The Two-Component System CpxRA Negatively Regulates the Locus of Enterocyte Effacement of Enterohemorrhagic Escherichia coli Involving σ32 and Lon protease

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a significant cause of serious human gastrointestinal disease worldwide. EHEC strains contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes virulence factors responsible for damaging the gut mucosa. The Cpx envelope stress response of E. coli is controlled by a two-component system (TCS) consisting of a sensor histidine kinase (CpxA) and a cytoplasmic response regulator (CpxR). In this study, we investigated the role of CpxRA in the expression of LEE-encoded virulence factors of EHEC. We found that a mutation in cpxA significantly affected adherence of EHEC to human epithelial cells. Analysis of this mutant revealed the presence of high levels of CpxR which repressed transcription of grlA and ler, the main positive virulence regulators of the LEE, and influenced negatively the production of the type 3 secretion system–associated EspABD translocator proteins. It is known that CpxR activates rpoH (Sigma factor 32), which in turns activates transcription of the lon protease gene. We found that transcription levels of ler and grlA were significantly increased in the lon and cpxA lon mutants suggesting that lon is involved in down-regulating LEE genes. In addition, the Galleria mellonella model of infection was used to analyze the effect of the loss of the cpx and lon genes in EHEC's ability to kill the larvae. We found that the cpxA mutant was significantly deficient at killing the larvae however, the cpxA lon mutant which overexpresses LEE genes in vitro, was unable to kill the larvae, suggesting that virulence in the G. mellonella model is T3SS independent and that CpxA modulates virulence through a yet unknown EHEC-specific factor. Our data provides new insights and broadens our scope into the complex regulatory network of the LEE in which the CpxA sensor kinase plays an important role in a cascade involving both global and virulence regulators.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection

“…Several other examples of post‐transcriptional effects mediated by CpxAR have been described in other organisms. For example, deletion of cpxA in EHEC (which activates the Cpx pathway due to loss of CpxA's phosphatase activity) causes decreased expression of LEE‐encoded T3SS proteins such as EspA, EspB and EspD (De la Cruz et al , ). The repression of T3SS expression in EHEC ΔcpxA is dependent on the presence of Lon protease, suggesting that activation of the Cpx response causes Lon to degrade a regulator required for T3SS gene expression (De la Cruz et al , ).…”

“…For example, deletion of cpxA in EHEC (which activates the Cpx pathway due to loss of CpxA's phosphatase activity) causes decreased expression of LEE‐encoded T3SS proteins such as EspA, EspB and EspD (De la Cruz et al , ). The repression of T3SS expression in EHEC ΔcpxA is dependent on the presence of Lon protease, suggesting that activation of the Cpx response causes Lon to degrade a regulator required for T3SS gene expression (De la Cruz et al , ). Lon is also required for Cpx‐mediated repression of T3SS‐1 expression in Salmonella Typhimurium; in this case, activation of the Cpx response causes Lon to degrade the regulatory protein HilD (De la Cruz et al , ).…”

“…Decreased pilus elaboration in the absence of Cpx pathway activity has been attributed to reduced expression of envelope‐localized protein folding factors, including DsbA, DegP and CpxP, which have been shown to promote stability of pilus component proteins (Zhang and Donnenberg, ; Vogt et al , ; Humphries et al , ). However, inactivation of cpxR has little effect on the transcription of the bfp genes or the expression and in vitro functionality of the T3SS in EPEC and EHEC (MacRitchie et al , ; De la Cruz et al , ). Conversely, constitutive Cpx activation dramatically reduces expression of the BFP in EPEC and the T3SS in both EPEC and EHEC (MacRitchie et al , ; Vogt et al , ; De la Cruz et al , ).…”

“…However, inactivation of cpxR has little effect on the transcription of the bfp genes or the expression and in vitro functionality of the T3SS in EPEC and EHEC (MacRitchie et al , ; De la Cruz et al , ). Conversely, constitutive Cpx activation dramatically reduces expression of the BFP in EPEC and the T3SS in both EPEC and EHEC (MacRitchie et al , ; Vogt et al , ; De la Cruz et al , ). Repression of the BFP when the Cpx response is activated occurs primarily at the level of transcription of the bfp genes, while repression of the T3SS involves both transcriptional and post‐translational effects (MacRitchie et al , ; ; Vogt et al , ).…”

Involvement of Two-Component Signaling on Bacterial Motility and Biofilm Development

“…A 2CSTS that acts upon the transcription of tier III genes is CpxA/CpxR. This 2CSTS responds to envelope stress, inhibits LEE gene transcription (82), and activates the synthesis pathway that leads to the production of an antimicrobial peptide that causes multidrug resistance (83). Direct binding of CpxR-P to the promoter regions of the motA and tsr operons has been shown (84) and so has transcriptional repression of csgD by CpxR (33,85).…”