IAI Accepts, published online ahead of print on 18 February 2014 Infect. Immun. doi:10.1128/IAI.01431-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Title

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The inhibitory effect of probiotic Escherichia coli Nissle 1917 on aEPEC infection: Role of

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F1C fimbriae, flagellae and secreted bacterial components

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Running title

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Effects of E. coli Nissle 1917 on aEPEC infection

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Authors

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Sylvia Kleta,a Marcel Nordhoff,a Karsten Tedin,a Lothar H. Wieler,a Rafal Kolendab,c, Sibylle

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Oswald,d Tobias A. Oelschlaeger,d Wilfried Bleiß,e Peter Schierack a,c

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Affiliations

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a

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Berlin, Germany

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b

Department of Biochemistry, Wrocáaw University of Environmental and Life Sciences

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c

Fakultät für Naturwissenschaften, Brandenburgische Technische Universität Cottbus-

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Senftenberg, Germany

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d

Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg, Germany

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e

Lehrstuhl für Molekulare Parasitologie, Humboldt-Universität zu Berlin, Germany

Institut für Mikrobiologie und Tierseuchen, Fachbereich Veterinärmedizin, Freie Universität

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Corresponding author

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Peter Schierack, [email protected]

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Abstract

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Enteropathogenic Escherichia coli (EPEC) are recognized as important intestinal pathogens

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that frequently cause acute and persistent diarrhea in humans and animals. The use of

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probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli

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strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastro-intestinal

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disorders. While both in vitro and in vivo studies have described strong inhibitory effects of

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EcN on enteropathogenic bacteria including pathogenic E. coli, the underlying molecular

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mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN

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on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli

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(aEPEC) with respect to single infection steps including adhesion, microcolony formation and

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attaching and effacing phenotype. We show that EcN drastically reduced the infection

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efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not

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the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN

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adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1

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flagellae, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere

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with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These

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components do not appear to be specific for EcN but we propose that the strong adhesion

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capacities enable EcN to secrete sufficient local concentrations of these inhibitory factor. The

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results of this study are consistent with a mode of action whereby EcN inhibits secretion of

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virulence associated proteins of EPEC, but not their expression.

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Introduction

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Enteropathogenic E. coli (EPEC) frequently cause acute and persistent diarrhea in animals

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and humans. EPEC leads to serious acute diarrhea in weaned pigs (1, 2). In humans, EPEC

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infections are particularly serious for infants and toddlers and are often accompanied by high

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mortality rates in developing countries (3, 4). However, it is now recognized that atypical

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EPECs (aEPECs) are more frequent in humans than typical EPECs in both developing and

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developed countries (5, 6). In animals, aEPECs are much more prevalent than EPECs and

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aEPEC strains isolated from cattle, sheep and pigs belong to the same serotypes also found in

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humans (7, 8). Furthermore, recent studies showed a close clonal relationship between human

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and animal aEPEC isolates, suggesting a possible zoonotic potential of animal aEPEC strains

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which could serve as a reservoir for human infections (9).

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Infection of intestinal epithelial cells by EPEC is a complex multistage process.

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Initially, EPEC loosely adhere to epithelial cells by diverse adhesins and subsequently

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translocate effector molecules, including the translocated intimin receptor (Tir), into host cells

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using a type 3 secretion system (T3SS). After integration of Tir into the host cell membrane,

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EPEC binds tightly through the adhesin intimin with Tir. EPEC is able to intimately adhere to

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epithelial cells and to form microcolonies with resulting typically associated histopathological

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alterations of the host cell surface known as attaching and effacing (AE) lesions.

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Rearrangement and massive accumulation of actin and other cytoskeletal proteins beneath the

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site of bacterial attachment lead to the formation of pedestal structures and destruction of

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microvilli (effacement). The pathogenesis is further characterized by loss of tight-junction

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integrity and barrier functions of the gut epithelium, destruction of microvilli (effacement)

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and the brush border that leads to diarrhea (4, 10-13).

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The non-pathogenic E. coli strain Nissle 1917 (EcN) is a widely employed probiotic

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strain and several in vivo studies have demonstrated its promising probiotic activity in humans

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and animals including the treatment of acute, chronic or frequent recurring diarrhea and 3

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inflammatory bowel disease (14-19). Proposed probiotic actions of EcN include effects on

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pathogens, host epithelial cells, host smooth muscle cell activity and the host immune system

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(20-28). In vitro, EcN has been shown to inhibit invasion of host cells by several enteric

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pathogens, including Salmonella, Yersinia, Shigella, Legionella, Listeria and adherent-

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invasive E. coli (29, 30). However, the underlying molecular mechanisms remain largely

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unknown. Here we characterize the effects of EcN on aEPEC infection to IPEC-J2 cells by

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means of confocal laser scanning microscopy and scanning electron microscopy as well as

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molecular and protein biochemical methods. Our data provide new insights into host-bacteria

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and inter-bacterial interactions, and show that EcN might be a promising tool in prophylactic

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defence against EPEC infections.

