292 results on '"James B. Kaper"'
Search Results
2. Phosphodiesterase 5 (PDE5) restricts intracellular cGMP accumulation during enterotoxigenic Escherichia coli infection
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Jennifer Foulke-Abel, Huimin Yu, Laxmi Sunuwar, Ruxian Lin, James M. Fleckenstein, James B. Kaper, and Mark Donowitz
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e. coli heat-stable enterotoxin ,e. coli heat-labile enterotoxin ,cyclic nucleotide ,etec ,pde5 ,mrp5 ,enteroid monolayer ,intestinal organoid ,host–pathogen interaction ,Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) has a continuing impact on residents and travelers in underdeveloped countries. Both heat-labile (LT) and heat-stable (ST) enterotoxins contribute to pathophysiology via induction of cyclic nucleotide synthesis, and previous investigations focused on intracellular signal transduction rather than possible intercellular second messenger signaling. We modeled ETEC infection in human jejunal enteroid/organoid monolayers (HEM) and evaluated cyclic nucleotide pools, finding that intracellular cAMP was significantly increased but also underwent apical export, whereas cGMP was minimally retained intracellularly and predominantly effluxed into the basolateral space. LT and virulence factors including EatA, EtpA, and CfaE promoted ST release and enhanced ST-stimulated cGMP production. Intracellular cGMP was regulated by MK-571-sensitive export in addition to degradation by phosphodiesterase 5. HEMs had limited ST-induced intracellular cGMP accumulation compared to T84 or Caco-2 models. Cyclic nucleotide export/degradation demonstrates additional complexity in the mechanism of ETEC infection and may redirect understanding of diarrheal onset.
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- 2020
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3. Enteropathogenic Escherichia coli EspH-Mediated Rho GTPase Inhibition Results in Desmosomal PerturbationsSummary
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Jennifer Lising Roxas, Ross Calvin Monasky, Bryan Angelo P. Roxas, Al B. Agellon, Asad Mansoor, James B. Kaper, Gayatri Vedantam, and V.K. Viswanathan
- Subjects
Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Background & Aims: The diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss. Methods: DSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell–cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model. Results: The secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo. Conclusions: Our studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens. Keywords: EPEC, Desmoglein, DSG2, Host–Pathogen Interaction
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- 2018
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4. Cross-Talk between Probiotic Nissle 1917 and Human Colonic Epithelium Affects the Metabolite Composition and Demonstrates Host Antibacterial Effect
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Karol Dokladny, John K. Crane, Alex J. Kassicieh, James B. Kaper, and Olga Kovbasnjuk
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human colonoid monolayers ,metabolome ,glutamine ,GABA ,spermine ,host anti-bacterial effect ,Microbiology ,QR1-502 - Abstract
Colonic epithelium–commensal interactions play a very important role in human health and disease development. Colonic mucus serves as an ecologic niche for a myriad of commensals and provides a physical barrier between the epithelium and luminal content, suggesting that communication between the host and microbes occurs mainly by soluble factors. However, the composition of epithelia-derived metabolites and how the commensal flora influences them is less characterized. Here, we used mucus-producing human adult stem cell-derived colonoid monolayers exposed apically to probiotic E. coli strain Nissle 1917 to characterize the host–microbial communication via small molecules. We measured the metabolites in the media from host and bacterial monocultures and from bacteria-colonoid co-cultures. We found that colonoids secrete amino acids, organic acids, nucleosides, and polyamines, apically and basolaterally. The metabolites from host-bacteria co-cultures markedly differ from those of host cells grown alone or bacteria grown alone. Nissle 1917 affects the composition of apical and basolateral metabolites. Importantly, spermine, secreted apically by colonoids, shows antibacterial properties, and inhibits the growth of several bacterial strains. Our data demonstrate the existence of a cross-talk between luminal bacteria and human intestinal epithelium via metabolites, which might affect the numbers of physiologic processes including the composition of commensal flora via bactericidal effects.
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- 2021
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5. PaxVax CVD 103-HgR single-dose live oral cholera vaccine
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Myron M. Levine, Wilbur H. Chen, James B. Kaper, Michael Lock, Lisa Danzig, and Marc Gurwith
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cholera ,cholera vaccines ,live oral vaccine ,travelers’ vaccines ,vaccine ,Internal medicine ,RC31-1245 - Abstract
Introduction: Cholera remains a problem in developing countries and a risk for travelers. Hypochlorhydria, blood group O, cardiac and renal disease increase the risk of developing cholera gravis. Oral vaccines containing inactivated Vibrio cholerae and requiring two doses are available in some countries. No cholera vaccine had been available for U.S. travelers for decades until 2016 when CVD 103-HgR (VAXCHORA™), an oral live attenuated vaccine, was licensed by the U.S. FDA. Areas covered: Enduring protection following wild-type cholera provided the rationale to develop a single-dose live oral vaccine. CVD 103-HgR is well-tolerated and protects against cholera caused by V. cholerae O1 of either serotype (Inaba, Ogawa) and biotype (El Tor, Classical). Since 90% vaccine efficacy is evident 10 days post-ingestion of a single dose, CVD 103-HgR can rapidly protect travelers. Vibriocidal antibody seroconversion correlates with protection; >90% of U.S. adult (including elderly) vaccinees seroconvert. The U.S. Public Health Service’s Advisory Committee on Immunization Practices recommends CVD 103-HgR for U.S. travelers to areas of ongoing cholera transmission. Expert commentary: Next steps include evaluations in children, post-licensure safety and effectiveness monitoring, diminishing cold chain constraints, optimizing a ‘high-dose’ formulation for developing countries, and diminishing/eliminating the need for water to administer a dose.
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- 2017
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- View/download PDF
6. Phosphotyrosine-Mediated Regulation of Enterohemorrhagic Escherichia coli Virulence
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Colin D. Robertson, Tracy H. Hazen, James B. Kaper, David A. Rasko, and Anne-Marie Hansen
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EHEC ,T3SS ,gene regulation ,tyrosine phosphorylation ,Microbiology ,QR1-502 - Abstract
ABSTRACT Enteric pathogens with low infectious doses rely on the ability to orchestrate the expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of ler, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) causes outbreaks of hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome. Successful host colonization by EHEC relies on the ability to coordinate the expression of virulence factors in response to environmental cues. A complex network that integrates environmental signals at multiple regulatory levels tightly controls virulence gene expression. We demonstrate that EHEC utilizes a previously uncharacterized phosphotyrosine signaling pathway through Cra to fine-tune the expression of virulence-associated genes to effectively control T3SS production. This study demonstrates that tyrosine phosphorylation negatively affects the DNA-binding capacity of Cra, which affects the expression of genes related to virulence and metabolism. We demonstrate for the first time that phosphotyrosine-mediated control affects global transcription in EHEC. Our data provide insight into a hitherto unexplored regulatory level of the global network controlling EHEC virulence gene expression.
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- 2018
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7. Enterohemorrhagic Escherichia coli Reduces Mucus and Intermicrovillar Bridges in Human Stem Cell-Derived ColonoidsSummary
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Julie In, Jennifer Foulke-Abel, Nicholas C. Zachos, Anne-Marie Hansen, James B. Kaper, Harris D. Bernstein, Marc Halushka, Sarah Blutt, Mary K. Estes, Mark Donowitz, and Olga Kovbasnjuk
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Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Background & Aims: Enterohemorrhagic Escherichia coli (EHEC) causes over 70,000 episodes of foodborne diarrhea annually in the United States. The early sequence of events that precede life-threatening hemorrhagic colitis and hemolytic uremic syndrome is not fully understood due to the initial asymptomatic phase of the disease and the lack of a suitable animal model. We determined the initial molecular events in the interaction between EHEC and human colonic epithelium. Methods: Human colonoids derived from adult proximal colonic stem cells were developed into monolayers to study EHEC-epithelial interactions. Monolayer confluency and differentiation were monitored by transepithelial electrical resistance measurements. The monolayers were apically infected with EHEC, and the progression of epithelial damage over time was assessed using biochemical and imaging approaches. Results: Human colonoid cultures recapitulate the differential protein expression patterns characteristic of the crypt and surface colonocytes. Mucus-producing differentiated colonoid monolayers are preferentially colonized by EHEC. Upon colonization, EHEC forms characteristic attaching and effacing lesions on the apical surface of colonoid monolayers. Mucin 2, a main component of colonic mucus, and protocadherin 24 (PCDH24), a microvillar resident protein, are targeted by EHEC at early stages of infection. The EHEC-secreted serine protease EspP initiates brush border damage through PCDH24 reduction. Conclusions: Human colonoid monolayers are a relevant pathophysiologic model that allow the study of early molecular events during enteric infections. Colonoid monolayers provide access to both apical and basolateral surfaces, thus providing an advantage over three-dimensional cultures to study hostâpathogen interactions in a controllable and tractable manner. EHEC reduces colonic mucus and affects the brush border cytoskeleton in the absence of commensal bacteria. Keywords: Human Colonoid Monolayers, Intestinal Organoids, Microvillar Effacement, Serine Protease EspP
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- 2016
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8. Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers
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C. Ming Tse, Julie G. In, Jianyi Yin, Mark Donowitz, Michele Doucet, Jennifer Foulke-Abel, Fernando Ruiz-Perez, James P. Nataro, Nicholas C. Zachos, James B. Kaper, and Olga Kovbasnjuk
- Subjects
EHEC ,serine protease EspP ,human colonoid monolayers ,SPATEs ,diarrhea ,short circuit current ,CFTR ,intracellular Ca2+ ,Medicine - Abstract
One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.
