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Berson Y, Khaitlina S, Tsaplina O. Involvement of Lipid Rafts in the Invasion of Opportunistic Bacteria Serratia into Eukaryotic Cells. Int J Mol Sci 2023; 24:ijms24109029. [PMID: 37240375 PMCID: PMC10361209 DOI: 10.3390/ijms24109029] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2023] [Revised: 05/16/2023] [Accepted: 05/19/2023] [Indexed: 05/28/2023] Open
Abstract
Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein-protein and lipid-protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces Serratia invasion.
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Affiliation(s)
- Yuliya Berson
- Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av. 4, 194064 St. Petersburg, Russia
| | - Sofia Khaitlina
- Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av. 4, 194064 St. Petersburg, Russia
| | - Olga Tsaplina
- Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av. 4, 194064 St. Petersburg, Russia
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Hall CP, Jadeja NB, Sebeck N, Agaisse H. Characterization of MxiE- and H-NS-Dependent Expression of ipaH7.8, ospC1, yccE, and yfdF in Shigella flexneri. mSphere 2022; 7:e0048522. [PMID: 36346241 PMCID: PMC9769918 DOI: 10.1128/msphere.00485-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Accepted: 10/10/2022] [Indexed: 11/09/2022] Open
Abstract
Shigella flexneri uses a type 3 secretion system (T3SS) apparatus to inject virulence effector proteins into the host cell cytosol. Upon host cell contact, MxiE, an S. flexneri AraC-like transcriptional regulator, is required for the expression of a subset of T3SS effector genes encoded on the large virulence plasmid. Here, we defined the MxiE regulon using RNA-seq. We identified virulence plasmid- and chromosome-encoded genes that are activated in response to type 3 secretion in a MxiE-dependent manner. Bioinformatic analysis revealed that similar to previously known MxiE-dependent genes, chromosome-encoded genes yccE and yfdF contain a regulatory element known as the MxiE box, which is required for their MxiE-dependent expression. The significant AT enrichment of MxiE-dependent genes suggested the involvement of H-NS. Using a dominant negative H-NS system, we demonstrate that H-NS silences the expression of MxiE-dependent genes located on the virulence plasmid (ipaH7.8 and ospC1) and the chromosome (yccE and yfdF). Furthermore, we show that MxiE is no longer required for the expression of ipaH7.8, ospC1, yccE, and yfdF when H-NS silencing is relieved. Finally, we show that the H-NS anti-silencer VirB is not required for ipaH7.8 and yccE expression upon MxiE/IpgC overexpression. Based on these genetic studies, we propose a model of MxiE-dependent gene regulation in which MxiE counteracts H-NS-mediated silencing. IMPORTANCE The expression of horizontally acquired genes, including virulence genes, is subject to complex regulation involving xenogeneic silencing proteins, and counter-silencing mechanisms. The pathogenic properties of Shigella flexneri mainly rely on the acquisition of the type 3 secretion system (T3SS) and cognate effector proteins, whose expression is repressed by the xenogeneic silencing protein H-NS. Based on previous studies, releasing H-NS-mediated silencing mainly relies on two mechanisms involving (i) a temperature shift leading to the release of H-NS at the virF promoter, and (ii) the virulence factor VirB, which dislodges H-NS upon binding to specific motifs upstream of virulence genes, including those encoding the T3SS. In this study, we provide genetic evidence supporting the notion that, in addition to VirB, the AraC family member MxiE also contributes to releasing H-NS-mediated silencing in S. flexneri.
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Affiliation(s)
- Chelsea P. Hall
- Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA
| | - Niti B. Jadeja
- Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA
| | - Natalie Sebeck
- Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA
| | - Hervé Agaisse
- Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA
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SepA Enhances Shigella Invasion of Epithelial Cells by Degrading Alpha-1 Antitrypsin and Producing a Neutrophil Chemoattractant. mBio 2021; 12:e0283321. [PMID: 34724811 PMCID: PMC8561385 DOI: 10.1128/mbio.02833-21] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Shigella spp. are highly adapted pathogens that cause bacillary dysentery in human and nonhuman primates. An unusual feature of Shigella pathogenesis is that this organism invades the colonic epithelia from the basolateral pole. Therefore, it has evolved the ability to disrupt the intestinal epithelial barrier to reach the basolateral surface. We have shown previously that the secreted serine protease A (SepA), which belongs to the family of serine protease autotransporters of Enterobacteriaceae, is responsible for the initial destabilization of the intestinal epithelial barrier that facilitates Shigella invasion. However, the mechanisms used by SepA to regulate this process remain unknown. To investigate the protein targets cleaved by SepA in the intestinal epithelium, we incubated a sample of homogenized human colon with purified SepA or with a catalytically inactive mutant of this protease. We discovered that SepA targets an array of 18 different proteins, including alpha-1 antitrypsin (AAT), a major circulating serine proteinase inhibitor in humans. In contrast to other serine proteases, SepA cleaved AAT without forming an inhibiting complex, which resulted in the generation of a neutrophil chemoattractant. We demonstrated that the products of the AAT-SepA reaction induce a mild but significant increase in neutrophil transepithelial migration in vitro. Moreover, the presence of AAT during Shigella infection stimulated neutrophil migration and dramatically enhanced the number of bacteria invading the intestinal epithelium in a SepA-dependent manner. We conclude that by cleaving AAT, SepA releases a chemoattractant that promotes neutrophil migration, which in turn disrupts the intestinal epithelial barrier to enable Shigella invasion. IMPORTANCE Shigella is the second leading cause of diarrheal death globally. In this study, we identified the host protein targets of SepA, Shigella's major protein secreted in culture. We demonstrated that by cleaving AAT, a serine protease inhibitor important to protect surrounding tissue at inflammatory sites, SepA releases a neutrophil chemoattractant that enhances Shigella invasion. Moreover, SepA degraded AAT without becoming inhibited by the cleaved product, and SepA catalytic activity was enhanced at higher concentrations of AAT. Activation of SepA by an excess of AAT may be physiologically relevant at the early stages of Shigella infection, when the amount of synthesized SepA is very low compared to the concentration of AAT in the intestinal lumen. This observation may also help to explain the adeptness of Shigella infectivity at low dose, despite the requirement of reaching the basolateral side to invade and colonize the colonic epithelium.
