1
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Mok CY, Chu HY, Lam WWL, Au SWN. Structural insights into the assembly pathway of the Helicobacter pylori CagT4SS outer membrane core complex. Structure 2024; 32:1725-1736.e4. [PMID: 39032488 DOI: 10.1016/j.str.2024.06.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Revised: 05/16/2024] [Accepted: 06/25/2024] [Indexed: 07/23/2024]
Abstract
Cag type IV secretion system (CagT4SS) translocates oncoprotein cytotoxin-associated gene A (CagA) into host cells and plays a key role in the pathogenesis of Helicobacter pylori. The structure of the outer membrane core complex (OMCC) in CagT4SS consists of CagX, CagY, CagM, CagT, and Cag3 in a stoichiometric ratio of 1:1:2:2:5 with 14-fold symmetry. However, the assembly pathway of OMCC remains elusive. Here, we report the crystal structures of CagT and Cag3-CagT complex, and the structural dynamics of Cag3 and CagT using hydrogen deuterium exchange-mass spectrometry (HDX-MS). The interwoven interaction of Cag3 and CagT involves conformational changes of CagT and β strand swapping. In conjunction with biochemical and biophysical assays, we further demonstrate the different oligomerization states of Cag3 and Cag3-CagT complex. Additionally, the association with CagM requires the pre-formation of Cag3-CagT complex. These results demonstrate the generation of different intermediate sub-assemblies and their structural flexibility, potentially representing different building blocks for OMCC assembly.
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Affiliation(s)
- Chin Yu Mok
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Hoi Yee Chu
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Wendy Wai Ling Lam
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Shannon Wing Ngor Au
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
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2
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Su Y, Zhang Z, Wang L, Zhang B, Su L. Whole-Genome Sequencing and Phenotypic Analysis of Streptococcus equi subsp. zooepidemicus Sequence Type 147 Isolated from China. Microorganisms 2024; 12:824. [PMID: 38674768 PMCID: PMC11051846 DOI: 10.3390/microorganisms12040824] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Revised: 04/15/2024] [Accepted: 04/16/2024] [Indexed: 04/28/2024] Open
Abstract
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the important zoonotic and opportunistic pathogens. In recent years, there has been growing evidence that supports the potential role of S. zooepidemicus in severe diseases in horses and other animals, including humans. Furthermore, the clinical isolation and drug resistance rates of S. zooepidemicus have been increasing yearly, leading to interest in its in-depth genomic analysis. In order to deepen the understanding of the S. zooepidemicus characteristics and genomic features, we investigated the genomic islands, mobile genetic elements, virulence and resistance genes, and phenotype of S. zooepidemicus strain ZHZ 211 (ST147), isolated from an equine farm in China. We obtained a 2.18 Mb, high-quality chromosome and found eight genomic islands. According to a comparative genomic investigation with other reference strains, ZHZ 211 has more virulence factors, like an iron uptake system, adherence, exoenzymes, and antiphagocytosis. More interestingly, ZHZ 211 has acquired a mobile genetic element (MGE), prophage Ph01, which was found to be in the chromosome of this strain and included two hyaluronidase (hyl) genes, important virulence factors of the strain. Moreover, two transposons and two virulence (virD4) genes were found to be located in the same genome island of ZHZ 211. In vitro phenotypic results showed that ZHZ 211 grows faster and is resistant to clarithromycin, enrofloxacin, and sulfonamides. The higher biofilm-forming capabilities of ZHZ 211 may provide a competitive advantage for survival in its niche. The results expand our understanding of the genomic, pathogenicity, and resistance characterization of Streptococcus zooepidemicus and facilitate further exploration of its molecular pathogenic mechanism.
