhoQ harbors an extracytoplasmic domain implicated in signal sensing, and a cytoplasmic domain controlling activation of the regulator PhoP. We now report that, surprisingly, a decrease in Salmonella’s own cytoplasmic pH induces transcription of PhoP-activated genes even when the extracytoplasmic pH remains neutral. Amino acid substitutions in PhoQ’s cytoplasmic domain hindered activation by acidic pH and attenuated virulence in mice, but did not abolish activation by low Mg2+ or the antimicrobial peptide C18G. Conversely, removal of PhoQ’s extracytoplasmic domains prevented the response to the latter PhoQ-activating signals but not to acidic pH. PhoP-dependent genes were minimally induced by acidic pH in the non-pathogenic species S. bongori but were activated by low Mg2+ and C18G as in pathogenic S. enterica. Our findings indicate that the sensor PhoQ enables S. enterica to respond to both host- and bacterial-derived signals that alter its cytoplasmic pH.