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Khaitlina S, Bozhokina E, Tsaplina O, Efremova T. Bacterial Actin-Specific Endoproteases Grimelysin and Protealysin as Virulence Factors Contributing to the Invasive Activities of Serratia. Int J Mol Sci 2020; 21:E4025. [PMID: 32512842 PMCID: PMC7311988 DOI: 10.3390/ijms21114025] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/02/2020] [Accepted: 06/02/2020] [Indexed: 12/25/2022] Open
Abstract
The article reviews the discovery, properties and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of Serratia grimesii and protealysin of Serratia proteamaculans, characterized by both a highly specific "actinase" activity and their ability to stimulate bacterial invasion. Grimelysin cleaves the only polypeptide bond Gly42-Val43 in actin. This bond is not cleaved by any other proteases and leads to a reversible loss of actin polymerization. Similar properties were characteristic for another bacterial protease, protealysin. These properties made grimelysin and protealysin a unique tool to study the functional properties of actin. Furthermore, bacteria Serratia grimesii and Serratia proteamaculans, producing grimelysin and protealysin, invade eukaryotic cells, and the recombinant Escherichia coli expressing the grimelysin or protealysins gene become invasive. Participation of the cellular c-Src and RhoA/ROCK signaling pathways in the invasion of eukaryotic cells by S. grimesii was shown, and involvement of E-cadherin in the invasion has been suggested. Moreover, membrane vesicles produced by S. grimesii were found to contain grimelysin, penetrate into eukaryotic cells and increase the invasion of bacteria into eukaryotic cells. These data indicate that the protease is a virulence factor, and actin can be a target for the protease upon its translocation into the host cell.
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Affiliation(s)
- Sofia Khaitlina
- Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia; (E.B.); (O.T.); (T.E.)
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Wong J, Chopra J, Chiang LLW, Liu T, Ho J, Wu WKK, Tse G, Wong SH. The Role of Connexins in Gastrointestinal Diseases. J Mol Biol 2019; 431:643-652. [PMID: 30639409 DOI: 10.1016/j.jmb.2019.01.007] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2018] [Revised: 10/03/2018] [Accepted: 01/04/2019] [Indexed: 12/13/2022]
Abstract
Gap junctions are hexagonal arrays of protein molecules in the plasma membrane and were first described in Mauthner cell synapses of goldfish. They form pathways for coupling between cells, allowing passive, electrotonic spread of ions and also passage of larger molecules such as amino acids and nucleotides. They are expressed in both excitable and non-excitable tissues. Each gap junction is made of two connexons, which are hexameric proteins of the connexin subunit. In this review, the roles that connexins play in gastrointestinal motility, the mechanisms of altered connexin expression leading to inflammatory bowel disease, gastrointestinal infections, and gastrointestinal symptoms in autistic spectrum disorder are discussed in detail.
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Affiliation(s)
- Jeremy Wong
- Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, PR China
| | - Jasmine Chopra
- Faculty of Arts and Science, University of Toronto, Toronto, Canada
| | | | - Tong Liu
- Tianjin Key Laboratory of Ionic-Molecular Function of Cardiovascular disease, Department of Cardiology, Tianjin Institute of Cardiology, Second Hospital of Tianjin Medical University, Tianjin 300211, PR China
| | - Jeffery Ho
- Department of Anaesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong, PR China; Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China
| | - William K K Wu
- Department of Anaesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong, PR China; Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China
| | - Gary Tse
- Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China; Li Ka Shing Institute of Health Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, PR China.
| | - Sunny Hei Wong
- Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China; Li Ka Shing Institute of Health Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, PR China.
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Maldonado-Contreras A, Birtley JR, Boll E, Zhao Y, Mumy KL, Toscano J, Ayehunie S, Reinecker HC, Stern LJ, McCormick BA. Shigella depends on SepA to destabilize the intestinal epithelial integrity via cofilin activation. Gut Microbes 2017; 8:544-560. [PMID: 28598765 PMCID: PMC5730386 DOI: 10.1080/19490976.2017.1339006] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Shigella is unique among enteric pathogens, as it invades colonic epithelia through the basolateral pole. Therefore, it has evolved the ability to breach the intestinal epithelial barrier to deploy an arsenal of effector proteins, which permits bacterial invasion and leads to a severe inflammatory response. However, the mechanisms used by Shigella to regulate epithelial barrier permeability remain unknown. To address this question, we used both an intestinal polarized model and a human ex-vivo model to further characterize the early events of host-bacteria interactions. Our results showed that secreted Serine Protease A (SepA), which belongs to the serine protease autotransporter of Enterobacteriaceae family, is responsible for critically disrupting the intestinal epithelial barrier. Such disruption facilitates bacterial transit to the basolateral pole of the epithelium, ultimately fostering the hallmarks of the disease pathology. SepA was found to cause a decrease in active LIM Kinase 1 (LIMK1) levels, a negative inhibitor of actin-remodeling proteins, namely cofilin. Correspondingly, we observed increased activation of cofilin, a major actin-polymerization factor known to control opening of tight junctions at the epithelial barrier. Furthermore, we resolved the crystal structure of SepA to elucidate its role on actin-dynamics and barrier disruption. The serine protease activity of SepA was found to be required for the regulatory effects on LIMK1 and cofilin, resulting in the disruption of the epithelial barrier during infection. Altogether, we demonstrate that SepA is indispensable for barrier disruption, ultimately facilitating Shigella transit to the basolateral pole where it effectively invades the epithelium.
