scholarly journals Probing Clostridium difficile infection in innovative human gut cellular models

2018 ◽  
Author(s):  
Blessing O. Anonye ◽  
Jack Hassall ◽  
Jamie Patient ◽  
Usanee Detamornrat ◽  
Afnan M. Aladdad ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Epithelial Cells ◽  
Gradual Increase ◽  
Controlled Environment ◽  
Human Gut ◽  
Epithelial Monolayers ◽  
Cellular Models ◽  
Gut Colonization ◽  
D Model

AbstractInteractions of anaerobic gut bacteria, such as Clostridium difficile, with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins and bacterial filaments. This 2-D dual chamber system was used to examine C. difficile interactions with the commensal Bacteroides dorei, and interestingly, C. difficile growth is significantly reduced in presence of B. dorei. Furthermore, in novel multilayer and 3-D gut models containing a myofibroblast layer, C. difficile adheres more efficiently to epithelial cells, as compared to the 2-D model, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host-anaerobe and pathogen-commensal interaction studies in vitro.

scholarly journals Bacteroidales Secreted Antimicrobial Proteins Target Surface Molecules Necessary for Gut Colonization and Mediate Competition In Vivo

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Kevin G. Roelofs ◽  
Michael J. Coyne ◽  
Rahul R. Gentyala ◽  
Maria Chatzidaki-Livanis ◽  
Laurie E. Comstock
Keyword(s):  
Sequence Similarity ◽  
Interference Competition ◽  
Sensitive Strain ◽  
Strain Level ◽  
Surface Molecule ◽  
Antimicrobial Proteins ◽  
Human Gut ◽  
Gut Colonization ◽  
Target Molecules

ABSTRACT We recently showed that human gut Bacteroidales species secrete antimicrobial proteins (BSAPs), and we characterized in vitro the first such BSAP produced by Bacteroides fragilis . In this study, we identified a second potent BSAP produced by the ubiquitous and abundant human gut species Bacteroides uniformis . The two BSAPs contain a membrane attack complex/perforin (MACPF) domain but share very little sequence similarity. We identified the target molecules of BSAP-sensitive cells and showed that each BSAP targets a different class of surface molecule: BSAP-1 targets an outer membrane protein of sensitive B. fragilis strains, and BSAP-2 targets the O-antigen glycan of lipopolysaccharide (LPS) of sensitive B. uniformis strains. Species-wide genomic and phenotypic analyses of B. fragilis and B. uniformis showed that BSAP-producing strains circumvent killing by synthesizing an orthologous nontargeted surface molecule. The BSAP genes are adjacent to the gene(s) encoding their target replacements, suggesting coacquisition. Using a gnotobiotic mouse competitive-colonization model, we found that the BSAP surface targets are important for colonization of the mammalian gut, thereby explaining why they are maintained in sensitive strains and why they were replaced rather than deleted in BSAP-producing strains. Using isogenic BSAP-producing, -sensitive, and -resistant strains, we show that a BSAP-producing strain outcompetes a sensitive strain but not a resistant strain in the mammalian gut. Human gut metagenomic datasets reveal that BSAP-1-sensitive strains do not cooccur with BSAP-1-producing strains in human gut microbiotas, further supporting the idea that BSAPs are important competitive factors with relevance to the strain-level composition of the human gut microbiota. IMPORTANCE We know relatively little about the ecology of the human intestinal microbiota and the combination of factors that dictate which strains and species occupy an individual’s gut microbial community. Interference competition, mediated by bacterial factors that directly harm other members, is beginning to be appreciated as important in contributing to species- and strain-level dynamics of abundant gut bacteria. Here, we show that gut Bacteroidales secrete antimicrobial proteins (BSAPs) that antagonize strains of the same species. We show that BSAPs target molecules of sensitive cells that are important for gut colonization and therefore are maintained in sensitive cells. In an experimental animal model of gut colonization, a BSAP-1-producing strain antagonized and outcompeted an isogenic sensitive strain. Furthermore, metagenomic analyses showed that BSAP-1-producing and -sensitive strains are not found together in human gut microbiotas. These data suggest that BSAPs are strong ecological drivers shaping the strain-level composition of gut communities.

scholarly journals Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

2019 ◽  
Vol 10 ◽  
Author(s):  
Blessing O. Anonye ◽  
Jack Hassall ◽  
Jamie Patient ◽  
Usanee Detamornrat ◽  
Afnan M. Aladdad ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Clostridium Difficile Infection ◽  
Human Gut ◽  
Cellular Models

scholarly journals Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip

2015 ◽  
Vol 113 (1) ◽  
pp. E7-E15 ◽  
Author(s):  
Hyun Jung Kim ◽  
Hu Li ◽  
James J. Collins ◽  
Donald E. Ingber
Keyword(s):  
Epithelial Cells ◽  
Pathogenic Bacteria ◽  
Intestinal Inflammation ◽  
Bacterial Overgrowth ◽  
Intestinal Barrier ◽  
Inflammatory Cells ◽  
Intestinal Barrier Function ◽  
Human Gut ◽  
Intestinal Bacterial Overgrowth