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- 2018
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9. Correction: Chromosomal and Plasmid-Encoded Factors of Induce Secretogenic Activity.
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Christina S. Faherty, Jill M. Harper, Terez Shea-Donohue, Eileen M. Barry, James B. Kaper, Alessio Fasano, and James P. Nataro
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Medicine ,Science - Published
- 2013
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10. Human Epithelial Stem Cell-Derived Colonoid Monolayers as a Model to Study Shiga Toxin-Producing Escherichia coli–Host Interactions
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Karol Dokladny, Julie G. In, Olga Kovbasnjuk, and James B. Kaper
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0301 basic medicine ,biology ,Host (biology) ,Shiga toxin ,Enteroendocrine cell ,medicine.disease_cause ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,biology.protein ,medicine ,Organoid ,Epithelial Physiology ,030211 gastroenterology & hepatology ,Stem cell ,Escherichia coli ,Shiga toxin-producing Escherichia coli - Abstract
Human intestinal organoid cultures established from crypt-derived stem cells truly revolutionized our approach to study intestinal epithelial physiology and pathologies as they can be propagated indefinitely and preserve the genetic signature of the donor and the gut segment specificity in culture. Here we describe human stem cell-derived colonoid monolayers as a reliable and reproducible model to study Shiga toxin-producing Escherichia coli (STEC) infection and STEC-caused pathologies of the whole colonic epithelium comprising a mixture of colonocytes, goblet, enteroendocrine, and other rare cells present in human colonic epithelial tissue.
- Published
- 2021
11. Coronin-1 is necessary for enteric pathogen-induced transcytosis across human ileal enteroid monolayers expressing M cells
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Mary K. Estes, Janet F. Staab, Michele Doucet, Rachel Latanich, Sun Lee, James B. Kaper, and Nicholas C. Zachos
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Gene knockdown ,biology ,Antigen ,Transcytosis ,Chemistry ,Coronin ,biology.protein ,Cytoskeleton ,biology.organism_classification ,Bacteria ,Function (biology) ,Microfold cell ,Cell biology - Abstract
In the intestine, luminal sampling by microfold (M) cells is crucial for inducing protective mucosal immune responses but can also serve as an entry pathway for pathogens, including bacteria and viruses. Enteric pathogens can influence intestinal M cell function; however, the molecular mechanisms involved in the regulation of uptake and transcytosis of gut cargo by human M cells remain to be determined. Understanding the mechanisms responsible for regulating human M cell function requires a relevant human model. In this study, human ileal enteroids established from healthy donors were grown as confluent monolayers on permeable supports and differentiated to express mature M cells. Enteric pathogens including enteropathogenicE. coli(EPEC), adherent invasive E. coli (AIEC), and human rotavirus were apically exposed to M cell enteroid monolayers. M cell-mediated uptake and transcytosis was compared in enteroids infected by pathogenic or commensal bacteria (HS strain). EPEC and AIEC, but not HS, stimulated M cell uptake and transcytosis. We discovered that this pathogenspecific effect was dependent on expression of coronin 1a, a cytoskeletal remodeling protein. Using stable coronin 1a knockdown (KD) enteroids, we observed that EPEC-stimulated transcytosis of fluorescent beads was lost and associated with a significant decrease in the number of glycoprotein-2 positive (Gp-2+ve) M cells. The results of these studies demonstrate that coronin 1a is required for uptake and transcytosis of luminal cargo across human M cells and that coronin 1a is necessary for differentiation of mature M cells that actively transcytose luminal gut antigens in response to pathogenic, but not commensal, microbes.
- Published
- 2020
12. Mechanical Stimuli Affect Escherichia coli Heat-Stable Enterotoxin-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Model
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Laxmi Sunuwar, James M. Fleckenstein, Mark Donowitz, James B. Kaper, Katia Karalis, Magdalena Kasendra, and Jianyi Yin
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0301 basic medicine ,Virulence Factors ,Bacterial Toxins ,Immunology ,Enterotoxin ,Biology ,medicine.disease_cause ,Microbiology ,Enterotoxins ,03 medical and health sciences ,0302 clinical medicine ,Enterotoxigenic Escherichia coli ,Intestine, Small ,medicine ,Humans ,Heat-stable enterotoxin ,Secretion ,Cyclic GMP ,Escherichia coli Infections ,Peristalsis ,Escherichia coli Proteins ,Molecular Pathogenesis ,Small intestine ,Cell biology ,Jejunum ,030104 developmental biology ,Infectious Diseases ,medicine.anatomical_structure ,Cell culture ,030211 gastroenterology & hepatology ,Parasitology ,Stress, Mechanical ,Intracellular ,Signal Transduction - Abstract
Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions.
- Published
- 2020
13. Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence
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Sabrina Secord, Landon J. Getz, Anne Marie Hansen, Akio Abe, Nikhil A. Thomas, Umang Jain, Asaomi Kuwae, James B. Kaper, Jason J. LeBlanc, Cameron S. Runte, and Andrew W. Stadnyk
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0301 basic medicine ,Effector ,030106 microbiology ,Virulence ,Chromosomal translocation ,Phenylalanine ,Biology ,Microbiology ,Cell biology ,03 medical and health sciences ,Chaperone (protein) ,biology.protein ,Secretion ,Tyrosine ,Enteropathogenic Escherichia coli ,Molecular Biology - Abstract
Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.
- Published
- 2018
14. Enteropathogenic Escherichia coli EspH-Mediated Rho GTPase Inhibition Results in Desmosomal Perturbations
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Ross Monasky, Jennifer Lising Roxas, Asad Mansoor, V. K. Viswanathan, Al Agellon, Gayatri Vedantam, James B. Kaper, and Bryan Roxas
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0301 basic medicine ,RHOA ,Hepatology ,biology ,Cadherin ,Chemistry ,Effector ,Gastroenterology ,Guanosine ,GTPase ,digestive system ,Cell junction ,3. Good health ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,parasitic diseases ,biology.protein ,lcsh:Diseases of the digestive system. Gastroenterology ,Secretion ,Guanine nucleotide exchange factor ,lcsh:RC799-869 - Abstract
Background & Aims: The diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss. Methods: DSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell–cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model. Results: The secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo. Conclusions: Our studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens. Keywords: EPEC, Desmoglein, DSG2, Host–Pathogen Interaction
- Published
- 2018
15. PaxVax CVD 103-HgR single-dose live oral cholera vaccine
- Author
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Michael Lock, Wilbur H. Chen, Myron M. Levine, James B. Kaper, Marc Gurwith, and Lisa Danzig
- Subjects
0301 basic medicine ,030106 microbiology ,Immunology ,Administration, Oral ,Disease ,Vaccines, Attenuated ,CVD 103-HgR ,03 medical and health sciences ,0302 clinical medicine ,Cholera ,Drug Discovery ,medicine ,Humans ,030212 general & internal medicine ,Vibrio cholerae ,Pharmacology ,business.industry ,Cholera Vaccines ,medicine.disease ,Antibodies, Bacterial ,Virology ,Molecular Medicine ,Cholera vaccine ,business - Abstract
Cholera remains a problem in developing countries and a risk for travelers. Hypochlorhydria, blood group O, cardiac and renal disease increase the risk of developing cholera gravis. Oral vaccines containing inactivated Vibrio cholerae and requiring two doses are available in some countries. No cholera vaccine had been available for U.S. travelers for decades until 2016 when CVD 103-HgR (VAXCHORA™), an oral live attenuated vaccine, was licensed by the U.S. FDA. Areas covered: Enduring protection following wild-type cholera provided the rationale to develop a single-dose live oral vaccine. CVD 103-HgR is well-tolerated and protects against cholera caused by V. cholerae O1 of either serotype (Inaba, Ogawa) and biotype (El Tor, Classical). Since 90% vaccine efficacy is evident 10 days post-ingestion of a single dose, CVD 103-HgR can rapidly protect travelers. Vibriocidal antibody seroconversion correlates with protection;90% of U.S. adult (including elderly) vaccinees seroconvert. The U.S. Public Health Service's Advisory Committee on Immunization Practices recommends CVD 103-HgR for U.S. travelers to areas of ongoing cholera transmission. Expert commentary: Next steps include evaluations in children, post-licensure safety and effectiveness monitoring, diminishing cold chain constraints, optimizing a 'high-dose' formulation for developing countries, and diminishing/eliminating the need for water to administer a dose.