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Materials and Methods

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Cell line and bacterial strains

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The porcine intestinal epithelial cell line IPEC-J2 (31) was grown to confluence in Dulbecco’s

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Modified Eagle Medium (DMEM)/Ham’s F-12 (1:1) (Biochrom, Berlin, Germany)

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supplemented with 5% fetal calf serum and maintained in an atmosphere of 5% CO2 at 37°C.

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Bacterial strains used in this study are listed in Table 1. E. coli Nissle 1917 (EcN) was kindly

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provided by G. Breves (Hannover, Germany). The EcN mutant EcN ΔfliA (this study) was

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generated using the method of Datsenko and Wanner (32). The fliA gene was replaced by a

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kanamycin-resistance (kan) antibiotic cassette generated using the plasmid pKD4 as template

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and

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AAGGTGTAATGGATAAACAGTGTAGGCTGGAGCTGCTTC-3´) and fliAH2P2 (5´-

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ACTTACCCAGTTTAGTGCGTAACCGTTTAATGCCTGGCTGTGCATATGAATATCC-

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TCCTTAG-3´). EcN ΔfliA was complemented using the plasmid pACYC177 harboring the

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sequence encoding fliA (strain EcN ΔfliA + fliA). E. coli MG1655 was a laboratory strain

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originally obtained from C. A. Cross (San Francisco, California, USA). E. coli IMT13962

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was from the collection of the Institute of Microbiology and Epizootics (Berlin, Germany)

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and was originally isolated from the colon of a clinical healthy piglet and chosen for to its

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strong adherence to IPEC-J2 cells. Strain IMT13962(pCosF1C6) was generated from strain

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IMT13962 by complementation with the foc operon cloned into the pSuperCos1 vector

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(Stratagene, Heidelberg, Germany). Strain P2005/03 (kindly provided by R. Bauerfeind,

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Gießen, Germany) was isolated from a piglet with diarrhea and classified as atypical

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enteropathogenic E. coli (aEPEC). Human EPEC E2348/69 was kindly provided by J. B.

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Kaper (Baltimore, MD, USA). E. coli strain H5316 is a microcin-sensitive indicator strain

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kindly provided by K. Hantke (Tübingen, Germany). Uropathogenic E. coli (UPEC) strain

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RZ525 was kindly provided by U. Dobrindt (Würzburg, Germany). Unless otherwise

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indicated, bacterial strains were grown in LB broth at 37°C with agitation at 200 rpm. For

primer

pair

fliAH1P1

(5´-GTGAATTCACTCTATACCGCTG-

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cultivation of strains P2005/03 and E2348/69, LB broth was supplemented with 20 μg/ml

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tetracycline and 30 μg/ml nalidixic acid, respectively.

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Preparation of bacterial supernatants

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Bacterial strains were grown in LB broth at 37°C with agitation at 200 rpm to an optical

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density at 600 nm (OD600) of 1.0, diluted 1:100 in DMEM/Ham’s F12 cell culture medium

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containing 5% fetal calf serum and grown again to OD600=1.0. Bacterial cultures were

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centrifuged at 8000×g at 4°C for 15 min. Supernatants were sterile filtered with 0.22 μm

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filters and kept until use at -20°C.

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Infection assay

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aEPEC P2005/03 and EPEC E2348/69 were grown to OD600=1.0, washed by centrifugation,

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re-suspended in cell culture media and adjusted by dilution to provide a multiplicity of

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infection (MOI) of 100:1 bacteria to host cells in wells of 12- or 24-well cell culture plates.

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Confluent monolayers of IPEC-J2 cells were infected with P2005/03 or E2348/69 and

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incubated at 37°C. After 3 h, non-adherent bacteria were removed by three washes with PBS.

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For P2005/03, incubation was continued for an additional 3 h. Infection efficiencies to

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epithelial cells were determined by washing and lysing cells with 0.1% Triton X-100 in

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ddH2O and plating serial dilutions to LB agar plates containing 5 μg/ml tetracycline or 30

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μg/ml nalidixic acid which allowed the selective growth of P2005/03 and E2348/69,

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respectively. Plates were incubated over night at 37°C and the resulting colony forming units

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(CFU) were determined. For pre-incubation experiments, epithelial cells were first incubated

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with the strains indicated for 2 h and washed three times with PBS prior to EPEC infections.

