Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection

Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the host's immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenicEscherichia coli(EPEC) strain E2348/69 (O127:H6) and the probiotic strainEscherichia coliNissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin-8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probioticE. colistrains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side.

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Apple Flavonoid Phloretin Inhibits Escherichia coli O157:H7 Biofilm Formation and Ameliorates Colon Inflammation in Rats

Infection and Immunity ◽

10.1128/iai.05580-11 ◽

2011 ◽

Vol 79 (12) ◽

pp. 4819-4827 ◽

Cited By ~ 94

Author(s):

Jin-Hyung Lee ◽

Sushil Chandra Regmi ◽

Jung-Ae Kim ◽

Moo Hwan Cho ◽

Hyungdon Yun ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Trinitrobenzene Sulfonic Acid ◽

Content Type ◽

E Coli ◽

Colon Inflammation ◽

K 12

ABSTRACTPathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagicEscherichia coliO157:H7. The antioxidant phloretin, which is abundant in apples, markedly reducedE. coliO157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensalE. coliK-12 biofilms. Also, phloretin reducedE. coliO157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyEandstx2), autoinducer-2 importer genes (lsrACDBF), curli genes (csgAandcsgB), and dozens of prophage genes inE. coliO157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production inE. coliO157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory responsein vitrousing human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor ofE. coliO157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensalE. colibiofilms.

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Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02085-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 459-470 ◽

Cited By ~ 7

Author(s):

Romina J. Fernandez-Brando ◽

Nao Yamaguchi ◽

Amin Tahoun ◽

Sean P. McAteer ◽

Trudi Gillespie ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Bacterial Colonization ◽

Effector Proteins ◽

Content Type ◽

E Coli ◽

Type Iii ◽

Bacterial Binding

ABSTRACTA subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagicEscherichia coli(EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment ofE. coliO157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing ofE. coliO157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS.

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Bacterial Secretions of Nonpathogenic Escherichia coli Elicit Inflammatory Pathways: a Closer Investigation of Interkingdom Signaling

mBio ◽

10.1128/mbio.00025-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 33

Author(s):

Amin Zargar ◽

David N. Quan ◽

Karen K. Carter ◽

Min Guo ◽

Herman O. Sintim ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Negative Feedback ◽

Cell Contact ◽

Bacterial Cells ◽

Content Type ◽

Phenotypic Differences ◽

E Coli ◽

Autoinducer 2 ◽

Human Epithelial Cells

ABSTRACTThere have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenicEscherichia colistrains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq).E. coliBL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.IMPORTANCEThe symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to “communicate” with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

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d-Serine induces distinct transcriptomes in diverse Escherichia coli pathotypes

Microbiology ◽

10.1099/mic.0.001093 ◽

2021 ◽

Vol 167 (10) ◽

Author(s):

James P. R. Connolly ◽

Natasha C. A. Turner ◽

Jennifer C. Hallam ◽

Patricia T. Rimbi ◽

Tom Flett ◽

...

Keyword(s):

