scholarly journals Increase in Campylobacter jejuni Invasion of Intestinal Epithelial Cells under Low-Oxygen Coculture Conditions That Reflect theIn VivoEnvironment

2012 ◽  
Vol 80 (5) ◽  
pp. 1690-1698 ◽  
Author(s):  
Dominic C. Mills ◽  
Ozan Gundogdu ◽  
Abdi Elmi ◽  
Mona Bajaj-Elliott ◽  
Peter W. Taylor ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Model System ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Low Oxygen ◽  
Aerobic Conditions ◽  
Content Type ◽  
Microaerobic Conditions

ABSTRACTCampylobacter jejuniinfection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. WhileC. jejuniinfection can be reproducedin vitrousing intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performingin vitroassays under atmospheric oxygen conditions neither is optimal for microaerophilicC. jejuninor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing anin vitrosystem that closely mimicsin vivoconditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellularC. jejuni11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotileC. jejuni11168HrpoNmutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent thein vivosituation and will allow novel insights intoC. jejunipathogenic mechanisms.

scholarly journals Cj1136 Is Required for Lipooligosaccharide Biosynthesis, Hyperinvasion, and Chick Colonization by Campylobacter jejuni

2012 ◽  
Vol 80 (7) ◽  
pp. 2361-2370 ◽  
Author(s):  
Muhammad Afzal Javed ◽  
Shaun A. Cawthraw ◽  
Abiyad Baig ◽  
Jianjun Li ◽  
Alan McNally ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Intestinal Epithelial Cells ◽  
Sodium Taurocholate ◽  
Sodium Deoxycholate ◽  
Targeted Mutagenesis ◽  
Intestinal Epithelial ◽  
Content Type

ABSTRACTCampylobacter jejuniis a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasiveC. jejunistrains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue ofcj1136, which encodes a putative galactosyltransferase according to the annotation of theC. jejuniNCTC11168 genome. In the current study, we investigated the role ofcj1136inC. jejunivirulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. Thecj1136mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation ofcj1136resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. Thecj1136mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded bycj1136is involved in LOS biosynthesis and is important forC. jejunivirulence, as disruption of this gene and the resultant truncation of LOS affect both colonizationin vivoand invasivenessin vitro.

scholarly journals Campylobacter jejuni Gene Expression in the Chick Cecum: Evidence for Adaptation to a Low-Oxygen Environment

2005 ◽  
Vol 73 (8) ◽  
pp. 5278-5285 ◽  
Author(s):  
C. A. Woodall ◽  
M. A. Jones ◽  
P. A. Barrow ◽  
J. Hinds ◽  
G. L. Marsden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Electron Transport ◽  
Campylobacter Jejuni ◽  
Metabolic Pathways ◽  
Transcriptional Profiling ◽  
Physiological State ◽  
Low Oxygen ◽  
Oxygen Environment

ABSTRACT Transcriptional profiling of Campylobacter jejuni during colonization of the chick cecum identified 59 genes that were differentially expressed in vivo compared with the genes in vitro. The data suggest that C. jejuni regulates electron transport and central metabolic pathways to alter its physiological state during establishment in the chick cecum.

scholarly journals Development of Heptylmannoside-Based Glycoconjugate Antiadhesive Compounds against Adherent-Invasive Escherichia coli Bacteria Associated with Crohn's Disease

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Adeline Sivignon ◽  
Xibo Yan ◽  
Dimitri Alvarez Dorta ◽  
Richard Bonnet ◽  
Julie Bouckaert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Content Type ◽  
Type 1 Pili