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Affiliation(s)
- Yan Su
- Department of Microbiology and Immunology, College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
| | - Zehua Zhang
- Department of Microbiology and Immunology, College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
| | - Li Wang
- Department of Microbiology and Immunology, College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
| | - Baojiang Zhang
- Department of Microbiology and Immunology, College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
| | - Lingling Su
- Xinjiang Academy of Animal Science, Urumqi 830000, China
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3
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Azizimoghaddam Y, Kermanpour S, Mirzaei N, Houri H, Nabavi-Rad A, Asadzadeh Aghdaei H, Yadegar A, Zali MR. Genetic diversity of Helicobacter pylori type IV secretion system cagI and cagN genes and their association with clinical diseases. Sci Rep 2023; 13:10264. [PMID: 37355714 PMCID: PMC10290643 DOI: 10.1038/s41598-023-37392-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Accepted: 06/21/2023] [Indexed: 06/26/2023] Open
Abstract
A number of cagPAI genes in the Helicobacter pylori genome are considered the most evolved genes under a diversifying selection and evolutionary pressure. Among them, cagI and cagN are described as a part of the two different-operon of cagPAI that are involved in the T4SS machinery, but the definite association of these factors with clinical manifestations is still unclear. A total of 70 H. pylori isolates were obtained from different gastroduodenal patients. All isolates were examined for the presence of primary H. pylori virulence genes by PCR analysis. Direct DNA sequence analysis was performed for the cagI and cagN genes. The results were compared with the reference strain. The cagI, cagN, cagA, cagL, vacA s1m1, vacA s1m2, vacA s2m2, babA2, sabA, and dupA genotypes were detected in 80, 91.4, 84, 91.4, 32.8, 42.8, 24.4, 97.1, 84.3, and 84.3% of the total isolates, respectively. The most variable codon usage in cagI was observed at residues 20-25, 55-60, 94, 181-199, 213-221, 241-268, and 319-320, while the most variable codon usage in CagN hypervariable motif (CagNHM) was observed at residues 53 to 63. Sequencing data analysis of cagN revealed a hypothetical hexapeptide motif (EAKDEN/K) in residues of 278-283 among six H. pylori isolates, which needs further studies to evaluate its putative function. The present study demonstrated a high prevalence of cagI and cagN genes among Iranian H. pylori isolates with gastroduodenal diseases. Furthermore, no significant correlation between cagI and cagN variants and clinical diseases was observed in the present study. However, all patients had a high prevalence of cagPAI genes including cagI, cagN, cagA, and cagL, which indicates more potential role of these genes in disease outcome.
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Affiliation(s)
- Yasaman Azizimoghaddam
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Sadaf Kermanpour
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Nasrin Mirzaei
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Hamidreza Houri
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Ali Nabavi-Rad
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Hamid Asadzadeh Aghdaei
- Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Abbas Yadegar
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
| | - Mohammad Reza Zali
- Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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4
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Miftahussurur M, Alfaray RI, Fauzia KA, Dewayani A, Doohan D, Waskito LA, Rezkitha YAA, Utomo DH, Somayana G, Fahrial Syam A, Lubis M, Akada J, Matsumoto T, Yamaoka Y. Low-grade intestinal metaplasia in Indonesia: Insights into the expression of proinflammatory cytokines during Helicobacter pylori infection and unique East-Asian CagA characteristics. Cytokine 2023; 163:156122. [PMID: 36640695 DOI: 10.1016/j.cyto.2022.156122] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2022] [Revised: 12/28/2022] [Accepted: 12/28/2022] [Indexed: 01/15/2023]
Abstract
Helicobacter pylori infection is a major cause of intestinal metaplasia. In this study, we aimed to understand the reason underlying the low grade and incidence of intestinal metaplasia in Indonesia, based on the expression of genes encoding proinflammatory cytokines in gastric biopsy specimens. The possible reasons for the lesser virulence of the East-Asian-type CagA in Indonesia than that of the Western-type CagA, which is not common in other countries, were also investigated. The mRNA expression of cytokines was evaluated using real-time PCR. CagA characteristics were analyzed using in silico analysis. The expression of cytokines was typically not robust, among H. pylori-infected subjects in Indonesia, despite them predominantly demonstrating the East-Asian-type CagA. This might partially be explained by the characteristics of the East-Asian-type CagA in Indonesia, which showed a higher instability index and required higher energy to interact with proteins related to the cytokine induction pathway compared with the other types (p < 0.001 and p < 0.05, respectively). Taken together, besides the low prevalence of H. pylori, the low inflammatory response of the host and low CagA virulence, even among populations with high infection rates, may play an essential role in the low grade and low incidence of intestinal metaplasia in Indonesia. We believe that these findings would be relevant for better understanding of intestinal metaplasia, which is closely associated with the development of gastric cancer.
