scholarly journals EatA, an Immunogenic Protective Antigen of Enterotoxigenic Escherichia coli, Degrades Intestinal Mucin

2013 ◽  
Vol 82 (2) ◽  
pp. 500-508 ◽  
Author(s):  
Pardeep Kumar ◽  
Qingwei Luo ◽  
Tim J. Vickers ◽  
Alaullah Sheikh ◽  
Warren G. Lewis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protective Antigen ◽  
Shigella Flexneri ◽  
Enterotoxigenic Escherichia Coli ◽  
Effective Vaccine ◽  
Major Protein ◽  
Passenger Domain ◽  
Content Type ◽  
Intestinal Mucin

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is a major cause of morbidity and mortality due to infectious diarrhea in developing countries for which there is presently no effective vaccine. A central challenge in ETEC vaccinology has been the identification of conserved surface antigens to formulate a broadly protective vaccine. Here, we demonstrate that EatA, an immunogenic secreted serine protease of ETEC, contributes to virulence by degrading MUC2, the major protein present in the small intestinal mucous layer, and that removal of this barrierin vitroaccelerates toxin access to the enterocyte surface. In addition, we demonstrate that vaccination with the recombinant secreted passenger domain of EatA (rEatAp) elicits high titers of antibody and is protective against intestinal infection with ETEC. These findings may have significant implications for development of both subunit and live-attenuated vaccines against ETEC and other enteric pathogens, includingShigella flexneri, that express similar proteins.

scholarly journals Immunogenicity and Protective Efficacy against Enterotoxigenic Escherichia coli Colonization following Intradermal, Sublingual, or Oral Vaccination with EtpA Adhesin

2016 ◽  
Vol 23 (7) ◽  
pp. 628-637 ◽  
Author(s):  
Qingwei Luo ◽  
Tim J. Vickers ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Enterotoxigenic Escherichia Coli ◽  
Oral Vaccination ◽  
Content Type ◽  
Degree Of Protection ◽  
Alternative Routes

EnterotoxigenicEscherichia coli(ETEC) strains are a common cause of diarrhea. Extraordinary antigenic diversity has prompted a search for conserved antigens to complement canonical approaches to ETEC vaccine development. EtpA, an immunogenic extracellular ETEC adhesin relatively conserved in the ETEC pathovar, has previously been shown to be a protective antigen following intranasal immunization. These studies were undertaken to explore alternative routes of EtpA vaccination that would permit use of a double mutant (R192G L211A) heat-labile toxin (dmLT) adjuvant. Here, oral vaccination with EtpA adjuvanted with dmLT afforded significant protection against small intestinal colonization, and the degree of protection correlated with fecal IgG, IgA, or total fecal antibody responses to EtpA. Sublingual vaccination yielded compartmentalized mucosal immune responses with significant increases in anti-EtpA fecal IgG and IgA, and mice vaccinated via this route were also protected against colonization. In contrast, while intradermal (i.d.) vaccination achieved high levels of both serum and fecal antibodies against both EtpA and dmLT, mice vaccinated via the i.d. route were not protected against subsequent colonization and the avidity of serum IgG and IgA EtpA-specific antibodies was significantly lower after i.d. immunization compared to other routes. Finally, we demonstrate that antiserum from vaccinated mice significantly impairs binding of LT to cognate GM1 receptors and shows near complete neutralization of toxin delivery by ETECin vitro. Collectively, these data provide further evidence that EtpA could complement future vaccine strategies but also suggest that additional effort will be required to optimize its use as a protective immunogen.

scholarly journals Maternal Vaccination with a Fimbrial Tip Adhesin and Passive Protection of Neonatal Mice against Lethal Human Enterotoxigenic Escherichia coli Challenge

