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Vijay-Kumar M, Bovilla VR, Yeoh BS, Golonka RM, Saha P, Joe B, Gewirtz AT. Bacterial flagellin is a dominant, stable innate immune activator in the gastrointestinal contents of mice and rats. Gut Microbes 2023; 15:2185031. [PMID: 36880647 PMCID: PMC10012918 DOI: 10.1080/19490976.2023.2185031] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/12/2022] [Accepted: 02/21/2023] [Indexed: 03/08/2023] Open
Abstract
Intestinal contents comprise the largest repository of immunogenic ligands of microbial origin. We undertook this study to assess the predominant microbe-associated molecular patterns (MAMPs) present therein and the receptors) that mediate the innate immune responses to them. Here, we demonstrated that intestinal contents from conventional, but not germ-free, mice and rats triggered robust innate immune responses in vitro and in vivo. Such immune responses were abrogated in the absence of either myeloid differentiation factor 88 (MyD88) or Toll-like receptor (TLR) 5, but not TLR4, suggesting that the stimuli was flagellin (i.e., protein subunit of flagella that drives bacterial motility). Accordingly, pre-treating intestinal extracts with proteinase, thereby degrading flagellin, was sufficient to block their ability to activate innate immune responses. Taken together, this work serves to underscore flagellin as a major, heat-stable and bioactive MAMP in the intestinal content that confers this milieu strong potential to trigger innate immune responses.
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Affiliation(s)
- Matam Vijay-Kumar
- UT Microbiome Consortium, Department of Physiology & Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
| | - Venugopal R. Bovilla
- UT Microbiome Consortium, Department of Physiology & Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
| | - Beng San Yeoh
- UT Microbiome Consortium, Department of Physiology & Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
| | - Rachel M. Golonka
- UT Microbiome Consortium, Department of Physiology & Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
| | - Piu Saha
- UT Microbiome Consortium, Department of Physiology & Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
| | - Bina Joe
- UT Microbiome Consortium, Department of Physiology & Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
| | - Andrew T. Gewirtz
- Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA
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Fulde M, van Vorst K, Zhang K, Westermann AJ, Busche T, Huei YC, Welitschanski K, Froh I, Pägelow D, Plendl J, Pfarrer C, Kalinowski J, Vogel J, Valentin-Weigand P, Hensel M, Tedin K, Repnik U, Hornef MW. SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo. Gut Microbes 2022; 13:1973836. [PMID: 34542008 PMCID: PMC8475570 DOI: 10.1080/19490976.2021.1973836] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.
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Affiliation(s)
- Marcus Fulde
- Department of Veterinary Medicine, Freie Universität Berlin, Institute of Microbiology and Epizootics, Berlin, Germany,CONTACT Mathias Hornef Institute for Medical Microbiology; RWTH University Hospital; Pauwelsstr. 30, Aachen, D-52074, Germany
| | - Kira van Vorst
- Department of Veterinary Medicine, Freie Universität Berlin, Institute of Microbiology and Epizootics, Berlin, Germany
| | - Kaiyi Zhang
- Institute of Medical Microbiology, Rwth University Hospital Aachen, Aachen, Germany
| | - Alexander J. Westermann
- Institute of Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany,Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany
| | - Tobias Busche
- Technology Platform Genomics, Center for Biotechnology (Cebitec), Bielefeld University, Bielefeld, Germany
| | - Yong Chiun Huei
- Institute of Medical Microbiology, Rwth University Hospital Aachen, Aachen, Germany
| | - Katharina Welitschanski
- Hannover Medical School, Institute for Medical Microbiology and Hospital Epidemiology, Hannover, Germany
| | - Isabell Froh
- Hannover Medical School, Institute for Medical Microbiology and Hospital Epidemiology, Hannover, Germany
| | - Dennis Pägelow
- Department of Veterinary Medicine, Freie Universität Berlin, Institute of Microbiology and Epizootics, Berlin, Germany
| | - Johanna Plendl
- Department of Veterinary Medicine, Freie Universität Berlin, Institute of Veterinary Anatomy, Berlin, Germany
| | - Christiane Pfarrer
- Institute for Anatomy, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
| | - Jörn Kalinowski
- Technology Platform Genomics, Center for Biotechnology (Cebitec), Bielefeld University, Bielefeld, Germany
| | - Jörg Vogel
- Institute of Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany,Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany
| | - Peter Valentin-Weigand
- Institute of Microbiology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
| | - Michael Hensel
- Division of Microbiology, University of Osnabrück, Osnabrück, Germany
| | - Karsten Tedin
- Department of Veterinary Medicine, Freie Universität Berlin, Institute of Microbiology and Epizootics, Berlin, Germany
| | - Urska Repnik
- Department of Biosciences, University of Oslo, Oslo, Norway
| | - Mathias W. Hornef
- Institute of Medical Microbiology, Rwth University Hospital Aachen, Aachen, Germany
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Molecular determinants of peaceful coexistence versus invasiveness of non-Typhoidal Salmonella: Implications in long-term side-effects. Mol Aspects Med 2021; 81:100997. [PMID: 34311996 DOI: 10.1016/j.mam.2021.100997] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2021] [Revised: 07/02/2021] [Accepted: 07/16/2021] [Indexed: 01/28/2023]
Abstract
The genus Salmonella represents a wide range of strains including Typhoidal and Non-Typhoidal Salmonella (NTS) isolates that exhibit illnesses of varied pathophysiologies. The more frequent NTS ensues a self-limiting enterocolitis with rare occasions of bacteremia or systemic infections. These self-limiting Salmonella strains are capable of subverting and dampening the host immune system to achieve a more prolonged survival inside the host system thus leading to chronic manifestations. Notably, emergence of new invasive NTS isolates known as invasive Non-Typhoidal Salmonella (iNTS) have worsened the disease burden significantly in some parts of the world. NTS strains adapt to attain persister phenotype intracellularly and cause relapsing infections. These chronic infections, in susceptible hosts, are also capable of causing diseases like IBS, IBD, reactive arthritis, gallbladder cancer and colorectal cancer. The present understanding of molecular mechanism of how these chronic infections are manifested is quite limited. The current work is an effort to review the prevailing knowledge emanating from a large volume of research focusing on various forms of NTS infections including those that cause localized, systemic and persistent disease. The review will further dwell into the understanding of how this pathogen contributes to the associated long term sequelae.
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Ryan D, Ojha UK, Jaiswal S, Padhi C, Suar M. The Small RNA DsrA Influences the Acid Tolerance Response and Virulence of Salmonella enterica Serovar Typhimurium. Front Microbiol 2016; 7:599. [PMID: 27199929 PMCID: PMC4844625 DOI: 10.3389/fmicb.2016.00599] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2016] [Accepted: 04/11/2016] [Indexed: 01/05/2023] Open
Abstract
The Gram-negative, enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is exposed to various stress conditions during pathogenesis, of which acid stress serves as a major defense mechanism in the host. Such environments are encountered in the stomach and Salmonella containing vacuole of phagocytic and non-phagocytic cells. It is only recently that small RNAs (sRNAs) have come to the forefront as major regulators of stress response networks. Consequently, the sRNA DsrA which regulates acid resistance in Escherichia coli, has not been characterized in the acid tolerance response (ATR) of Salmonella. In this study, we show dsrA to be induced two and threefold under adaptation and challenge phases of the ATR, respectively. Additionally, an isogenic mutant lacking dsrA (ΔDsrA) displayed lower viability under the ATR along with reduced motility, feeble adhesion and defective invasion efficacy in vitro. Expression analysis revealed down regulation of several Salmonella pathogenicity island-1 (SPI-1) effectors in ΔDsrA compared to the wild-type, under SPI-1 inducing conditions. Additionally, our in vivo data revealed ΔDsrA to be unable to cause gut inflammation in C57BL/6 mice at 72 h post infection, although intracellular survival and systemic dissemination remained unaffected. A possible explanation may be the significantly reduced expression of flagellin structural genes fliC and fljB in ΔDsrA, which have been implicated as major proinflammatory determinants. This study serves to highlight the role of sRNAs such as DsrA in both acid tolerance and virulence of S. Typhimurium. Additionally the robust phenotype of non-invasiveness could be exploited in developing SPI-I attenuated S. Typhimurium strains without disrupting SPI-I genes.
