Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri

The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the inhibition of the signalling via NF-κB that in turn led to the attenuation of the inflammatory response.

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Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells

BIO-PROTOCOL ◽

10.21769/bioprotoc.3293 ◽

2019 ◽

Vol 9 (13) ◽

Author(s):

Atin Sharma ◽

Andrea Puhar

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Shigella Flexneri ◽

Plaque Assay ◽

Intestinal Epithelial ◽

Human Intestinal Epithelial Cells

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Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

Cellular Microbiology ◽

10.1111/j.1462-5822.2005.00535.x ◽

2005 ◽

Vol 7 (8) ◽

pp. 1109-1116 ◽

Cited By ~ 30

Author(s):

Rafika Athman ◽

Maria-Isabel Fernandez ◽

Pierre Gounon ◽

Philippe Sansonetti ◽

Daniel Louvard ◽

...

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Shigella Flexneri ◽

Primary Cultures ◽

Intestinal Epithelial ◽

Mouse Intestine

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Transient Suppression of Shigella flexneri Type 3 Secretion by a Protective O-Antigen-Specific Monoclonal IgA

mBio ◽

10.1128/mbio.00042-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 22

Author(s):

Stephen J. Forbes ◽

Tia Bumpus ◽

Elizabeth A. McCarthy ◽

Blaise Corthésy ◽

Nicholas J. Mantis

Keyword(s):

Epithelial Cells ◽

Mucosal Immunity ◽

Intestinal Epithelial Cells ◽

Shigella Flexneri ◽

Secretory Iga ◽

Bacterial Invasion ◽

Intestinal Epithelial ◽

Content Type ◽

O Antigen ◽

Type 3

ABSTRACTMucosal immunity to the enteric pathogenShigella flexneriis mediated by secretory IgA (S-IgA) antibodies directed against the O-antigen (O-Ag) side chain of lipopolysaccharide. While secretory antibodies against the O-Ag are known to prevent bacterial invasion of the intestinal epithelium, the mechanisms by which this occurs are not fully understood. In this study, we report that the binding of a murine monoclonal IgA (IgAC5) to the O-Ag ofS. flexneriserotype 5a suppresses activity of the type 3 secretion (T3S) system, which is necessary forS. flexnerito gain entry into intestinal epithelial cells. IgAC5’s effects on the T3S were rapid (5 to 15 min) and were coincident with a partial reduction in the bacterial membrane potential and a decrease in intracellular ATP levels. Activity of the T3S system returned to normal levels 45 to 90 min following antibody treatment, demonstrating that IgAC5’s effects were transient. Nonetheless, these data suggest a model in which the association of IgA with the O-Ag ofS. flexneripartially de-energizes the T3S system and temporarily renders the bacterium incapable of invading intestinal epithelial cells.IMPORTANCESecretory IgA (S-IgA) serves as the first line of defense against enteric infections. However, despite its well-recognized role in mucosal immunity, relatively little is known at the molecular level about how this class of antibody functions to prevent pathogenic bacteria from penetrating the epithelial barrier. It is generally assumed that S-IgA functions primarily by “immune exclusion,” a phenomenon in which the antibody binds to microbial surface antigens and thereby promotes bacterial agglutination, entrapment in mucus, and physical clearance from the gastrointestinal tract via peristalsis. The results of the present study suggest that in addition to serving as a physical barrier, S-IgA may have a direct impact on the ability of microbial pathogens to secrete virulence factors required for invasion of intestinal epithelial cells.

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The NleE/OspZ Family of Effector Proteins Is Required for Polymorphonuclear Transepithelial Migration, a Characteristic Shared by Enteropathogenic Escherichia coli and Shigella flexneri Infections

Infection and Immunity ◽

10.1128/iai.00684-07 ◽

2007 ◽

Vol 76 (1) ◽

pp. 369-379 ◽

Cited By ~ 36

Author(s):

Daniel V. Zurawski ◽

Karen L. Mumy ◽

Luminita Badea ◽

Julia A. Prentice ◽

Elizabeth L. Hartland ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Deletion Mutant ◽

Intestinal Epithelial Cells ◽

Shigella Flexneri ◽

Host Cells ◽

Apical Surface ◽

Intestinal Epithelial ◽

Wild Type ◽

Secretion Systems

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and Shigella flexneri are human host-specific pathogens that infect intestinal epithelial cells. However, each bacterial species employs a different infection strategy within this environmental niche. EPEC attaches to the apical surface of small intestine enterocytes, causing microvillus effacement and rearrangement of the host cell cytoskeleton beneath adherent bacteria. In contrast, S. flexneri invades the large intestine epithelium at the basolateral membrane, replicates, and spreads cell to cell. Both EPEC and S. flexneri rely on type three secretion systems (T3SS) to secrete effectors into host cells, and both pathogens recruit polymorphonuclear leukocytes (PMNs) from the submucosa to the lumen of the intestine. In this report, we compared the virulence functions of the EPEC T3SS effector NleE and the homologous Shigella protein Orf212. We discovered that Orf212 was secreted by the S. flexneri T3SS and renamed this protein OspZ. Infection of polarized T84 intestinal epithelial cells with an ospZ deletion mutant of S. flexneri resulted in reduced PMN transepithelial migration compared to infection by the wild type. An nleE deletion mutant of EPEC showed a similar reduction of PMN migration. The ability to induce PMN migration was restored in both mutants when either ospZ or nleE was expressed from a plasmid. An infection of T84 cells with the ΔospZ mutant resulted in reduced extracellular signal-related kinase phosphorylation and NF-κB activation compared to infection with the wild type. Therefore, we conclude that OspZ and NleE have similar roles in the upstream induction of host signaling pathways required for PMN transepithelial migration in Shigella and EPEC infections.

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