- Published
- 2017
16. Mechanical Stimuli Affect E. Coli Heat Stable Enterotoxin (ST)-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model
- Author
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Katia Karalis, Laxmi Sunuwar, Magdalena Kasendra, Mark Donowitz, James M. Fleckenstein, James B. Kaper, and Jianyi Yin
- Subjects
Cyclic nucleotide ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Cell culture ,medicine ,Secretion ,Enterotoxin ,Epithelium ,Small intestine ,Intracellular ,Peristalsis ,Cell biology - Abstract
Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a pathogen virulence factor, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips), exposed to enterotoxigenic E. coli heat-stable enterotoxin A (ST), and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height, transcription of the cyclic nucleotide transporting protein MRP4, and apical and basolateral secretion of cGMP under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to static conditions, but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to static culture conditions under both baseline conditions and with exposure to ST enterotoxin, and suggests that further investigations of application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resembles intact intestine than study under static conditions.
- Published
- 2019
17. Phosphodiesterase 5 (PDE5) restricts intracellular cGMP accumulation during enterotoxigenic Escherichia coli infection
- Author
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Mark Donowitz, Jennifer Foulke-Abel, Ruxian Lin, James M. Fleckenstein, Huimin Yu, James B. Kaper, and Laxmi Sunuwar
- Subjects
0301 basic medicine ,Microbiology (medical) ,enteroid monolayer ,Virulence ,Biology ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,Cyclic nucleotide ,chemistry.chemical_compound ,0302 clinical medicine ,cyclic nucleotide ,Enterotoxigenic Escherichia coli ,etec ,Organoid ,medicine ,lcsh:RC799-869 ,e. coli heat-stable enterotoxin ,Gastroenterology ,e. coli heat-labile enterotoxin ,intestinal organoid ,pde5 ,Intracellular signal transduction ,030104 developmental biology ,Infectious Diseases ,chemistry ,cGMP-specific phosphodiesterase type 5 ,mrp5 ,Second messenger system ,lcsh:Diseases of the digestive system. Gastroenterology ,030211 gastroenterology & hepatology ,host–pathogen interaction ,Intracellular - Abstract
Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) has a continuing impact on residents and travelers in underdeveloped countries. Both heat-labile (LT) and heat-stable (ST) enterotoxins contribute to pathophysiology via induction of cyclic nucleotide synthesis, and previous investigations focused on intracellular signal transduction rather than possible intercellular second messenger signaling. We modeled ETEC infection in human jejunal enteroid/organoid monolayers (HEM) and evaluated cyclic nucleotide pools, finding that intracellular cAMP was significantly increased but also underwent apical export, whereas cGMP was minimally retained intracellularly and predominantly effluxed into the basolateral space. LT and virulence factors including EatA, EtpA, and CfaE promoted ST release and enhanced ST-stimulated cGMP production. Intracellular cGMP was regulated by MK-571-sensitive export in addition to degradation by phosphodiesterase 5. HEMs had limited ST-induced intracellular cGMP accumulation compared to T84 or Caco-2 models. Cyclic nucleotide export/degradation demonstrates additional complexity in the mechanism of ETEC infection and may redirect understanding of diarrheal onset.
- Published
- 2020
18. Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence
- Author
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Cameron S, Runte, Umang, Jain, Landon J, Getz, Sabrina, Secord, Asaomi, Kuwae, Akio, Abe, Jason J, LeBlanc, Andrew W, Stadnyk, James B, Kaper, Anne-Marie, Hansen, and Nikhil A, Thomas
- Subjects
Virulence ,Polymers ,Virulence Factors ,Escherichia coli Proteins ,Escherichia coli O157 ,Organophosphates ,Mice, Inbred C57BL ,Disease Models, Animal ,Enteropathogenic Escherichia coli ,Intestinal Diseases ,Mice ,Animals ,Humans ,Tyrosine ,Female ,Escherichia coli Infections ,HeLa Cells ,Molecular Chaperones - Abstract
Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.
- Published
- 2018
19. Tandem tyrosine residues in the EPEC multicargo chaperone CesT support differential type III effector translocation and early host colonization
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Akio Abe, Andrew W. Stadnyk, Umang Jain, Landon J. Getz, Nikhil A. Thomas, Sabrina Secord, Asaomi Kuwae, Cameron S. Runte, James B. Kaper, Jason J. LeBlanc, and Anne Marie Hansen
- Subjects
chemistry.chemical_classification ,Innate immune system ,biology ,Effector ,Chromosomal translocation ,biochemical phenomena, metabolism, and nutrition ,Amino acid ,Cell biology ,chemistry ,Chaperone (protein) ,biology.protein ,Secretion ,Enteropathogenic Escherichia coli ,Tyrosine - Abstract
Enteropathogenic Escherichia coli (EPEC) are worldwide human enteric pathogens inflicting significant morbidity and causing large economic losses. A type 3 secretion system (T3SS) is critical for EPEC intestinal colonization, and injection of effectors into host cells contributes to cellular subversion and innate immune evasion. Here, we demonstrate that two strictly conserved C-terminal tyrosine residues, Y152 and Y153, within the multicargo T3SS chaperone CesT serve differential roles in regulating effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine attenuated EPEC type 3 effector injection into host cells, and significantly limited Tir effector mediated intimate adherence, a key feature of attaching and effacing pathogenesis. Whereas CesT Y153 supported normal levels of Tir translocation, CesT Y152 was strictly required for the effector NleA to be expressed and subsequently translocated into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y152, Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating Y152 in CesT functionality. A mouse infection model of EPEC intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for strictly conserved tandem tyrosine residues within CesT. Tyrosine 152 of CesT is implicated in NleA expression, providing functional relevance for localized amino acid conservation. Therefore, CesT via its novel C-terminal domain, has relevant roles beyond typical T3SC that interact and stabilize effector proteins.
- Published
- 2018
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20. Enterotoxigenic Escherichia coli is phagocytosed by macrophages underlying villus-like intestinal epithelial cells: modeling ex vivo innate immune defenses of the human gut
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Nicholas C. Zachos, James B. Kaper, James P. Nataro, Gaelle Noel, Marcela F. Pasetti, and Michele Doucet
- Subjects
0301 basic medicine ,Microbiology (medical) ,030106 microbiology ,Biology ,medicine.disease_cause ,Microbiology ,Proinflammatory cytokine ,Pathogenesis ,03 medical and health sciences ,Enterotoxigenic Escherichia coli ,enteroid model ,medicine ,Macrophage ,intestine ,Innate immune system ,Gastroenterology ,Enterotoxigenic E. coli ,Phenotype ,Epithelium ,3. Good health ,Addendum ,macrophages ,030104 developmental biology ,Infectious Diseases ,medicine.anatomical_structure ,Ex vivo - Abstract
There is a paucity of information on diarrheagenic enterotoxigenic Escherichia coli (ETEC)’s interaction with innate immune cells, in part due to the lack of reliable models that recapitulate infection in human gut. In a recent publication, we described the development of an ex vivo enteroid-macrophage co-culture model using human primary cells. We reported that macrophages residing underneath the epithelial monolayer acquired “resident macrophage” phenotype characterized by lower production of inflammatory cytokines and strong phagocytic activity. These macrophages extended projections across the epithelium, which captured ETEC applied to the apical side of the epithelium and reduced luminal bacterial load. Additional evidence presented in this addendum confirms these findings and further demonstrates that macrophage adaptation occurs regardless of the stage of differentiation of epithelial cells, and that ETEC uptake arises rapidly after infection. The enteroid-macrophage co-culture represents a novel and relevant tool to study host-cell interactions and pathogenesis of enteric infections in humans.
- Published
- 2017
21. Comparative Genomics Provides Insight into the Diversity of the Attaching and Effacing Escherichia coli Virulence Plasmids
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James P. Nataro, James B. Kaper, David A. Rasko, and Tracy H. Hazen
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Molecular Sequence Data ,Immunology ,Virulence ,Molecular Genomics ,Biology ,medicine.disease_cause ,Microbiology ,Genome ,Enteropathogenic Escherichia coli ,Plasmid ,Genomic island ,medicine ,Humans ,Escherichia coli ,Escherichia coli Infections ,Phylogeny ,Genetics ,Escherichia coli Proteins ,Genetic Variation ,Shiga toxin ,Genomics ,biochemical phenomena, metabolism, and nutrition ,Infectious Diseases ,biology.protein ,Parasitology ,Plasmids ,Locus of enterocyte effacement - Abstract
Attaching and effacing Escherichia coli (AEEC) strains are a genomically diverse group of diarrheagenic E. coli strains that are characterized by the presence of the locus of enterocyte effacement (LEE) genomic island, which encodes a type III secretion system that is essential to virulence. AEEC strains can be further classified as either enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC), or atypical EPEC, depending on the presence or absence of the Shiga toxin genes or bundle-forming pilus (BFP) genes. Recent AEEC genomic studies have focused on the diversity of the core genome, and less is known regarding the genetic diversity and relatedness of AEEC plasmids. Comparative genomic analyses in this study demonstrated genetic similarity among AEEC plasmid genes involved in plasmid replication conjugative transfer and maintenance, while the remainder of the plasmids had sequence variability. Investigation of the EPEC adherence factor (EAF) plasmids, which carry the BFP genes, demonstrated significant plasmid diversity even among isolates within the same phylogenomic lineage, suggesting that these EAF-like plasmids have undergone genetic modifications or have been lost and acquired multiple times. Global transcriptional analyses of the EPEC prototype isolate E2348/69 and two EAF plasmid mutants of this isolate demonstrated that the plasmid genes influence the expression of a number of chromosomal genes in addition to the LEE. This suggests that the genetic diversity of the EAF plasmids could contribute to differences in the global virulence regulons of EPEC isolates.