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Affiliation(s)
- Daniel Ryan
- Infection Biology Laboratory, School of Biotechnology, KIIT University Bhubaneswar, India
| | - Urmesh K Ojha
- Infection Biology Laboratory, School of Biotechnology, KIIT University Bhubaneswar, India
| | - Sangeeta Jaiswal
- Infection Biology Laboratory, School of Biotechnology, KIIT University Bhubaneswar, India
| | - Chandrashekhar Padhi
- Infection Biology Laboratory, School of Biotechnology, KIIT University Bhubaneswar, India
| | - Mrutyunjay Suar
- Infection Biology Laboratory, School of Biotechnology, KIIT University Bhubaneswar, India
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Fröhlich EE, Mayerhofer R, Holzer P. Reevaluating the hype: four bacterial metabolites under scrutiny. Eur J Microbiol Immunol (Bp) 2015; 5:1-13. [PMID: 25883790 DOI: 10.1556/eujmi-d-14-00030] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2014] [Accepted: 11/06/2014] [Indexed: 12/20/2022] Open
Abstract
With microbiome research being a fiercely contested playground in science, new data are being published at tremendous pace. The review at hand serves to critically revise four microbial metabolites widely applied in research: butyric acid, flagellin, lipoteichoic acid, and propionic acid. All four metabolites are physiologically present in healthy humans. Nevertheless, all four are likewise involved in pathologies ranging from cancer to mental retardation. Their inflammatory potential is equally friend and foe. The authors systematically analyze positive and negative attributes of the aforementioned substances, indicating chances and dangers with the use of pre- and probiotic therapeutics. Furthermore, the widespread actions of microbial metabolites on distinct organs and diseases are reconciled. Moreover, the review serves as critical discourse on scientific methods commonly employed in microbiome research and comparability as well as reproducibility issues arising thereof.
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Affiliation(s)
- E E Fröhlich
- Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical Pharmacology, Medical University of Graz Universitätsplatz 4, 8010 Graz Austria
| | - R Mayerhofer
- Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical Pharmacology, Medical University of Graz Universitätsplatz 4, 8010 Graz Austria
| | - P Holzer
- Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical Pharmacology, Medical University of Graz Universitätsplatz 4, 8010 Graz Austria
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Abstract
The ability to create and maintain a specialized organelle that supports bacterial replication is an important virulence property for many intracellular pathogens. Living in a membrane-bound vacuole presents inherent challenges, including the need to remodel a plasma membrane-derived organelle into a novel structure that will expand and provide essential nutrients to support replication, while also having the vacuole avoid membrane transport pathways that target bacteria for destruction in lysosomes. It is clear that pathogenic bacteria use different strategies to accomplish these tasks. The dynamics by which host Rab GTPases associate with pathogen-occupied vacuoles provide insight into the mechanisms used by different bacteria to manipulate host membrane transport. In this review we highlight some of the strategies bacteria use to maintain a pathogen-occupied vacuole by focusing on the Rab proteins involved in biogenesis and maintenance of these novel organelles.
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Affiliation(s)
- Racquel Kim Sherwood
- Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA
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Enhancement of host immune responses by oral vaccination to Salmonella enterica serovar Typhimurium harboring both FliC and FljB flagella. PLoS One 2013; 8:e74850. [PMID: 24069357 PMCID: PMC3775770 DOI: 10.1371/journal.pone.0074850] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2013] [Accepted: 08/06/2013] [Indexed: 01/17/2023] Open
Abstract
Flagellin, the structural component of the flagellar filament in various motile bacteria, can contribute to the activation of NF-κB and proinflammatory cytokine expression during the innate immune response in host cells. Thus, flagellin proteins represent a particularly attractive target for the development of vaccine candidates. In this study, we investigated the immune response by increasing the flagella number in the iacP mutant strain and the adjuvant activity of the flagellin component FljB of Salmonella enterica serovar Typhimurium. We found that the iacP mutant strain expresses two flagellin proteins (FliC and FljB), which result in increased NF-κB-dependent gene expression in bone marrow derived macrophages. Using an oral immunization mouse model, we observed that the administration of a live attenuated S. typhimurium BRD509 strain expressing the FliC and FljB flagellins induced significantly enhanced flagellin-specific IgG responses in the systemic compartment. The mice immunized with the recombinant attenuated S. typhimurium strain that has two types of flagella were protected from lethal challenge with the Salmonella SL1344 strain. These results indicate that overexpression of flagella in the iacP mutant strain enhance the induction of an antigen-specific immune responses in macrophage cell, and both the FliC and FljB flagellar filament proteins-producing S. typhimurium can induce protective immune responses against salmonellosis.
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Anwar N, Sem XH, Rhen M. Oxidoreductases that act as conditional virulence suppressors in Salmonella enterica serovar Typhimurium. PLoS One 2013; 8:e64948. [PMID: 23750221 PMCID: PMC3672137 DOI: 10.1371/journal.pone.0064948] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2012] [Accepted: 04/19/2013] [Indexed: 11/28/2022] Open
Abstract
In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium.
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Affiliation(s)
- Naeem Anwar
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
| | - Xiao Hui Sem
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore, Republic of Singapore
| | - Mikael Rhen
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
- * E-mail:
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9
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Yoshino Y, Kitazawa T, Ikeda M, Tatsuno K, Yanagimoto S, Okugawa S, Yotsuyanagi H, Ota Y. Clostridium difficile flagellin stimulates toll-like receptor 5, and toxin B promotes flagellin-induced chemokine production via TLR5. Life Sci 2012; 92:211-7. [PMID: 23261530 DOI: 10.1016/j.lfs.2012.11.017] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2012] [Revised: 11/05/2012] [Accepted: 11/24/2012] [Indexed: 02/04/2023]
Abstract
AIMS Clostridium difficile is an important pathogen in nosocomial infections. Although C. difficile toxins are considered to be major virulence factors, pathogenesis of C. difficile associated diseases remains to be determined. In this study, we investigated whether C. difficile flagellin is involved in the pathogenesis of C. difficile-associated diseases. MAIN METHODS C. difficile flagellin was extracted from bacterial body by using a combination of ultracentrifugation and low speed centrifugation. Extracted C. difficile flagellin was added to HEK293T cells transiently transfected with pUNO-mcs (empty vector) or pUNO-hTLR5, and NF-kappaB activation was compared by a dual-luciferase assay. The amount of C. difficile flagellin-induced inflammatory mediators such as interleukin-8 and CCL20 was measured by ELISA assay in the culture media of intestinal epithelial cell lines, HT29 cells and Caco-2 cells. Flagellin induced phosphorylation of p38 mitogen-activated protein kinase was examined by Western blotting analysis in Caco-2 cells. The amount of C. difficile flagellin-induced inflammatory mediators in the presence, or absence of C. difficile toxin B was also measured by ELISA assay. KEY FINDINGS C. difficile flagellin induced activation of NF-kappaB in HEK293T cells via toll-like receptor 5. C. difficile flagellin also induced activation of p38 mitogen-activated protein kinase, and promoted the production of interleukin-8 and CCL20 in intestinal epithelial cells via toll-like receptor 5. Pretreatment with toxin B enhanced flagellin-induced cytokine productions. SIGNIFICANCE Our results indicate that toxin B promotes flagellin-induced activation of intestinal epithelial cells, and that C. difficile flagellin may play a role in the occurrence of C. difficile-associated diseases.
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Affiliation(s)
- Yusuke Yoshino
- Department of Infectious Diseases, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Tokyo 173-8606, Japan
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Duerr CU, Hornef MW. The mammalian intestinal epithelium as integral player in the establishment and maintenance of host-microbial homeostasis. Semin Immunol 2011; 24:25-35. [PMID: 22138188 DOI: 10.1016/j.smim.2011.11.002] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Only one single layer of epithelial cells separates the densely colonized and environmentally exposed intestinal lumen from the largely sterile subepithelial tissue. Together with the overlaying mucus and the subepithelial mucosal immune system the epithelium has evolved to maintain homeostasis in the presence of the enteric microbiota. It also contributes to rapid and efficient antimicrobial host defence in the event of infection with pathogenic microorganisms. Both, epithelial antimicrobial host defence and homeostasis rely on signalling pathways induced by innate immune receptors demonstrating the active role of epithelial cells in the host-microbial interplay. The interaction of epithelial cells with professional immune cells illustrates the integrated function within the mucosal tissue. In the present review we focus on structural and functional changes of the intestinal epithelium during the fetal-neonatal transition and infancy and try to delineate its role in the induction and maintenance of host-microbial homeostasis. We also address factors that impair epithelial functions and may lead to disruption of the mucosal barrier, tissue damage and the development of symptomatic disease.