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Affiliation(s)
- Ana Maldonado-Contreras
- Department of Microbiology and Physiological Systems, University of Massachusetts, Medical School, Worcester, MA, USA,CONTACT Beth A. McCormick ; Ana Maldonado-Contreras 55 Lake Ave N, Worcester, MA, 01655
| | - James R. Birtley
- Department of Pathology, University of Massachusetts, Medical School, Worcester, MA, USA
| | - Erik Boll
- Statens Serum Institut, Copenhagen, Denmark
| | - Yun Zhao
- Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Karen L. Mumy
- Naval Medical Research Unit Dayton, Wright-Patterson Air Force Base, Dayton, OH, USA
| | - Juan Toscano
- Department of Microbiology and Physiological Systems, University of Massachusetts, Medical School, Worcester, MA, USA
| | | | - Hans-Christian Reinecker
- Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Lawrence J. Stern
- Department of Pathology, University of Massachusetts, Medical School, Worcester, MA, USA
| | - Beth A. McCormick
- Department of Microbiology and Physiological Systems, University of Massachusetts, Medical School, Worcester, MA, USA,CONTACT Beth A. McCormick ; Ana Maldonado-Contreras 55 Lake Ave N, Worcester, MA, 01655
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Agaisse H. Molecular and Cellular Mechanisms of Shigella flexneri Dissemination. Front Cell Infect Microbiol 2016; 6:29. [PMID: 27014639 PMCID: PMC4786538 DOI: 10.3389/fcimb.2016.00029] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2015] [Accepted: 02/26/2016] [Indexed: 11/13/2022] Open
Abstract
The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. The disease is characterized by bacterial invasion of intestinal cells, dissemination within the colonic epithelium through direct spread from cell to cell, and massive inflammation of the intestinal mucosa. Here, we review the mechanisms supporting S. flexneri dissemination. The dissemination process primarily relies on actin assembly at the bacterial pole, which propels the pathogen throughout the cytosol of primary infected cells. Polar actin assembly is supported by polar expression of the bacterial autotransporter family member IcsA, which recruits the N-WASP/ARP2/3 actin assembly machinery. As motile bacteria encounter cell-cell contacts, they form plasma membrane protrusions that project into adjacent cells. In addition to the ARP2/3-dependent actin assembly machinery, protrusion formation relies on formins and myosins. The resolution of protrusions into vacuoles occurs through the collapse of the protrusion neck, leading to the formation of an intermediate membrane-bound compartment termed vacuole-like protrusions (VLPs). VLP formation requires tyrosine kinase and phosphoinositide signaling in protrusions, which relies on the integrity of the bacterial type 3 secretion system (T3SS). The T3SS is also required for escaping double membrane vacuoles through the activity of the T3SS translocases IpaB and IpaC, and the effector proteins VirA and IcsB. Numerous factors supporting envelope biogenesis contribute to IcsA exposure and maintenance at the bacterial pole, including LPS synthesis, membrane proteases, and periplasmic chaperones. Although less characterized, the assembly and function of the T3SS in the context of bacterial dissemination also relies on factors supporting envelope biogenesis. Finally, the dissemination process requires the adaptation of the pathogen to various cellular compartments through transcriptional and post-transcriptional mechanisms.
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Affiliation(s)
- Hervé Agaisse
- Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine Charlottesville, VA, USA
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Kuehl CJ, Dragoi AM, Agaisse H. The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial dissemination. PLoS One 2014; 9:e112738. [PMID: 25405985 PMCID: PMC4236203 DOI: 10.1371/journal.pone.0112738] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2014] [Accepted: 10/14/2014] [Indexed: 01/29/2023] Open
Abstract
Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.
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Affiliation(s)
- Carole J. Kuehl
- Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut, United States of America
| | - Ana-Maria Dragoi
- Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut, United States of America
| | - Hervé Agaisse
- Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut, United States of America
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The serine/threonine kinase STK11 promotes Shigella flexneri dissemination through establishment of cell-cell contacts competent for tyrosine kinase signaling. Infect Immun 2014; 82:4447-57. [PMID: 25114112 DOI: 10.1128/iai.02078-14] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Shigella flexneri is an intracellular pathogen that disseminates in the intestinal epithelium by displaying actin-based motility. We found that although S. flexneri displayed comparable actin-based motilities in the cytosols of HeLa229 and HT-29 epithelial cell lines, the overall dissemination process was much more efficient in HT-29 cells. Time-lapse microscopy demonstrated that as motile bacteria reached the cell cortex in HT-29 cells, they formed membrane protrusions that resolved into vacuoles, from which the bacteria escaped and gained access to the cytosol of adjacent cells. In HeLa229 cells, S. flexneri also formed membrane protrusions that extended into adjacent cells, but the protrusions rarely resolved into vacuoles. Instead, the formed protrusions collapsed and retracted, bringing the bacteria back to the cytosol of the primary infected cells. Silencing the serine/threonine kinase STK11 (also known as LKB1) in HT-29 cells decreased the efficiency of protrusion resolution into vacuoles. Conversely, expressing STK11 in HeLa229 cells, which lack the STK11 locus, dramatically increased the efficiency of protrusion resolution into vacuoles. S. flexneri dissemination in HT-29 cells led to the local phosphorylation of tyrosine residues in protrusions, a signaling event that was not observed in HeLa229 cells but was restored in STK11-expressing HeLa229 cells. Treatment of HT-29 cells with the tyrosine kinase inhibitor imatinib abrogated tyrosine kinase signaling in protrusions, which correlated with a severe decrease in the efficiency of protrusion resolution into vacuoles. We suggest that the formation of STK11-dependent lateral cell-cell contacts competent for tyrosine kinase signaling promotes S. flexneri dissemination in epithelial cells.
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Abstract
Shigella flexneri is an enteropathogenic bacterium responsible for approximately 100 million cases of severe dysentery each year. S. flexneri colonization of the human colonic epithelium is supported by direct spread from cell to cell, which relies on actin-based motility. We have recently uncovered that, in intestinal epithelial cells, S. flexneri actin-based motility is regulated by the Bruton's tyrosine kinase (Btk). Consequently, treatment with Ibrutinib, a specific Btk inhibitor currently used in the treatment of B-cell malignancies, effectively impaired S. flexneri spread from cell to cell. Thus, therapeutic intervention capitalizing on drugs interfering with host factors supporting the infection process may represent an effective alternative to treatments with antimicrobial compounds.
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Fisher ML, Sun W, Curtiss R. The route less taken: pulmonary models of enteric Gram-negative infection. Pathog Dis 2013; 70:99-109. [PMID: 24259516 DOI: 10.1111/2049-632x.12109] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2013] [Accepted: 10/16/2013] [Indexed: 11/29/2022] Open
Abstract
Many pathogens are capable of causing a fulminant infection in pulmonary tissues of mammals. Animal models have provided an extensive understanding of the genetic and molecular mechanisms of bacterial pathogenesis as well as host immune response in the lungs. Many clinically relevant Gram-negative bacteria are host-restricted. Thus, the powerful, informative tools of mouse models are not available for study with these organisms. However, over the past 30 years, enterprising work has demonstrated the utility of pulmonary infection with enteric pathogens. Such infection models have increased our understanding host-pathogen interactions in these organisms. Here, we provide a review and comparison of lung models of infection with enteric, Gram-negative bacteria relative to naturally occurring lung pathogens.