- Published
- 2015
22. Safety, Immunogenicity, and Transmissibility of Single-Dose Live Oral Cholera Vaccine Strain CVD l03-HgR in 24- to 59-Month-Old Indonesian Children
- Author
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Genevieve Losonsky, Gary Pazzaglia, Peter O'Hanley, Cyrus H. Simanjuntak, James B. Kaper, Stanley J. Cryz, Bradford A. Kay, Patricia Dykstra, Myron M. Levine, Fernando R. Noriega, Steven S. Wasserman, Suharyono, Aswitha Budiarso, Narain H. Punjabi, and Atti R. Rifai
- Subjects
medicine.medical_specialty ,business.industry ,Immunogenicity ,medicine.disease ,medicine.disease_cause ,Placebo ,Cholera ,Vaccination ,Diarrhea ,Infectious Diseases ,Vibrio cholerae ,Internal medicine ,Immunology ,medicine ,Vomiting ,Immunology and Allergy ,medicine.symptom ,business ,Cholera vaccine - Abstract
Recombinant A-B+ Vibrio cholerae O1 strain CVD 103-HgR is a safe, highly immunogenic, single-dose live oral vaccine in adults in industrialized countries. Safety, excretion, immunogenicity, vaccine transmissibility, and environmental introduction of CVD 103-HgR were investigated among 24- to 59-month-old children in Jakarta. In 81 households, 1 child was randomly allocated a single dose of vaccine (5 x 10(9) cfu) and another, placebo. Additionally, 139 unpaired children were randomly allocated vaccine or placebo. During 9 days of follow-up, diarrhea or vomiting did not occur more often among vaccines than controls. Vaccine was minimally excreted and was isolated from no controls and from 1 (0.6%) of 177 unvaccinated family contacts. A 4-fold or higher rise in serum vibriocidal antibody was observed in 75% of vaccines (10-fold rise in geometric mean titer over baseline). Of 135 paired placebo recipients or household contacts, 5 had vibriocidal seroconversions. Moore swabs placed in sewers and latrines near 97 households failed to detect vaccine. These observations pave the way for a large-scale field trial of efficacy.
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- 2017
23. Safety and Immunogenicity of Live Oral Cholera Vaccine Candidate CVD 110, a ΔctxA ctxAzot zotace Derivative of El Tor Ogawa Vibrio cholerae
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Myron M. Levine, Genevieve Losonsky, Stanley J. Cryz, Jane Michalski, Alessio Fasano, James P. Nataro, Carol O. Tacket, Robert R. Edelman, and James B. Kaper
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biology ,Cholera toxin ,Enterotoxin ,medicine.disease ,biology.organism_classification ,medicine.disease_cause ,Cholera ,El Tor ,Virology ,Microbiology ,Vaccination ,Infectious Diseases ,Vibrionaceae ,Vibrio cholerae ,medicine ,Immunology and Allergy ,Cholera vaccine - Abstract
The current pandemic of cholera is caused primarily by Vibrio cholerae O1 of the El Tor biotype. Live attenuated classical biotype V. cholerae vaccine strains prevent severe and moderate cholera due to either biotype in challenged volunteers but may provide less protection against mild cholera due to El Tor organisms. CVD 110, a new ctxA-deleted vaccine strain derived from an El Tor Ogawa parent, lacks zona occludens toxin (Zot), accessory cholera enterotoxin (Ace), and hemolysin/enterotoxin. Ten healthy adult volunteers were given 10(8) cfu of CVD 110 with buffer; 7 developed diarrhea (mean stool volume, 861 mL). Vaccine organisms were shed in stool by all vaccines and were recovered from duodenal fluid in three-quarters of vaccinees. After vaccination, the geometric mean peak reciprocal vibriocidal titer among vaccinees was 17,829. CVD 110 is a powerful immunogen but insufficiently attenuated despite the absence of known potential enterotoxins of V. cholerae. Another unrecognized toxin or colonization alone may be responsible for diarrhea after ingestion of this strain.
- Published
- 2017
24. The Presence of the pAA Plasmid in the German O104:H4 Shiga Toxin Type 2a (Stx2a)–Producing Enteroaggregative Escherichia coli Strain Promotes the Translocation of Stx2a Across an Epithelial Cell Monolayer
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Anne-Marie Hansen, Alison D. O'Brien, Nadia Boisen, Ninell P. Mortensen, Angela R. Melton-Celsa, Tonia Zangari, James B. Kaper, and James P. Nataro
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Genotype ,Virulence Factors ,Fimbria ,Virulence ,Serogroup ,medicine.disease_cause ,Shiga Toxin 2 ,Bacterial Adhesion ,Cell Line ,Microbiology ,Plasmid ,Germany ,Escherichia coli ,medicine ,Humans ,Immunology and Allergy ,Escherichia coli Infections ,biology ,Escherichia coli Proteins ,Interleukin-8 ,Epithelial Cells ,Shiga toxin ,biology.organism_classification ,Enterobacteriaceae ,Virology ,Bacterial adhesin ,Protein Transport ,Infectious Diseases ,Enteroaggregative Escherichia coli ,Trans-Activators ,biology.protein ,Gene Deletion ,Plasmids - Abstract
(See the editorial commentary by Steiner on pages 1860–2.) Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic Escherichia coli that is a cause of acute and persistent diarrhea in many settings [1–7]. EAEC strains express a heterogeneous array of putative virulence factors [8–12] encoded on the bacterial chromosome or on the EAEC-specific pAA plasmid. EAEC strains often harbor a transcriptional activator of the AraC/XylS class, called “AggR” [13], which controls genes on both the plasmid and the chromosome. Among the genes under AggR control include those that encode the aggregative adherence fimbriae (AAF), of which at least 4 variants exist [14–18]. AAF adhesins have been shown to be essential for EAEC adherence to human intestinal explants and to elicit both cytokine release and opening of epithelial tight junctions in a polarized epithelial model [19, 20]. EAEC strains also often harbor a variable number of serine protease autotransporters of Enterobacteriaceae (SPATEs) that are implicated in immune evasion, mucosal damage, secretogenicity, and colonization [21]. In 2011, an outbreak of foodborne hemorrhagic colitis originated in Germany, spreading to other European countries. Over 4000 individuals were affected, including primary and secondary cases [22]. Hemolytic uremic syndrome (HUS) developed in approximately 22% of the cases [22], and 54 people died [23, 24]. The implicated pathogen was an EAEC strain of the rare serotype O104:H4 [23], which was lysogenized with an Stx2a-converting phage. Genomic analysis [25] demonstrated that the outbreak strain contained the genes required to produce the AAF/I variant and 3 SPATEs (Pic, SigA, and SepA). Although much is known about the pathogenesis of serotype O157:H7 Shiga toxin (Stx)–producing E. coli, it is unclear how an EAEC strain would be able to elicit severe hemorrhagic colitis and HUS, even when harboring an Stx-encoding gene. In this study, we tested the hypothesis that the plasmid-borne virulence factors of EAEC contributed to the high pathogenicity of the German outbreak strain by promoting strong adherence to the epithelium and/or by opening epithelial tight junctions.