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Affiliation(s)
- Claudia U Duerr
- Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Carl-Neuberg Str. 1, D-30625 Hannover, Germany
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Vijay-Kumar M, Carvalho FA, Aitken JD, Fifadara NH, Gewirtz AT. TLR5 or NLRC4 is necessary and sufficient for promotion of humoral immunity by flagellin. Eur J Immunol 2011; 40:3528-34. [PMID: 21072873 DOI: 10.1002/eji.201040421] [Citation(s) in RCA: 103] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The fact that some TLR-based vaccine adjuvants maintain function in TLR-deficient hosts highlights that their mechanism of function remains incompletely understood. Thus, we examined the ability of flagellin to induce cytokines and elicit/promote murine antibody responses upon deletion of the flagellin receptors TLR5 and/or NLRC4 (also referred to as IPAF) using a prime/boost regimen. In TLR5-KO mice, flagellin failed to induce NF-κB-regulated cytokines such as keratinocyte-derived chemokine (CXCL1) but induced WT levels of the inflammasome cytokine IL-18 (IL-1F4). Conversely, in NLRC4-KO mice, flagellin induced keratinocyte-derived chemokine, but not IL-18, whereas TLR5/NLRC4-DKO lacked induction of all cytokines measured. Flagellin/ovalbumin treatment resulted in high-antibody titers to both flagellin and ovalbumin in WT, TLR5-KO and DKO mice but did not elicit antibodies to either in TLR5/NLRC4-DKO mice. Thus, flagellin's ability to elicit/promote humoral immunity requires a germ-line-encoded receptor capable of recognizing this molecule. Such promotion of adaptive immunity can be effectively driven by either TLR5-mediated activation of NF-κB or NLRC4-mediated activation of the inflammasome.
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12
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Intestinal epithelial cells and their role in innate mucosal immunity. Cell Tissue Res 2010; 343:5-12. [PMID: 21104188 DOI: 10.1007/s00441-010-1082-5] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2010] [Accepted: 10/22/2010] [Indexed: 01/15/2023]
Abstract
The mucosal surfaces of the respiratory, gastrointestinal and urogenital tracts are covered by a layer of epithelial cells that are responsible for sensing and promoting a host immune response in order to establish the limits not only for commensal microorganisms but also for foreign organisms or particles. This is a remarkable task as the human body represents a composite of about 10 trillion human-self cells plus non-self cells from autochthonous or indigenous microbes that outnumber human cells 10:1. Hence, the homeostasis of epithelial cells that line mucosal surfaces relies on a fine-tuned immune system that patrols the boundaries between human and microbial cells. In the case of the intestine, the epithelial layer is composed of at least six epithelial cell lineages that act as a physiological barrier in addition to aiding digestion and the absorption of nutrients, water and electrolytes. In this review, we highlight the immense role of the intestinal epithelium in coordinating the mucosal innate immune response.
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13
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Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat Rev Microbiol 2010; 8:171-84. [PMID: 20157338 DOI: 10.1038/nrmicro2297] [Citation(s) in RCA: 684] [Impact Index Per Article: 45.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
How can probiotic bacteria transduce their health benefits to the host? Bacterial cell surface macromolecules are key factors in this beneficial microorganism-host crosstalk, as they can interact with host pattern recognition receptors (PRRs) of the gastrointestinal mucosa. In this Review, we highlight the documented signalling interactions of the surface molecules of probiotic bacteria (such as long surface appendages, polysaccharides and lipoteichoic acids) with PRRs. Research on host-probiotic interactions can benefit from well-documented host-microorganism studies that span the spectrum from pathogenicity to mutualism. Distinctions and parallels are therefore drawn with the interactions of similar molecules that are presented by gastrointestinal commensals and pathogens.
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Sukumaran SK, Fu NY, Tin CB, Wan KF, Lee SS, Yu VC. A Soluble Form of the Pilus Protein FimA Targets the VDAC-Hexokinase Complex at Mitochondria to Suppress Host Cell Apoptosis. Mol Cell 2010; 37:768-83. [DOI: 10.1016/j.molcel.2010.02.015] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2009] [Revised: 10/28/2009] [Accepted: 12/24/2009] [Indexed: 11/16/2022]
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Gao N, Kumar A, Jyot J, Yu FS. Flagellin-induced corneal antimicrobial peptide production and wound repair involve a novel NF-kappaB-independent and EGFR-dependent pathway. PLoS One 2010; 5:e9351. [PMID: 20195469 PMCID: PMC2829077 DOI: 10.1371/journal.pone.0009351] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2009] [Accepted: 01/29/2010] [Indexed: 01/17/2023] Open
Abstract
Background The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs). Methodology/Principal Findings Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-κB activation, but were potent in eliciting EGFR signaling in a TGF-α–related pathway in HCECs. Concomitant with the lack of NF-κB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling. Conclusions/Significance Our data suggest that inflammatory response mediated by NF-κB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues.
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Affiliation(s)
- Nan Gao
- Departments of Ophthalmology, Anatomy, and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States of America
| | - Ashok Kumar
- Departments of Ophthalmology, Anatomy, and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States of America
| | - Jeevan Jyot
- Department of Medicine/Infectious Diseases, University of Florida, Gainesville, Florida, United States of America
| | - Fu-Shin Yu
- Departments of Ophthalmology, Anatomy, and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States of America
- * E-mail:
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Bu HF, Wang X, Tang Y, Koti V, Tan XD. Toll-like receptor 2-mediated peptidoglycan uptake by immature intestinal epithelial cells from apical side and exosome-associated transcellular transcytosis. J Cell Physiol 2010; 222:658-68. [PMID: 20020500 DOI: 10.1002/jcp.21985] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Peptidoglycan (PGN) is a potent immune adjuvant derived from bacterial cell walls. Previous investigations suggest that intestinal epithelium may absorb PGN from the lumen. Nonetheless, how PGN is taken up and crosses intestinal epithelium remains largely unclear. Here, we first characterized PGN transport in vitro using IEC-18 and HT29-CL19A cells, which represent less mature epithelial cells in intestinal crypts. With fluorescent microscopy, we visualized internalization of dual-labeled PGN by enterocytes. Engulfed PGN was found to form a complex with PGN recognition protein-3, which may facilitate delivering PGN in vivo. Utilizing electronic microscopy, we revealed that uptake of apical PGN across intestinal epithelial monolayers was involved in phagocytosis, multivesicular body formation, and exosome secretion. We also studied transport of PGN using the transwell system. Our data indicated that apically loaded PGN was exocytosed to the basolateral compartment with exosomes by HT29-CL19A cells. The PGN-contained basolateral exosome extracts induced macrophage activation. Through gavaging mice with labeled PGN, we found that luminal PGN was taken up by columnar epithelial cells in crypts of the small intestine. Furthermore, we showed that pre-confluent immature but not post-confluent mature C2BBe1 cells engulfed PGN via a toll-like receptor 2-dependent manner. Together, our findings suggest that (1) crypt-based immature intestinal epithelial cells play an important role in transport of luminal PGN over the intestinal epithelium; and (2) luminal PGN is transcytosed across intestinal epithelia via a toll-like receptor 2-mediated phagocytosis-multivesicular body-exosome pathway. The absorbed PGN and its derivatives may facilitate maintenance of intestinal immune homeostasis.
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Affiliation(s)
- Heng-Fu Bu
- Center for Digestive Diseases and Immunobiology, Children's Memorial Research Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60614, USA
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17
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Yasuda Y, Matsumura Y, Kasahara K, Ouji N, Sugiura S, Mikasa K, Kita E. Microbial exposure early in life regulates airway inflammation in mice after infection with Streptococcus pneumoniae with enhancement of local resistance. Am J Physiol Lung Cell Mol Physiol 2010; 298:L67-78. [DOI: 10.1152/ajplung.00193.2009] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The immunological explanation for the “hygiene hypothesis” has been proposed to be induction of T helper 1 (Th1) responses by microbial products. However, the protective results of hygiene hypothesis-linked microbial exposures are currently shown to be unlikely to result from a Th1-skewed response. Until now, effect of microbial exposure early in life on airway innate resistance remained unclear. We examined the role of early life exposure to microbes in airway innate resistance to a respiratory pathogen. Specific pathogen-free weanling mice were nasally exposed to the mixture of microbial extracts or PBS (control) every other day for 28 days and intratracheally infected with Streptococcus pneumoniae 10 days after the last exposure. Exposure to microbial extracts facilitated colonization of aerobic gram-positive bacteria, anaerobic microorganisms, and Lactobacillus in the airway, compared with control exposure. In pneumococcal pneumonia, the exposure prolonged mouse survival days by suppressing bacterial growth and by retarding pneumococcal blood invasion, despite significantly low levels of leukocyte recruitment in the lung. Enhancement of airway resistance was associated with a significant decrease in production of leukocyte chemokine (KC) and TNFα, and suppression of matrix metalloproteinase (MMP-9) expression/activation with enhancement of tissue inhibitor of MMP (TIMP-3) activation. The exposure increased production of IFN-γ, IL-4, and monocyte chemoattractant-1 following infection. Furthermore, expression of Toll-like receptor 2, 4, and 9 was promoted by the exposure but no longer upregulated upon pneumococcal infection. Thus, we suggest that hygiene hypothesis is more important in regulating the PMN-dominant inflammatory response than in inducing a Th1-dominant response.
