E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells

BACKGROUND: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. CONCLUSIONS: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion

Molecular investigations of the probiotic Escherichia coli strain DSM 6601 and development of the indigenous plasmids as cloning vectors

Der apathogene E. coli Stamm DSM 6601 (E. coli Nissle 1917) kann als Modellorganismus für die Verwendung eines kommensalen Gram-negativen Bakterienstammes als Probiotikum angesehen werden. Dieser E. coli Stamm wurde intensiv erforscht und seine Eigenschaften sind daher gut charakterisiert. Der probiotische Charakter dieses Bakterienstammes ist auf gute Kolonisierungseigenschaften des menschlichen Darms, immunmodulatorische Effekte und antagonistische Wirkungen zurückzuführen. Der E. coli Stamm DSM 6601 wird seit einigen Jahrzehnten zur Behandlung verschiedener gastrointestinaler Erkrankungen eingesetzt und seine therapeutische Wirksamkeit ist wissenschaftlich bewiesen. Daher eignet sich dieser Stamm als Modellstamm für die Entwicklung eines bakteriellen Lebendvektors, der für mukosale Immunisierungen oder die zielgerichtete Lieferung von therapeutischen Molekülen in den Darm eingesetzt werden könnte. Ein Ziel dieser Arbeit war die Charakterisierung der kryptischen Plasmide pMUT1 und pMUT2 des probiotischen E. coli Stammes DSM 6601 durch Analyse der DNA-Sequenz. Die Analyse ergab, dass das Plasmid pMUT1 ein Replikationssystem vom ColE1-Typ, ein Mobilisierungssystem sowie eine Stabilitätsregion enthält, während das Plasmid pMUT2 ein ColE2-ähnliches Replikationssystem und ein anderes Mobilisierungssystem besitzt. In beiden Plasmiden konnten keine weiteren offenen Leserahmen mit bekannter Funktion identifiziert werden. Des Weiteren wurde ein spezifisches PCR-Nachweissystem für den E. coli Stamm DSM 6601 etabliert, das auf einer Methode zur direkten DNA-Isolierung aus Stuhlproben und einem optimierten PCR-Protokoll für auf den kryptischen Plasmiden basierende Primerkombinationen beruht. Dadurch konnte eine Sensitivität von 10(3)-10(4) Bakterien/0,1 g Stuhl erreicht werden, die vergleichbar mit den Nachweisgrenzen anderer beschriebener PCR-Nachweissysteme ist. Durch Analysen von Patientenstuhlproben wurde die Spezifität und der diagnostische Nutzen dieses PCR-Nachweissystems bestätigt. Darüber hinaus wurde eine plasmidfreie Variante des E. coli Stammes DSM 6601 hergestellt. Durch funktionelle Untersuchungen dieses Stammes konnten keine Unterschiede im Vergleich zu dem Wildtyp festgestellt werden, wodurch eine mögliche Funktion der beiden kryptischen Plasmide weiterhin unklar bleibt. Diese plasmidfreie Variante kann als Lebendvektor für rekombinante Plasmide auf Basis der Plasmide pMUT1 und pMUT2 verwendet werden. Ein weiteres Ziel dieser Arbeit war die Entwicklung von stabilen Klonierungsvektoren für den probiotischen E. coli Stamm DSM 6601. Durch Integration von Antibiotika-Resistenzkassetten in die Plasmide pMUT1 und pMUT2 wurden Klonierungsvektoren konstruiert, die auch nach Insertion weiterer DNA-Fragmente ohne Antibiotika-Selektionsdruck stabil in diesem Stamm beibehalten werden. Zusätzlich wurde durch die stabile Expression von fluoreszierenden Proteinen ein visuelles Nachweissystem etabliert, das bei in vivo Experimenten verwendet werden kann. Dadurch wird die Möglichkeit geboten, Erkenntnisse über Kolonisierungseigenschaften sowie Interaktionen des E. coli Stammes DSM 6601 mit endogenen Mikroorganismen und Zellen des Darmimmunsystems zu erlangen, was zur Aufklärung der Wirkungsweise dieses Stammes beitragen könnte. Im Hinblick auf die Entwicklung eines Lebendvakzins auf der Basis des probiotischen E. coli Stammes DSM 6601 wurden Adhäsine von humanpathogenen enterohämorrhagischen E. coli und von tierpathogenen enterotoxischen E. coli in diesem Stamm exprimiert. Bei ersten Immunisierungsversuchen in Mäusen konnte jedoch keine Induktion einer spezifischen Immunantwort gegen diese Adhäsine nachgewiesen werden. Weiterhin wurde die inhibitorische Wirkung des E. coli Stammes DSM 6601 auf die Invasivität von Salmonellen in vitro und in vivo untersucht. Es konnte gezeigt werden, dass Typ 1- und F1C-Fimbrien keine Rolle bei dem inhibitorischen Effekt in vitro spielen und dass durch diesen E. coli Stamm in konventionellen Mäusen keine inhibitorischen Wirkungen