Enterotoxigenic Escherichia coli Secretes a Highly Conserved Mucin-Degrading Metalloprotease To Effectively Engage Intestinal Epithelial Cells

Luo, Qingwei; Kumar, Pardeep; Vickers, T. G.; Sheikh, Alaullah; Lewis, Warren G.; Rasko, David A.; Sistrunk, Jeticia R.; Fleckenstein, James M.

doi:10.1128/iai.01106-13

Cited by 118 publications

(109 citation statements)

References 59 publications

(67 reference statements)

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“…Thus far, the only SPATE identified as an ETEC virulence factor is EatA (ETEC autotransporter A), which does not function directly as an adhesin but, rather, influences the toxigenic effect by degrading EtpA via proteolytic cleavage and colonization by degrading the main component of the intestinal human mucus layer, Muc2 (27,28). Additionally, another nonautotransporter secreted protein, the metalloprotease YghJ, has recently been described as a mucinase secreted by ETEC strains (29).…”

Section: Discussionmentioning

confidence: 99%

TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

et al. 2015

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e Enterotoxigenic Escherichia coli (ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avian E. coli strains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with a tleA mutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression of tleA conferred the capacity for adherence to nonadherent E. coli HB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.

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Section: Discussionmentioning

confidence: 99%

TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

et al. 2015

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“…We therefore questioned whether, in addition to deploying recently described mucin-degrading enzymes (12,13), this pathogen might be equipped to engage MUC2 to establish intestinal colonization. Because the EtpA adhesin promotes effective small intestinal colonization (20) and early studies demonstrated binding to goblet cells (15), we first examined the interaction of this secreted protein with MUC2.…”

Section: Resultsmentioning

confidence: 99%

“…Confocal microscopy images were captured with a Zeiss LSM 510 Meta confocal laser scanning microscope (CLSM). Data were then analyzed with Volocity three-dimensional image analysis software (version 6.2; PerkinElmer, Inc.) as previously described (13).…”

Section: Real-time Quantitative Pcr (Qpcr)mentioning

confidence: 99%

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Dynamic Interactions of a Conserved Enterotoxigenic Escherichia coli Adhesin with Intestinal Mucins Govern Epithelium Engagement and Toxin Delivery

et al. 2016

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e At present, there is no vaccine for enterotoxigenic Escherichia coli (ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design. Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in the developing world (1), where this organism causes millions of infections and hundreds of thousands of deaths, particularly in young children (2). This organism contributes substantially to the large global burden of diarrheal illness that has been associated with a number of important but poorly understood sequelae, including "environmental enteropathy" and compromised growth and cognitive development (3).ETEC was discovered more than 40 years ago in cases of diarrheal illness clinically indistinguishable from cholera (4) and is defined by the production of plasmid-encoded heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). LT and ST must successfully engage cognate receptors (GM-1 ganglioside and guanylyl cyclase C, respectively) on the surface of epithelial cells to activate the production of cyclic nucleotides. The respective increases in intracellular cyclic AMP (cAMP) and cGMP trigger signaling events culminating in altered salt and water absorption in the small intestine accompanied by voluminous watery diarrhea (5).Because of their substantial impact on global health, these pathogens are a principal target for vaccine development. Unfortunately, there is currently no vaccine that affords significant broad-based protection against ETEC (6). Despite decades of research, the current understanding of the pathogenesis of this complex and hig...

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“…These results indicate that (i) pathogenicity is rigorously regulated by host environmental stimulation (19,20) and (ii) the expression and secretion of virulence factors are two separate processes that affect the pathogenicity of microorganisms at different levels. Previously, most functional and pathology studies that were focused on ETEC were performed under aerobic conditions (21)(22)(23); however, in the intestine, the target position of ETEC infection is anoxic or anaerobic. In addition, the environment of the intestine (i.e., pH, nutrient availability, and osmolality) is distinctly different from in vitro conditions (24).…”

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confidence: 99%

Electron Acceptors Induce Secretion of Enterotoxigenic Escherichia coli Heat-Labile Enterotoxin under Anaerobic Conditions through Promotion of GspD Assembly

Lü

Xie

et al. 2016

Infect Immun

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Heat-labile enterotoxin (LT), the major virulence factor of enterotoxigenic Escherichia coli (ETEC), can lead to severe diarrhea and promotes ETEC adherence to intestinal epithelial cells. Most previous in vitro studies focused on ETEC pathogenesis were conducted under aerobic conditions, which do not reflect the real situation of ETEC infection because the intestine is anoxic. In this study, the expression and secretion of LT under anaerobic or microaerobic conditions were determined; LT was not efficiently secreted into the supernatant under anaerobic or microaerobic conditions unless terminal electron acceptors (trimethylamine N-oxide dihydrate [TMAO] or nitrate) were available. Furthermore, we found that the restoration effects of TMAO and nitrate on LT secretion could be inhibited by amytal or ⌬torCAD and ⌬narG E. coli strains, indicating that LT secretion under anaerobic conditions was dependent on the integrity of the respiratory chain. At the same time, electron acceptors increase the ATP level of ETEC, but this increase was not the main reason for LT secretion. Subsequently, the relationship between the integrity of the respiratory chain and the function of the type II secretion system was determined. The GspD protein, the secretin of ETEC, was assembled under anaerobic conditions and was accompanied by LT secretion when TMAO or nitrate was added. Our data also demonstrated that TMAO and nitrate could not induce the GspD assembly and LT secretion in ⌬torCAD and ⌬narG strains, respectively. Moreover, GspD assembly under anaerobic conditions was assisted by the pilot protein YghG. Enterotoxigenic Escherichia coli (ETEC) is an important enteric pathogen that causes cholera-like diarrhea in humans and porcines (1). ETEC is a leading cause of mortality in children in less developed countries (2) and is the most common cause of travelers' diarrhea worldwide (3), accounting for more than 200 million cases of diarrhea and approximately 400,000 deaths of children annually (4, 5). A complete understanding of the virulence factors of secretion under the complex intestinal environment is a key event in diarrheal pathogenesis.Heat-labile enterotoxin (LT) is one of the critical ETEC enterotoxins, and the pathogenesis is well established. After release into the host small intestine, a pentamer of the B subunit of LT binds to epithelial cell surface receptor gangliosides (e.g., GM1). After the holotoxin is internalized by the Golgi apparatus, the A subunit exerts NAD-glycohydrolase and ADP-ribosyltransferase activities, which irreversibly activate adenylate cyclase and induce an increase in intracellular cyclic AMP (cAMP) (6). Subsequently, the chloride ion channel of the cystic fibrosis transmembrane regulator is opened, causing chloride ions and water to enter the intestinal lumen (7). LT also plays multiple roles in modulating host cell function and bacterial adherence (8-11). For example, LT activates both the NF-B and mitogen-activated protein kinase pathways, which promote ETEC adherence to intestinal epithelial ...

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Enterotoxigenic Escherichia coli Secretes a Highly Conserved Mucin-Degrading Metalloprotease To Effectively Engage Intestinal Epithelial Cells

Cited by 118 publications

References 59 publications

TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

Dynamic Interactions of a Conserved Enterotoxigenic Escherichia coli Adhesin with Intestinal Mucins Govern Epithelium Engagement and Toxin Delivery

Electron Acceptors Induce Secretion of Enterotoxigenic Escherichia coli Heat-Labile Enterotoxin under Anaerobic Conditions through Promotion of GspD Assembly

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