ABSTRACTThe ileal lesions of Crohn's disease (CD) patients are colonized by adherent-invasiveEscherichia coli(AIEC) bacteria. These bacteria adhere to mannose residues expressed by CEACAM6 on host cells in a type 1 pilus-dependent manner. In this study, we investigated different antagonists of FimH, the adhesin of type 1 pili, for their ability to block AIEC adhesion to intestinal epithelial cells (IEC). Monovalent and multivalent derivatives ofn-heptyl α-d-mannoside (HM), a nanomolar antagonist of FimH, were testedin vitroin IEC infected with the AIEC LF82 strain andin vivoby oral administration to CEACAM6-expressing mice infected with LF82 bacteria.In vitro, multivalent derivatives were more potent than the monovalent derivatives, with a gain of efficacy superior to their valencies, probably owing to their ability to form bacterial aggregates. Of note, HM and the multi-HM glycoconjugates exhibited lower efficacyin vivoin decreasing LF82 gut colonization. Interestingly, HM analogues functionalized with an isopropylamide (1A-HM) or β-cyclodextrin pharmacophore at the end of the heptyl tail (1CD-HM) exerted beneficial effectsin vivo. These two compounds strongly decreased the amount of LF82 bacteria in the feces of mice and that of bacteria associated with the gut mucosa when administered orally at a dose of 10 mg/kg of body weight after infection. Importantly, signs of colitis and intestinal inflammation induced by LF82 infection were also prevented. These results highlight the potential of the antiadhesive compounds to treat CD patients abnormally colonized by AIEC bacteria and point to an alternative to the current approach focusing on blocking proinflammatory mediators.IMPORTANCECurrent treatments for Crohn's disease (CD), including immunosuppressive agents, anti-tumor necrosis factor alpha (anti-TNF-α) and anti-integrin antibodies, focus on the symptoms but not on the cause of the disease. Adherent-invasiveEscherichia coli(AIEC) bacteria abnormally colonize the ileal mucosa of CD patients via the interaction of the mannose-specific adhesin FimH of type 1 pili with CEACAM6 mannosylated proteins expressed on the epithelial cell surface. Thus, we decided to develop an antiadhesive strategy based on synthetic FimH antagonists specifically targeting AIEC bacteria that would decrease intestinal inflammation. Heptylmannoside (HM)-based glycocompounds strongly inhibit AIEC adhesion to intestinal epithelial cellsin vitro. The antiadhesive effect of two of these compounds of relatively simple chemical structure was also observedin vivoin AIEC-infected CEACAM6-expressing mice and was associated with a reduction in the signs of colitis. These results suggest a new therapeutic approach for CD patients colonized by AIEC bacteria, based on the development of synthetic FimH antagonists.

scholarly journals Narrow-Spectrum Inhibitors of Campylobacter jejuni Flagellar Expression and Growth

2015 ◽  
Vol 59 (7) ◽  
pp. 3880-3886 ◽  
Author(s):  
Jeremiah G. Johnson ◽  
Caroline Yuhas ◽  
Thomas J. McQuade ◽  
Martha J. Larsen ◽  
Victor J. DiRita
Keyword(s):  
Small Molecules ◽  
Campylobacter Jejuni ◽  
Campylobacter Coli ◽  
Content Type ◽  
Food Borne ◽  
Food Borne Illness ◽  
Inhibitor Screen

ABSTRACTCampylobacter jejuniis a major cause of food-borne illness due to its ability to reside within the gastrointestinal tracts of chickens. Multiple studies have identified the flagella ofC. jejunias a major determinant of chicken colonization. An inhibitor screen of approximately 147,000 small molecules was performed to identify compounds that are able to inhibit flagellar expression in a reporter strain ofC. jejuni. Several compounds that modestly inhibited motility of wild-typeC. jejuniin standard assays were identified, as were a number of small molecules that robustly inhibitedC. jejunigrowth,in vitro. Examination of similar bacterial screens found that many of these small molecules inhibited only the growth ofC. jejuni. Follow-up assays demonstrated inhibition of other strains ofC. jejuniandCampylobacter colibut no inhibition of the closely relatedHelicobacter pylori. The compounds were determined to be bacteriostatic and nontoxic to eukaryotic cells. Preliminary results from a day-of-hatch chick model of colonization suggest that at least one of the compounds demonstrates promise for reducingCampylobactercolonization loadsin vivo, although further medicinal chemistry may be required to enhance bioavailability.

scholarly journals Virulent Shigella flexneri Affects Secretion, Expression, and Glycosylation of Gel-Forming Mucins in Mucus-Producing Cells

2013 ◽  
Vol 81 (10) ◽  
pp. 3632-3643 ◽  
Author(s):  
Brice Sperandio ◽  
Natalie Fischer ◽  
Marie Joncquel Chevalier-Curt ◽  
Yannick Rossez ◽  
Pascal Roux ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Protein Glycosylation ◽  
Apical Surface ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Content Type ◽  
Secretion Expression ◽  
Mucus Barrier ◽  
Biochemical Analyses

ABSTRACTMucin glycoproteins are secreted in large amounts by the intestinal epithelium and constitute an efficient component of innate immune defenses to promote homeostasis and protect against enteric pathogens. In this study, our objective was to investigate how the bacterial enteropathogenShigella flexneri, which causes bacillary dysentery, copes with the mucin defense barrier. We report that uponin vitroinfection of mucin-producing polarized human intestinal epithelial cells, virulentS. flexnerimanipulates the secretion of gel-forming mucins. This phenomenon, which is triggered only by virulent strains, results in accumulation of mucins at the cell apical surface, leading to the appearance of a gel-like structure that favors access of bacteria to the cell surface and the subsequent invasion process. We identify MUC5AC, a gel-forming mucin, as a component of this structure. Formation of this gel does not depend on modifications of electrolyte concentrations, induction of trefoil factor expression, endoplasmic reticulum stress, or response to unfolded proteins. In addition, transcriptional and biochemical analyses of infected cells reveal modulations of mucin gene expression and modifications of mucin glycosylation patterns, both of which are induced by virulent bacteria in a type III secretion system-dependent manner. Thus,S. flexnerihas developed a dedicated strategy to alter the mucus barrier by targeting key elements of the gel-forming capacity of mucins: gene transcription, protein glycosylation, and secretion.