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Affiliation(s)
- Muhammad Miftahussurur
- Division of Gastroentero-Hepatology, Department of Internal Medicine, Faculty of Medicine-Dr. Soetomo Teaching Hospital, Universitas Airlangga, Jalan Mayjend Prof, Dr. Moestopo, No. 6-8, Surabaya, Surabaya 60131, Indonesia; Helicobacter pylori and Microbiota Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia.
| | - Ricky Indra Alfaray
- Helicobacter pylori and Microbiota Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia; Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, 1-1, Idaigaoka, Hasama-machi, Yufu Oita 879-5593, Japan.
| | - Kartika Afrida Fauzia
- Helicobacter pylori and Microbiota Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia; Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, 1-1, Idaigaoka, Hasama-machi, Yufu Oita 879-5593, Japan; Department of Public Health and Preventive Medicine, Faculty of Medicine, Universitas Airlangga, Surabaya 60132, Indonesia.
| | - Astri Dewayani
- Helicobacter pylori and Microbiota Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia; Department of Infectious Disease Control, Oita University Faculty of Medicine, 1-1, Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japan; Department of Anatomy, Histology and Pharmacology, Universitas Airlangga, Surabaya 60131, Indonesia.
| | - Dalla Doohan
- Helicobacter pylori and Microbiota Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia; Department of Anatomy, Histology and Pharmacology, Universitas Airlangga, Surabaya 60131, Indonesia.
| | - Langgeng Agung Waskito
- Helicobacter pylori and Microbiota Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia; Department of Physiology and Medical Biochemistry, Faculty of Medicine, Universitas Airlangga, Surabaya, 60132, Indonesia; Department of Internal Medicine, Faculty of Medicine, Universitas Airlangga, Surabaya 60132, Indonesia.
| | - Yudith Annisa Ayu Rezkitha
- Helicobacter pylori and Microbiota Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia; Department of Internal Medicine, Faculty of Medicine, University of Muhammadiyah, Surabaya, Surabaya 60113, Indonesia.
| | - Didik Huswo Utomo
- Research and Education Center for Bioinformatics, Indonesia Institute of Bioinformatics, Malang 65162, Indonesia.
| | - Gde Somayana
- Gastroentero Hepatology Division, Department of Internal Medicine, Faculty of Medicine-Sanglah Hospital, Udayana University, Denpasar, Bali 80114, Indonesia.
| | - Ari Fahrial Syam
- Division of Gastroenterology, Department of Internal Medicine, Faculty of Medicine-Cipto Mangunkusumo Teaching Hospital, University of Indonesia, Jakarta 10430, Indonesia.
| | - Masrul Lubis
- Division of Gastroenterology, Department of Internal Medicine, Faculty of Medicine-Cipto Mangunkusumo Teaching Hospital, Universitas Sumatera Utara, Medan 20222, Indonesia
| | - Junko Akada
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, 1-1, Idaigaoka, Hasama-machi, Yufu Oita 879-5593, Japan.
| | - Takashi Matsumoto
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, 1-1, Idaigaoka, Hasama-machi, Yufu Oita 879-5593, Japan.
| | - Yoshio Yamaoka
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, 1-1, Idaigaoka, Hasama-machi, Yufu Oita 879-5593, Japan; Department of Medicine, Gastroenterology and Hepatology Section, Baylor College of Medicine, Houston, TX 77030, USA.
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5
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Gobert AP, Wilson KT. Induction and Regulation of the Innate Immune Response in Helicobacter pylori Infection. Cell Mol Gastroenterol Hepatol 2022; 13:1347-1363. [PMID: 35124288 PMCID: PMC8933844 DOI: 10.1016/j.jcmgh.2022.01.022] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 01/25/2022] [Accepted: 01/25/2022] [Indexed: 12/30/2022]
Abstract
Gastric cancer (GC) is the fifth most common cancer and the fourth most common cause of cancer-related death worldwide. The intestinal type of GC progresses from acute to chronic gastritis, multifocal atrophic gastritis, intestinal metaplasia, dysplasia, and carcinoma. Infection of the stomach by Helicobacter pylori, a Gram-negative bacterium that infects approximately 50% of the world's population, is the causal determinant that initiates the gastric inflammation and then disease progression. In this context, the induction of the innate immune response of gastric epithelial cells and myeloid cells by H. pylori effectors plays a critical role in the outcome of the infection. However, only 1% to 3% of infected patients develop gastric adenocarcinoma, emphasizing that other mechanisms regulate the localized non-specific response, including the gastric microbiota and genetic factors. This review summarizes studies describing the factors that induce and regulate the mucosal innate immune response during H. pylori infection.