nachzuweisen sind. Die Ergebnisse dieser Arbeit bilden durch die Entwicklung von stabilen Klonierungsvektoren und die Etablierung von Nachweissystemen für den probiotischen E. coli Stamm DSM 6601 die Grundlage für den Einsatz dieses Stammes als Lebendvektor und für in vivo Untersuchungen, die zur Aufklärung der Wirkungsmechanismen dieses Stammes beitragen könnten.The nonpathogenic E. coli strain DSM 6601 (E. coli Nissle 1917) can be considered as model organism for the employment of a commensal Gram-negative bacterial strain as a probiotic. This E. coli strain has been intensively investigated and therefore its properties are well characterized. The probiotic character of this strain is due to excellent colonization properties of the human gut, immunomodulatory effects and antagonistic activities. The E. coli strain DSM 6601 has been used for decades for the treatment of various gastrointestinal diseases and its therapeutic efficacy is scientifically proved. Therefore, this strain is suited as a model strain for the development of a bacterial live vector, which might be used for mucosal immunization or localized delivery of therapeutic molecules into the intestine. One major aim of this work was the characterization of the cryptic plasmids pMUT1 and pMUT2 of probiotic E. coli strain DSM 6601 by DNA sequence analysis. The analysis showed that plasmid pMUT1 carries a replication system of ColE1-type, a mobilization system as well as a stabilization region, whereas plasmid pMUT2 contains a ColE2-like replication system and another mobilization system. Further open reading frames with known function were not identified in both plasmids. Furthermore, a specific PCR detection system for E. coli strain DSM 6601 was established, which uses a method for direct isolation of DNA from faecal samples and an optimized PCR protocol for primer combinations based on the cryptic plasmids. Thereby, a sensitivity of 10(3)-10(4) bacteria/0,1 g faeces was achieved that is comparable with detection limits of other described PCR detection systems. The specifity and diagnostic utility of this PCR detection system was confirmed by analysis of faecal samples from patients. In addition, a plasmid-free variant of E. coli strain DSM 6601 was constructed. Functional analyses of this strain detected no differences compared to the wildtype, whereby the possible function of both cryptic plasmids still remains unclear. This plasmid-free variant can be used as live vector for recombinant plasmids based on the plasmids pMUT1 and pMUT2. Another aim of this work was the development of stable cloning vectors for the probiotic E. coli strain DSM 6601. Cloning vectors were constructed by integration of antibiotic resistance cassettes in the plasmids pMUT1 and pMUT2, which are still stably maintained following insertion of additional DNA fragments without selection pressure by antibiotics. Furthermore, a visual detection system was established by the stable expression of fluorescent proteins that can be used in in vivo experiments. This offers the opportunity to gain knowledge of colonization properties as well as interactions of E. coli strain DSM 6601 with endogenous microorganisms and cells of the gut’s immune system, which might contribute to explain the mode of action of this strain. With regard to the development of a live vaccine based on the probiotic E. coli strain DSM 6601, adhesins of human pathogenic enterohaemorrhagic E. coli and animal pathogenic enterotoxigenic E. coli were expressed in this strain. Induction of specific immune responses to these adhesins were not demonstrated by first immunization experiments in mice. Moreover, the inhibitory effect of the E. coli strain DSM 6601 on Salmonella invasion was investigated in vivo and in vitro. It was demonstrated that type 1 and F1C fimbriae have no influence on the inhibitory effect in vitro and that no inhibitory effects could be established in conventional mice by this E. coli strain. By the development of stable cloning vectors and the establishment of detection systems for the E. coli strain DSM 6601, the results of this work provide the basis for the employment of this strain as live vector and for in vivo investigations, which might contribute to explain this strain’s mode of action

<i>Salmonella</i> invasion gene regulation by <i>E. coli</i> supernatants.

