c-di-GMP signaling regulates E. coli O157:H7 adhesion to colonic epithelium

Abstract

Escherichia coli O157:H7 is an important foodborne pathogen that causes serious illness in humans at low infectious doses. The main source of infections is beef or greens contaminated with E. coli O157:H7 shed by cattle. Here we investigated the role of c-di-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 levels reduced adhesion of E. coli O157:H7 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, long polar fimbriae 1, hemorrhagic coli pilus, as well as intimin and tir. We further observed decreased curli fimbriae synthesis in the strain with decreased c-di-GMP levels, which was supported by the reduction in the transcription of curli large subunit gene csgA and the curli expression regulator gene csgD. Genes for enterocyte effacement encoded regulator (Ler) and type III secretion system effectors, EspA and EspB, were also down-regulated. Collectively, data indicated that c-di-GMP signaling positively regulates E. coli O157:H7 intestinal epithelial cell and tissue colonization and expression of associated adhesion factors.

Introduction

E. coli O157:H7 is the most common bacterial etiologic agent in outbreaks associated with beef product and fresh produce. E. coli O157:H7 produces Shiga toxins which cause hemorrhagic colitis, hemolytic uremic syndrome (HUS) and acute renal failure in humans (Karmali, 2004, Nataro and Kaper, 1998). In addition, it can intimately attach to the intestinal epithelial cells causing attaching and effacing (A/E) lesions (Dean-Nystrom et al., 1998, Nataro and Kaper, 1998). E. coli O157:H7 interaction with gut tissues depends on its ability to adhere to the host cells. Multiple factors have been identified to regulate E. coli O157:H7 adherence and colonization of epithelial cells in the gastrointestinal (GI) tract (Farfan and Torres, 2012). Intimin, tir (translocated bacterially encoded intimin receptor), and effectors secreted through the type III secretion system (T3SS) mediate intimate contact between E. coli O157:H7 and gut epithelial cells, contributing to the formation of A/E lesions on intestinal epithelium (Cornick et al., 2002, Dean-Nystrom et al., 1998, Farfan and Torres, 2012, Ritchie et al., 2003). Other adhesins such as E. coli common pili (ECP) (Rendon et al., 2007), curli (Saldana et al., 2009, Uhlich et al., 2002), long polar fimbriae (LPF) (Jordan et al., 2004, Lloyd et al., 2012a, Torres et al., 2008), hemorrhagic coli pilus (HCP) (Xicohtencatl-Cortes et al., 2007) and H7 flagella (Mahajan et al., 2009) are also important mediators in E. coli O157:H7 intestinal epithelial adhesion.

Cyclic diguanylate (c-di-GMP) is a common second messenger in bacteria that is synthesized by diguanylate cyclases and degraded by c-di-GMP-specific phosphodiesterases (PDEs) (Hengge, 2009, Romling and Simm, 2009). It regulates numerous processes, including bacterial transition from planktonic to sessile. The elevated levels of c-di-GMP inhibit motility and promote biofilm formation (Hengge, 2009, Romling and Simm, 2009, Simm et al., 2004). In addition, c-di-GMP signaling pathways regulate bacterial virulence. The elevated levels of c-di-GMP are generally associated with decreased virulence but also reported to enhance virulence, and our understanding of these mechanisms is far from complete. For examples, in the mouse model of Yersinia pestis infection (Bobrov et al., 2011), c-di-GMP-induced exopolysaccharides are deleterious for virulence. In V. cholerae, low intracellular c-di-GMP is required for the expression of cholera toxin genes (Tischler and Camilli, 2005). Consistently, high c-di-GMP level inhibits Salmonella enterica serovar Typhimurium invasion of HT-29 cells (Lamprokostopoulou et al., 2010). On the other hand, in Ehrlichia chaffeensis (Kumagai et al., 2011) and Anaplasma phagocytophilum (Lai et al., 2009), c-di-GMP signaling is required for virulence. A recent study characterized an E. coli O157:H7-specific c-di-GMP PDE, VmpA, and showed that it affects biofilm formation and motility (Branchu et al., 2012), which indicate that c-di-GMP likely involves in the regulation of attachment and virulence of E. coli O157:H7 to epithelial cells, considering the intrinsic association between biofilm formation and colonization. The objective of this study was to assess the impact of c-di-GMP signaling on E. coli O157:H7 adherence to colonic epithelial cells and explant tissues.