Escherichia Coli ◽

Type Species ◽

Pathogenic Bacteria ◽

Neonatal Meningitis ◽

Published Data ◽

Toxic Metabolite ◽

Transcriptional Responses ◽

Content Type ◽

E Coli ◽

Link Type

Appropriate interpretation of environmental signals facilitates niche specificity in pathogenic bacteria. However, the responses of niche-specific pathogens to common host signals are poorly understood. d-Serine (d-ser) is a toxic metabolite present in highly variable concentrations at different colonization sites within the human host that we previously found is capable of inducing changes in gene expression. In this study, we made the striking observation that the global transcriptional response of three Escherichia coli pathotypes – enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis-associated E. coli (NMEC) – to d-ser was highly distinct. In fact, we identified no single differentially expressed gene common to all three strains. We observed the induction of ribosome-associated genes in extraintestinal pathogens UPEC and NMEC only, and the induction of purine metabolism genes in gut-restricted EHEC, and UPEC indicating distinct transcriptional responses to a common signal. UPEC and NMEC encode dsdCXA – a genetic locus required for detoxification and hence normal growth in the presence of d-ser. Specific transcriptional responses were induced in strains accumulating d-ser (WT EHEC and UPEC/NMEC mutants lacking the d-ser-responsive transcriptional activator DsdC), corroborating the notion that d-ser is an unfavourable metabolite if not metabolized. Importantly, many of the UPEC-associated transcriptome alterations correlate with published data on the urinary transcriptome, supporting the hypothesis that d-ser sensing forms a key part of urinary niche adaptation in this pathotype. Collectively, our results demonstrate distinct pleiotropic responses to a common metabolite in diverse E. coli pathotypes, with important implications for niche selectivity.

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Comparative Analysis of EspF Variants in Inhibition of Escherichia coli Phagocytosis by Macrophages and Inhibition of E. coli Translocation through Human- and Bovine-Derived M Cells

Infection and Immunity ◽

10.1128/iai.00023-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4716-4729 ◽

Cited By ~ 19

Author(s):

Amin Tahoun ◽

Gabriella Siszler ◽

Kevin Spears ◽

Sean McAteer ◽

Jai Tree ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cellular Localization ◽

M Cells ◽

Binding Assays ◽

M Cell ◽

Content Type ◽

E Coli ◽

Coculture System

ABSTRACTThe EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagicEscherichia coli(EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspFO127is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspFO127has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of differentespFalleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. TheespFgene fromE. coliserotype O157 (espFO157) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibitE. colitranslocation through a human-derivedin vitroM-cell coculture system in comparison toespFO127andespFO26. In contrast,espFO157was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

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Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase

mBio ◽

10.1128/mbio.02018-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

Author(s):

Homer Pantua ◽

Elizabeth Skippington ◽

Marie-Gabrielle Braun ◽

Cameron L. Noland ◽

Jingyu Diao ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Susceptibility ◽

Pathogenic Bacteria ◽

Susceptibility Testing ◽

Regulatory Elements ◽

Signal Peptidase ◽

Type Ii ◽

Mechanisms Of Resistance ◽

Content Type ◽

E Coli

ABSTRACT Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use. IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.

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The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Stimulating Expression of Toll-Like Receptor 5, CD14, MyD88, and TRIF Together with the Induction of Interleukin-8 Expression via the Mitogen-Activated Protein Kinase Pathway in Epithelial Cells

Infection and Immunity ◽

10.1128/iai.01406-09 ◽

2010 ◽

Vol 78 (5) ◽

pp. 2153-2162 ◽

Cited By ~ 26

Author(s):

Mohamed Hafez ◽

Kelly Hayes ◽

Marie Goldrick ◽

Richard K. Grencis ◽

Ian S. Roberts

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Map Kinase ◽

Interleukin 8 ◽

Mitogen Activated Protein Kinase ◽

Probiotic Properties ◽

Toll Like Receptor ◽

Mitogen Activated Protein ◽

Map Kinase Pathway ◽

Kinase Pathway

ABSTRACT Escherichia coli strain Nissle 1917, which has been widely used as a probiotic for the treatment of inflammatory bowel disorders, expresses a K5 capsule, the expression of which is often associated with extraintestinal and urinary tract isolates of E. coli. Previously, it had been shown that the expression of a K5 capsule by Nissle 1917 was important in mediating interactions with epithelial cells and the extent of chemokine expression. In this paper, we show that infection with Nissle 1917 induces expression of Toll-like receptor 4 (TLR4) and TLR5 in Caco-2 cells and that maximal induction of TLR5 required the K5 capsule. In addition, purified K5 polysaccharide was capable of inducing expression of TLR5 and mCD14 and potentiated the activity of both TLR4 and TLR5 agonists to increase the proinflammatory response. Infection with Nissle 1917 also increased the expression of the adaptor molecules MyD88 and TRIF, which was K5 capsule dependent. By Western blot analysis, it was possible to show that induction of interleukin-8 by Nissle 1917 was predominantly through the mitogen-activated protein (MAP) kinase pathway and that expression of the K5 capsule was important for activation of the MAP kinase pathway. This paper provides new information on the function of the K5 capsule in mediating interactions between Nissle 1917 and epithelial cells and the mechanisms that underlie the probiotic properties of Nissle 1917.