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Affiliation(s)
| | | | | | | | | | - Keiichi Mikasa
- Center for Infectious Diseases, Nara Medical University, School of Medicine, Kashihara, Nara, Japan
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Nicoletti C, Arques JL, Bertelli E. CX₃CR1 is critical for Salmonella-induced migration of dendritic cells into the intestinal lumen. Gut Microbes 2010; 1:131-4. [PMID: 21327020 PMCID: PMC3023593 DOI: 10.4161/gmic.1.3.11711] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/16/2009] [Revised: 02/26/2010] [Accepted: 03/04/2010] [Indexed: 02/03/2023] Open
Abstract
We have demonstrated that direct antigen sampling of bacteria by intestinal dendritic cells (DCs) is accompanied by a rapid migration of CD11c+CX3CR1+MHCII+CD8α-CD11b− DCs into the intestinal lumen upon exposure to non-invasive ΔSPI1-Salmonella. Importantly, intraluminal DCs internalized Salmonella but were not able to cross the epithelium to return into tissue, thus showing that these DCs do not function as antigen-presenting cells and participate in the conventional regulation of immune responses to intestinal pathogens. Here we show that the presence of the chemokine receptor CX3CR1, that plays a vital role in DC-mediated antigen sampling and clearance in the gut, is also instrumental for the transepithelial migration of DCs. The latter observation, along with the notion that CX3CR1-deficient mice displayed higher susceptibility to Salmonella infection compared to wild-type mice raises the possibility that Salmonella-induced migration of “bacteria-capturing” DCs into the lumen may be an important mechanism of mucosal defence and clearance.
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Affiliation(s)
- Claudio Nicoletti
- Laboratory of Mucosal Immunology; Programme of Integrated Biology of the GI Tract; Institute of Food Research; Norwich, UK
| | - Juan L Arques
- Laboratory of Mucosal Immunology; Programme of Integrated Biology of the GI Tract; Institute of Food Research; Norwich, UK,Department Tecnología de Alimentos; INIA; Madrid, Spain
| | - Eugenio Bertelli
- Department of Pharmacology “G. Segre”; University of Siena; Siena, Italy
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Stevens MP, Humphrey TJ, Maskell DJ. Molecular insights into farm animal and zoonotic Salmonella infections. Philos Trans R Soc Lond B Biol Sci 2009; 364:2709-23. [PMID: 19687040 PMCID: PMC2865095 DOI: 10.1098/rstb.2009.0094] [Citation(s) in RCA: 113] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Salmonella enterica is a facultative intracellular pathogen of worldwide importance. Infections may present in a variety of ways, from asymptomatic colonization to inflammatory diarrhoea or typhoid fever depending on serovar- and host-specific factors. Human diarrhoeal infections are frequently acquired via the food chain and farm environment by virtue of the ability of selected non-typhoidal serovars to colonize the intestines of food-producing animals and contaminate the avian reproductive tract and egg. Colonization of reservoir hosts often occurs in the absence of clinical symptoms; however, some S. enterica serovars threaten animal health owing to their ability to cause acute enteritis or translocate from the intestines to other organs causing fever, septicaemia and abortion. Despite the availability of complete genome sequences of isolates representing several serovars, the molecular mechanisms underlying Salmonella colonization, pathogenesis and transmission in reservoir hosts remain ill-defined. Here we review current knowledge of the bacterial factors influencing colonization of food-producing animals by Salmonella and the basis of host range, differential virulence and zoonotic potential.
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Affiliation(s)
- Mark P Stevens
- Division of Microbiology, Institute for Animal Health, Compton, Berkshire RG20 7NN, UK.
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20
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Uchiya KI, Sugita A, Nikai T. Involvement of SPI-2-encoded SpiC in flagellum synthesis in Salmonella enterica serovar Typhimurium. BMC Microbiol 2009; 9:179. [PMID: 19706157 PMCID: PMC2782266 DOI: 10.1186/1471-2180-9-179] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2009] [Accepted: 08/25/2009] [Indexed: 01/07/2023] Open
Abstract
BACKGROUND SpiC encoded within Salmonella pathogenicity island 2 on the Salmonella enterica serovar Typhimurium chromosome is required for survival within macrophages and systemic infection in mice. Additionally, SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages by affecting the expression of FliC, a component of flagella filaments. Here, we show the contribution of SpiC in flagellum synthesis. RESULTS Quantitative RT-PCR shows that the expression levels of the class 3 fliD and motA genes that encode for the flagella cap and motor torque proteins, respectively, were lower for a spiC mutant strain than for the wild-type Salmonella. Further, this mutant had lower expression levels of the class 2 genes including the fliA gene encoding the flagellar-specific alternative sigma factor. We also found differences in flagella assembly between the wild-type strain and the spiC mutant. Many flagella filaments were observed on the bacterial surface of the wild-type strain, whereas the spiC mutant had only few flagella. The absence of spiC led to reduced expression of the FlhD protein, which functions as the master regulator in flagella gene expression, although no significant difference at the transcription level of the flhDC operon was observed between the wild-type strain and the spiC mutant. CONCLUSION The data show that SpiC is involved in flagella assembly by affecting the post-transcription expression of flhDC.
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Affiliation(s)
- Kei-ichi Uchiya
- Department of Microbiology, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya, Japan.
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21
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Arques JL, Hautefort I, Ivory K, Bertelli E, Regoli M, Clare S, Hinton JCD, Nicoletti C. Salmonella induces flagellin- and MyD88-dependent migration of bacteria-capturing dendritic cells into the gut lumen. Gastroenterology 2009; 137:579-87, 587.e1-2. [PMID: 19375423 DOI: 10.1053/j.gastro.2009.04.010] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/04/2008] [Revised: 02/15/2009] [Accepted: 04/09/2009] [Indexed: 12/24/2022]
Abstract
BACKGROUND & AIMS Intestinal dendritic cells (DCs) sample bacteria, such as Salmonella, by extending cellular processes into the lumen to capture bacteria and shuttle them across the epithelium; however, direct evidence of bacteria-loaded DCs travelling back into the tissue is lacking. We hypothesized that sampling is paralleled by migration of DCs into the lumen prior to or following the internalization of Salmonella. METHODS The small intestine and the colon of BALB/c and C57BL/6 mice were challenged with noninvasive Salmonella enterica serovar Typhimurium SL1344-DeltaSalmonella pathogenicity island (SPI) 1 or Escherichia coli DH5alpha by using isolated loops or oral administration by gavage. Transepithelial migration of DCs was documented by immunohistochemistry, microscopy, and flow cytometry. The role of flagellin was determined by using flagellin (DeltafliC DeltafljB)- and SPI1-SPI2 (DeltaSPI1 DeltassrA)-deficient Salmonella, flagellated E coli K12, and MyD88 mice. RESULTS Salmonella DeltaSPI1 induced migration of CD11c(+)CX(3)CR1(+)MHCII(+)CD11b(-)CD8alpha(-) DCs into the small intestine, whereas flagellin- and SPI1-SPI2-deficient Salmonella, soluble flagellin, and E coli DH5alpha or flagellated K12, failed to do so. DC migration did not occur in the colon; it was not observed in MyD88 mice, and intraluminal DCs internalized Salmonella but did not cross the epithelium to return into tissues. Finally, DC migration was not linked to Salmonella-induced damage of the epithelium. CONCLUSIONS DC-mediated sampling of Salmonella is accompanied by flagellin- and MyD88-dependent migration of Salmonella-capturing DCs into the intestinal lumen. We suggest that the rapid intraluminal migration of Salmonella-capturing DCs may play a role in the protection of the intestinal mucosa against bacterial infection.
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Affiliation(s)
- Juan L Arques
- Programme of GI Tract Biology, Institute of Food Research, Norwich, United Kingdom
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22
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Vijay-Kumar M, Gewirtz AT. Role of flagellin in Crohn's disease: emblematic of the progress and enigmas in understanding inflammatory bowel disease. Inflamm Bowel Dis 2009; 15:789-95. [PMID: 19107795 DOI: 10.1002/ibd.20734] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Elevated immune responses to the enteric microbiota have long been associated with inflammatory bowel disease (IBD), especially Crohn's disease. In recent years there has been considerable progress in identifying a number of the specific bacterial and host molecules whose interactions mediate these responses. However, deciphering the role of these interactions in the pathophysiology of IBD remains a difficult challenge, in part due to the very complex nature of the epithelial cell / microbial / immune cell interactions that play a central role in maintaining the gut's well-being. This article reviews such progress and discusses these challenges in the context of focusing on 1 particular protein, bacterial flagellin.