- Published
- 2014
25. Su1943 – Long-Term Ehec Infection Stimulates Mucus Production in Human Stem Cell-Derived Colonoids Possibly Via Atoh1 Activation
- Author
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Olga Kovbasnjuk, Anne-Marie Hansen, Karol Dokladny, and James B. Kaper
- Subjects
ATOH1 ,Hepatology ,Mucus production ,Gastroenterology ,biology.protein ,Biology ,Stem cell ,Term (time) ,Cell biology - Published
- 2019
26. Su1012 – Mechanical Stimuli Affect Enterotoxigenic E. Coli (ETEC) Heat Stable Enterotoxin (ST)-Cyclic Nucleotide Signaling by Altering Mrp4 Expression in a Human Enteroid Monolayer Model of Etec Diarrhea
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Jianyi Yin, Mark Donowitz, Katia Karalis, Magdalena Kasendra, James B. Kaper, and Laxmi Sunuwar
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Enterotoxigenic E. coli ,Cyclic nucleotide ,chemistry.chemical_compound ,Diarrhea ,Hepatology ,chemistry ,Monolayer ,Gastroenterology ,medicine ,Heat-stable enterotoxin ,medicine.symptom ,Microbiology - Published
- 2019
27. Editorial: Infection and immunity research at the University of Maryland, Baltimore
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James B. Kaper, Harry L. T. Mobley, and Martin F. Flajnik
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0301 basic medicine ,Microbiology (medical) ,Gerontology ,Microbial pathogenesis ,Infectious Disease Medicine ,General Immunology and Microbiology ,Universities ,business.industry ,Library science ,General Medicine ,Translational Research, Biomedical ,03 medical and health sciences ,030104 developmental biology ,Infectious Diseases ,Infectious disease (medical specialty) ,Allergy and Immunology ,Baltimore ,Immunology and Allergy ,Medicine ,Humans ,business - Abstract
This special issue of Pathogens and Disease focuses on the research in understanding, treating and preventing a variety of infectious diseases that is being conducted at the University of Maryland, Baltimore (UMB). This university features three internationally renowned research institutes founded by three of the most highly cited investigators in the area of microbiology and the immunology of infectious diseases: The Center for Vaccine Development (CVD) (Myron M. Levine, Founder), The Institute of Human Virology (Robert C. Gallo, Founder) and the Institute for Genome Sciences (Claire M. Fraser, Founder). Two academic departments (Microbiology and Immunology in the UM School of Medicine and Microbial Pathogenesis in the UM School of Dentistry) house additional researchers in this area. UMB is located in the Baltimore–Washington corridor, which is intellectually rich with numerous other institutions conducting infectious disease research including the National Institutes of Health, the Johns Hopkins University, Uniformed Services University, the Walter Reed Army Institute of Research, the US Army Medical Research Institute of Infectious Diseases and the … [↵][1]* Corresponding author: Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St. HSF I-Rm 380, Baltimore, MD 21201, USA. Tel: +410-706-2344; Fax: +410-706-6970; E-mail: jkaper{at}medicine.umaryland.edu [1]: #xref-corresp-1-1
- Published
- 2016
28. Oral Cholera Vaccines
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Jan Holmgren and James B. Kaper
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business.industry ,Medicine ,Cholera vaccine ,business ,Virology - Published
- 2016
29. Enterohemorrhagic
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Julie, In, Jennifer, Foulke-Abel, Nicholas C, Zachos, Anne-Marie, Hansen, James B, Kaper, Harris D, Bernstein, Marc, Halushka, Sarah, Blutt, Mary K, Estes, Mark, Donowitz, and Olga, Kovbasnjuk
- Subjects
MUC2, extracellular mucin 2 ,SPATE, serine protease autotransporters of Enterobacteriaceae ,PBS, phosphate-buffered saline ,PCDH24, protocadherin 24 ,HCM, human colonoid monolayers ,Microvillar Effacement ,IEC, intestinal epithelial cell ,TER, transepithelial electrical resistance ,NHE2, sodium-hydrogen exchanger isoform 2 ,PCR, polymerase chain reaction ,CM, complete medium ,CCS, cold chelating solution ,EM, expansion medium ,Original Research ,Serine Protease EspP ,EHEC, enterohemorrhagic Escherichia coli ,LGR5, leucine-rich repeat containing G protein-coupled receptor 5 ,MLPCDH, mucin-like protocadherin ,3D, three dimensional ,TBS, Tris-buffered saline ,HUS, hemolytic uremic syndrome ,TJ, tight junction ,Human Colonoid Monolayers ,NHERF3, sodium-hydrogen exchanger regulatory factor 3 ,BB, brush border ,Stx, Shiga toxins ,Intestinal Organoids ,A/E, attaching and effacing - Abstract
Background & Aims Enterohemorrhagic Escherichia coli (EHEC) causes over 70,000 episodes of foodborne diarrhea annually in the United States. The early sequence of events that precede life-threatening hemorrhagic colitis and hemolytic uremic syndrome is not fully understood due to the initial asymptomatic phase of the disease and the lack of a suitable animal model. We determined the initial molecular events in the interaction between EHEC and human colonic epithelium. Methods Human colonoids derived from adult proximal colonic stem cells were developed into monolayers to study EHEC-epithelial interactions. Monolayer confluency and differentiation were monitored by transepithelial electrical resistance measurements. The monolayers were apically infected with EHEC, and the progression of epithelial damage over time was assessed using biochemical and imaging approaches. Results Human colonoid cultures recapitulate the differential protein expression patterns characteristic of the crypt and surface colonocytes. Mucus-producing differentiated colonoid monolayers are preferentially colonized by EHEC. Upon colonization, EHEC forms characteristic attaching and effacing lesions on the apical surface of colonoid monolayers. Mucin 2, a main component of colonic mucus, and protocadherin 24 (PCDH24), a microvillar resident protein, are targeted by EHEC at early stages of infection. The EHEC-secreted serine protease EspP initiates brush border damage through PCDH24 reduction. Conclusions Human colonoid monolayers are a relevant pathophysiologic model that allow the study of early molecular events during enteric infections. Colonoid monolayers provide access to both apical and basolateral surfaces, thus providing an advantage over three-dimensional cultures to study host–pathogen interactions in a controllable and tractable manner. EHEC reduces colonic mucus and affects the brush border cytoskeleton in the absence of commensal bacteria.
- Published
- 2016
30. Genomic diversity of EPEC associated with clinical presentations of differing severity
- Author
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James P. Nataro, Anita K. M. Zaidi, Boubou Tamboura, Anowar Hossain, Shahida Qureshi, Michael S. Donnenberg, David A. Rasko, Karen L. Kotloff, John B. Ochieng, Thandavarayan Ramamurthy, Martin Antonio, Farheen Quadri, Tracy H. Hazen, James B. Kaper, Myron M. Levine, Inacio Mandomando, Sandra Panchalingam, and Eileen M. Barry
- Subjects
Diarrhea ,0301 basic medicine ,Microbiology (medical) ,Virulence Factors ,030106 microbiology ,Immunology ,Virulence ,Biology ,digestive system ,Applied Microbiology and Biotechnology ,Microbiology ,Asymptomatic ,Article ,Virulence factor ,Pilus ,Enteropathogenic Escherichia coli ,03 medical and health sciences ,Plasmid ,parasitic diseases ,Genetics ,medicine ,Humans ,Pathogen ,Escherichia coli Infections ,Phylogeny ,Comparative genomics ,Genetic Variation ,Genomics ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,030104 developmental biology ,Genes, Bacterial ,bacteria ,medicine.symptom - Abstract
Enteropathogenic Escherichia coli (EPEC) are diarrhoeagenic E. coli, and are a significant cause of gastrointestinal illness among young children in developing countries. Typical EPEC are identified by the presence of the bundle-forming pilus encoded by a virulence plasmid, which has been linked to an increased severity of illness, while atypical EPEC lack this feature. Comparative genomics of 70 total EPEC from lethal (LI), non-lethal symptomatic (NSI) or asymptomatic (AI) cases of diarrhoeal illness in children enrolled in the Global Enteric Multicenter Study was used to investigate the genomic differences in EPEC isolates obtained from individuals with various clinical outcomes. A comparison of the genomes of isolates from different clinical outcomes identified genes that were significantly more prevalent in EPEC isolates of symptomatic and lethal outcomes than in EPEC isolates of asymptomatic outcomes. These EPEC isolates exhibited previously unappreciated phylogenomic diversity and combinations of virulence factors. These comparative results highlight the diversity of the pathogen, as well as the complexity of the EPEC virulence factor repertoire. Comparative genomics of 70 lethal, non-lethal symptomatic and asymptomatic enteropathogenic Escherichia coli (EPEC) isolates identifies the virulence-associated genes that are significantly more prevalent in symptomatic and lethal infections.
- Published
- 2016
31. The ribosome binding site of a mini-ORF protects a T3SS mRNA from degradation by RNase E
- Author
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Ping-kun Hsieh, Joel G. Belasco, Patricia B. Lodato, and James B. Kaper
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AU-rich element ,Messenger RNA ,Effector ,RNase P ,Protein biosynthesis ,Biology ,Translocon ,Molecular Biology ,Microbiology ,Ribosome ,Molecular biology ,Ribosomal binding site - Abstract
Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.