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Affiliation(s)
- Alain P Gobert
- Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Nashville, Tennessee; Center for Mucosal Inflammation and Cancer, Nashville, Tennessee; Program in Cancer Biology, Nashville, Tennessee.
| | - Keith T Wilson
- Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Nashville, Tennessee; Center for Mucosal Inflammation and Cancer, Nashville, Tennessee; Program in Cancer Biology, Nashville, Tennessee; Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee; Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee.
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6
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Lee HA, Kim JY, Kim J, Nam B, Kim O. Anti- Helicobacter pylori activity of acomplex mixture of Lactobacillus paracasei HP7 including the extract of Perilla frutescens var. acuta and Glycyrrhiza glabra. Lab Anim Res 2020; 36:40. [PMID: 33134159 PMCID: PMC7594437 DOI: 10.1186/s42826-020-00073-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2020] [Accepted: 10/18/2020] [Indexed: 12/31/2022] Open
Abstract
The effect of standard therapeutic strategies on Helicobacter pylori infection is diminished over time owing to the emergence of drug resistant strains. In this study, we would like to confirm the enhanced effect of L. paracasei HP7, which has been reported to exert antibacterial and gastric mucosal protective effects, in combination with Perilla frutescens var. acuta (P. frutescens)and Glycyrrhiza glabra (G. glabra) extracts. P. frutescens extract and G. glabra extract were found to inhibit the growth of H. pylori in a concentration-dependent manner, and the combination of L. paracasei HP7 and P. frutescens extract and G. glabra extract effectively inhibited H. pylori from attaching to AGS a gastric epithelial cells. Moreover, L. paracasei HP7 complex mixture containing P. frutescens and G. glabra extracts has been shown to inhibit H. pylori virulence genes such as AlpA, CagA, FlaA and UreA. When H. pylori-infected mice were administered a complex mixture of L. paracasei HP7 containing P. frutescens and G. glabra extract, the infection rate of H. pylori was significantly reduced. In addition, the L. paracasei HP7 complex mixture significantly reduced serum IL-8 levels and stomach inflammation in H. pylori infected mice. These results suggest that a complex mixture of L. paracasei HP7 containing P. frutescens and G. glabra extracts may be an alternative to treating diseases caused by H. pylori infection.
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Affiliation(s)
- Hyun-A Lee
- Center for Animal Resources Development, Wonkwang University, 460 Iksandae-ro, Iksan, 54538 Republic of Korea
| | - Joo-Yun Kim
- R&BD Center, Korea Yakult Co., Ltd., 22 Giheungdanji-ro 24 beon-gil, Yongin-si, Gyeonggi-do 17086 Republic of Korea
| | - Jisoo Kim
- R&BD Center, Korea Yakult Co., Ltd., 22 Giheungdanji-ro 24 beon-gil, Yongin-si, Gyeonggi-do 17086 Republic of Korea
| | - Bora Nam
- R&BD Center, Korea Yakult Co., Ltd., 22 Giheungdanji-ro 24 beon-gil, Yongin-si, Gyeonggi-do 17086 Republic of Korea
| | - Okjin Kim
- Center for Animal Resources Development, Wonkwang University, 460 Iksandae-ro, Iksan, 54538 Republic of Korea
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7
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Rizzato C, Torres J, Obazee O, Camorlinga-Ponce M, Trujillo E, Stein A, Mendez-Tenorio A, Bravo MM, Canzian F, Kato I. Variations in cag pathogenicity island genes of Helicobacter pylori from Latin American groups may influence neoplastic progression to gastric cancer. Sci Rep 2020; 10:6570. [PMID: 32300197 PMCID: PMC7162905 DOI: 10.1038/s41598-020-63463-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Accepted: 03/20/2020] [Indexed: 12/11/2022] Open
Abstract
