Development of an HTS system to identify natural chemicals that specifically inhibit Escherichia coli O157:H7 adhesion to host cells
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.
Section snippets
Bacterial strains, plasmids, culture conditions, and chemicals
The strains and plasmids used in this study are listed in Table 1. Unless noted otherwise, the E. coli strains were grown in a Luria-Bertani (LB) medium at 37 °C. Total 897 plant-derived natural chemicals were obtained from Korea Chemical Bank (http://www.chembank.org).
Generation of the E. coli ler deletion mutant
The ler gene was inactivated in vitro by the deletion of the ler ORF using the temperature-sensitive plasmid pRedET (Gene Bridges, Dresden, Germany). The pRedET encodes a λ red recombinase, as described previously (Datsenko &
Generation of the Δler::neo mutant
To examine the role of Ler, an E. coli O157:H7 ler isogenic mutant was constructed using the linear recombination (λ Red) method. The wild-type ler on the E. coli O157:H7 chromosome was replaced with an internal 290-bp fragment deleted ler gene fused to neo gene ORF (△ler::neo allele). Double cross-overs were confirmed using PCR as shown in Fig. S1. PCR analysis of genomic DNA from the wild type using primers C1 and C4 (Table S1) produced a 556-bp fragment (Fig. S1). PCR amplification of
Discussion
The annual cost of illness due to E. coli O157:H7 infections transmitted by food is $405 million in the USA, including $370 million for premature deaths, $30 million for medical care, and $5 million in lost productivity (Frenzen, Drake, Angulo, & Emerging Infections Program FoodNet Working Group, 2005). The high cost of illness is caused by the high virulence with a low infectious dose of E. coli O157:H7. The attachment to host intestinal epithelial cells is one of the most essential virulence
Conclusions
This study revealed the characteristics of E. coli O157:H7 Ler in its ability to adhere to intestinal epithelial cells. With this information, we developed an effective HTS system to identify active natural compounds having anti-adhesion activity via inhibition of the Ler activity for preventing E. coli O157:H7 infections. Using the system, we identified a natural chemical compound with therapeutic potential, called yomogin, a component of Artemisia princeps. The data presented here extend our
Acknowledgements
This research was supported by a grant from Korea Food Research Institute. The chemical library used in this study was kindly provided by Korea Chemical Bank (http://www.chembank.org/) of Korea Research Institute of Chemical Technology.
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