2015 ◽  
Vol 83 (12) ◽  
pp. 4555-4564 ◽  
Author(s):  
Wilson B. Luiz ◽  
Juliana F. Rodrigues ◽  
Joseph H. Crabb ◽  
Stephen J. Savarino ◽  
Luis C. S. Ferreira
Keyword(s):  
Escherichia Coli ◽  
Protective Antigen ◽  
Survival Rates ◽  
Small Animal ◽  
Enterotoxigenic Escherichia Coli ◽  
Vaccine Antigen ◽  
Effective Vaccine ◽  
Content Type ◽  
Protective Antigens ◽  
Pup Survival

Globally, enterotoxigenicEscherichia coli(ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 × 107bacteria resulted in 50% lethal doses (LD50) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD50challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.

scholarly journals Transcriptional Modulation of Enterotoxigenic Escherichia coli Virulence Genes in Response to Epithelial Cell Interactions

2012 ◽  
Vol 81 (1) ◽  
pp. 259-270 ◽  
Author(s):  
Rita Kansal ◽  
David A. Rasko ◽  
Jason W. Sahl ◽  
George P. Munson ◽  
Koushik Roy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antigen Expression ◽  
Toxin Production ◽  
Enterotoxigenic Escherichia Coli ◽  
Receptor Protein ◽  
Effective Vaccine ◽  
Cell Contact ◽  
Content Type ◽  
Host Interactions

EnterotoxigenicEscherichia coli(ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cellsin vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens.

scholarly journals Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

2012 ◽  
Vol 19 (10) ◽  
pp. 1603-1608 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Colonization ◽  
Content Type ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

scholarly journals Characterization and Functional Analysis of AatB, a Novel Autotransporter Adhesin and Virulence Factor of Avian Pathogenic Escherichia coli

2013 ◽  
Vol 81 (7) ◽  
pp. 2437-2447 ◽  
Author(s):  
Xiangkai ZhuGe ◽  
Shaohui Wang ◽  
Hongjie Fan ◽  
Zihao Pan ◽  
Jianluan Ren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Large Family ◽  
Extracellular Proteins ◽  
Dimensional Structure ◽  
Phylogenetic Groups ◽  
Passenger Domain ◽  
Content Type

ABSTRACTAutotransporter (AT) proteins constitute a large family of extracellular proteins that contribute to bacterial virulence. A novel AT adhesin gene,aatB, was identified in avian pathogenicEscherichia coli(APEC) DE205B via genomic analyses. The open reading frame ofaatBwas 1,017 bp, encoding a putative 36.3-kDa protein which contained structural motifs characteristic for AT proteins: a signal peptide, a passenger domain, and a translocator domain. The predicted three-dimensional structure of AatB consisted of two distinct domains, the C-terminal β-barrel translocator domain and an N-terminal passenger domain. The prevalence analyses ofaatBin APEC indicated thataatBwas detected in 26.4% (72/273) of APEC strains and was strongly associated with phylogenetic groups D and B2. Quantitative real-time reverse transcription-PCR analyses revealed that AatB expression was increased during infectionin vitroandin vivo. Moreover, AatB could elicit antibodies in infected ducks, suggesting that AatB is involved in APEC pathogenicity. Thus, APEC DE205B strains with a mutatedaatBgene and mutated strains complemented with theaatBgene were constructed. Inactivation ofaatBresulted in a reduced capacity to adhere to DF-1 cells, defective virulence capacityin vivo, and decreased colonization capacity in lung during systemic infection compared with the capacities of the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatB makes a significant contribution to APEC virulence through bacterial adherence to host tissuesin vivoandin vitro. In addition, biofilm formation assays with strain AAEC189 expressing AatB indicated that AatB mediates biofilm formation.

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

2013 ◽  
Vol 82 (2) ◽  
pp. 509-521 ◽  
Author(s):  
Qingwei Luo ◽  
Pardeep Kumar ◽  
Tim J. Vickers ◽  
Alaullah Sheikh ◽  
Warren G. Lewis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterotoxigenic Escherichia Coli ◽  
Effective Vaccine ◽  
Type Ii Secretion ◽  
Intestinal Epithelial ◽  
Content Type ◽  
E Coli ◽  
Type Ii Secretion System ◽  
Efficient Access ◽  
Genes Encoding