Helicobacter pylori (HP) colonizes the human stomach and induces acute gastritis, peptic ulcer disease, atrophic gastritis, and gastric adenocarcinoma. Increased virulence in HP isolates derives from harboring the cag (cytotoxin-associated genes) pathogenicity island (cagPAI). We analyzed the microvariants in cagPAI genes with the hypothesis that they may play an important role in determining HP virulence. We tested DNAs from cagA positive patients HP isolates; a total of 74 patients with chronic gastritis (CG, N = 37), intestinal metaplasia (IM, N = 21) or gastric cancer (GC, N = 16) from Mexico and Colombia. We selected 520 non-synonymous variants with at least 7.5% frequency in the original sequence outputs or with a minimum of 5 isolates with minor allele. After adjustment for multiple comparisons, no variants were statistically significantly associated with IM or GC. However, 19 non-synonymous showed conventional P-values < 0.05 comparing the frequency of the alleles between the isolates from subjects with gastritis and isolates from subjects with IM or GC; 12 of these showed a significant correlation with the severity of the disease. The present study revealed that several cagPAI genes from Latin American Western HP strains contains a number of non-synonymous variants in relatively high frequencies which could influence on the clinical outcome. However, none of the associations remained statistically significant after adjustment for multiple comparison.
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Affiliation(s)
- Cosmeri Rizzato
- Department of Translation Research and of New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy.
| | - Javier Torres
- Unidad de Investigación en Enfermedades Infecciosas, UMAE Pediatría, Instituto Mexicano del Seguro Social, Mexico City, Mexico
| | - Ofure Obazee
- Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Margarita Camorlinga-Ponce
- Unidad de Investigación en Enfermedades Infecciosas, UMAE Pediatría, Instituto Mexicano del Seguro Social, Mexico City, Mexico
| | - Esperanza Trujillo
- Grupo de Investigación en Biología del Cáncer. Instituto Nacional de Cancerología, Bogotá, Colombia
| | - Angelika Stein
- Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Alfonso Mendez-Tenorio
- Laboratorio de Biotecnología y Bioinformática Genómica, ENCB, Instituto Politécnico Nacional, México City, México
| | - Maria Mercedes Bravo
- Grupo de Investigación en Biología del Cáncer. Instituto Nacional de Cancerología, Bogotá, Colombia
| | - Federico Canzian
- Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Ikuko Kato
- Department of Oncology and Pathology, Wayne State University School of Medicine, Detroit, MI, USA
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8
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Fujii Y, Murata-Kamiya N, Hatakeyama M. Helicobacter pylori CagA oncoprotein interacts with SHIP2 to increase its delivery into gastric epithelial cells. Cancer Sci 2020; 111:1596-1606. [PMID: 32198795 PMCID: PMC7226221 DOI: 10.1111/cas.14391] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Revised: 03/11/2020] [Accepted: 03/12/2020] [Indexed: 12/13/2022] Open
Abstract
Chronic infection with Helicobacter pylori cagA‐positive strains is causally associated with the development of gastric diseases, most notably gastric cancer. The cagA‐encoded CagA protein, which is injected into gastric epithelial cells by bacterial type IV secretion, undergoes tyrosine phosphorylation at the Glu‐Pro‐Ile‐Tyr‐Ala (EPIYA) segments (EPIYA‐A, EPIYA‐B, EPIYA‐C, and EPIYA‐D), which are present in various numbers and combinations in its C‐terminal polymorphic region, thereby enabling CagA to promiscuously interact with SH2 domain‐containing host cell proteins, including the prooncogenic SH2 domain‐containing protein tyrosine phosphatase 2 (SHP2). Perturbation of host protein functions by aberrant complex formation with CagA has been considered to contribute to the development of gastric cancer. Here we show that SHIP2, an SH2 domain‐containing phosphatidylinositol 5′‐phosphatase, is a hitherto undiscovered CagA‐binding host protein. Similar to SHP2, SHIP2 binds to the Western CagA‐specific EPIYA‐C segment or East Asian CagA‐specific EPIYA‐D segment through the SH2 domain in a tyrosine phosphorylation‐dependent manner. In contrast to the case of SHP2, however, SHIP2 binds more strongly to EPIYA‐C than to EPIYA‐D. Interaction with CagA tethers SHIP2 to the plasma membrane, where it mediates production of phosphatidylinositol 3,4‐diphosphate [PI(3,4)P2]. The CagA‐SHIP2 interaction also potentiates the morphogenetic activity of CagA, which is caused by CagA‐deregulated SHP2. This study indicates that initially delivered CagA interacts with SHIP2 and thereby strengthens H. pylori‐host cell attachment by altering membrane phosphatidylinositol compositions, which potentiates subsequent delivery of CagA that binds to and thereby deregulates the prooncogenic phosphatase SHP2.
