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 and immunological characterisation of Acanthocheilonema viteae chitinase

Chitinasen sind wichtigen Moleküle im Lebenszyklus parasitischer Fadenwürmer und bedeutende Medikamenten- und Impfstoffziele. Hier wurden drei Chitinasesequenzen (I, II und III) durch Charakterisierung von 9 Klonen aus einer Genbank von Acanthocheilonema viteae gefunden. Die Anzahl der drei sehr homologen Chitinasegene wurde durch Southern-Blot bestätigt. Die größten Unterschiede sind in den Exons zu finden, die die Serin-Threonin-reiche Domäne der Chitinasen codieren. Diese Domäne ist in Sequenz III ca. 10fach länger als in Sequenz I. Sequenz I und III haben Eigenschaften eines transkribierten Genes: ein Startcodon, ein offener Leserahmen, ein Stopcodon und ein Polyadenylierungssignal. Sequenz II fehlt das erste Exon mit dem Startcodon. Bei Durchmusterung einer cDNA-Bibliothek adulter A. viteae Würmer bzw. L3 Stadien, sowie durch Reverser Transkriptase-PCR (RT-PCR) wurden in den Mikrofilarien, L3 und L4 Transkripte für Gen I gefunden, jedoch nicht für Gen III. Das N-terminale Fragment der A. viteae-Chitinase wurde in ein Expressionsplasmid ligiert und in E. coli exprimiert. Ungefähr 80 % der exprimierten Chitinase lagen in Einschluss-Körpern vor. Dieser unlösliche Anteil konnte unter denaturierenden, der lösliche Anteil unter nativen Bedingungen aufgereinigt werden. Die aus den Einschluss-Körpern aufgereinigte Chitinase zeigte im Vergleich zur löslichen Chitinase eine 13fach verminderte Aktivität. Vom aktiven Zentrum der Chitinase wurden Peptide synthetisiert und parallel mit Chitinase aus den Einschluss-Körpern für Vakzinierungsexperimente im A. viteae / Meriones unguiculatus-Filarien-Modell genutzt. Die Reduzierung der Wurmlast um 29 % nach einer Immunisierung mit rekombinantem Protein zeigte eine tendenziell schützende Kapazität des Proteins. In zwei unabhängigen Experimenten konnte nach Immunisierung mit zwei synthetischen Peptiden (P1 und P2) eine bedeutende Reduktion der Wurmlast beobachtet werden. In der P1-Gruppe gab es eine gleichmäßige Reduktion der Mf-Last, die nur im zweiten Experiment signifikant war (39 %, p > 0,05 und 45 %, p 0.05 and 45%,

<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 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

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> 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>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

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