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is a leading cause of death due to diarrheal illness among young children in developing countries, and there is currently no effective vaccine. Many elements of ETEC pathogenesis are still poorly defined. Here we demonstrate that YghJ, a secreted ETEC antigen identified in immunoproteomic studies using convalescent patient sera, is required for efficient access to small intestinal enterocytes and for the optimal delivery of heat-labile toxin (LT). Furthermore, YghJ is a highly conserved metalloprotease that influences intestinal colonization of ETEC by degrading the major mucins in the small intestine, MUC2 and MUC3. Genes encoding YghJ and its cognate type II secretion system (T2SS), which also secretes LT, are highly conserved in ETEC and exist in other enteric pathogens, including other diarrheagenicE. coliandVibrio choleraebacteria, suggesting that this mucin-degrading enzyme may represent a shared virulence feature of these important pathogens.

scholarly journals The EtpA Exoprotein of Enterotoxigenic Escherichia coli Promotes Intestinal Colonization and Is a Protective Antigen in an Experimental Model of Murine Infection

2008 ◽  
Vol 76 (5) ◽  
pp. 2106-2112 ◽  
Author(s):  
Koushik Roy ◽  
David Hamilton ◽  
Kenneth P. Allen ◽  
Mildred P. Randolph ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Vaccine Development ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Enterotoxigenic Escherichia Coli ◽  
Deletion Mutants ◽  
Diverse Group ◽  
Intestinal Colonization

ABSTRACT The enterotoxigenic Escherichia coli (ETEC) strains are major causes of morbidity and mortality due to diarrheal illness in developing countries. At present, there is no broadly protective vaccine for this diverse group of pathogens. The EtpA protein, identified in ETEC H10407 in a recent search for candidate immunogens, is a large glycosylated exoprotein secreted via two-partner secretion (TPS). Similar to structurally related molecules, EtpA functions in vitro as an adhesin. The studies reported here use a recently developed murine model of ETEC intestinal colonization to examine the immunogenicity and protective efficacy of EtpA. We report that mice repeatedly exposed to ETEC are protected from subsequent colonization and that they mount immune responses to both EtpA and its presumed two-partner secretion transporter (EtpB) during the course of experimental infection. Furthermore, isogenic etpA deletion mutants were impaired in the colonization of mice, and intranasal immunization of mice with recombinant EtpA conferred protection against ETEC H10407 in this model. Together, these data suggest that EtpA is required for optimal colonization of the intestine, findings paralleling those of previous in vitro studies demonstrating its role in adherence. EtpA and other TPS proteins may be viable targets for ETEC vaccine development.

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

2016 ◽  
Vol 84 (10) ◽  
pp. 2748-2757 ◽  
Author(s):  
Xi Lu ◽  
Enqing Fu ◽  
Yonghong Xie ◽  
Faguang Jin
Keyword(s):  
Escherichia Coli ◽  
Respiratory Chain ◽  
Electron Acceptors ◽  
Enterotoxigenic Escherichia Coli ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Severe Diarrhea ◽  
Heat Labile ◽  
Microaerobic Conditions

Heat-labile enterotoxin (LT), the major virulence factor of enterotoxigenicEscherichia coli(ETEC), can lead to severe diarrhea and promotes ETEC adherence to intestinal epithelial cells. Most previousin vitrostudies 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 (trimethylamineN-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 ΔtorCADand ΔnarGE. colistrains, 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 ΔtorCADand ΔnarGstrains, respectively. Moreover, GspD assembly under anaerobic conditions was assisted by the pilot protein YghG.

scholarly journals Neutralizing Anti-Heat-Stable Toxin (STa) Antibodies Derived from Enterotoxigenic Escherichia coli Toxoid Fusions with STa Proteins Containing N12S, L9A/N12S, or N12S/A14T Mutations Show Little Cross-Reactivity with Guanylin or Uroguanylin

2017 ◽  
Vol 84 (2) ◽  
Author(s):  
Qiangde Duan ◽  
Jiachen Huang ◽  
Nan Xiao ◽  
Hyesuk Seo ◽  
Weiping Zhang
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Vaccine Development ◽  
Cross Reactivity ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Renal Epithelial Cells ◽  
Severe Diarrhea ◽  
Heat Stable