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Affiliation(s)
- Michael L Fisher
- Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ, USA
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Bruton's tyrosine kinase regulates Shigella flexneri dissemination in HT-29 intestinal cells. Infect Immun 2012; 81:598-607. [PMID: 23230296 DOI: 10.1128/iai.00853-12] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Shigella flexneri is a Gram-negative intracellular pathogen that infects the intestinal epithelium and utilizes actin-based motility to spread from cell to cell. S. flexneri actin-based motility has been characterized in various cell lines, but studies in intestinal cells are limited. Here we characterized S. flexneri actin-based motility in HT-29 intestinal cells. In agreement with studies conducted in various cell lines, we showed that S. flexneri relies on neural Wiskott-Aldrich Syndrome protein (N-WASP) in HT-29 cells. We tested the potential role of various tyrosine kinases involved in N-WASP activation and uncovered a previously unappreciated role for Bruton's tyrosine kinase (Btk) in actin tail formation in intestinal cells. We showed that Btk depletion led to a decrease in N-WASP phosphorylation which affected N-WASP recruitment to the bacterial surface, decreased the number of bacteria displaying actin-based motility, and ultimately affected the efficiency of spread from cell to cell. Finally, we showed that the levels of N-WASP phosphorylation and Btk expression were increased in response to infection, which suggests that S. flexneri infection not only triggers the production of proinflammatory factors as previously described but also manipulates cellular processes required for dissemination in intestinal cells.
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Hensley CT, Kamneva OK, Levy KM, Labahn SK, Africa LA, Wing HJ. Two promoters and two translation start sites control the expression of the Shigella flexneri outer membrane protease IcsP. Arch Microbiol 2011; 193:263-74. [PMID: 21225241 PMCID: PMC3071033 DOI: 10.1007/s00203-010-0669-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2010] [Revised: 12/10/2010] [Accepted: 12/15/2010] [Indexed: 11/26/2022]
Abstract
The Shigella flexneri outer membrane protease IcsP proteolytically cleaves the actin-based motility protein IcsA from the bacterial surface. The icsP gene is monocistronic and lies downstream of an unusually large intergenic region on the Shigella virulence plasmid. In silico analysis of this region predicts a second transcription start site 84 bp upstream of the first. Primer extension analyses and beta-galactosidase assays demonstrate that both transcription start sites are used. Both promoters are regulated by the Shigella virulence gene regulator VirB and both respond similarly to conditions known to influence Shigella virulence gene expression (iron concentration, pH, osmotic pressure, and phase of growth). The newly identified promoter lies upstream of a Shine-Dalgarno sequence and second 5'-ATG-3', which is in frame with the annotated icsP gene. The use of either translation start site leads to the production of IcsP capable of proteolytically cleaving IcsA. A bioinformatic scan of the Shigella genome reveals multiple occurrences of in-frame translation start sites associated with putative Shine-Dalgarno sequences, immediately upstream and downstream of annotated open reading frames. Taken together, our observations support the possibility that the use of in-frame translation start sites may generate different protein isoforms, thereby expanding the proteome encoded by bacterial genomes.
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Affiliation(s)
| | - Olga K. Kamneva
- Department of Molecular Biology, University of Wyoming, Laramie, WY, 82071
| | - Karen M. Levy
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada 89154-4004
| | - Stephanie K. Labahn
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada 89154-4004
| | - Lia A. Africa
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada 89154-4004
| | - Helen J. Wing
- School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada 89154-4004
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Raja SB, Murali MR, Kumar NK, Devaraj SN. Isolation and partial characterisation of a novel lectin from Aegle marmelos fruit and its effect on adherence and invasion of Shigellae to HT29 cells. PLoS One 2011; 6:e16231. [PMID: 21283697 PMCID: PMC3025011 DOI: 10.1371/journal.pone.0016231] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2010] [Accepted: 12/09/2010] [Indexed: 11/19/2022] Open
Abstract
Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 µg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.
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Affiliation(s)
- Subramaniya Bharathi Raja
- Department of Biochemistry, School of Life Sciences, University of Madras, Chennai, Tamilnadu, India
| | - Malliga Raman Murali
- Department of Biochemistry, School of Life Sciences, University of Madras, Chennai, Tamilnadu, India
| | - Nirmal Kasinathan Kumar
- Department of Biochemistry, School of Life Sciences, University of Madras, Chennai, Tamilnadu, India
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15
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Bagchi AK, Sinha AK, Adhikari R, Maiti P, Mukherjee J, Panda A, Saha DR. Selective deletion of CD8(+) cells upregulated by caspases-1 via IL-18 in mice immunized with major outer membrane protein of Shigella dysenteriae 1 following infection. J Clin Immunol 2010; 30:408-18. [PMID: 20084439 DOI: 10.1007/s10875-009-9359-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2009] [Accepted: 12/08/2009] [Indexed: 01/25/2023]
Abstract
INTRODUCTION Mucosal lymphoid changes were observed in cryopreserved rectal tissues obtained from BALB/c mice infected with Shigella dysenteriae 1, immunized with 57-kDa major antigenic outer membrane protein, and infection after immunization. DISCUSSION Our data suggested that caspase-3 is downregulated in CD4(+) cells of immunized BALB/c mice following infection with substantial increased expression of interleukin (IL)-2 and interferon (IFN)-gamma, while caspase-1 is upregulated in CD8(+) cells with decreased expression of IL-4 and IL-10. This indicated an involvement of Fas-mediated lytic pathway for selective deletion of CD8(+) cells out of CD3(+) T cells. IL-18 promotes inflammation and induces IFN-gamma and tumor necrosis factor (TNF)-alpha as the expression of IFN-gamma and TNF-alpha cytokines was evident in this study. It is assumed that the role of caspase-1 in inducing the CD4+ T cell activity increased with IL-18 rather than CD8+ suppressor cell activity. Bcl-2 is capable of inhibiting the Fas/Fas-L-mediated cell death for helper cells. Overall, the findings indicate that majority of the apoptotic cells were CD8(+) T cells in the groups of infection following immunization, and there might be a selective deletion of T lymphocytes mediated by caspase-1 via IL-18.
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Affiliation(s)
- Ashim Kumar Bagchi
- Research & Development Division, Nutratech, Inc., Winnipeg, MB, R3Y 1M5, Canada.