- Published
- 2012
32. Escherichia, Shigella , and Salmonella
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James P. Nataro, Cheryl A. Bopp, Patricia I. Fields, James B. Kaper, and Nancy A. Strockbine
- Published
- 2011
33. Functional and Phylogenetic Analysis of ureD in Shiga Toxin-Producing Escherichia coli
- Author
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David A. Rasko, James B. Kaper, and Susan R. Steyert
- Subjects
Molecular Sequence Data ,Mutation, Missense ,Biology ,medicine.disease_cause ,Polymorphism, Single Nucleotide ,Microbiology ,Bacterial Proteins ,medicine ,Humans ,Missense mutation ,Molecular Biology ,Pathogen ,Escherichia coli ,Phylogeny ,chemistry.chemical_classification ,Genetics ,Shiga-Toxigenic Escherichia coli ,Escherichia coli Proteins ,Point mutation ,biology.organism_classification ,Urease ,Stop codon ,Amino acid ,chemistry ,Multilocus sequence typing ,Population Genetics and Evolution ,Bacteria - Abstract
Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG , the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD , encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro . We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.
- Published
- 2011
34. Determination of spatial and temporal colonization of enteropathogenicE. coliand enterohemorrhagicE. coliin mice using bioluminescent in vivo imaging
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Cara E. Morin, Antoneicka L. Harris, Ki Jong Rhee, Gail Hecht, Hao Cheng, and James B. Kaper
- Subjects
Male ,Microbiology (medical) ,Colon ,Porins ,Human pathogen ,Biology ,digestive system ,Microbiology ,Enteropathogenic Escherichia coli ,Mice ,In vivo ,parasitic diseases ,medicine ,Citrobacter rodentium ,Animals ,Humans ,Luciferases ,Cecum ,Pathogen ,Intimin ,Luminescent Agents ,Enterobacteriaceae Infections ,Gastroenterology ,Bioluminescent bacteria ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,Virology ,Mice, Inbred C57BL ,Disease Models, Animal ,Diarrhea ,Infectious Diseases ,Enterohemorrhagic Escherichia coli ,Luminescent Measurements ,bacteria ,medicine.symptom ,Ex vivo ,Research Paper ,Plasmids - Abstract
Infectious diarrhea is a major contributor of child morbidity and mortality in developing nations. Murine models to study the pathogenesis of infectious diarrhea caused by organisms such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are not fully characterized. More emphasis has been placed on infection of mice with the murine specific pathogen Citrobacter rodentium. While these three organisms are genetically related they are not identical. Our goal was to better characterize the murine model of EPEC and EHEC infection by using bioluminescent bacteria to determine temporal and spatial colonization of these two human pathogens. EPEC and EHEC were transformed with a bacterial luciferase expression plasmid containing the constitutive OmpC promoter. C57BL/6 mice were orally inoculated with bioluminescent EPEC or EHEC and bacterial localization in the intestine was monitored ex vivo and in vivo by IVIS. At 3 days after infection, EPEC, EHEC and Citrobacter rodentium were all localized in the cecum and colon. EPEC colonization peaked at day 2-3 and was undetectable by day 7. The bioluminescent EPEC adheres to the cecum and colon of the mouse intestine. However, when EPEC infected mice were administered xylazine/ketamine for in vivo live imaging, the EPEC persisted at high densities for up to 31 days. This is the first report of a bioluminescent imaging of luciferase expressing EPEC in a mouse model.
- Published
- 2011
35. The LysR-Type Transcriptional Regulator QseD Alters Type Three Secretion in Enterohemorrhagic Escherichia coli and Motility in K-12 Escherichia coli
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Jennifer Smart, Vanessa Sperandio, Benjamin J. Habdas, and James B. Kaper
- Subjects
Molecular Biology of Pathogens ,Gene isoform ,Escherichia coli K12 ,Transcription, Genetic ,biology ,Operon ,Escherichia coli Proteins ,Quorum Sensing ,Repressor ,Shiga toxin ,Gene Expression Regulation, Bacterial ,Saccharomyces cerevisiae ,medicine.disease_cause ,Microbiology ,Molecular biology ,Repressor Proteins ,Enterohemorrhagic Escherichia coli ,medicine ,Transcriptional regulation ,biology.protein ,Autoinducer ,Molecular Biology ,Escherichia coli ,Locus of enterocyte effacement - Abstract
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 responds to the host-produced epinephrine and norepinephrine, and bacterially produced autoinducer 3 (AI-3), through two-component systems. Further integration of multiple regulatory signaling networks, involving regulators such as the LysR-type transcriptional regulator (LTTR) QseA, promotes effective regulation of virulence factors. These include the production of flagella, a phage-encoded Shiga toxin, and genes within the locus of enterocyte effacement (LEE) responsible for attaching and effacing (AE) lesion formation. Here, we describe a new member of this signaling cascade, an LTTR heretofore renamed QseD (quorum-sensing E. coli regulator D). QseD is present in all enterobacteria but exists almost exclusively in O157:H7 isolates as a helix-turn-helix (HTH) truncated isoform. This “short” isoform (sQseD) is still able to regulate gene expression through a different mechanism than the full-length K-12 E. coli “long” QseD isoform (lQseD). The EHEC Δ qseD mutant exhibits increased expression of all LEE operons and deregulation of AE lesion formation. The loss of qseD in EHEC does not affect motility, but the K-12 Δ qseD mutant is hypermotile. While the lQseD directly binds to the ler promoter, encoding the LEE master regulator, to repress LEE transcription, the sQseD isoform does not. LTTRs bind to DNA as tetramers, and these data suggest that sQseD regulates ler by forming heterotetramers with another LTTR. The LTTRs known to regulate LEE transcription, QseA and LrhA, do not interact with sQseD, suggesting that sQseD acts as a dominant-negative partner with a yet-unidentified LTTR.
- Published
- 2010
36. 789 - Enteric Pathogens Induce M Cell Transcytosis in a Human Enteroid Model of the Follicle Associated Epithelium
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Eileen M. Barry, Michele Doucet, Nicholas C. Zachos, Olga Kovbasnjuk, James B. Kaper, Sridevi Ranganathan, and Janet F. Staab
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Hepatology ,Transcytosis ,Gastroenterology ,Biology ,Follicle associated epithelium ,Cell biology - Published
- 2018
37. 267 - Rho Gtpase Inhibition Contributes to Enteropathogenic Escherichia Coli Esph-Induced Desmosomal Perturbations
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Ross Monasky, Al Agellon, Asad Mansoor, V. K. Viswanathan, Gayatri Vedantam, Jennifer Lising Roxas, Bryan Roxas, and James B. Kaper
- Subjects
Hepatology ,Chemistry ,Gastroenterology ,GTPase ,Enteropathogenic Escherichia coli ,Cell biology - Published
- 2018
38. Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation
- Author
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Jean Marc Gabastou, Sophie Kernéis, James B. Kaper, Marie Françoise Bernet-Camard, Alain L. Servin, Marie Helene Coconnier, and Alain Barbat
- Subjects
Microscopy, Electron, Scanning Transmission ,Cancer Research ,Enterocyte ,Cellular differentiation ,Cell ,Down-Regulation ,Biology ,digestive system ,Article ,Bacterial Adhesion ,Epithelium ,Microbiology ,medicine ,Escherichia coli ,Tumor Cells, Cultured ,Intestinal epithelial cell differentiation ,Animals ,Humans ,Enteropathogenic Escherichia coli ,Molecular Biology ,Epithelial cell differentiation ,Microvilli ,Carcinoma ,Cell Differentiation ,Epithelial Cells ,Cell Biology ,Embryonic stem cell ,Up-Regulation ,Intestines ,medicine.anatomical_structure ,Cell culture ,Colonic Neoplasms ,Microscopy, Electron, Scanning ,Rabbits ,Developmental Biology - Abstract
Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc−/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and tranmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc−/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occured in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated. Our results provide evidence that the intestinal cell differentiation could play a dual role in EPEC pathogenesis: it up-regulates intestinal cell colonization and down-regulates intestinal cell invasion.
- Published
- 2009
39. Hfq affects the expression of the LEE pathogenicity island in enterohaemorrhagicEscherichia coli
- Author
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James B. Kaper and Anne-Marie Hansen
- Subjects
Genetics ,Genomic Islands ,Escherichia coli Proteins ,Mutant ,Wild type ,Repressor ,Virulence ,Gene Expression Regulation, Bacterial ,Host Factor 1 Protein ,biochemical phenomena, metabolism, and nutrition ,Biology ,Phosphoproteins ,bacterial infections and mycoses ,Microbiology ,Pathogenicity island ,Article ,Virulence factor ,Repressor Proteins ,RNA, Bacterial ,Enterohemorrhagic Escherichia coli ,Trans-Activators ,RNA Processing, Post-Transcriptional ,Molecular Biology ,Gene ,Locus of enterocyte effacement - Abstract
Colonization of the intestinal epithelium by enterohaemorrhagic Escherichia coli (EHEC) is characterized by an attaching and effacing (A/E) histopathology. The locus of enterocyte effacement (LEE) pathogenicity island encodes many genes required for the A/E phenotype including the global regulator of EHEC virulence gene expression, Ler. The LEE is subject to a complex regulatory network primarily targeting ler transcription. The RNA chaperone Hfq, implicated in post-transcriptional regulation, is an important virulence factor in many bacterial pathogens. Although post-transcriptional regulation of EHEC virulence genes is known to occur, a regulatory role of Hfq in EHEC virulence gene expression has yet to be defined. Here, we show that an hfq mutant expresses increased levels of LEE-encoded proteins prematurely, leading to earlier A/E lesion formation relative to wild type. Hfq indirectly affects LEE expression in exponential phase independent of Ler by negatively controlling levels of the regulators GrlA and GrlR through post-transcriptional regulation of the grlRA messenger. Moreover, Hfq negatively affects LEE expression in stationary phase independent of GrlA and GrlR. Altogether, Hfq plays an important role in co-ordinating the temporal expression of the LEE by controlling grlRA expression at the post-transcriptional level.