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Affiliation(s)
- Yumiko Fujii
- Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.,Max-Planck Center for Integrative Inflammology, The University of Tokyo, Tokyo, Japan.,Division of Tumor Pathology, Asahikawa Medical University, Asahikawa, Japan
| | - Naoko Murata-Kamiya
- Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Masanori Hatakeyama
- Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.,Max-Planck Center for Integrative Inflammology, The University of Tokyo, Tokyo, Japan
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9
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Silbergleit M, Vasquez AA, Miller CJ, Sun J, Kato I. Oral and intestinal bacterial exotoxins: Potential linked to carcinogenesis. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2020; 171:131-193. [PMID: 32475520 DOI: 10.1016/bs.pmbts.2020.02.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Growing evidence suggests that imbalances in resident microbes (dysbiosis) can promote chronic inflammation, immune-subversion, and production of carcinogenic metabolites, thus leading to neoplasia. Yet, evidence to support a direct link of individual bacteria species to human sporadic cancer is still limited. This chapter focuses on several emerging bacterial toxins that have recently been characterized for their potential oncogenic properties toward human orodigestive cancer and the presence of which in human tissue samples has been documented. These include cytolethal distending toxins produced by various members of gamma and epsilon Proteobacteria, Dentilisin from mammalian oral Treponema, Pasteurella multocida toxin, two Fusobacterial toxins, FadA and Fap2, Bacteroides fragilis toxin, colibactin, cytotoxic necrotizing factors and α-hemolysin from Escherichia coli, and Salmonella enterica AvrA. It was clear that these bacterial toxins have biological activities to induce several hallmarks of cancer. Some toxins directly interact with DNA or chromosomes leading to their breakdowns, causing mutations and genome instability, and others modulate cell proliferation, replication and death and facilitate immune evasion and tumor invasion, prying specific oncogene and tumor suppressor pathways, such as p53 and β-catenin/Wnt. In addition, most bacterial toxins control tumor-promoting inflammation in complex and diverse mechanisms. Despite growing laboratory evidence to support oncogenic potential of selected bacterial toxins, we need more direct evidence from human studies and mechanistic data from physiologically relevant experimental animal models, which can reflect chronic infection in vivo, as well as take bacterial-bacterial interactions among microbiome into consideration.
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Affiliation(s)
| | - Adrian A Vasquez
- Department of Civil and Environmental Engineering, Wayne State University, Healthy Urban Waters, Detroit, MI, United States
| | - Carol J Miller
- Department of Civil and Environmental Engineering, Wayne State University, Healthy Urban Waters, Detroit, MI, United States
| | - Jun Sun
- Department of Medicine, College of Medicine, University of Illinois at Chicago, Chicago, IL, United States
| | - Ikuko Kato
- Department of Oncology, Wayne State University School of Medicine, Detroit, MI, United States; Department of Pathology, Wayne State University School of Medicine, Detroit, MI, United States.
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10
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Zeng B, Chen C, Yi Q, Zhang X, Wu X, Zheng S, Li N, She F. N-terminal region of Helicobacter pylori CagA induces IL-8 production in gastric epithelial cells via the β1 integrin receptor. J Med Microbiol 2020; 69:457-464. [DOI: 10.1099/jmm.0.001088] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Introduction.
