The c-di-gmp signaling and its potential receptor pnpase in the pathogenesis of escherichia coli o157:h7

Escherichia coli O157:H7 is an important foodborne pathogen that causes serious illness in humans at low infective doses. The main source of infections is beef or greens contaminated with E. coli O157:H7 shed by cattle. Two key virulence strategies used by E. coli O157: H7 once passed through the stomach are Shiga toxin production and colonization of the intestine (Karmali et al., 2010). Colonization of large intestine is a key step in E. coli O157: H7 pathogenesis (Yin et al., 2011b). c-di-GMP signaling plays important roles in biofilm formation, motility, and pathogenesis of bacteria. Here we investigated the role of cdi- GMP-dependent signal transduction in cattle gut colonization of E. coli O157:H7. To manipulate intracellular c-di-GMP levels, we introduced into E. coli O157:H7 a c-di-GMP specific phosphodiesterase (PDE). Liquid chromatography tandem mass spectrometry analysis confirmed that in E. coli O157:H7, over-expression of PDE decreased c-di-GMP level. Consistent with the altered c-di-GMP level, PDE overexpression resulted in decreased biofilm formation in E. coli O157:H7. Furthermore, this diminished c-di-GMP level decreased E. coli O157:H7 adhesion to both cultured HT-29 cells and cattle colon explants. Consistently, mRNA levels of genes involved in adhesion were down-regulated including genes encoding E. coli common pili, lpf1A, and hcp, as well as intimin and tir. We further observed decreased curli fimbriae synthesis in the strain with low c-di-GMP concentration, which were supported by the reduction in the transcription of curli large subunit gene csgA and the curli expression regulator gene csgD. The genes for positive regulator of the locus of enterocyte effacement and type III secretion system, ler, and its effectors espA and espB, were also down-regulated. Collectively, data indicated that c-di-GMP signaling positively 2 regulates E. coli O157:H7 colonization on intestinal epithelial cells and tissue, and the expression of associated adhesion factors.;Polynucleotide phosphorylase (PNPase) is one of potential c-di-GMP receptors in E. coli O157:H7. Previous studies indicate that PNPase regulates virulence in several examined pathogens. Our data demonstrated that PNPase are essential for Shiga toxin production, Stx2 prophage activation, and colonization of E. coli O157:H7. PNPase represses T3SS by controlling the master regulator ler. The T3SS regulation system is conserved among strains EDL933, 86-24 and MIO335, but the regulation of Shiga toxin production is strain specific.;The growth of E. coli O157:H7 in contaminated dairy and other refrigerated food products due to temperature fluctuation poses a major food safety threat. Effective control or inhibition of E. coli O157:H7 growth depends on our understanding of mechanisms that regulate its growth at low temperature. PNPase was previously reported to be involved in cold adaption in generic E. coli, thus we hypothesized that polynucleotide phosphorylase (PNPase) plays a critical role in E. coli O157:H7 low temperature growth. Comparing the growth of E. coli O157:H7 wild type strain and pnp, we further investigated the role of pnp in E. coli O157:H7 growth and survival at different temperatures in LB media as well as milk. Results indicated that PNPase is required for the growth of E. coli O157:H7 at low temperature. The deletion of pnp impaired its growth in LB at 10 °C and 22°C. During 14 days of 10°C storage in both LB and milk, WT grew and reached >8 Log10CFU/ml after 4 days of 10°C storage, while Deltapnp gradually died off with effects more pronounced in milk, which were again mitigated by pnp overexpression. In addition, pnp deletion impaired the motility of E. coli O157:H7 but did not affect its resistance to H2O2.;In summary, these studies explored the role of c-di-GMP signaling in E. coli O157:H7 gut colonization, indicating that c-di-GMP signaling positively regulates E. coli 3 O157:H7 intestinal epithelial cell and tissue colonization and expression of associated adhesion factors. The roles of potential c-di-GMP receptor, PNPase in E. coli O157:H7 were further examined. Results showed that PNPase regulate T3SS expression, Shiga toxin production, and colonization of E. coli O157:H7 to gut epithelium. In addition, we found that PNPase is essential for E. coli O157:H7 growth at low temperatures, which might have important practical applications in dairy and other food industry.