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For co- and post-incubation experiments, strains were added simultaneously or 1 h after

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aEPEC infection. For growth kinetics of adherent aEPEC on epithelial cells, cell culture

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medium was changed every 30 min 2 h after the beginning of aEPEC infection to remove

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non-adherent bacteria and to exchange exhausted cell culture medium.

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Adhesion assay

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Bacterial strains were prepared and added to IPEC-J2 cell cultures as described for infection

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assays. At the indicated time points, non-adherent bacteria were removed by washing cells

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three times with PBS. Adherent bacteria were determined by lysing cells with 0.1 Triton X-

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100 and plating serial dilutions to LB agar plates.

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Fluorescence microscopy

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For fluorescence microscopy, epithelial cells were grown to confluence on glass coverslips

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and fixed with acetone for 2 min at -20°C (confocal laser scanning microscopy) or 4%

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paraformaldehyde for 30 min at 4°C (epi-fluorescence microscopy), after performing

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infection or adhesion experiments. All incubation steps during staining with fluorescent dyes

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were performed in the dark. Strain P2005/03 was detected by immuno-histochemical staining.

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Samples were incubated with polyclonal antibodies against serotype O108 raised in rabbit

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(Federal Institute for Risk Assessment, Berlin, Germany; diluted 1:50 in PBS with 0.5%

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BSA) for 30 min at room temperature followed by incubation with goat anti-rabbit TRITC-

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labeled secondary monoclonal antibodies (Sigma-Aldrich, Munich, Germany; diluted 1:200 in

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PBS with 0.5% BSA) for 30 min at room temperature. Samples were washed three times with

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PBS after each antibody labeling.

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The ability to produce attaching and effacing lesions (AE lesions) was indirectly

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examined by the fluorescent actin staining (FAS) according to Knutton et al. (33). F-Actin

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was stained with FITC phalloidin (5 μg/ml; Invitrogen, Karlsruhe, Germany) for 30 min at

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room temperature. Samples were washed three times with PBS. Cell nuclei and bacteria in

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general were visualized by staining DNA with 0.3 μg/ml propidium iodide (Invitrogen, 7

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Karlsruhe, Germany) for 3 min at room temperature. Before staining, PFA-fixed cells were

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first incubated with 1 ml ice-cold 0.1% Triton X-100 in PBS for 4 min on ice and washed

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three times with PBS in order to permeabilise cells. Images were acquired with the confocal

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laser scanning microscope DMIRE 2 TCS SP2 or the epi-fluorescence microscope DMBL

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(both Leica, Wetzlar, Germany).

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EcN flagellae were stained with polyclonal antibodies against flagellae H1 kindly

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provided by L. Beutin (National Reference Laboratory for E. coli, Federal Institute for Risk

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Assessment, Berlin, Germany). Antibodies were diluted 1:50 in PBS with 0.5% BSA and

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applied for 30 min at room temperature followed by incubation with FITC-labeled secondary

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monoclonal antibodies against rabbit (Sigma-Aldrich, Munich, Germany) for 30 min at room

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temperature. Samples were washed three times with PBS after each antibody labeling.

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Scanning electron microscopy

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Infected cells grown on glass coverslips were fixed with 2% glutaraldehyde and 0.05%

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calcium chloride in 0.1 M sodium cacodylate buffer, pH 7.4 for 24 h at 4°C. Afterwards, the

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samples were rinsed three times with ice-cold 0.1 M sodium cacodylate buffer, pH 7.4, fixed a

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second time with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 3 h at 4°C and

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subsequently rinsed again. For raster preparation, samples were dehydrated in a graduated

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series of ethanol solutions and finally 100% acetone, critical point dried with liquid carbon

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dioxide using the point dryer CPD 030 (BAL-TEC, Witten, Germany) and sputtered with 20

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nm gold particles using the sputter coater SCD 005 (BAL-TEC, Witten, Germany). Samples

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were examined with a LEO 1430 scanning electron microscope (LEO Elektronenmikroskopie,

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Oberkochen, Germany).

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Microcin test

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The expression as well as the sensitivity of E. coli strains against microcins were tested as

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described by Kleta et al. (34).