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Affiliation(s)
- Matam Vijay-Kumar
- Department of Pathology, Emory University School of Medicine, Atlanta, Georgia 30322, USA
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23
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Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis. J Transl Med 2008; 88:1215-26. [PMID: 18810251 DOI: 10.1038/labinvest.2008.86] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Crohn's disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv-) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of beta1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv+ Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P<0.01) and ileal mucosa (268+/-47% of control; P<0.01), whereas inv bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size- and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of beta1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv+ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of beta1-integrin and stimulated uptake of nanoparticles via invasin-dependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.
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24
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Uchiya KI, Nikai T. Salmonella virulence factor SpiC is involved in expression of flagellin protein and mediates activation of the signal transduction pathways in macrophages. Microbiology (Reading) 2008; 154:3491-3502. [DOI: 10.1099/mic.0.2008/021667-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Affiliation(s)
- Kei-ichi Uchiya
- Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan
| | - Toshiaki Nikai
- Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan
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25
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Subramanian S, Rhodes JM, Hart AC, Tam B, Roberts CL, Smith SL, Corkill JE, Winstanley C, Virji M, Campbell BJ. Characterization of epithelial IL-8 response to inflammatory bowel disease mucosal E. coli and its inhibition by mesalamine. Inflamm Bowel Dis 2008; 14:162-75. [PMID: 17941093 PMCID: PMC7108638 DOI: 10.1002/ibd.20296] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Mucosally adherent E. coli are found in inflammatory bowel disease (IBD) and colon cancer. They promote release of the proinflammatory cytokine interleukin-8 (IL-8). We explored mechanisms for this release and its inhibition by drugs. METHODS IL-8 release from colon epithelial cells in response to mucosal E. coli isolates from IBD, colon cancer, and controls was characterized at the cellular and molecular level. RESULTS IL-8 response of HT29 cells was greater with Crohn's disease (689 +/- 298 [mean +/- SD] pg IL-8/mL at 4 hours, n = 7) and colon cancer isolates (532 +/- 415 pg/mL, n = 14) than with ulcerative colitis (236 +/- 58 pg/mL, n = 6) or control isolates (236 +/- 100 pg/mL, n = 6, P < 0.0001). Bacterial supernatants contained shed flagellin that triggered IL-8 release. For whole bacteria the IL-8 response to E. coli that agglutinate red blood cells (548 +/- 428 pg IL-8/mL, n = 16), a function that correlates with epithelial invasion, was greater than for nonhemagglutinators (281 +/- 253 pg/mL, n = 17; P < 0.0001). This was particularly marked among E. coli that, although flagellate, could not release IL-8 from TLR5-transfected HEK293 cells. IL-8 release was mediated by extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and inhibited by mesalamine, but not hydrocortisone, at therapeutic concentrations. CONCLUSIONS Mucosa-associated E. coli shed flagellin that elicits epithelial IL-8 release but this may only become relevant when the mucosal barrier is weakened to expose basolateral TLR5. Adherent and invasive IBD and colon cancer E. coli isolates also elicit a flagellin-independent IL-8 response that may be relevant when the mucosal barrier is intact. The IL-8 release is MAPK-dependent and inhibited by mesalamine.
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Affiliation(s)
- Sreedhar Subramanian
- Division of Gastroenterology, University School of Clinical Science, Liverpool, UK
| | - Jonathan M. Rhodes
- Division of Gastroenterology, University School of Clinical Science, Liverpool, UK
| | - Anthony C. Hart
- Division of Medical Microbiology, University School of Infection and Host Defence, Liverpool, UK
| | - Ben Tam
- Division of Gastroenterology, University School of Clinical Science, Liverpool, UK
| | - Carol L. Roberts
- Division of Gastroenterology, University School of Clinical Science, Liverpool, UK
| | - Shirley L. Smith
- Division of Gastroenterology, University School of Clinical Science, Liverpool, UK
| | - John E. Corkill
- Division of Medical Microbiology, University School of Infection and Host Defence, Liverpool, UK
| | - Craig Winstanley
- Division of Medical Microbiology, University School of Infection and Host Defence, Liverpool, UK
| | - Mumtaz Virji
- Department of Cellular and Molecular Medicine, University of Bristol, UK
| | - Barry J. Campbell
- Division of Gastroenterology, University School of Clinical Science, Liverpool, UK
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26
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Decreased potency of the Vibrio cholerae sheathed flagellum to trigger host innate immunity. Infect Immun 2008; 76:1282-8. [PMID: 18174340 DOI: 10.1128/iai.00736-07] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Vibrio cholerae is a monoflagellated gram-negative bacterium that causes the severe diarrheal disease cholera. In contrast to Salmonella enterica serovar Typhimurium infection, which is accompanied by both acute diarrhea and high-level inflammation, V. cholerae infection is largely noninflammatory in human hosts. Bacterial flagella are composed of flagellin, a highly conserved protein that is also a target of the innate immune response. Because the V. cholerae flagellum is covered by a sheath, we hypothesized that it might be less prone to activation of the innate immune response than nonsheathed flagella, such as those produced by Salmonella serovar Typhimurium. Indeed, compared with Salmonella serovar Typhimurium flagella, V. cholerae flagella demonstrated significantly reduced NF-kappaB activation in A549 human pulmonary epithelial cells. However, V. cholerae flagellin monomers, FlaD and FlaC, were almost equally potent with purified FliC, a monomer derived from Salmonella serovar Typhimurium flagella, in NF-kappaB activation. Heat- and acid-induced dissociation assays showed that Salmonella serovar Typhimurium flagella disassembled far more readily into monomeric flagellins than V. cholerae flagella, suggesting that the differential levels of NF-kappaB activation by V. cholerae and Salmonella serovar Typhimurium flagella are likely attributable to the difference in their flagellin shedding. Our results suggest that monomer dissociation of V. cholerae flagella is suppressed likely due to the presence of the sheath and that this unique structural feature of V. cholerae flagella may have evolved as a strategy to evade flagellin-triggered host innate immune responses in various host species.
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Toll like receptor-5: protecting the gut from enteric microbes. Semin Immunopathol 2007; 30:11-21. [PMID: 18066550 DOI: 10.1007/s00281-007-0100-5] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2007] [Accepted: 11/05/2007] [Indexed: 01/01/2023]
Abstract
The intestine is normally colonized by a large and diverse commensal microbiota and is occasionally exposed to a variety of potential pathogens. In recent years, there has been substantial progress made in identifying molecular mechanisms that normally serve to protect the intestine from such enteric bacteria and which may go awry in chronic idiopathic inflammatory diseases of the gut. One specific molecular interaction that appears to play a key role in governing bacterial-intestinal interactions is that of the bacterial protein flagellin with toll-like receptor 5. This article reviews studies performed in vitro, in mice, and in humans that indicate an important role for the flagellin-TLR5 interaction in regulating both the innate and adaptive immune responses in the intestine.
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Volf J, Boyen F, Faldyna M, Pavlova B, Navratilova J, Rychlik I. Cytokine response of porcine cell lines to Salmonella enterica serovar typhimurium and its hilA and ssrA mutants. Zoonoses Public Health 2007; 54:286-93. [PMID: 17894638 DOI: 10.1111/j.1863-2378.2007.01064.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular bacterium which can infect and colonize pigs. After contact with enterocytes and macrophages, S. Typhimurium induces production of cytokines thus triggering the innate immune response. In this study we evaluated the cytokine response of two porcine cell lines, IPI-2I and 3D4/31, of epithelial or macrophage origins, respectively, to the wild-type S. Typhimurium and its hilA and ssrA mutants. We observed that the 3D4/31 cell line essentially did not respond to S. Typhimurium infection when a medium with foetal calf serum was used. However when the 3D4 cell line was incubated overnight in the presence of porcine serum, it efficiently responded to the wild-type strain and the ssrA mutant but not to the noninvasive hilA mutant as measured by mRNA quantification of TNF-alpha, IL-8 and GM-CSF by the real-time RT-PCR. In IPI-2I, all the cytokines were also induced by the wild-type S. Typhimurium and the ssrA mutant although the induction of TNF-alpha was lower than that induced by the wild-type strain. The hilA mutant was unable to induce any of the cytokines tested. The ssrA mutant can therefore be considered as more suitable for further vaccine development as the stimulation of innate immune response is important for animal protection against a challenge with virulent strains.
