Development of an HTS system to identify natural chemicals that specifically inhibit Escherichia coli O157:H7 adhesion to host cells

Highlights

• We performed a high-throughput screening (HTS) against Escherichia coli O157:H7 Ler.

• The HTS system is a rapid and cost-effective tool for probing Ler inhibition.

• Yomogin specifically inhibits the expression of virulence factors.

• Yomogin has potential as a novel anti-pathogenic agent for E. coli O157:H7.

• Yomogin can overcome the issue of antibiotic resistance.

Abstract

Adhesion to intestinal epithelium is critical for virulence and infection of Escherichia coli O157:H7, which is a major cause of foodborne illnesses. Targeting adhesion but not growth of the pathogen is an effective approach to control bacterial infections. In this study, a high-throughput screening system was developed to identify anti-adhesive natural chemicals using a reporter strain whose activity entirely depends on Ler since genes encoding the locus of enterocyte effacement (LEE) which forms attaching and effacing lesions are known to be activated by Ler. A selected hit, yomogin, suppressed the expression of virulent genes encoded within the LEE without affecting bacterial growth itself. Yomogin significantly reduced the adherence of E. coli O157:H7 to Caco-2 epithelial cells and cytotoxicity. These results indicated that the developed system allows for rapid and cost-effective discovery of novel anti-adhesive agents and yomogin can prevent early stage of E. coli O157:H7 pathogenesis.

Introduction

Escherichia coli O157:H7 was first recognized as an enteric pathogen in 1982 (Riley et al., 1983). Since then, more than 30 countries have reported E. coli O157:H7 outbreaks in humans. The Centers for Disease Control and Prevention (CDC) has estimated that E. coli O157:H7 infections cause 73,000 illnesses annually in the United States, resulting in more than 2000 hospitalizations and 60 deaths (Scallan et al., 2011). Also in Korea, pathogenic E. coli including E. coli O157:H7 was the second most common causative agent for foodborne outbreaks from 2007 to 2009 (Gwack et al., 2010). E. coli O157:H7 outbreaks are often associated with the consumption of raw or undercooked meat (Su & Brandt, 1995) as well as foods such as fresh produce (Rodríguez et al., 2011). As fresh produce is frequently consumed raw and do not undergo any preservation or inactivation treatments during processing, consumers could be exposed directly to foodborne pathogens such as E. coli O157:H7. In addition, the low infectious dose (<100 cells) and high virulence of E. coli O157:H7 make infections severe and life threatening, particularly for young children, the elderly, and those with weakened immune systems (Kaper, Nataro, & Mobley, 2004). Hence, additional efforts have been required to control this pathogen.

The food industry has tended to use plant-derived antimicrobials due to their antimicrobial activity without side effects often associated with use of synthetic chemicals, their nontoxic nature, and their affordability. Numerous research groups have sought to elucidate their antimicrobial mechanisms of action. They revealed that the plant-derived antimicrobials affect membrane stability, cellular metabolism, biofilm formation, bacterial capsule production, toxin production, host immune system, and adhesion activity (Cushnie and Lamb, 2005, Upadhyay et al., 2014). As an enteric pathogen, adhesion of E. coli O157:H7 to intestinal epithelium is critical for virulence and infection (Frankel et al., 1998). In this aspects, to identify the novel types of plant-derived antimicrobials that inhibit adhesions to intestinal epithelium can be a promising way to block pathogenic infections thereby to control the foodborne illness due to E. coli O157:H7.

Adhesion of E. coli O157:H7 to intestinal epithelial cells causes serious diseases such as severe diarrhea and hemorrhagic colitis, which can lead to the life-threatening hemolytic-uremic syndrome (HUS) (Tarr, Gordon, & Chandler, 2005). A distinctive histopathology in host intestinal cells is attaching and effacing (A/E) lesions which are characterized by the loss of microvilli, an intimate adherence of bacteria adjacent to the host cell membrane, and the generation of an organized cytoskeletal structure containing filamentous actin beneath adherent bacteria, which is called an actin pedestal (Donnenberg, Kaper, & Finlay, 1997). The genes involved in the formation of these lesions are encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which is 30,919-bp in size. The LEE region contains the following three segments that encode five major polycistronic operons (Frankel et al., 1998, Perna et al., 1998): (i) the first segment includes the LEE1, LEE2, and LEE3 operons, which contain genes that encode the type III secretion system (TTSS); (ii) the second segment includes the translocated intimin receptor (Tir) (LEE5) operon, which contains genes that encode bacterial adhesion proteins such as intimin (EaeA) as well as Tir; and (iii) the third segment includes the LEE4 operon, which contains genes that encode for secreted proteins such as EspA, EspB, and EspD. The EspABD complex forms the translocation apparatus that transfers the effector proteins of the TTSS.

The regulatory mechanisms of the operons encoded within LEE are known to be complicated (Mellies, Barron, & Carmona, 2007). The LEE-encoded regulator (Ler) encoded by the first gene in the LEE1 operon has been known as a central regulator of the expression of genes involved in the formation of A/E lesions (Elliott et al., 2000). In late-log phase cells, thirty-nine genes were found to be transcriptionally activated by Ler (increases in expression of 2-fold or more). Of these thirty-nine genes, thirty-five were within the LEE region (increases in expression between 4- and 32-fold) (Bingle et al., 2014). These findings indicate that the Ler acts as a major activator in LEE expression. Accordingly, the objectives of this study were to clarify the role of Ler, to construct an effective high-throughput phenotypic screening (HTS) system, and to identify plant-derived antimicrobials that inhibit the expression of virulence factors by hindering the activity of Ler. Our results indicated that Ler plays an essential role in E. coli O157:H7 adhesion, and thus, targeting Ler is crucial for preventing E. coli O157:H7 infections.