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Isolation of secreted and intra-cellular EPEC proteins

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The human EPEC strain E2348/69 was grown in 6 ml DMEM/Ham’s F12 cell culture

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medium to an OD600 of approximately 1.0. Bacteria were centrifuged for 5 min at 8000×g at

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room temperature. The pellet was re-suspended in 6 ml DMEM/Ham’s F12 cell culture

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medium. The bacterial suspension was then diluted 1:100 in 50 ml DMEM/Ham’s F12 in a

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250 ml Erlenmeyer flask, and grown again to an OD600 of 1.0 with shaking at 200 rpm at

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37°C. 45 ml of the bacterial culture was centrifuged (15 min, 8000×g, 4°C) and the resulting

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supernatants were sterile-filtered using 0.22 μm filters. Secreted proteins of supernatants were

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precipitated with trichloroacetic acid (TCA, final concentration 10%) overnight at 4°C. The

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next day, the TCA precipitates were centrifuged for 30 min. at 10000×g, 4°C. The resulting

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protein pellet was dried for 5 min at room temperature, resuspended in 1 ml 0.2% SDS in 25

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mM Tris-HCl, pH 8.0, mixed with ice-cold acetone (1:4) and incubated for 1 h at -20°C. After

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centrifugation (30 min, 10000×g, 4°C) the protein pellet was dried (5 min, room temperature)

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and diluted in 25 mM Tris-HCl (pH 8.0) and 4 M urea. Secreted proteins (1000-fold

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concentrated) were stored at -20°C.

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In order to extract proteins from EPEC bacteria, 1 ml of the 50 ml bacterial suspension

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of the 250 ml Erlenmeyer flask was centrifuged for 5 min at 8000×g at room temperature. The

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bacterial pellet was re-suspended in 4× sample buffer (1 M Tris-HCl, pH 6.8, 4% SDS, 8% ß-

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mercaptoethanol, 20% glycerin, 0.025% bromophenol blue). Proteins were denatured by

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heating at 99°C for 5 min. Tubes were incubated on ice for 5 min, centrifuged for 5 sec and

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kept at -20°C until further use.

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To determine the effects of EcN and MG1655 on protein secretion of EPEC, E2348/69

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was grown in culture supernatants of EcN and MG1655, respectively. Bacterial supernatants

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were obtained from DMEM/Ham’s medium as described but without added FCS. For dilution

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of bacterial supernatants, DMEM/Ham’s cell culture medium was used.

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Western blot analysis

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Proteins were separated by SDS PAGE according to Laemmli (35). For non-specific protein

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detection, SDS PAGE gels were stained with the Silver Stain Plus Kit (Bio-Rad, München,

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Germany) according to the manufacturer’s instructions. Proteins EspA, EspB and Tir were

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detected using Western blot analysis according to Towbin et al. (36). Briefly, proteins were

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transferred to nitrocellulose membrane (Sartorius, Göttingen, Germany) using a tank blot

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apparatus (Bio-Rad Laboratories, Munich, Germany). The membrane was blocked in 5%

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skimmed milk powder in TBS buffer for 1 h. Primary antibodies against EspA, EspB and Tir

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were kindly provided by J. B. Kaper (University of Maryland, Baltimore, USA). Membranes

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were incubated with primary antibodies diluted 1:5000 in blocking buffer overnight at 4°C

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with shaking, washed three times with TBS for 10 min and incubated with 1:2000 diluted

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secondary antibodies labeled with horseradish peroxidase (Sigma-Aldrich, Munich, Germany)

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in blocking buffer for 1 h, and washed as described. Proteins were visualized by enhanced

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chemiluminescence.

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Statistical methods

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Statistical calculations were performed using SPSS 11.5 (SPSS, Chicago, USA).

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Significances (p-values) were calculated using the student’s t test for normally distributed

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data or Mann-Whitney U test for non-normal distributed data. A p-value of less than 0.05 was

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considered to indicate a statistically significant difference.

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Results

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Effects of EcN on aEPEC adhesion, microcolony formation and formation of attaching

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and effacing lesions

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The probiotic strain E. coli Nissle 1917 (EcN) and two non-pathogenic E. coli control strains

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(IMT13962 and MG1655) were tested for their ability to inhibit infection of IPEC-J2 cells

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with the atypical enteropathogenic E. coli (aEPEC) strain P2005/03. In mono-infection

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experiments, the infection efficiencies of aEPEC strain P2005/03 varied from approximately

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2.2x104 to 1.7x105 bacteria per well or 0.1 to 0.65 bacteria per epithelial cell, respectively.

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Pre-incubation of EcN resulted in a reduction of aEPEC infection by 83% (p