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Affiliation(s)
- J Volf
- Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic
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29
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Abstract
Type III secretion systems (T3SSs) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu. Although the effector proteins vary among different T3SS pathogens, common pathogenic mechanisms emerge, including interference with the host cell cytoskeleton to promote attachment and invasion, interference with cellular trafficking processes, cytotoxicity and barrier dysfunction, and immune system subversion. The activity of the T3SSs correlates closely with infection progression and outcome, both in animal models and in human infection. Therefore, to facilitate patient care and improve outcomes, it is important to understand the T3SS-mediated virulence processes and to target T3SSs in therapeutic and prophylactic development efforts.
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30
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Hautefort I, Thompson A, Eriksson-Ygberg S, Parker ML, Lucchini S, Danino V, Bongaerts RJM, Ahmad N, Rhen M, Hinton JCD. During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems. Cell Microbiol 2007; 10:958-84. [PMID: 18031307 PMCID: PMC2343689 DOI: 10.1111/j.1462-5822.2007.01099.x] [Citation(s) in RCA: 189] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
The biogenesis of the Salmonella-containing vacuole within mammalian cells has been intensively studied over recent years. However, the ability of Salmonella to sense and adapt to the intracellular environment of different types of host cells has received much less attention. To address this issue, we report the transcriptome of Salmonella enterica serovar Typhimurium SL1344 within epithelial cells and show comparisons with Salmonella gene expression inside macrophages. We report that S. Typhimurium expresses a characteristic intracellular transcriptomic signature in response to the environments it encounters within different cell types. The signature involves the upregulation of the mgtBC, pstACS and iro genes for magnesium, phosphate and iron uptake, and Salmonella pathogenicity island 2 (SPI2). Surprisingly, in addition to SPI2, the invasion-associated SPI1 pathogenicity island and the genes involved in flagellar biosynthesis were expressed inside epithelial cells at later stages of the infection, while they were constantly downregulated in macrophage-like cells. To our knowledge, this is the first report of the simultaneous transcription of all three Type Three Secretion Systems (T3SS) within an intracellular Salmonella population. We discovered that S. Typhimurium strain SL1344 was strongly cytotoxic to epithelial cells after 6 h of infection and hypothesize that the time-dependent changes in Salmonella gene expression within epithelial cells reflects the bacterial response to host cells that have been injured by the infection process.
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Affiliation(s)
- I Hautefort
- Molecular Microbiology Group, Institute of Food Research, Norwich NR4 7UA, UK.
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31
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Sun YH, Rolán HG, Tsolis RM. Injection of flagellin into the host cell cytosol by Salmonella enterica serotype Typhimurium. J Biol Chem 2007; 282:33897-901. [PMID: 17911114 DOI: 10.1074/jbc.c700181200] [Citation(s) in RCA: 141] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Bacterial flagellins are potent inducers of innate immunity. Three signaling pathways have been implicated in the sensing of flagellins; these involve toll-like receptor 5 (TLR5) and the cytosolic proteins Birc1e/Naip5 and Ipaf. Although the structural basis of TLR5-flagellin interaction is known, little is known about how flagellin enters the host cell cytosol to induce signaling via Birc1e/Naip5 and Ipaf. Here we demonstrate for the first time the translocation of bacterial flagellin into the cytosol of host macrophages by the vacuolar pathogen, Salmonella enterica serotype Typhimurium. Translocation of flagellin into the host cell cytosol was directly demonstrated using beta-lactamase reporter constructs. Flagellin translocation required the Salmonella Pathogenicity Island 1 Type III secretion system (SPI-1 T3SS) but not the flagellar T3SS.
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Affiliation(s)
- Yao-Hui Sun
- Department of Medical Microbiology and Immunology, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
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Srikanth CV, Cherayil BJ. Intestinal innate immunity and the pathogenesis of Salmonella enteritis. Immunol Res 2007; 37:61-78. [PMID: 17496347 PMCID: PMC3199302 DOI: 10.1007/bf02686090] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/1999] [Revised: 11/30/1999] [Accepted: 11/30/1999] [Indexed: 12/30/2022]
Abstract
Acute gastroenteritis caused by Salmonella typhimurium infection is a clinical problem with significant public health impact. The availability of several experimental models of this condition has allowed detailed investigation of the cellular and molecular interactions involved in its pathogenesis. Such studies have shed light on the roles played by bacterial virulence factors and host innate immune mechanisms in the development of intestinal inflammation.
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Affiliation(s)
- Chittur V. Srikanth
- Mucosal Immunology Laboratory, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129
| | - Bobby J. Cherayil
- Mucosal Immunology Laboratory, Division of Pediatric Gastroenterology and Nutrition, Room 3400, Massachusetts General Hospital East, Building 114, 16 Street, Charlestown, MA 02129.
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Sano GI, Takada Y, Goto S, Maruyama K, Shindo Y, Oka K, Matsui H, Matsuo K. Flagella facilitate escape of Salmonella from oncotic macrophages. J Bacteriol 2007; 189:8224-32. [PMID: 17873035 PMCID: PMC2168665 DOI: 10.1128/jb.00898-07] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
The intracellular parasite Salmonella enterica serovar Typhimurium causes a typhoid-like systemic disease in mice. Whereas the survival of Salmonella in phagocytes is well understood, little has been documented about the exit of intracellular Salmonella from host cells. Here we report that in a population of infected macrophages Salmonella induces "oncosis," an irreversible progression to eukaryotic cell death characterized by swelling of the entire cell body. Oncotic macrophages (OnMphis) are terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling negative and lack actin filaments (F-actin). The plasma membrane of OnMphis filled with bacilli remains impermeable, and intracellular Salmonella bacilli move vigorously using flagella. Eventually, intracellular Salmonella bacilli intermittently exit host cells in a flagellum-dependent manner. These results suggest that induction of macrophage oncosis and intracellular accumulation of flagellated bacilli constitute a strategy whereby Salmonella escapes from host macrophages.
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Affiliation(s)
- Gen-ichiro Sano
- Department of Microbiology and Immunology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
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Layton AN, Galyov EE. Salmonella-induced enteritis: molecular pathogenesis and therapeutic implications. Expert Rev Mol Med 2007; 9:1-17. [PMID: 17605831 DOI: 10.1017/s1462399407000373] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Salmonella-induced enteritis is a gastrointestinal disease that causes major economic and welfare problems throughout the world. Although the infection is generally self-limiting, subgroups of the population such as immunocompromised individuals, the young and the elderly are susceptible to developing more severe systemic infections. The emergence of widespread antibiotic resistance and the lack of a suitable vaccine against enteritis-causing Salmonella have led to a search for alternative therapeutic strategies. This review focuses on how Salmonella induces enteritis at the molecular level in terms of bacterial factors, such as the type III secretion systems used to inject a subset of bacterial proteins into host cells, and host factors, such as Toll-like receptors and cytokines. The type III secreted bacterial proteins elicit a variety of responses in host cells that contribute to enteritis. Cytokines form part of the host defence mechanism, but in combination with bacterial factors can contribute to Salmonella-induced enteritis. We also discuss animal and cell culture models currently used to study Salmonella-induced enteritis, and how understanding the mechanisms of the disease has impacted on the development of Salmonella therapeutics.
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Affiliation(s)
- Abigail N Layton
- Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK.
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35
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Badie G, Heithoff DM, Sinsheimer RL, Mahan MJ. Altered levels of Salmonella DNA adenine methylase are associated with defects in gene expression, motility, flagellar synthesis, and bile resistance in the pathogenic strain 14028 but not in the laboratory strain LT2. J Bacteriol 2006; 189:1556-64. [PMID: 17172341 PMCID: PMC1855711 DOI: 10.1128/jb.01580-06] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response to dam levels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed between Salmonella serovars that are closely related at the DNA level.