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Molecular Characterization of the EhaG and UpaG Trimeric Autotransporter Proteins from Pathogenic Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.06680-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2179-2189 ◽

Cited By ~ 47

Author(s):

Makrina Totsika ◽

Timothy J. Wells ◽

Christophe Beloin ◽

Jaione Valle ◽

Luke P. Allsopp ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cell Surface ◽

Specific Binding ◽

Negative Regulator ◽

Passenger Domain ◽

Content Type ◽

E Coli ◽

Ecm Proteins

ABSTRACTTrimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagicEscherichia coli(EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenicE. coli(UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from severalE. coligenomes revealed grouping of the proteins in clades almost exclusively represented by distinctE. colipathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in anhnsisogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

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Plasmids shape the diverse accessory resistomes of Escherichia coli ST131

Access Microbiology ◽

10.1099/acmi.0.000179 ◽

2020 ◽

Author(s):

Arun Gonzales Decano ◽

Nghia Tran ◽

Hawriya Al-Foori ◽

Buthaina Al-Awadi ◽

Leigh Campbell ◽

...

Keyword(s):

Escherichia Coli ◽

Type Species ◽

Pathogenic Bacteria ◽

Gastrointestinal Microbiome ◽

Content Type ◽

E Coli ◽

Link Type ◽

Type Iv ◽

Clade C

The human gut microbiome includes beneficial, commensal and pathogenic bacteria that possess antimicrobial resistance (AMR) genes and exchange these predominantly through conjugative plasmids. Escherichia coli is a significant component of the gastrointestinal microbiome and is typically non-pathogenic in this niche. In contrast, extra-intestinal pathogenic E. coli (ExPEC) including ST131 may occupy other environments like the urinary tract or bloodstream where they express genes enabling AMR and host cell adhesion like type 1 fimbriae. The extent to which commensal E. coli and uropathogenic ExPEC ST131 share AMR genes remains understudied at a genomic level, and we examined this here using a preterm infant resistome. We found that individual ST131 had small differences in AMR gene content relative to a larger shared resistome. Comparisons with a range of plasmids common in ST131 showed that AMR gene composition was driven by conjugation, recombination and mobile genetic elements. Plasmid pEK499 had extended regions in most ST131 Clade C isolates, and it had evidence of a co-evolutionary signal based on protein-level interactions with chromosomal gene products, as did pEK204 that had a type IV fimbrial pil operon. ST131 possessed extensive diversity of selective type 1, type IV, P and F17-like fimbriae genes that was highest in subclade C2. The structure and composition of AMR genes, plasmids and fimbriae vary widely in ST131 Clade C and this may mediate pathogenicity and infection outcomes.

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Role of Host Xanthine Oxidase in Infection Due to Enteropathogenic and Shiga-Toxigenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.01124-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1129-1139 ◽

Cited By ~ 28

Author(s):

John K. Crane ◽

Tonniele M. Naeher ◽

Jacqueline E. Broome ◽

Edgar C. Boedeker

Keyword(s):

Escherichia Coli ◽

Xanthine Oxidase ◽

Host Defense ◽

Pathogenic Bacteria ◽

Intestinal Cell ◽

Intestinal Loop ◽

Loop Model ◽

Content Type ◽

E Coli

ABSTRACTXanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenicEscherichia coli(EPEC) and Shiga-toxigenicE. coli(STEC), also known as enterohemorrhagicE. coli(EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H2O2) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H2O2generated by XO activity was effective at killing laboratory strains ofE. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains.In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

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