ABSTRACT Heat-stable toxin (STa)-producing enterotoxigenic Escherichia coli (ETEC) strains are a top cause of moderate-to-severe diarrhea in children from developing countries and a common cause of travelers' diarrhea. Recent progress in using STa toxoids and toxoid fusions to induce neutralizing anti-STa antibodies has accelerated ETEC vaccine development. However, concern remains regarding whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two guanylate cyclase C (GC-C) ligands regulating fluid and electrolyte transportation in human intestinal and renal epithelial cells. To further divert STa from guanylin and uroguanylin structurally and antigenically and to eliminate anti-STa antibody cross-reactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the double and triple mutants STaL9A/N12S, STaL9A/A14H, STaN12S/A14T, and STaL9A/N12S/A14H. We then fused each STa mutant (three copies) to a monomeric heat-labile toxin (LT) mutant (mnLTR192G/L211A) for the toxoid fusions 3×STaL9A/N12S-mnLTR192G/L211A, 3×STaL9A/A14H-mnLTR192G/L211A, 3×STaN12S/A14T-mnLTR192G/L211A, and 3×STaL9A/N12S/A14H-mnLTR192G/L211A; examined each fusion for anti-STa immunogenicity; and assessed the derived antibodies for in vitro neutralization activity against STa toxicity and for cross-reactivity with guanylin and uroguanylin. Mice subcutaneously immunized with each fusion protein developed anti-STa antibodies, and the antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Currently, there is no licensed vaccine against ETEC diarrhea. One key challenge is to identify safe antigens to induce antibodies neutralizing the key STa without cross-reacting with guanylin and uroguanylin, two important ligands controlling homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies that neutralize STa enterotoxicity in vitro and do not cross-react with guanylin or uroguanylin. These fusions have become the preferred antigens for the development of ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.

scholarly journals Characterization of Mucosal Immune Responses to Enterotoxigenic Escherichia coli Vaccine Antigens in a Human Challenge Model: Response Profiles after Primary Infection and Homologous Rechallenge with Strain H10407

2015 ◽  
Vol 23 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Subhra Chakraborty ◽  
Clayton Harro ◽  
Barbara DeNearing ◽  
Malathi Ram ◽  
Andrea Feller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Response ◽  
Immune Responses ◽  
Vaccine Development ◽  
Lavage Fluid ◽  
Antibody Responses ◽  
Enterotoxigenic Escherichia Coli ◽  
Effective Vaccine ◽  
Content Type ◽  
Mucosal Immune Responses

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) bacteria are the most common bacterial cause of diarrhea in children in resource-poor settings as well as in travelers. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. A significant challenge to successful vaccine development is our poor understanding of the immune responses that correlate best with protection against ETEC illness. In this study, ETEC-specific mucosal immune responses were characterized and compared in subjects challenged with ETEC strain H10407 and in subjects rechallenged with the homologous organism. IgA responses to lipopolysaccharide (LPS), heat-labile toxin B subunit (LTB), and colonization factor antigen I (CFA/I) in antibody in lymphocyte supernatant (ALS), feces, lavage fluid, and saliva samples were evaluated. In all assay comparisons, ALS was the most sensitive indicator of a local immune response, but serum IgA was also a useful indirect marker of immune response to oral antigens. Volunteers challenged and then rechallenged with strain H10407 were protected from illness following rechallenge. Comparing mucosal antibody responses after primary and homologous rechallenge, protection against disease was reflected in reduced antibody responses to key ETEC antigens and in reduced fecal shedding of the H10407 challenge strain. Subjects challenged with strain H10407 mounted stronger antibody responses to LPS and LTB than subjects in the rechallenge group, while responses to CFA/I in the rechallenge group were higher than in the challenge group. We anticipate that this study will help provide an immunological benchmark for the evaluation of ETEC vaccines and immunization regimens in the future.

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