<p><i>E. coli</i> were cultivated in cell culture medium (DMEM HAM'S/F-12) until an OD_600nm = 1. Supernatants were collected by centrifugation with subsequent sterile filtration. Subsequently, SL1344 fusion strains (SL1344 <i>hilC</i>-<i>lacZ</i>, SL1344 <i>hilD</i>-<i>lacZ</i>, SL1344 <i>hilA</i>-<i>lacZ</i>, SL1344 <i>icgA</i>(<i>siiE</i>)-<i>lacZ</i>) were cultivated in supernatants of EcN, <i>E. coli</i> 140815 or <i>E. coli</i> MG1655. B-Galactosidase activity was measured as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014712#pone.0014712-Thompson1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014712#pone.0014712-Hernandez1" target="_blank">[26]</a>. The results shown are representative of at least two independent experiments. White bar: <i>Salmonella</i> grown in EcN supernatant, gray bar: <i>Salmonella</i> grown in <i>E. coli</i> 140815 supernatant, black bar: <i>Salmonella</i> grown in <i>E. coli</i> MG1655 supernatant, patterned bar: <i>Salmonella</i> grown in pure cell culture medium.</p

Invasion efficiency of <i>Salmonella Typhimurium</i> into IPEC-J2 cells after incubation with <i>E. coli</i> culture supernatants.

<p>Confluent monolayers of IPEC-J2 cells were pre-incubated (SN before) and/or co-incubated (SN simultaneously) with <i>E. coli</i> supernatants (SN). Cells were infected with <i>Salmonella Typhimurium</i> using an MOI of 100∶1 <i>Salmonella</i> to host cells. Invasion levels in percent (%) are expressed as invasion of <i>Salmonella</i> relative to invasion without pre- and/or co-incubation with <i>E. coli</i> SN. The data are the mean ± S.E.M. of at least three separate experiments in duplicate wells. * = p<0.05 compared to <i>Salmonella</i> infection without influence of <i>E. coli</i> SN. EcN: <i>E. coli</i> Nissle 1917.</p

Invasion efficiency of <i>Salmonella Typhimurium</i> into IPEC-J2 cells after pre-incubation with <i>E. coli</i> mixed cultures.

<p>Confluent monolayers of IPEC-J2 cells were pre-incubated with <i>E. coli</i> Nissle 1917 (EcN) monocultures or mixed cultures using an MOI of 100∶1 or 10∶1 <i>E. coli</i> to host cells. After two hours, cells were washed and infected with <i>Salmonella Typhimurium</i> using an MOI of 100∶1 <i>Salmonella</i> to host cells. Invasion levels in percent (%) are expressed as invasion of <i>Salmonella</i> relative to invasion without pre-incubation with <i>E. coli</i> (<i>Salmonella</i> mono-infection). The data are the mean ± S.E.M. of at least three separate experiments in duplicate wells. * = p<0.01 compared to <i>Salmonella</i> mono-infection.</p

Inhibitory effects of <i>focA</i>-positive and <i>focA</i>-negative <i>E. coli</i> isolates on <i>Salmonella Typhimurium</i> invasion.

<p>Confluent monolayers of IPEC-J2 cells were pre-incubated with <i>E. coli</i> Nissle 1917 (EcN, <i>focA</i>-positive strain), <i>E. coli</i> WS15C1 (<i>focA</i>-positive strain), <i>E. coli</i> WS30C1 (<i>focA</i>-negative strain) and <i>E. coli</i> WS46C1 (<i>focA</i>-negative strain) using an MOI of 100∶1 <i>E. coli</i> to host cells. After two hours, cells were washed and adhesion efficiencies of <i>E. coli</i> isolates were determined (left side); alternatively, cells were washed after two hours and infected with <i>Salmonella Typhimurium</i> using an MOI of 100∶1 <i>Salmonella</i> to host cells (right side). Adhesion levels in percent (%) were expressed relative to adhesion of EcN. Invasion levels in percent (%) were expressed relative to <i>Salmonella</i> invasion without pre-incubation with bacteria (<i>Salmonella</i> mono-infection). The data are the mean ± S.E.M. of at least three separate experiments in duplicate wells. * = p<0.01 compared to EcN adhesion (left side) or <i>Salmonella</i> mono-infection (right side).</p

Intracellular growth of <i>Salmonella Typhimurium</i> in IPEC-J2 cells after post-incubation with <i>E. coli</i> Nissle 1917.