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16
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Clair C, Combettes L, Pierre F, Sansonetti P, Tran Van Nhieu G. Extracellular-loop peptide antibodies reveal a predominant hemichannel organization of connexins in polarized intestinal cells. Exp Cell Res 2008; 314:1250-65. [PMID: 18267319 DOI: 10.1016/j.yexcr.2007.12.021] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2007] [Revised: 11/19/2007] [Accepted: 12/20/2007] [Indexed: 01/18/2023]
Abstract
Shigella, the causative agent of bacillary dysentery, invades colonic epithelial cells to elicit an intense inflammatory reaction leading to destruction of the mucosa. ATP-dependent paracrine signalling induced by connexin (Cx) hemichannel opening was previously shown to favor Shigella flexneri invasion and dissemination in transfectants of HeLa cells [G. Tran Van Nhieu, C. Clair, R. Bruzzone, M. Mesnil, P. Sansonetti and L. Combettes. (2003). Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat. Cell Biol. 5, 720-726.]. However, although Cxs have been described in polarized epithelial cells, little is known about their structural organization and the role of hemichannels during S. flexneri invasion. We show here that polarized Caco-2/TC7 cells express significant amounts of Cx26, Cx32 and Cx43, but that unexpectedly, cell-cell coupling assessed by dye-transfer experiments is inefficient. Consistent with a predominant Cx organization in hemichannels, dye loading induced by low calcium was readily observed, with preferential loading at the basolateral side. Antibodies (Abs) against connexin extracellular loop peptides (CELAbs) demonstrated the importance of hemichannel signalling since they inhibited dye uptake at low calcium and at physiological calcium concentrations during S. flexneri invasion. Importantly, CELAbs allowed the visualization of hemichannels at the surface of epithelial cells, as structures distinct from gap intercellular junctions.
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Affiliation(s)
- Caroline Clair
- Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris Cedex 15, France
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17
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Raqib R, Sarker P, Bergman P, Ara G, Lindh M, Sack DA, Nasirul Islam KM, Gudmundsson GH, Andersson J, Agerberth B. Improved outcome in shigellosis associated with butyrate induction of an endogenous peptide antibiotic. Proc Natl Acad Sci U S A 2006; 103:9178-83. [PMID: 16740661 PMCID: PMC1482586 DOI: 10.1073/pnas.0602888103] [Citation(s) in RCA: 235] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Shigella is a major cause of morbidity, mortality, and growth retardation for children in developing countries. Emergence of antibiotic resistance among Shigellae demands the development of effective medicines. Previous studies found that the endogenous antimicrobial peptide LL-37 is down-regulated in the rectal epithelium of patients during shigellosis and that butyrate up-regulates the expression of LL-37 in colonic epithelial cells in vitro and decreases severity of inflammation in experimental shigellosis. In this study, Shigella-infected dysenteric rabbits were treated with butyrate (0.14 mmol/kg of body weight) twice daily for 3 days, and the expression levels of the rabbit homologue to LL-37, CAP-18, were monitored in the colon. Butyrate treatment resulted in (i) reduced clinical illness, severity of inflammation in the colon, and bacterial load in the stool, (ii) significant up-regulation of CAP-18 in the surface epithelium, and (iii) disappearance of CAP-18-positive cells in lamina propria. The active CAP-18 peptide was released in stool from its proform by butyrate treatment. In healthy controls, CAP-18 expression was localized predominantly to the epithelial surface of the colon. In infected rabbits, CAP-18 expression was localized to immune and inflammatory cells in the colon, whereas the ulcerated epithelium was devoid of CAP-18 expression. The combination of CAP-18 and butyrate was more efficient in killing Shigella in vitro than CAP-18 alone. Our findings indicate that oral butyrate treatment in shigellosis may be of clinical value because of induction of the endogenous cathelicidin CAP-18 in the colonic epithelium, stimulation of the release of the active peptide CAP-18, and promoting elimination of Shigella.
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Affiliation(s)
- Rubhana Raqib
- International Centre for Diarrhoeal Disease Research Bangladesh, Centre for Health and Population Research, Dhaka 1212, Bangladesh.
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18
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Malík R, Ivan J, Javorský P, Pristas P. Seasonal dynamics of antibiotic-resistantEnterobacteriaceae in the gastrointestinal tract of domestic sheep. Folia Microbiol (Praha) 2005; 50:349-52. [PMID: 16408855 DOI: 10.1007/bf02931417] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Considerable variation in counts of antibiotic-resistant enterobacteria in the ovine gastrointestinal tract was observed. The occurrence of ruminal and fecal isolates resistant to ampicillin (Ap), kanamycin (Km) and tetracycline (Tc) culminated in summer months, followed by rapid decline in subsequent months. Using PCR the tem1bla (Apr), aphA1 (Kmr) and tetB (Tcr) genes were found to be predominant. Under in vitro conditions all resistance genes were transferable into laboratory Escherichia coli strain with relatively high frequency (10(-3) transconjugants per recipient).
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Affiliation(s)
- R Malík
- Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovakia.
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19
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Chen J, Tsang LL, Ho LS, Rowlands DK, Gao JY, Ng CP, Chung YW, Chan HC. Modulation of human enteric epithelial barrier and ion transport function by Peyer’s patch lymphocytes. World J Gastroenterol 2004; 10:1594-9. [PMID: 15162532 PMCID: PMC4572761 DOI: 10.3748/wjg.v10.i11.1594] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the role of Peyer’s patch lymphocytes in the regulation of enteric epithelial barrier and ion transport function in homeostasis and host defense.
METHODS: Mouse Peyer’s patch lymphocytes were co-cultured with human intestinal epithelial cell line Caco-2 either in the mixed or separated (isolated but permeable compartments) culture configuration. Barrier and transport functions of the Caco-2 epithelial monolayers were measured with short-circuit current (Isc) technique. Release of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA expression was analyzed by semi-quantitative RT-PCR. Barrier and iontransport functions of both culture conditions following exposure to Shigella lipopolysaccharide (LPS) were also examined.
RESULTS: The transepithelial resistance (TER) of the epithelial monolayers co-cultured with Peyer’s patch lymphocytes was maintained whereas that of the Caco-2 monolayers alone significantly decreased after eight days in culture. The forskolin-induced anion secretion, in either absence or presence of LPS, was significantly suppressed in the both co-cultures as compared with the Caco-2 cells alone. Furthermore, only the mixed co-culture condition induced the expression and release of mIL-6 from Peyer’s patch lymphocytes, which could be further enhanced by LPS. However, both co-culture conditions suppressed expression and release of epithelial hIL-8 under the unstimulated conditions, while the treatment with LPS stimulated their hIL-8 expression and release.