scholarly journals Prolonged NF-κB Activation by a Macrophage Inhibitory Cytokine 1-Linked Signal in Enteropathogenic Escherichia coli-Infected Epithelial Cells

2013 ◽  
Vol 81 (6) ◽  
pp. 1860-1869 ◽  
Author(s):  
Hye Jin Choi ◽  
Juil Kim ◽  
Kee Hun Do ◽  
Seong-Hwan Park ◽  
Yuseok Moon
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Watery Diarrhea ◽  
Intestinal Epithelial ◽  
Severe Injuries ◽  
Epithelial Integrity ◽  
Content Type

ABSTRACTIntestinal epithelial activation of nuclear factor kappa B (NF-κB) exerts both detrimental and beneficial functions in response to various luminal insults, including ones associated with mucosa-associated pathogens. Gastrointestinal infection with enteropathogenicEscherichia coli(EPEC) causes severe injuries in epithelial integrity and leads to watery diarrhea. The present study was conducted to investigate the prolonged epithelial responses to persistent EPEC infection via NF-κB activation. EPEC infection led to sustained activation of NF-κB signal in mouse intestinal epithelial cellsin vivoandin vitro, which was positively associated with a type III secretion system, whereas early NF-κB is regulated. Moreover, prolonged NF-κB activation was found to be a part of macrophage inhibitory cytokine 1 (MIC-1)-mediated signaling activation, a novel link between NF-κB signaling and infection-associated epithelial stress. EPEC infection induced gene expression of MIC-1, a member of the transforming growth factor β (TGF-β) superfamily, which then activated TGF-β-activated kinase 1 and consequently led to NF-κB activation. Functionally, both EPEC-induced MIC-1 and NF-κB signaling mediated epithelial survival by enhancing the expression of cyclin D1, a target of NF-κB. In summary, the results of the present study suggest that MIC-1 serves as a mediator of prolonged NF-κB activation, which is critical in maintaining gut epithelial integrity in response to infection-induced injuries.

scholarly journals Identification and Analysis of Staphylococcus aureus Components Expressed by a Model System of Growth in Serum

2001 ◽  
Vol 69 (8) ◽  
pp. 5198-5202 ◽  
Author(s):  
Michael D. Wiltshire ◽  
Simon J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Model System ◽  
Surface Proteins ◽  
Lysine Biosynthesis ◽  
Genes Encoding ◽  
Peptide Biosynthesis ◽  
Microaerobic Conditions

ABSTRACT A model system mimicking Staphylococcus aureusbacteremia was developed by growth in serum under microaerobic conditions. Eight genes induced by growth in serum were identified, including an antimicrobial peptide biosynthesis locus, amino acid biosynthetic loci, and genes encoding putative surface proteins. Nine independent insertions were found in the major lysine biosynthesis operon, which encodes eight genes, is repressed by lysine in vitro, and is expressed in vivo.

scholarly journals Intestinal Epithelial CD98 Directly Modulates the Innate Host Response to Enteric Bacterial Pathogens

2013 ◽  
Vol 81 (3) ◽  
pp. 923-934 ◽  
Author(s):  
Moiz A. Charania ◽  
Hamed Laroui ◽  
Hongchun Liu ◽  
Emilie Viennois ◽  
Saravanan Ayyadurai ◽  
...  
Keyword(s):  
Intestinal Inflammation ◽  
Ex Vivo ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Extracellular Loop ◽  
Ki 67 ◽  
Content Type ◽  
Direct Binding

ABSTRACTCD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells (IECs) during intestinal inflammation. EnteropathogenicEscherichia coli(EPEC) is a food-borne human pathogen that attaches to IECs and injects effector proteins directly into the host cells, thus provoking an inflammatory response. In the present study, we investigated CD98 and EPEC interactionsin vitroandex vivoand examined FVB wild-type (WT) and villin-CD98 transgenic mice overexpressing human CD98 in IECs (hCD98 Tg mice) and infected withCitrobacter rodentiumas anin vivomodel.In vivostudies indicated that CD98 overexpression, localized to the apical domain of colonic cells, increased the attachment ofC. rodentiumin mouse colons and resulted in increased expression of proinflammatory markers and decreased expression of anti-inflammatory markers. The proliferative markers Ki-67 and cyclin D1 were significantly increased in the colonic tissue ofC. rodentium-infected hCD98 Tg mice compared to that of WT mice.Ex vivostudies correlate with thein vivodata. Small interfering RNA (siRNA) studies with Caco2-BBE cells showed a decrease in adherence of EPEC to Caco2 cells in which CD98 expression was knocked down.In vitrosurface plasmon resonance (SPR) experiments showed direct binding between recombinant hCD98 and EPEC/C. rodentiumproteins. We also demonstrated that the partial extracellular loop of hCD98 was sufficient for direct binding to EPEC/C. rodentium. These findings demonstrate the importance of the extracellular loop of CD98 in the innate host defense response to intestinal infection by attaching and effacing (A/E) pathogens.