- Published
- 2009
40. TheEscherichia coli ycbQRSToperon encodes fimbriae with laminin-binding and epithelial cell adherence properties in Shiga-toxigenicE. coliO157:H7
- Author
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Juan Xicohtencatl-Cortes, Partha Samadder, Jorge A. Girón, James B. Kaper, Phillip I. Tarr, Zeus Saldaña, and Dianna M. Murphy Jordan
- Subjects
Swine ,Operon ,Molecular Sequence Data ,Fimbria ,Biology ,Flagellum ,medicine.disease_cause ,Microbiology ,Article ,Bacterial Adhesion ,Cell Line ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Laminin binding ,Escherichia coli ,Peptide sequence ,Ecology, Evolution, Behavior and Systematics ,Sequence Homology, Amino Acid ,Shiga-Toxigenic Escherichia coli ,Escherichia coli Proteins ,Epithelial Cells ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,Molecular biology ,Bacterial adhesin ,Fimbrin ,bacteria ,Cattle ,Fimbriae Proteins ,Laminin ,Protein Binding - Abstract
The human pathogen Shiga-toxigenic Escherichia coli (STEC) O157:H7 contains a ycbQRST fimbrial-like operon, which shares significant homology to the family of F17 fimbrial biogenesis genes f17ADCG found in enterotoxigenic E. coli. We report that growth of STEC O157:H7 strain EDL933 in minimal Minca medium at 37°C and during adherence to epithelial cells led to the production of fine peritrichous fimbriae, which were found to be composed of a major subunit of 18 kDa whose N-terminal amino acid sequence matched the predicted protein product of the ycbQ gene; and showed significant homology to the F17a-A fimbrin. Similar to the F17 fimbriae, the purified STEC fimbriae and the recombinant YcbQ protein fused to a His peptide tag bound laminin, but not fibronectin or collagen. Thus, we propose the name E. coli YcbQ laminin-binding fimbriae (ELF) to designate the fimbriae encoded by the ycbQRST operon. The role of ELF as an adherence factor of STEC to cultured epithelial cells was investigated. We provide compelling evidence demonstrating that ELF contributes to adherence of STEC to human intestinal epithelial cells and to cow and pig gut tissue in vitro. Deletion in the fimbrin subunit gene elfA (or ycbQ) in STEC strain EDL933 led to an isogenic strain, which showed significant reduction (60%) in adherence to HEp-2 cells in comparison with the parental strain. In addition, antibodies against the purified ELF also partially blocked adherence of two STEC O157:H7 strains. These observations suggest that ELF functions as an accessory adherence factor that, along with other known redundant adhesins, contributes to the overall adhesive properties of STEC O157:H7 providing these organisms with ecological advantages to survive in different hosts and in the environment.
- Published
- 2009
41. Theex vivoresponse of human intestinal mucosa to enteropathogenicEscherichia coliinfection
- Author
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Jorge A. Girón, James B. Kaper, Alan D. Phillips, Stephanie Schüller, and Mark Lucas
- Subjects
Immunology ,Organ culture ,Microbiology ,Article ,Enteropathogenic Escherichia coli ,Organ Culture Techniques ,Intestinal mucosa ,Virology ,medicine ,Humans ,Intestinal Mucosa ,Epithelial polarity ,biology ,Escherichia coli Proteins ,Gene Expression Profiling ,Interleukin-8 ,biochemical phenomena, metabolism, and nutrition ,Epithelium ,Toll-Like Receptor 5 ,medicine.anatomical_structure ,TLR5 ,biology.protein ,bacteria ,Gene Deletion ,Flagellin ,Ex vivo - Abstract
In vitro organ culture (IVOC) represents a gold standard model to study enteropathogenic E. coli (EPEC) infection of human intestinal mucosa. However, the optimal examination of the bacterial-host cell interaction requires a directional epithelial exposure, without serosal or cut surface stimulation. A polarized IVOC system (pIVOC) was developed in order to overcome such limitations: apical EPEC infection produced negligible bacterial leakage via biopsy edges, resulted in enhanced colonization compared with standard IVOC, and showed evidence of bacterial detachment, as in natural rabbit EPEC infections. Examination of mucosal innate immune responses in pIVOC showed both interleukin (IL)-8 mRNA and protein levels were significantly increased after apical EPEC infection. Increased IL-8 levels mainly depended on flagellin expression as fliC-negative EPEC did not elicit a significant IL-8 response despite increased mucosal colonization compared with wild-type EPEC. In addition, apical application of purified flagella significantly increased IL-8 protein levels over non-infected controls. Immunofluorescence staining of EPEC-infected small intestinal biopsies revealed apical and basolateral distribution of Toll-like receptor (TLR) 5 on epithelium, suggesting that EPEC can trigger mucosal IL-8 responses by apical flagellin/TLR5 interaction ex vivo and does not require access to the basolateral membrane as postulated in cell culture models.
- Published
- 2009
42. An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per
- Author
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Carl Brinkley, Marie-Pierre Castanie-Cornet, Sooan Shin, J. Adam Crawford, John W. Foster, and James B. Kaper
- Subjects
Plasmid ,Regulon ,Virulence ,Promoter ,Biology ,Molecular Biology ,Microbiology ,Molecular biology ,Pathogenicity island ,Derepression ,Locus of enterocyte effacement ,Intimin - Abstract
Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.
- Published
- 2008
43. Genetic Diversity of the Gene Cluster Encoding Longus, a Type IV Pilus of Enterotoxigenic Escherichia coli
- Author
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Evgeni V. Sokurenko, Oscar G. Gómez-Duarte, Jorge A. Girón, Scott J. Weissman, Sujay Chattopadhyay, and James B. Kaper
- Subjects
DNA, Bacterial ,Sequence analysis ,Bacterial Toxins ,Molecular Sequence Data ,Fimbria ,medicine.disease_cause ,Microbiology ,Pilus ,Enterotoxigenic Escherichia coli ,Gene cluster ,medicine ,Molecular Biology ,Escherichia coli ,Genetics ,Sequence Homology, Amino Acid ,biology ,Escherichia coli Proteins ,Genetic Variation ,Sequence Analysis, DNA ,musculoskeletal system ,biology.organism_classification ,Enterobacteriaceae ,Fimbriae, Bacterial ,Multigene Family ,Horizontal gene transfer ,Population Genetics and Evolution - Abstract
Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.
- Published
- 2007
44. The possible influence of LuxS in the in vivo virulence of rabbit enteropathogenic Escherichia coli
- Author
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Edgar C. Boedeker, Venessa Sperandio, James B. Kaper, Chengru Zhu, Timothy E. Thate, Shuzhang Feng, and Zhuolu Yang
- Subjects
DNA, Bacterial ,Male ,Mutant ,Virulence ,Biology ,medicine.disease_cause ,Microbiology ,Article ,Bacterial Adhesion ,Enteropathogenic Escherichia coli ,Feces ,Lactones ,Bacterial Proteins ,Intestinal mucosa ,Homoserine ,medicine ,Animals ,Humans ,Intestinal Mucosa ,Escherichia coli ,Escherichia coli Infections ,General Veterinary ,Quorum Sensing ,General Medicine ,biochemical phenomena, metabolism, and nutrition ,Pathogenicity island ,Carbon-Sulfur Lyases ,Intestinal Diseases ,Mutagenesis, Insertional ,Quorum sensing ,bacteria ,Autoinducer ,Rabbits ,HeLa Cells - Abstract
Attaching and effacing (A/E) organisms, such as rabbit enteropathogenic Escherichia coli (EPEC), human EPEC or enterohemorrhagic E. coli (EHEC) share attaching and effacing phenotype and LEE pathogenicity island responsible for A/E. The present study was undertaken to investigate the impact of the LuxS quorum sensing (QS) signaling system in vitro and in vivo pathogenicity of A/E organisms using rabbit EPEC (rEPEC) strain E22 (O103:H2). Analysis of the bioluminescence indicated abolished production of the QS signal AI-2 by luxS mutant (E22DeltaluxS). Strain E22Deltalux also exhibited impaired expression of several normally secreted proteins and reduced adherence to cultured HeLa cells. Complementation of the intact luxS gene to E22DeltaluxS restored secreted protein expression comparable to the WT type but not adherence to HeLa cells. In experimentally infected rabbits, the isogenic luxS mutant induced clinical illness and intimate adherence to the intestinal mucosa, albeit to a less extent, comparable to that seen with the parent virulent strain. It is worth noting that reduced fecal bacterial shedding, mucosal adherence and improved cumulative weight gain were seen for the mutant strain complemented with luxS when compared to the WT. It appears that the luxS gene is not essential for in vivo pathogenicity by rEPEC where exogenous QS signals are present in the gut. The impact of AI-2 provided by multicopy plasmid on bacterial virulence is discussed.
