Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo.

Cheng-Ju Kuo,Ting-Chen Chou,Hao-Chieh Chiu,Jenn-Wei Chen,Ching-Hao Teng,Pei-Jung Lu,Wan-Jr Syu,Tai-I Hsu,Chang-Shi Chen,Sin-Tian Wang

doi:10.3389/fcimb.2016.00082

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection.

Highlights

Enterohemorrhagic Escherichia coli (EHEC), the most recognized serotype of which is O157:H7, is a gram-negative foodborne pathogen which can cause diarrhea and other severe symptoms, like hemorrhagic colitis (HC), hemolytic-uremic syndrome (HUS) and acute renal failure in humans (Pennington, 2010)
From a genetic screen for identification of attenuated EHEC mutants in C. elegans, we have identified an EHEC mutant strain YQ033 (EDL933 rfaD::Tn5) in which the rfaD gene was disrupted by a Tn5 transposon insertion in the EHEC O157:H7 strain EDL933
C. elegans N2 animals fed with EHEC O157:H7 strain EDL933 had significantly shorter life spans than if fed with E. coli strain OP50 (P < 0.001), the standard nonpathogenic food source for C. elegans. When fed with both EDL933:∆rfaD (P < 0.001) and EDL933 rfaD::Tn5 (P < 0.001), C. elegans N2 animals lived significantly longer than animals fed with wild-type EDL933. These results suggest that disruption of the core LPS biosynthesis genes, including rfaD, confer less virulence of EHEC toward C. elegans

Summary

Introduction

Enterohemorrhagic Escherichia coli (EHEC), the most recognized serotype of which is O157:H7, is a gram-negative foodborne pathogen which can cause diarrhea and other severe symptoms, like hemorrhagic colitis (HC), hemolytic-uremic syndrome (HUS) and acute renal failure in humans (Pennington, 2010). Treatment of EHEC infection with antibiotics is not recommended because more EHEC virulence factors, such as Shiga toxins and endotoxins, may be released and exacerbate severe syndromes in patients (Pacheco and Sperandio, 2012; Freedman et al, 2016). The development of a novel therapeutic strategy for EHEC infection is urgently needed. Several virulence factors expressed by EHEC contribute to its pathogenicity (Nguyen and Sperandio, 2012). The Shiga toxins (Stxs) produced by EHEC are responsible for HUS and HC. Many virulence factors and mechanisms of EHEC have been reported, therapeutic strategies targeting these specific virulence factors remain under investigation (Nguyen and Sperandio, 2012; Pacheco and Sperandio, 2012)

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