<p>Confluent monolayers of IPEC-J2 cells were infected with <i>Salmonella Typhimurium</i> for one hour using an MOI of 1∶1 <i>Salmonella</i> to host cells. After one additional hour of incubation in media containing gentamicin, IPEC-J2 cells were incubated with <i>E. coli</i> Nissle 1917 (EcN), <i>E. coli</i> 140815 or <i>E. coli</i> MG1655 for two hours using an MOI of 100∶1 <i>E. coli</i> to host cells, followed by incubation in media with gentamicin. Intracellular <i>Salmonella</i> numbers are presented per well of a 24-well plate. The data are the mean ± S.E.M. of at least three separate experiments in duplicate wells.</p

Invasion efficiency of <i>Salmonella Typhimurium</i> into IPEC-J2 cells after pre-incubation with <i>E. coli</i> Nissle 1917.

<p>Confluent monolayers of IPEC-J2 cells were pre-incubated with <i>E. coli</i> Nissle 1917 (EcN), <i>E. coli</i> 140815 or <i>E. coli</i> MG1655 at an MOI of 100∶1 or 10∶1 bacteria to host cells. After two hours, cells were washed and infected with <i>Salmonella Typhimurium</i> using an MOI of 100∶1 <i>Salmonella</i> to host cells. Invasion levels in percent (%) are expressed as invasion of <i>Salmonella</i> relative to invasion without pre-incubation with <i>E. coli</i> (<i>Salmonella</i> mono-infection). The data are the mean ± S.E.M. of at least three separate experiments in duplicate wells. * = p<0.01 compared to <i>Salmonella</i> mono-infection.</p

Inhibitory effects of <i>E. coli</i> Nissle 1917 on <i>Salmonella Typhimurium</i> invasion is dependent on adhesion.

<p>Confluent monolayers of IPEC-J2 cells were pre-incubated with <i>E. coli</i> Nissle 1917 (EcN), EcN Δ<i>focA</i>, EcN Δ<i>fim</i> or EcN Δ<i>fliA</i> using an MOI of 100∶1 <i>E. coli</i> to host cells. After two or six hours, cells were washed and infected with <i>Salmonella Typhimurium</i> using an MOI of 100∶1 <i>Salmonella</i> to host cells. Invasion levels in percent (%) are expressed as invasion of <i>Salmonella</i> relative to invasion without pre-incubation with <i>E. coli</i> (<i>Salmonella</i> mono-infection). The data are the mean ± S.E.M. of at least three separate experiments in duplicate wells. * = p<0.01 compared to <i>Salmonella</i> mono-infection. A) Effects of EcN Δ<i>focA</i> and EcN Δ<i>fim</i> mutants on <i>Salmonella</i> invasion after a 2 or 6 hours pre-incubation period. B) Effects of EcN Δ<i>focA</i> and EcN Δ<i>fliA</i> mutants and their respective strains complemented with the plasmid pACYC 177 containing the relevant gene on <i>Salmonella</i> invasion after 2 hours pre-incubation.</p

Adhesion efficiency of <i>Salmonella Typhimurium</i> to IPEC-J2 cells after pre-incubation with <i>E. coli</i>.

<p>Confluent monolayers of IPEC-J2 cells were pre-incubated with <i>E. coli</i> Nissle 1917 (EcN), <i>E. coli</i> 140815 or <i>E. coli</i> MG1655 using an MOI of 100∶1 <i>E. coli</i> to host cells. After two or six hours, cells were washed and infected with non-invasive <i>Salmonella Typhimurium</i> SL1344 <i>hilA</i>-339::<i>kan</i> or SL1344 pEGFP <i>invG</i>-339::<i>kan</i> using an MOI of 100∶1 <i>Salmonella</i> to host cells. Adhesion levels in percent (%) are expressed as adhesion of <i>Salmonella</i> relative to adhesion without pre-incubation with <i>E. coli</i> (<i>Salmonella</i> mono-infection). The data are the mean ± S.E.M. of at least three separate experiments in duplicate wells. * = p<0.01 compared to <i>Salmonella</i> mono-infection.</p