A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models.

scholarly journals Evaluation of linezolid for the treatment of Clostridium difficile infection caused by epidemic strains using an in vitro human gut model

2011 ◽  
Vol 66 (7) ◽  
pp. 1537-1546 ◽  
Author(s):  
S. D. Baines ◽  
A. R. Noel ◽  
G. S. Huscroft ◽  
S. L. Todhunter ◽  
R. O'Connor ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Clostridium Difficile Infection ◽  
Human Gut

scholarly journals Evaluation of antimicrobial activity of ceftaroline against Clostridium difficile and propensity to induce C. difficile infection in an in vitro human gut model

2013 ◽  
Vol 68 (8) ◽  
pp. 1842-1849 ◽  
Author(s):  
S. D. Baines ◽  
C. H. Chilton ◽  
G. S. Crowther ◽  
S. L. Todhunter ◽  
J. Freeman ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Clostridium Difficile ◽  
Human Gut

scholarly journals Mecillinam: a low-risk antimicrobial agent for induction of Clostridium difficile infection in an in vitro human gut model

2009 ◽  
Vol 63 (4) ◽  
pp. 838-839 ◽  
Author(s):  
S. D. Baines ◽  
R. O'Connor ◽  
G. Huscroft ◽  
K. Saxton ◽  
J. Freeman ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Antimicrobial Agent ◽  
Clostridium Difficile Infection ◽  
Low Risk ◽  
Human Gut

scholarly journals Regulated and constitutive protein targeting can be distinguished by secretory polarity in thyroid epithelial cells.

1991 ◽  
Vol 112 (3) ◽  
pp. 365-376 ◽  
Author(s):  
P Arvan ◽  
J Lee
Keyword(s):  
Epithelial Cells ◽  
Protein Targeting ◽  
Secretory Protein ◽  
Residual Fraction ◽  
Secretory Proteins ◽  
Apical Surface ◽  
Epithelial Monolayers ◽  
Apical Polarity ◽  
Intracellular Storage

We have studied concurrent apical/basolateral and regulated/constitutive secretory targeting in filter-grown thyroid epithelial monolayers in vitro, by following the exocytotic routes of two newly synthesized endogenous secretory proteins, thyroglobulin (Tg) and p500. Tg is a regulated secretory protein as indicated by its acute secretory response to secretagogues. Without stimulation, pulse-labeled Tg exhibits primarily two kinetically distinct routes: less than or equal to 80% is released in an apical secretory phase which is largely complete by 6-10 h, with most of the remaining Tg retained in intracellular storage from which delayed apical discharge is seen. The rapid export observed for most Tg is unlikely to be because of default secretion, since its apical polarity is preserved even during the period (less than or equal to 10 h) when p500 is released basolaterally by a constitutive pathway unresponsive to secretagogues. p500 also exhibits a second, kinetically distinct secretory route: at chase times greater than 10 h, a residual fraction (less than or equal to 8%) of p500 is secreted with an apical preponderance similar to that of Tg. It appears that this fraction of p500 has failed to be excluded from the regulated pathway, which has a predetermined apical polarity. From these data we hypothesize that a targeting hierarchy may exist in thyroid epithelial cells such that initial sorting to the regulated pathway may be a way of insuring apical surface delivery from one of two possible exocytotic routes originating in the immature storage compartment.

scholarly journals Evaluation of NVB302 versus vancomycin activity in an in vitro human gut model of Clostridium difficile infection

2012 ◽  
Vol 68 (1) ◽  
pp. 168-176 ◽  
Author(s):  
G. S. Crowther ◽  
S. D. Baines ◽  
S. L. Todhunter ◽  
J. Freeman ◽  
C. H. Chilton ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Clostridium Difficile Infection ◽  
Human Gut

scholarly journals Flagella methylation promotes bacterial adhesion and host cell invasion

2019 ◽  
Author(s):  
Julia A. Horstmann ◽  
Michele Lunelli ◽  
Hélène Cazzola ◽  
Johannes Heidemann ◽  
Caroline Kühne ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Host Cell ◽  
Posttranslational Modifications ◽  
Surface Hydrophobicity ◽  
Lysine Methylation ◽  
Gut Colonization ◽  
Successful Infection ◽  
Lysine Residues

AbstractThe flagellum is the motility device of many bacteria and the long external filament is made of several thousand copies of a single protein, flagellin. While posttranslational modifications of flagellin are common among bacterial pathogens, the role of lysine methylation remained unknown. Here, we show that both flagellins of Salmonella enterica, FliC and FljB, are methylated at surface-exposed lysine residues. A Salmonella mutant deficient in flagellin methylation was outcompeted for gut colonization in a gastroenteritis mouse model. In support, methylation of flagellin promoted invasion of epithelial cells in vitro. Lysine methylation increased the surface hydrophobicity of flagellin and enhanced flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization and successful infection of the host.

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