Helicobacter pylori
is associated with gastrointestinal disease, most notably gastric cancer. Cytotoxin-associated antigen A (CagA), an important virulence factor for
H. pylori
pathogenicity, induces host cells to release inflammatory factors, especially interleukin-8 (IL-8). The mechanism by which C-terminal CagA induces IL-8 production has been studied extensively, but little is known about the role of the N-terminus.
Aim. To investigate the effect of CagA303–456aa (a peptide in the N-terminal CagA) on IL-8 production by gastric epithelial cells.
Methodology. CagA303-456aa was produced by a prokaryotic expression system and purified by Strep-tag affinity chromatography. An integrin β1 (ITGB1)-deficient AGS cell line was constructed using the CRISPR/Cas9 technique, and NCTC 11637 cagA and/or cagL knockout mutants were constructed via homologous recombination. The levels of IL-8 production were determined by enzyme-linked immunosorbent assay (ELISA), and p38 and ERK1/2 phosphorylation were examined by Western blot.
Results. CagA303-456aa induced IL-8 expression by AGS cells. IL-8 induction by CagA303-456aawas specifically inhibited by ITGB1 deficiency. Notably, CagA303-456aa activated the phosphorylation of both p38 and ERK1/2, and blocking p38 and ERK1/2 activity significantly reduced IL-8 induction by CagA303-456aa. ITGB1 deficiency also inhibited the activation of p38 phosphorylation by CagA303-456aa. Finally, experiments in CagA and/or CagL knockout bacterial lines demonstrated that extracellular CagA might induce IL-8 production by AGS cells.
Conclusion. Residues 303–456 of the N-terminal region of CagA induce IL-8 production via a CagA303-456–ITGB1–p38–IL-8 pathway, and ERK1/2 is also involved in the release of IL-8. Extracellular CagA might induce IL-8 production before translocation into AGS cells.
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Affiliation(s)
- Bangwei Zeng
- Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, Fujian Province 350001, PR China
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
| | - Chu Chen
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
| | - Qingfeng Yi
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
| | - Xiaoyan Zhang
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
| | - Xiangyan Wu
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
| | - Shurong Zheng
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
| | - Neng Li
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
| | - Feifei She
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
- Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, 1 Xuefu North Road, University Town, Fuzhou, Fujian Province 350122, PR China
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Zhang H, Zhang Y, Song Z, Li R, Ruan H, Liu Q, Huang X. sncRNAs packaged by Helicobacter pylori outer membrane vesicles attenuate IL-8 secretion in human cells. Int J Med Microbiol 2020; 310:151356. [DOI: 10.1016/j.ijmm.2019.151356] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2019] [Revised: 08/06/2019] [Accepted: 09/15/2019] [Indexed: 02/07/2023] Open
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12
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Pachathundikandi SK, Gutiérrez-Escobar AJ, Tegtmeyer N. Tailor-Made Detection of Individual Phosphorylated and Non-Phosphorylated EPIYA-Motifs of Helicobacter pylori Oncoprotein CagA. Cancers (Basel) 2019; 11:cancers11081163. [PMID: 31412675 PMCID: PMC6721621 DOI: 10.3390/cancers11081163] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2019] [Revised: 07/25/2019] [Accepted: 08/08/2019] [Indexed: 02/07/2023] Open
Abstract
The gastric pathogen and carcinogen Helicobacter pylori(H. pylori) encodes a type IV secretion system for translocation of the effector protein CagA into host cells. Injected CagA becomes tyrosine-phosphorylated at the five amino acid residue Glutamate-Proline- Isoleucine-Tyrosine-Alanine (EPIYA)-sequence motifs. These phosphorylated EPIYA-sites represent recognition motifs for binding of multiple host factors, which then manipulate signaling pathways to trigger gastric disease. Thus, efficient detection of single phosphorylated EPIYA-motifs in CagA is required. Detection of phospho-CagA is primarily performed using commercial pan-phosphotyrosine antibodies. However, those antibodies were originally generated to recognize many phosphotyrosines in various mammalian proteins and are not optimized for use in bacteria. To address this important limitation, we synthesized 11-mer phospho- and non-phospho-peptides from EPIYA-motifs A, B, and C, and produced three phospho-specific and three non-phospho-specific rabbit polyclonal CagA antibodies. These antibodies specifically recognized the corresponding phosphorylated and non-phosphorylated EPIYA-motifs, while the EPIYA-C antibodies also recognized the related East-Asian EPIYA-D motif. Otherwise, no cross-reactivity of the antibodies among EPIYAs was observed. Western blotting demonstrated that each EPIYA-motif can be predominantly phosphorylated during H. pylori infection. This represents the first complete set of phospho-specific antibodies for an effector protein in bacteria, providing useful tools to gather information for the categorization of CagA phosphorylation, cancer signaling, and gastric disease progression.
