Inhibition of Escherichia coli O157:H7 motility and biofilm by β-Sitosterol glucoside

https://doi.org/10.1016/j.bbagen.2013.07.022Get rights and content

Highlights

  • β-Sitosterol glucoside (SG) inhibits EHEC biofilm and motility.

  • SG represses flagellar master regulators flhDC.

  • SG inhibits biofilm and motility by repressing flhDC.

  • Repression of flhDC by SG is dependent upon rssAB and hns.

Abstract

Background

Escherichia coli O157:H7 (EHEC) is a food borne pathogen, which causes diarrhea and hemolytic uremic syndrome (HUS). There is an urgent need of novel antimicrobials for treatment of EHEC as conventional antibiotics enhance shiga toxin production and potentiate morbidity and mortality.

Methods

Six bioactive compounds were isolated, identified from citrus and evaluated for the effect on EHEC biofilm and motility. To determine the possible mode of action, a series of genes known to affect biofilm and motility were overexpressed and the effect on biofilm/motility was assessed. Furthermore, the relative expression of genes involved in motility and biofilm formation was measured by qRT-PCR in presence and absence of phytochemicals, to examine the repression caused by test compounds.

Results

The β-sitosterol glucoside (SG) was identified as the most potent inhibitor of EHEC biofilm formation and motility without affecting the cell viability. Furthermore, SG appears to inhibit the biofilm and motility through rssAB and hns mediated repression of flagellar master operon flhDC.

Conclusion

SG may serve as novel lead compound for further development of anti-virulence drugs.

General significance

Plant sterols constitute significant part of diet and impart various health benefits. Here we present the first evidence that SG, a plant sterol has significant effect on EHEC motility, a critical virulence factor, and may have potential application as antivirulence strategy.

Introduction

Emergence of antibiotic resistance has produced substantial medical challenges and a sense of urgency to identify and develop novel antimicrobials [1]. In order to overcome the virulence of antibiotic resistant strains, novel targets are constantly being searched and identified. Efforts in laboratories across the globe have identified several processes and microbial structures such as type three secretion system, quorum sensing, host–pathogen interaction, efflux systems and bacterial membrane functions as significant targets for development of novel antimicrobials [1].

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a Gram-negative foodborne pathogen that causes bloody diarrhea. EHEC colonizes the large intestine and produces attaching and effacing (AE) lesions and Shiga toxin. EHEC and other Shiga toxin producing E. coli strains harbor the gene encoding the toxin on a lysogenic phage. Antibiotic treatment often results in phage induction and hyper-production of toxin, leading to fatal hemorrhagic colitis and hemolytic uremic syndrome [2]. Therefore, the treatment options for EHEC infections are limited to intravenous fluid supplementation. Although several strategies such as antibodies against toxin, toxin binders, and Stx trafficking blockers are being tested at different levels [3], none of these have entered the clinical trials. Another potential prophylactic or therapeutic approach could be disruption or prevention of bacterial attachment to host surface.

Bacterial attachment to the host surface is mediated by a multitude of factors. Two key structures, namely flagella and locus of enterocyte effacement (LEE) help in colonization and attachment of EHEC to the epithelial cell surface [4], [5]. Once ingested, EHEC traverses to large intestine by the guided flagellar motility [4] and attaches to the epithelial cell layer with the help of flagella, LEE and other surface structures such as type 1 pili and hemorrhagic coli pilus [6], [7], [8]. In the colon, a two component system involving Quorum Sensing E. coli Regulators (QseBC) regulates the expression of flagellar transcriptional regulators flhDC in response to the autoinducers (AI-2/AI-3) and endogenous hormones epinephrine (EPI), norepinephrine (NE) [5], [9]. QseB, the response regulator directly binds to and induces expression of flagellar master regulators flhD and flhC [8].

Biofilm communities show better tolerance to antimicrobials and are a source of chronic infections [12]. Therefore, biofilm formation and/or attachment of EHEC to biotic/abiotic surfaces are considered potential targets for development of antimicrobials. Flagella are important regulators of biofilm formation in E. coli under natural settings and help in initial attachment and development of the complex community [10], [11]. The importance of flagella in EHEC pathogenesis as well as in biofilm formation makes it an attractive target for development of alternative intervention strategies. One potential strategy of intervention is identification of small molecules that target and inhibit biofilm formation and attachment.

Natural products are the end products of secondary metabolism in plants and microorganisms. They demonstrate diverse biological activities including antimicrobial properties [13]. By virtue of their diversity, natural products provide an excellent source for identification of novel molecules targeting bacterial virulence and pathogenesis. Numerous plant secondary metabolites with wide-ranging activities have been identified [13]. In our earlier studies, we identified several citrus limonoids and flavonoids that interfere with quorum sensing and virulence gene expression [14], [15], [16], [17]. Additionally, the antimicrobial activity of citrus peel oils and extracts has been extensively reported [18], [19]. However, the mechanism by which the essential oils exert their antimicrobial action is not completely understood [18]. Citrus peel is very complex matrix and contains many different classes of bioactive compounds including flavonoids, limonoids, carotenoids, coumarins, sterols and phenolic compounds [20]. We and others have reported diverse activities including bactericidal, bacteriostatic and anti-quorum sensing activity for some of the compounds present in citrus peel [14], [15], [16], [21]. The present report describes the anti-virulence activities of coumarins, polymethoxyflavones and a phytosterol isolated from citrus on the biofilm formation and motility of the devastating pathogen EHEC.

Section snippets

Isolation of bioactive compounds

Bergamottin [22], β-sitosterol glucoside (SG) [23], nobiletin, sinensetin [24], heptamethoxyflavone [25], and imperatorin [26] were purified in our lab. A 5 mM stock solution in DMSO was prepared and used for the studies. Briefly, clementine peel was dried, powdered and extracted with hexane using a Soxhlet type apparatus for 8 h. Vacuum concentrated hexane extract was separated over a silica gel (particle size 35–60 μm) flash column on Teledyne Isco CombiFlash Rf 4 × system (Lincoln, NE).

Identification of bioactive compounds

The isolated six compounds were identified by LC-MS with high resolution accurate mass spectral data. Fig. 1 shows the ESI–TOF mass spectra of the purified compounds from citrus. The compounds were identified by their high resolution accurate TOF–MS/MS data as heptamethoxy flavone 433.1508 [M + H]+, nobiletin 403.1386 [M + H]+, sinensitin 373.3735 [M + H]+, β-sitosterol glucoside 599.4257 [M + Na]+, bergamottin 339.3931 [M + H]+, and imperatorin 271.0991 [M + H]+.

Effect of citrus bioactives on EHEC growth and biofilm formation

The EHEC biofilms were grown in rich and

Discussion

The present study investigated the effect of secondary metabolites present in citrus on EHEC biofilm and motility. The flavedo is the outermost part of citrus fruit and provide defense against biotic and abiotic stress [36]. To aid in its function, the flavedo contains numerous structural adaptations and protective chemical constituents. The bioactive compounds present in flavedo or produced in oil glands, such as β-sitosterol, polymethoxyflavones and coumarins, are speculated to aid in defense

Acknowledgements

We would like to thank Dr. V. Sperandio (University of Texas Southwestern Medical Center, Dallas, TX) for generously providing AI-3 reporter strains harboring chromosomal LEE1:lacZ (TEVS232), LEE2:lacZ (TEVS21) and EHEC mutants VS145, VS151, VS138, VS179. This project is based upon the work supported by the USDA-NIFA No. 2010-34402-20875, “Designing Foods for Health” through the Vegetable & Fruit Improvement Center.

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