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Affiliation(s)
- Golnaz Badie
- Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106, USA
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36
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Tobar JA, Carreño LJ, Bueno SM, González PA, Mora JE, Quezada SA, Kalergis AM. Virulent Salmonella enterica serovar typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells. Infect Immun 2006; 74:6438-48. [PMID: 17057096 PMCID: PMC1695529 DOI: 10.1128/iai.00063-06] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Dendritic cells (DCs) constitute the link between innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) in bacteria and by presenting bacterial antigens to T cells. Recognition of PAMPs renders DCs as professional antigen-presenting cells able to prime naïve T cells and initiate adaptive immunity against bacteria. Therefore, interfering with DC function would promote bacterial survival and dissemination. Understanding the molecular mechanisms that have evolved in virulent bacteria to evade activation of adaptive immunity requires the characterization of virulence factors that interfere with DC function. Salmonella enterica serovar Typhimurium, the causative agent of typhoid-like disease in the mouse, can prevent antigen presentation to T cells by avoiding lysosomal degradation in DCs. Here, we show that this feature of virulent Salmonella applies in vivo to prevent activation of adaptive immunity. In addition, this attribute of virulent Salmonella requires functional expression of a type three secretion system (TTSS) and effector proteins encoded within the Salmonella pathogenicity island 2 (SPI-2). In contrast to wild-type virulent Salmonella, mutant strains carrying specific deletions of SPI-2 genes encoding TTSS components or effectors proteins are targeted to lysosomes and are no longer able to prevent DCs from activating T cells in vitro or in vivo. SPI-2 mutant strains are attenuated in vivo, showing reduced tissue colonization and enhanced T-cell activation, which confers protection against a challenge with wild-type virulent Salmonella. Our data suggest that impairment of DC function by the activity of SPI-2 gene products is crucial for Salmonella pathogenesis.
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Affiliation(s)
- Jaime A Tobar
- Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda #340, Santiago, Chile
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37
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Abstract
Salmonella species cause substantial morbidity, mortality and burden of disease globally. Infections with Salmonella species cause multiple clinical syndromes. Central to the pathophysiology of all human salmonelloses is the induction of a strong host innate immune/inflammatory response. Whether this ultimately reflects an adaptive advantage to the host or pathogen is not clear. However, it is evident that both the host and pathogen have evolved mechanisms of triggering host responses that are detrimental to the other. In this review, we explore some of the host and pathogenic mechanisms mobilized in the two predominant clinical syndromes associated with infection with Salmonella enterica species: enterocolitis and typhoid.
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Affiliation(s)
- Bryan Coburn
- Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada
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Affiliation(s)
- Theodore S Steiner
- Division of Infectious Diseases, Vancouver Coastal Health Research Institute and University of British Columbia, Vancouver, BC, Canada.
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Tükel C, Raffatellu M, Chessa D, Wilson RP, Akçelik M, Bäumler AJ. Neutrophil influx during non-typhoidal salmonellosis: who is in the driver's seat? ACTA ACUST UNITED AC 2006; 46:320-9. [PMID: 16553804 DOI: 10.1111/j.1574-695x.2006.00051.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
A massive neutrophil influx in the intestine is the histopathological hallmark of Salmonella enterica serovar Typhimurium-induced enterocolitis in humans. Two major hypotheses on the mechanism leading to neutrophil infiltration in the intestinal mucosa have emerged. One hypothesis suggests that S. enterica serovar Typhimurium takes an active role in triggering this host response by injecting proteins, termed effectors, into the host cell cytosol which induce a proinflammatory gene expression profile in the intestinal epithelium. The second hypothesis suggests a more passive role for the pathogen by proposing that bacterial invasion stimulates the innate pathways of inflammation because the pathogen-associated molecular patterns of S. enterica serovar Typhimurium are recognized by pathogen recognition receptors on cells in the lamina propria. A review of the current literature reveals that, while pathogen recognition receptors are clearly involved in eliciting neutrophil influx during S. enterica serovar Typhimurium infection, a direct contribution of effectors in triggering proinflammatory host cell responses cannot currently be ruled out.
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Affiliation(s)
- Cagla Tükel
- Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA 95616-8645, USA
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Gewirtz AT, Vijay-Kumar M, Brant SR, Duerr RH, Nicolae DL, Cho JH. Dominant-negative TLR5 polymorphism reduces adaptive immune response to flagellin and negatively associates with Crohn's disease. Am J Physiol Gastrointest Liver Physiol 2006; 290:G1157-63. [PMID: 16439468 DOI: 10.1152/ajpgi.00544.2005] [Citation(s) in RCA: 158] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Crohn's disease (CD) is associated with elevated adaptive immunity to commensal microbes, with flagellin being a dominant antigen. In light of heightened awareness of the importance of innate immunity in regulating adaptive immunity and ambiguity as to the role of CD-associated immune responses in CD pathophysiology, we sought to determine whether natural acquisition of immune responses to flagellin were regulated by the innate immune flagellin receptor toll-like receptor 5 (TLR5) and determine whether persons carrying a recently defined common dominant-negative TLR5 polymorphism (TLR5-stop) might be protected from developing CD. Carriage rates of a recently defined dominant-negative TLR5 polymorphism (TLR5-stop) and levels of serum immunoreactivity to bacterial products were measured in inflammatory bowel disease patients, first-degree relatives, and unrelated controls. We observed that, in healthy subjects, persons carrying TLR5-stop had significantly lower levels of flagellin-specific IgG and IgA but had similar levels of total and LPS-specific Ig. Moreover, we observed that, among Jewish subjects, the carriage rate of TLR5-stop (in heterozygous state) was significantly less in CD patients, but not ulcerative colitis (UC) patients, compared with unaffected relatives and unrelated controls (5.4, 0.9, 6.0, and 6.5% for unaffected relatives, CD, UC, and unrelated Jewish controls, respectively, n = 296, 215, 185, and 416, respectively; P = 0.037 by likelihood calculation for CD vs. controls), indicating that TLR5-stop can protect persons of Jewish ethnicity against CD. We did not observe a significant association of TLR5-stop with CD in a non-Jewish cohort (11.1, 10.4, and 11.7% for unaffected relatives, CD, and UC, respectively; n = 841, 543, and 300 for unaffected relatives, respectively). These results demonstrate that natural acquisition of immune responses to flagellin are regulated by TLR5 and suggest that immune responses to flagellin are not merely associated with CD but rather promote the pathogenic response.
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Affiliation(s)
- Andrew T Gewirtz
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology Unit, Emory University School of Medicine, Atlanta, GA 30322, USA.
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Rochon M, Römling U. Flagellin in combination with curli fimbriae elicits an immune response in the gastrointestinal epithelial cell line HT-29. Microbes Infect 2006; 8:2027-33. [PMID: 16807036 DOI: 10.1016/j.micinf.2006.03.003] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2005] [Revised: 01/31/2006] [Accepted: 03/02/2006] [Indexed: 11/28/2022]
Abstract
Flagellin is the major cytokine-releasing factor when Salmonella enterica serovar Typhimurium (S. Typhimurium) infects intestinal epithelial cells. In this work it is shown that curli, an adhesive proteinaceous surface component of Enterobacteriaceae involved in biofilm formation of S. Typhimurium and Escherichia coli strains can bind flagellin and thus elicit an immune response by the intestinal epithelial cell line HT-29.
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Affiliation(s)
- Maike Rochon
- Microbiology and Tumor Biology Center, Karolinska Institutet, Box 280, SE-171 77 Stockholm, Sweden
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42
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Abstract
The human gastrointestinal (GI) tract is colonized by non-pathogenic commensal microflora and frequently exposed to many pathogenic organisms. For the maintenance of GI homeostasis, the host must discriminate between pathogenic and non-pathogenic organisms and initiate effective and appropriate immune and inflammatory responses. Mammalian toll-like receptors (TLRs) are members of the pattern-recognition receptor (PRR) family that plays a central role in the initiation of innate cellular immune responses and the subsequent adaptive immune responses to microbial pathogens. Recent studies have shown that gastrointestinal epithelial cells express almost all TLR subtypes characterized to date and that the expression and activation of TLRs in the GI tract are tightly and coordinately regulated. This review summarizes the current understanding of the crucial dual roles of TLRs in the development of host innate and adaptive immune responses to GI infections and the maintenance of the immune tolerance to commensal bacteria through down-regulation of surface expression of TLRs in intestinal epithelial cells.
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Abstract
PURPOSE OF REVIEW To consider observations suggesting that the bacterial protein flagellin, the primary structural component of flagella, plays a major role in mediating gut inflammation associated with infection by enteric pathogens and in inflammatory bowel disease. RECENT FINDINGS Biochemical dissection of an in-vitro model of bacterial-epithelial interactions revealed flagellin, via ligation of Toll-like receptor 5, to be a major means of activating the innate immune responses defining active intestinal inflammation. Application of the novel technique of serologic expression cloning to murine models of colitis discovered that flagellin is also a dominant target of the adaptive immune responses that drive colitis in such models. Human studies observed that flagellin was also a major antigenic target of immune responses associated with Crohn's disease. Carriers of dominant-negative Toll-like receptor 5 gene exhibit reduced natural acquisition of immunity to flagellin, indicating that the adaptive immune response to flagellin is likely driven, in part, by Toll-like receptor 5. In some genetic backgrounds dominant-negative Toll-like receptor 5 associated negatively with Crohn's disease, suggesting that immune responses to flagellin are not only associated with Crohn's disease, but can promote the pathogenic response. SUMMARY Flagellin is a major activator of innate immunity thus driving pathogen-induced acute inflammation and, perhaps, the active flares of inflammatory bowel disease. Flagellin is also a dominant antigen of the Crohn's disease-associated adaptive immune response, thus placing this molecule at the crossroads of the innate and adaptive immune responses that are the hallmark of intestinal inflammation.