CONCLUSION: Peyer’s patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact and cytokine signaling.
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Affiliation(s)
- Jie Chen
- Department of Physiology, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
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20
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Tran Van Nhieu G, Clair C, Bruzzone R, Mesnil M, Sansonetti P, Combettes L. Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat Cell Biol 2003; 5:720-6. [PMID: 12844145 DOI: 10.1038/ncb1021] [Citation(s) in RCA: 136] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2003] [Accepted: 05/07/2003] [Indexed: 11/09/2022]
Abstract
Shigella flexneri, the causative agent of bacillar dystentery, invades the colonic mucosa where it elicits an intense inflammatory reaction responsible for destruction of the epithelium. During cell invasion, contact with host cells activates the type-III secretion of the Shigella IpaB and IpaC proteins. IpaB and IpaC are inserted into host cell plasma membranes and trigger initial signals that result in actin polymerization, while allowing cytosolic access of other bacterial effectors that further reorganize the cytoskeleton. After internalization, Shigella moves intracellularly and forms protrusions that infect neighbouring cells, promoting bacterial dissemination across the epithelium. Here, we show that during cell invasion, Shigella induces transient peaks in intracellular calcium concentration that are dependent on a functional type-III secretory apparatus. In addition, Shigella invasion induces the opening of Connexin 26 (Cx26) hemichannels in an actin- and phospholipase-C-dependent manner, allowing release of ATP into the medium. The released ATP, in turn, increases bacterial invasion and spreading, as well as calcium signalling induced by Shigella. These results provide evidence that pathogen-induced opening of connexin channels promotes signalling events that favour bacterial invasion and dissemination.
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Affiliation(s)
- Guy Tran Van Nhieu
- Unité de Pathogénie Microbienne Moléculaire, Inserm U389, Institut Pasteur, 75724, Paris, France
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21
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22
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Raqib R, Ekberg C, Sharkar P, Bardhan PK, Zychlinsky A, Sansonetti PJ, Andersson J. Apoptosis in acute shigellosis is associated with increased production of Fas/Fas ligand, perforin, caspase-1, and caspase-3 but reduced production of Bcl-2 and interleukin-2. Infect Immun 2002; 70:3199-207. [PMID: 12011015 PMCID: PMC127995 DOI: 10.1128/iai.70.6.3199-3207.2002] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Shigella dysenteriae type 1-induced apoptotic cell death in rectal tissues from patients infected with Shigella dysenteriae type 1 was studied by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and annexin V staining. Expression of proteins and cytokines participating in the apoptotic process (caspase-1, caspase-3, Fas [CD95], Fas ligand [Fas-L], perforin, granzyme A, Bax, WAF-1, Bcl-2, interleukin-2 [IL-2], IL-18, and granulocyte-macrophage colony-stimulating factor) in tissue in the acute and convalescent stages of dysentery was quantified at the single-cell level by in situ immunostaining. Apoptotic cell death in the lamina propria was markedly up-regulated at the acute stage (P < 0.05), where an increased number of necrotic cells were also seen. Phenotypic analysis of apoptotic cells revealed that 43% of T cells (CD3), 10% of granulocytes (CD15), and 5% of macrophages (CD56) underwent apoptosis. Increased activity of caspase-1 persisted in the rectum up to 1 month after onset. More-extensive expression of Fas, Fas-L, perforin, caspase-3, and IL-18, but not IL-2, at the acute stage than at the convalescent stage was observed. Increased expression of caspase-3 and IL-18 in tissues with severe inflammation compared to expression in those with mild inflammation was evident, implying a possible role in the perpetuation of inflammation. Significantly reduced cell death during convalescence was associated with a significant up-regulation of Bcl-2, Bax, and WAF-1 expression in the rectum compared to that in the acute phase of infection. Thus, induction of apoptosis at the local site in the early phase of S. dysenteriae type 1 infection was associated with a significant up-regulation of Fas/Fas-L and perforin and granzyme A expression and a down-regulation of Bcl-2 and IL-2, which promote cell survival.
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Affiliation(s)
- Rubhana Raqib
- International Centre for Diarrhoeal Diseases Research, Bangladesh, Dhaka, Bangladesh.
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23
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Kuwae A, Yoshida S, Tamano K, Mimuro H, Suzuki T, Sasakawa C. Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane. Functional analysis of IpaC. J Biol Chem 2001; 276:32230-9. [PMID: 11413141 DOI: 10.1074/jbc.m103831200] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake of Escherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117-169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial.
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Affiliation(s)
- A Kuwae
- Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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24
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Varela G, Schelotto F, di Conza J, Ayala JA. Analysis of the O-antigen chain length distribution during extracellular and intracellular growth of Shigella flexneri. Microb Pathog 2001; 31:21-7. [PMID: 11427033 DOI: 10.1006/mpat.2001.0443] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
This study has established that there were no changes in the general structure of LPS of Shigella flexneri M90T either when the bacteria grew free in the cytoplasm of the eucaryotic host cell or during extracellular growth in liquid LB medium at 37 degrees C. In both cases there was a similar bi-modal O-antigen chain length distribution with the mean modal values between 1 and 2, and between 11 and 14 subunits. This suggests that the intracellular localization is not a significant stimulus perceived by Shigella to regulate the length of its O-side chains. However, when the bacteria grew under extracellular conditions (liquid medium) at 30 degrees C, even though there were no changes in the modal values, the pattern of the O-antigen chain length distribution of LPS was different, with an increase in the amount of the long chains relative to the short chains.