scholarly journals Genetic Dissection of the Fermentative and Respiratory Contributions Supporting Vibrio cholerae Hypoxic Growth

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Emilio Bueno ◽  
Brandon Sit ◽  
Matthew K. Waldor ◽  
Felipe Cava
Keyword(s):  
Vibrio Cholerae ◽  
Respiratory Chain ◽  
Antimicrobial Agents ◽  
Specific Interaction ◽  
Infection Model ◽  
Genetic Dissection ◽  
Low Oxygen ◽  
Content Type

ABSTRACT Both fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors, such as nitrate (NO3−). During canonical NO3− respiration, NO3− is reduced in a stepwise manner to N2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, requires only a single periplasmic NO3− reductase (NapA) to undergo NO3− respiration, suggesting that the pathogen possesses a noncanonical NO3− respiratory chain. In this study, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3− respiration in V. cholerae. We found that while the V. cholerae NO3− respiratory chain is primarily composed of homologues of established NO3− respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae. Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo. Our findings reveal the genetic basis of a specific interaction between disparate energy production pathways that supports pathogen fitness under shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions. IMPORTANCE Bacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways: fermentation and respiration. In this study, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3− that consists largely of components found in other NO3− respiratory systems but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3− respiration were required for efficient pathogen growth under both laboratory conditions and in an animal infection model. These observations provide a specific example of fermentative respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.

Incomplete free fatty acid oxidation by ascites tumor cells under low oxygen tension

1983 ◽  
Vol 244 (1) ◽  
pp. R84-R92
Author(s):  
M. Ookhtens ◽  
N. Baker
Keyword(s):  
Fatty Acid ◽  
Maximum Rate ◽  
Acid Oxidation ◽  
Ascites Tumor ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Oxidation Rates

We tried to understand why our earlier estimates of fatty acid (FA) oxidation rates under the nearly anaerobic state of the Ehrlich ascites tumor (EAT) in vivo were even greater than those found in vitro under aerobic conditions. Using tracers [1–14C]linoleate, [1–14C]-, and [9,10–3H]palmitate, and NaH14CO3, we estimated essential and nonessential FA oxidation rates to CO2 + H2O by EAT in living mice and in vitro under aerobic and anaerobic conditions. Sequestration of intraperitoneally (ip)-injected 14C-FFA allowed a selective labeling of the tumor versus the host; thus, breath 14CO2 could be used to estimate the maximum rate of FA oxidation in vivo by the tumor. Initially, we measured breath 14CO2 following NaH14CO3 injections and developed a multicompartmental model to simulate the tumor-host HCO-3-CO2 system. This model was integrated with our earlier model for tumor FA turnover. The integrated model was fitted to breath 14CO2 data from mice injected ip with 14C-FFA to compute tumor FA oxidation rates. Both essential and nonessential FA were oxidized to CO2 at similar rates. The maximum rate of total FA oxidation to CO2 was 5–6 nmol FA X min-1 X 7-ml tumor-1, about 5–10 times lower than all previous estimates obtained in vitro and in vivo. To resolve this dilemma we used doubly labeled [1–14C; 9,10–3H]palmitate and found that under aerobic conditions, in vitro, EAT formed 3H2O and 14CO2 at nearly equal rates. These rates were suppressed markedly but unequally at low PO2. Anaerobic suppression of 14CO2 formation greatly exceeded that of 3H2O formation. As a result 3H2O/14CO2 reached a value of congruent to 10 at low PO2. Our data indicate that under the nearly anaerobic conditions of a growing EAT in vivo, the partial beta-oxidation of FA to 2C + H2O takes place at a 5 to 10 times faster rate than the complete oxidation of FA to CO2 + H2O. This finding can account for earlier apparent inconsistencies in the literature, since aerobic studies of 14C-FA oxidation to 14CO2 in vitro and of 3H-FA oxidation to 3H2O under nearly anaerobic conditions would both overestimate greatly the rate of FA oxidation to CO2 by EAT in vivo.

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