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Affiliation(s)
- Andrew T Gewirtz
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology Unit, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
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Harrington SM, Strauman MC, Abe CM, Nataro JP. Aggregative adherence fimbriae contribute to the inflammatory response of epithelial cells infected with enteroaggregative Escherichia coli. Cell Microbiol 2005; 7:1565-78. [PMID: 16207244 DOI: 10.1111/j.1462-5822.2005.00588.x] [Citation(s) in RCA: 102] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Enteroaggregative Escherichia coli (EAEC) causes watery diarrhoea that is often mildly inflammatory. Previous studies have reported that the flagellin of EAEC induces IL-8 from intestinal epithelial cells (IECs) in culture. To characterize more fully the inflammatory response to EAEC, we infected IECs with EAEC prototype strain 042 and assessed cellular responses by macroarray and reverse transcriptase polymerase chain reaction (RT-PCR). Genes upregulated in 042-infected non-polarized T84 cells included IL-8, IL-6, TNF-alpha, GRO-alpha, GRO-gamma, ICAM-1, GM-CSF and IL-1alpha. RT-PCR analyses performed with cDNA from T84 and HT-29 cells infected with an aflagellar mutant (042fliC) suggested that these responses were primarily mediated by flagellin. To better reproduce the conditions of the infection for this non-invasive pathogen, we assessed the responses of polarized IECs to strain 042 infection. As expected, 042 induced IL-8 production from both polarized T84 and HT-29 cells. However, significant IL-8 secretion was induced in polarized T84 cells infected with 042fliC, suggesting that a factor other than flagellin contributes to inflammation in this model. This non-flagellar IL-8 response required expression of the aggregative adherence fimbria (AAF) adhesin, and was related to the presence of the minor fimbria-associated protein AafB. Our data suggest that multiple factors contribute to EAEC-induced inflammation, and further characterization of the nature of EAEC proinflammatory factors will greatly advance our understanding of this emerging pathogen.
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Affiliation(s)
- Susan M Harrington
- Department of Microbiology, The University of Maryland, Baltimore, MD 21201, USA
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45
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Kelly D, Conway S, Aminov R. Commensal gut bacteria: mechanisms of immune modulation. Trends Immunol 2005; 26:326-33. [PMID: 15922949 DOI: 10.1016/j.it.2005.04.008] [Citation(s) in RCA: 249] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2005] [Revised: 03/30/2005] [Accepted: 04/15/2005] [Indexed: 12/13/2022]
Abstract
Mucosal immune responses to pathogenic gut bacteria and the mechanisms that govern disease progression and outcome have been researched intensely for decades. More recently, the influence of the resident non-pathogenic or 'commensal' microflora on mucosal immune function and gut health has emerged as an area of scientific and clinical importance. Major differences occur in the mucosal immune response to pathogens and commensals. In part, this functional dichotomy is explained by the presence of virulence factors in pathogenic species, which are generally absent in commensals. Additionally, immunological 'unresponsiveness' towards the resident commensal microflora is thought to permit their successful colonisation and co-existence within the host gut. However, evidence of an active dialogue between members of the commensal microflora and the host mucosal immune system is rapidly unfolding. This crosstalk is likely to affect immunological tolerance and homeostasis within the gut and to explain some of the differential host responses to commensal and pathogenic bacteria.
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Affiliation(s)
- Denise Kelly
- Gut Immunology Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK.
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46
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Rimoldi M, Chieppa M, Larghi P, Vulcano M, Allavena P, Rescigno M. Monocyte-derived dendritic cells activated by bacteria or by bacteria-stimulated epithelial cells are functionally different. Blood 2005; 106:2818-26. [PMID: 16030185 DOI: 10.1182/blood-2004-11-4321] [Citation(s) in RCA: 129] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Dendritic cells (DCs) are able to open the tight junctions between adjacent epithelial cells (ECs) and to take up both invasive and noninvasive bacteria directly from the intestinal lumen. In this study, we describe a tight cross talk between ECs and human monocyte-derived DCs (MoDCs) in bacterial handling across epithelial monolayers. We show that the release of proinflammatory mediators by ECs in response to bacteria is dependent on bacterial invasiveness and on the presence of flagella. This correlates with the capacity of EC-derived factors to modulate MoDC function. MoDCs incubated with supernatants of bacteria-treated ECs are "noninflammatory" as they release interleukin-10 (IL-10) but not IL-12 and can drive only T helper (Th)-2 type T cells. Moreover, noninflammatory MoDCs release chemokines aimed at recruiting Th2 and T-regulatory cells. In contrast, when MoDCs are incubated with ECs and bacteria in a transwell coculture system, and can contact directly the bacteria across stimulated EC monolayers, they are more inflammatory as they release IL-12 and IL-10 and induce both Th1 and Th2 responses. These results suggest that ECs are not simply a barrier to bacteria entering via the oral route, but they actively influence the activating properties of DCs.
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Affiliation(s)
- Monica Rimoldi
- Department of Experimental Oncology, European Institute of Oncology, Istituto di Ricerche Farmacologiche, Mario Negri, Milan, Italy
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47
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Coburn B, Li Y, Owen D, Vallance BA, Finlay BB. Salmonella enterica serovar Typhimurium pathogenicity island 2 is necessary for complete virulence in a mouse model of infectious enterocolitis. Infect Immun 2005; 73:3219-27. [PMID: 15908346 PMCID: PMC1111876 DOI: 10.1128/iai.73.6.3219-3227.2005] [Citation(s) in RCA: 156] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Salmonella species cause a wide range of disease in multiple hosts. Salmonella enterica serovar Typhimurium causes self-limited intestinal disease in humans and systemic typhoid-like illness in susceptible mice. The prevailing dogma in murine S. enterica serovar Typhimurium pathogenesis is that distinct virulence mechanisms-Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2)-perform distinct roles in pathogenesis, the former being important for invasion and intestinal disease and the latter important for intracellular survival and systemic persistence and disease. Although evidence from bovine infections has suggested that SPI2 has a role in ileal disease, there is no evidence that SPI2 is important for inflammation in a disease that more closely recapitulates human colitis. Using S. enterica serovar Typhimurium strains that lack functional type III secretion systems, we demonstrate that SPI2 is essential for complete virulence in murine infectious enterocolitis. Using a recently characterized murine model (M. Barthel,S. Hapfelmeier, L. Quintanilla-Martinez, M. Kremer, M. Rohde, M. Hogardt, K. Pfeffer, H. Russmann, and W. D. Hardt, Infect. Immun. 71:2839-2858, 2003), we demonstrate that SPI1 mutants are unable to cause intestinal disease 48 h after infection and that SPI2-deficient bacteria also cause significantly attenuated typhlitis. We show that at the peak of inflammation in the cecum, SPI2 mutants induce diminished intercellular adhesion molecule 1 expression and neutrophil recruitment but that wild-type and mutant Salmonella are similarly distributed in the lumen of the infected organ. Finally, we demonstrate that attenuation of intestinal inflammation is accompanied by resolution of typhlitis in the mutant, but not wild-type, infections. Collectively, these results indicate that SPI2 is needed for enterocolitis, as well as for systemic disease.
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Affiliation(s)
- Bryan Coburn
- Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada
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48
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Mumy KL, McCormick BA. Events at the host-microbial interface of the gastrointestinal tract. II. Role of the intestinal epithelium in pathogen-induced inflammation. Am J Physiol Gastrointest Liver Physiol 2005; 288:G854-9. [PMID: 15826933 DOI: 10.1152/ajpgi.00565.2004] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
An immense number of bacteria reside within the intestinal lumen. The task of appropriately identifying and responding to microbial threats lies primarily with the single layer of cells that line the intestinal tract. Intestinal epithelial cells have developed a number of strategies aimed at identifying microorganisms and eliciting the appropriate inflammatory response. The pathogen recognition mechanisms and the signaling and inflammatory events that ensue within the intestine are the focus of this review.
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Affiliation(s)
- Karen L Mumy
- Mucosal Immunology Laboratory, Massachusetts General Hospital, Charlestown, MA 02129, USA
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