- Published
- 2007
45. Shiga toxin binding in normal and inflamed human intestinal mucosa
- Author
-
Alan D. Phillips, James B. Kaper, Robert Heuschkel, Stephanie Schüller, and Franco Torrente
- Subjects
Paneth Cells ,Immunology ,Inflammation ,Shiga Toxin 1 ,Shiga Toxin 2 ,Microbiology ,chemistry.chemical_compound ,Shiga-like toxin ,Intestinal mucosa ,medicine ,Humans ,Antigens, Tumor-Associated, Carbohydrate ,Intestinal Mucosa ,Receptor ,biology ,Shiga toxin ,Inflammatory Bowel Diseases ,Intestinal epithelium ,Epithelium ,Infectious Diseases ,medicine.anatomical_structure ,chemistry ,Paneth cell ,biology.protein ,medicine.symptom - Abstract
Shiga toxins are associated with haemolytic uraemic syndrome but human intestinal epithelium does not express the Gb3 receptor. We describe Gb3 expression and Shiga toxin binding in histologically normal intestine and demonstrate that the pattern is unaltered in inflammatory disease states. Gb3 expression and Shiga toxin binding were identified in Paneth cells in both normal and inflamed mucosae.
- Published
- 2007
46. RNA-Seq analysis of isolate- and growth phase-specific differences in the global transcriptomes of enteropathogenic Escherichia coli prototype isolates
- Author
-
Tracy H Hazen, Sean C Daughtery, Amol eShetty, Anup A Mahurkar, Owen eWhite, James B Kaper, and David A Rasko
- Subjects
Microbiology (medical) ,RNA-sequencing ,lcsh:QR1-502 ,Virulence ,RNA-Seq ,Pathogenesis ,Biology ,digestive system ,Microbiology ,lcsh:Microbiology ,Pilus ,diversity ,Transcriptome ,Enteropathogenic Escherichia coli ,parasitic diseases ,Gene expression ,Gene ,Original Research ,1. No poverty ,regulation ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,3. Good health ,bacteria ,Locus of enterocyte effacement - Abstract
Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness among infants in developing countries. E. coli isolates classified as typical EPEC are identified by the presence of the locus of enterocyte effacement (LEE) and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin. Comparative genomic analyses have demonstrated that EPEC isolates belong to diverse evolutionary lineages and possess lineage- and isolate-specific genomic content. To investigate whether this genomic diversity results in significant differences in global gene expression, we used an RNA sequencing (RNA-Seq) approach to characterize the global transcriptomes of the prototype typical EPEC isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate E110019. The global transcriptomes were characterized during laboratory growth in two different media and three different growth phases, as well as during adherence of the EPEC isolates to human cells using in vitro tissue culture assays. Comparison of the global transcriptomes during these conditions was used to identify isolate- and growth phase-specific differences in EPEC gene expression. These analyses resulted in the identification of genes that encode proteins involved in survival and metabolism that were coordinately expressed with virulence factors. These findings demonstrate there are isolate- and growth phase-specific differences in the global transcriptomes of EPEC prototype isolates, and highlight the utility of comparative transcriptomics for identifying additional factors that are directly or indirectly involved in EPEC pathogenesis.
- Published
- 2015
47. Escherichia,Shigella, andSalmonella
- Author
-
Nancy A. Strockbine, Cheryl A. Bopp, Patricia I. Fields, James B. Kaper, and James P. Nataro
- Subjects
Serotype ,Salmonella ,biology ,Escherichia ,medicine ,Virulence ,Shigella ,Mobile genetic elements ,biology.organism_classification ,medicine.disease_cause ,Escherichia coli ,Shigella boydii ,Microbiology - Abstract
Members of the genus Shigella are phenotypically similar to Escherichia coli and, with the exception of Shigella boydii serotype 13, would be considered the same species by DNA-DNA hybridization analysis and whole-genome sequence analysis. The dynamic nature of the Shiga toxin-converting phages has implications for diagnostic testing for Shiga toxin-producing E. coli (STEC). Since STEC strains can lose critical virulence genes, some researchers have proposed that multiple virulence-associated genes, as well as conserved genes, be used to diagnose infections by these bacteria. This concept would also apply to other pathotypes of E. coli, as most of them carry critical virulence genes on mobile genetic elements. A preliminary report can be issued as soon as a presumptive identification of Salmonella is obtained. In most situations, a presumptive identification is based on phenotypic traits determined by either traditional or commercial systems or by reactivity with Salmonella O grouping antisera. A confirmed identification requires both phenotypic identification and O group or serotype determination. As national surveillance systems depend on the receipt of serotype information for Salmonella strains isolated in the United States, laboratories should follow the procedures recommended by their state health departments for submitting Salmonella isolates for further characterization, including complete serotyping. The antimicrobial susceptibilities of typhoidal Salmonella strains and strains from normally sterile sites should be determined, and the strains should be forwarded to a reference or public health laboratory for complete phenotypic identification and serotyping.
- Published
- 2015
48. Overview and Historical Perspectives
- Author
-
James B. Kaper and Alison D. O'Brien
- Subjects
0303 health sciences ,03 medical and health sciences ,030306 microbiology ,030304 developmental biology ,3. Good health - Published
- 2015
49. Identification and characterization of 'pathoadaptive mutations' of the cadBA operon in several intestinal Escherichia coli
- Author
-
Lothar H. Wieler, James B. Kaper, Joerg Jores, Christopher B. Tutt, Alfredo G. Torres, and Sylke Wagner
- Subjects
Microbiology (medical) ,Amino Acid Transport Systems ,Carboxy-Lyases ,Operon ,Virulence ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Microbiology ,Antiporters ,Bacterial Adhesion ,Plasmid ,Escherichia coli ,medicine ,Shigella ,Enteroinvasive Escherichia coli ,Genetics ,Lysine decarboxylase ,Escherichia coli Proteins ,Lysine ,Nucleic Acid Hybridization ,General Medicine ,Intestines ,Complementation ,Infectious Diseases ,Genes, Bacterial ,Mutation - Abstract
The dysenteric Shigella spp. and enteroinvasive Escherichia coli (EIEC) have evolved from commensal E. coli by the acquisition of a virulence plasmid and inactivation of genes of the cad locus encoding lysine decarboxylase (LDC) by so-called pathoadaptive mutation. As horizontal gene transfer and recombination occurs frequently in E. coli we were interested to see if similar pathoadaptive mutations are commonly present in other intestinal pathotypes. Therefore, we examined 140 intestinal E. coli strains of various pathotypes and the ECOR collection for their ability to decarboxylate lysine, and identified 25 strains that were unable to do so. Complementation of a Shiga toxin-producing E. coli and two enteropathogenic E. coli strains, both LDC-negative, with the intact cad locus restored LDC activity and resulted in a reduction in adherence to tissue culture cells. We investigated the cad locus for possible alterations by using hybridization and PCR techniques and compared the results with the alterations reported for Shigella spp. and EIEC strains. Interestingly, the alterations of the cad genes were similar to those previously reported, pointing towards a parallel evolution of LDC silencing in different intestinal E. coli pathotypes.
- Published
- 2006
50. Nucleotide Sequence Analysis of the Enteropathogenic Escherichia coli Adherence Factor Plasmid pMAR7
- Author
-
Adam T. Boutin, Rogéria Keller, Valerie Burland, Debra J. Rose, Carl Brinkley, Frederick R. Blattner, Sara A. Klink, and James B. Kaper
- Subjects
Molecular Sequence Data ,Immunology ,Virulence ,Molecular Genomics ,medicine.disease_cause ,Microbiology ,Bacterial Adhesion ,Pilus ,Plasmid ,Escherichia coli ,medicine ,Enteropathogenic Escherichia coli ,Gene ,Genetics ,Base Sequence ,biology ,Escherichia coli Proteins ,Nucleic acid sequence ,Sequence Analysis, DNA ,biology.organism_classification ,Enterobacteriaceae ,Infectious Diseases ,Genes, Bacterial ,Conjugation, Genetic ,Fimbriae, Bacterial ,Parasitology ,Fimbriae Proteins ,Plasmids - Abstract
The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per . Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. IS Ec13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.
- Published
- 2006
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