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Affiliation(s)
- G Varela
- Departamento de Bacteriología y Virología, Instituto de Higiene, Universidad de la República, Montevideo, Uruguay
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25
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Smajs D, Weinstock GM. The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake. J Bacteriol 2001; 183:3958-66. [PMID: 11395459 PMCID: PMC95278 DOI: 10.1128/jb.183.13.3958-3966.2001] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
A cosmid library of DNA from colicin Js-sensitive enteroinvasive Escherichia coli (EIEC) strain O164 was made in colicin Js-resistant strain E. coli VCS257, and colicin Js-sensitive clones were identified. Sensitivity to colicin Js was associated with the carriage of a three-gene operon upstream of and partially overlapping senB. The open reading frames were designated cjrABC (for colicin Js receptor), coding for proteins of 291, 258, and 753 amino acids, respectively. Tn7 insertions in any of them led to complete resistance to colicin Js. A near-consensus Fur box was found upstream of cjrA, suggesting regulation of the cjr operon by iron levels. CjrA protein was homologous to iron-regulated Pseudomonas aeruginosa protein PhuW, whose function is unknown; CjrB was homologous to the TonB protein from Pseudomonas putida; and CjrC was homologous to a putative outer membrane siderophore receptor from Campylobacter jejuni. Cloning experiments showed that the cjrB and cjrC genes are sufficient for colicin Js sensitivity. Uptake of colicin Js into sensitive bacteria was dependent on the ExbB protein but not on the E. coli K-12 TonB and TolA, -B, and -Q proteins. Sensitivity to colicin Js is positively regulated by temperature via the VirB protein and negatively controlled by the iron source through the Fur protein. Among EIEC strains, two types of colicin Js-sensitive phenotypes were identified that differed in sensitivity to colicin Js by 1 order of magnitude. The difference in sensitivity to colicin Js is not due to differences between the sequences of the CjrB and CjrC proteins.
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Affiliation(s)
- D Smajs
- Department of Microbiology and Molecular Genetics and Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, Texas 77030, USA
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26
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Schuch R, Maurelli AT. Spa33, a cell surface-associated subunit of the Mxi-Spa type III secretory pathway of Shigella flexneri, regulates Ipa protein traffic. Infect Immun 2001; 69:2180-9. [PMID: 11254573 PMCID: PMC98145 DOI: 10.1128/iai.69.4.2180-2189.2001] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2000] [Accepted: 12/22/2000] [Indexed: 01/11/2023] Open
Abstract
The Mxi-Spa type III secretion system of Shigella flexneri directs the host cell contact-induced secretion of a set of invasins, referred to as Ipas. In this study, we examined the role of Spa33 in Ipa secretion. A spa33-null mutant was both noninvasive and unable to translocate the Ipas from inner membrane to outer membrane (OM) positions of the Mxi-Spa transmembrane channel. Spa33 was found to be a Mxi-Spa substrate that is translocated to the bacterial cell surface upon the induction of Ipa secretion. This mobility may serve to drive Ipa translocation within Mxi-Spa toward OM positions. Consistent with a second distinct role in regulating Ipa traffic, the overexpression of Spa33 also blocked Ipa secretion and resulted in Ipa accumulation at the OM. Co-overexpression of Spa33 and another OM-associated element, Spa32, did not disrupt Ipa secretion, suggesting an interaction between the two proteins and an effect on the mechanism which serves to regulate Ipa release from the OM. These findings indicate that Spa33 is a mobile element within Mxi-Spa, which is required to control Ipa translocation into and out of OM positions of the secretory structure.
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Affiliation(s)
- R Schuch
- Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA
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27
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Godaly G, Bergsten G, Frendéus B, Hang L, Hedlund M, Karpman D, Samuelsson P, Svensson M, Otto G, Wullt B, Svanborg C. Innate defences and resistance to gram negative mucosal infection. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2001; 485:9-24. [PMID: 11109082 DOI: 10.1007/0-306-46840-9_2] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- G Godaly
- Department of MIG, Lund University, Sweden
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28
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Islam D, Bandholtz L, Nilsson J, Wigzell H, Christensson B, Agerberth B, Gudmundsson G. Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator. Nat Med 2001; 7:180-5. [PMID: 11175848 DOI: 10.1038/84627] [Citation(s) in RCA: 300] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Antibacterial peptides are active defense components of innate immunity. Several studies confirm their importance at epithelial surfaces as immediate barrier effectors in preventing infection. Here we report that early in Shigella spp. infections, expression of the antibacterial peptides LL-37 and human beta-defensin-1 is reduced or turned off. The downregulation is detected in biopsies from patients with bacillary dysenteries and in Shigella- infected cell cultures of epithelial and monocyte origin. This downregulation of immediate defense effectors might promote bacterial adherence and invasion into host epithelium and could be an important virulence parameter. Analyses of bacterial molecules causing the downregulation indicate Shigella plasmid DNA as one mediator.
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Affiliation(s)
- D Islam
- Laboratory Sciences Division, International Center for Diarrhoeal Disease Research, 1000 Dhaka, Bangladesh
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29
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Tran Van Nhieu G, Bourdet-Sicard R, Duménil G, Blocker A, Sansonetti PJ. Bacterial signals and cell responses during Shigella entry into epithelial cells. Cell Microbiol 2000; 2:187-93. [PMID: 11207575 DOI: 10.1046/j.1462-5822.2000.00046.x] [Citation(s) in RCA: 108] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Shigella invades epithelial cells by inducing cytoskeletal reorganization localized at the site of bacterial-host cell interaction. During entry, the Shigella type III secretion apparatus allows the insertion of a pore that contains the IpaB and IpaC proteins into cell membranes. Insertion of this complex is thought to allow translocation of the carboxy-terminus moiety of IpaC, but also of other Shigella effectors, such as IpaA, into the cell cytosol. IpaC triggers actin polymerization and the formation of filopodial and lamellipodial extensions dependent on the Cdc42 and Rac GTPases. IpaA, on the other hand, binds to the focal adhesion protein vinculin and induces depolymerization of actin filaments. IpaA and the GTPase Rho are not required for actin polymerization at the site of bacterial contact with the cell membrane, but allow the transformation of the IpaC-induced extensions into a structure that is productive for bacterial entry. Rho is required for the recruitment at entry foci of ezrin, a cytoskeletal linker required for Shigella entry, and also of the Src tyrosine kinase. The Src tyrosine kinase activity, which is required for Shigella-induced actin polymerization, also appears to be involved in a negative regulatory loop that downregulates Rho at the site of entry.
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Affiliation(s)
- G Tran Van Nhieu
- Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, Paris, France.
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30
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Bourdet-Sicard R, Egile C, Sansonetti PJ, Tran Van Nhieu G. Diversion of cytoskeletal processes by Shigella during invasion of epithelial cells. Microbes Infect 2000; 2:813-9. [PMID: 10955962 DOI: 10.1016/s1286-4579(00)90366-6] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Shigella, the causative agent of bacillar dysentery, invades colonic epithelial cells and moves intracellularly to spread from cell to cell. The processes of Shigella entry, determined by the Ipa proteins, and of actin-based motility, dependent on the IcsA/VirG protein, represent different levels of bacterial manipulation of the cell cytoskeleton.
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Affiliation(s)
- R Bourdet-Sicard
- Unité de pathogénie microbienne moléculaire, Inserm U389, Institut Pasteur, Paris, France
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Iwahashi H, Takeshita A, Hanazawa S. Prostaglandin E2 stimulates AP-1-mediated CD14 expression in mouse macrophages via cyclic AMP-dependent protein kinase A. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2000; 164:5403-8. [PMID: 10799905 DOI: 10.4049/jimmunol.164.10.5403] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
PGs play a functional role in the early stage of Gram-negative bacterial infections, because this prostanoid is produced rapidly by epithelial cells after a bacterial infection. CD14, one of the LPS receptors, is a key molecule in triggering the response to bacterial LPS in association with a Toll-like molecule. Therefore, in this study, we investigated the effect of PG on CD14 expression in mouse macrophages. PGE1, PGE2, and PGA1 among the PGs tested strongly stimulated the expression of the CD14 gene in the cells. The stimulatory action also was observed by Western blot analysis. cAMP-elevating agents stimulated expression of CD14 gene as well. Protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not protein kinase C inhibitor 3-(1-[3-(dimethylamino)propyl]-1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-py rrole-2,5-dione (GF109203X), abolished the stimulated expression of CD14. A run-on assay showed that PGE2 stimulated the CD14 gene expression at the transcriptional level via protein kinase A. PGE2 also stimulated activation of AP-1, a heterodimer of c-Jun and c-Fos, because the prostanoid increased specific binding of nuclear proteins to the AP-1 consensus sequence and stimulated AP-1-promoted luciferase activity. PGE2-stimulated expression of CD14 was inhibited by antisense c-fos and c-jun oligonucleotides, but not by their sense oligonucleotides. Finally, PGE2 pretreatment synergistically stimulated LPS-induced expression of IL-1beta and IL-6 genes in mouse macrophages. Therefore, the present study demonstrates that PGE2 has the ability to stimulate AP-1-mediated expression of CD14 in mouse macrophages via cAMP-dependent protein kinase A.
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Affiliation(s)
- H Iwahashi
- Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama, Japan
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Ramachandran A, Madesh M, Balasubramanian KA. Apoptosis in the intestinal epithelium: its relevance in normal and pathophysiological conditions. J Gastroenterol Hepatol 2000; 15:109-20. [PMID: 10735533 DOI: 10.1046/j.1440-1746.2000.02059.x] [Citation(s) in RCA: 150] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Apoptosis is now recognized as an important process responsible for maintenance of the cellular balance between proliferation and death. Apoptosis is distinct from necrosis in that it is a programmed form of cell death and occurs without any accompanying inflammation. This form of cell death can be induced by a wide range of cellular signals, which leads to activation of cell death machinery within the cell and is characterized by distinct morphological changes. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes a process of continual cell turnover that is essential for maintenance of normal function. Cell proliferation is confined to the crypts, while differentiation occurs during a rapid, orderly migration up to the villus. The differentiated enterocytes, which make up the majority of the cells, then undergo a process of programmed cell death (apoptosis). Although apoptosis is essential for the maintenance of normal gut epithelial function, dysregulated apoptosis is seen in a number of pathological conditions in the gastrointestinal tract. The cellular mechanisms regulating this tightly regimented process have not been clearly defined and this topic represents an area of active investigation as delineation of this process will lead to a better understanding of normal gut mucosal growth.
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Affiliation(s)
- A Ramachandran
- Department of Gastrointestinal Sciences, Christian Medical College and Hospital, Vellore, India
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Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP. Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun 1999; 67:5587-96. [PMID: 10531204 PMCID: PMC96930 DOI: 10.1128/iai.67.11.5587-5596.1999] [Citation(s) in RCA: 260] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis.
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Affiliation(s)
- I R Henderson
- Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
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Schuch R, Sandlin RC, Maurelli AT. A system for identifying post-invasion functions of invasion genes: requirements for the Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination. Mol Microbiol 1999; 34:675-89. [PMID: 10564508 DOI: 10.1046/j.1365-2958.1999.01627.x] [Citation(s) in RCA: 89] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Invasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion system (Mxi-Spa) and its cognate set of secreted Ipa invasins. Once intracellular, the IcsA protein directs a form of actin-based motility that helps to drive intracellular bacterial movement, formation of cellular protrusions and cell-to-cell spread. Work in our laboratory has focused on identifying additional factors required for this intercellular form of dissemination. In this study, we sought to identify novel contributions of the type III secretion pathway to post-invasion-specific processes, distinct from its previously characterized roles in invasion. Studies of post-invasion Ipa and Mxi-Spa functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system called TIER (for test of intracellular expression requirements), whereby specific ipa, mxi or spa loci are transiently expressed before infection of tissue culture cell monolayers (thus supporting invasion), but then repressed after invasion in the intracellular environment. Such invasive type III secretion mutants (called TIER mutants) were severely restricted in their ability to spread intercellularly and form plaques in confluent tissue culture cell monolayers. Intercellular spread defects were associated with the repression of most type III pathway components examined, including structural (MxiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elements (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell monolayers showed that each of the TIER mutants was defective with respect to long-term intracellular proliferation and viability. Examination of TIER mutant-infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread - bacterial escape from protrusion-derived, double-membrane-bound vacuoles. The TIER mutants were eventually degraded in a process involving vacuolar acidification. Based on these findings, we propose that Ipa secretion via Mxi-Spa is required in the protrusion vacuole for double-membrane lysis.
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Affiliation(s)
- R Schuch
- Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA
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Tran Van Nhieu G, Caron E, Hall A, Sansonetti PJ. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells. EMBO J 1999; 18:3249-62. [PMID: 10369666 PMCID: PMC1171406 DOI: 10.1093/emboj/18.12.3249] [Citation(s) in RCA: 191] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Shigella proteins that are targeted to host cells by a type III secretion apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.
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Affiliation(s)
- G Tran Van Nhieu
- Unité de Pathogénie Microbienne Moléculaire INSERM U389, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France.
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Sebo P. A meeting of good friends: when the cell biology of prokaryotes and eukaryotes meet. Folia Microbiol (Praha) 1998; 43:235-8. [PMID: 9717249 DOI: 10.1007/bf02818607] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- P Sebo
- Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
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