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Affiliation(s)
- Suneesh Kumar Pachathundikandi
- Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstraße 5, D-91058 Erlangen, Germany
| | - Andrés Julián Gutiérrez-Escobar
- Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstraße 5, D-91058 Erlangen, Germany
| | - Nicole Tegtmeyer
- Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Staudtstraße 5, D-91058 Erlangen, Germany.
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13
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Arya T, Oudouhou F, Casu B, Bessette B, Sygusch J, Baron C. Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system. Sci Rep 2019; 9:6474. [PMID: 31019200 PMCID: PMC6482174 DOI: 10.1038/s41598-019-42876-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Accepted: 04/05/2019] [Indexed: 01/16/2023] Open
Abstract
Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.
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Affiliation(s)
- Tarun Arya
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Québec, Canada
| | - Flore Oudouhou
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Québec, Canada
| | - Bastien Casu
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Québec, Canada
| | - Benoit Bessette
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Québec, Canada
| | - Jurgen Sygusch
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Québec, Canada
| | - Christian Baron
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Québec, Canada.
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14
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α-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY. Proc Natl Acad Sci U S A 2019; 116:5077-5085. [PMID: 30804204 DOI: 10.1073/pnas.1814497116] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.
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15
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Javed S, Skoog EC, Solnick JV. Impact of Helicobacter pylori Virulence Factors on the Host Immune Response and Gastric Pathology. Curr Top Microbiol Immunol 2019; 421:21-52. [PMID: 31123884 DOI: 10.1007/978-3-030-15138-6_2] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Helicobacter pylori chronically infects nearly half the world's population, yet most of those infected remain asymptomatic throughout their lifetime. The outcome of infection-peptic ulcer disease or gastric cancer versus asymptomatic colonization-is a product of host genetics, environmental influences, and differences in bacterial virulence factors. Here, we review the current understanding of the cag pathogenicity island (cagPAI), the vacuolating cytotoxin (VacA), and a large family of outer membrane proteins (OMPs), which are among the best understood H. pylori virulence determinants that contribute to disease. Each of these virulence factors is characterized by allelic and phenotypic diversity that is apparent within and across individuals, as well as over time, and modulates inflammation. From the bacterial perspective, inflammation is probably a necessary evil because it promotes nutrient acquisition, but at the cost of reduction in bacterial load and therefore decreases the chance of transmission to a new host. The general picture that emerges is one of a chronic bacterial infection that is dependent on both inducing and carefully regulating the host inflammatory response. A better understanding of these regulatory mechanisms may have implications for the control of chronic inflammatory diseases that are increasingly common causes of human morbidity and mortality.
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Affiliation(s)
- Sundus Javed
- Department of Medicine, Department of Microbiology & Immunology, Center for Comparative Medicine, University of California, Davis School of Medicine, Davis, CA, 95616, USA.,Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
| | - Emma C Skoog
- Department of Medicine, Department of Microbiology & Immunology, Center for Comparative Medicine, University of California, Davis School of Medicine, Davis, CA, 95616, USA
| | - Jay V Solnick
- Department of Medicine, Department of Microbiology & Immunology, Center for Comparative Medicine, University of California, Davis School of Medicine, Davis, CA, 95616, USA. .,Center for Comparative Medicine, University of California, Davis, Davis, CA, 95616, USA.
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16
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Bonsor DA, Sundberg EJ. Roles of Adhesion to Epithelial Cells in Gastric Colonization by Helicobacter pylori. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1149:57-75. [PMID: 31016628 DOI: 10.1007/5584_2019_359] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Helicobacter pylori adherence to host epithelial cells is essential for its survival against the harsh conditions of the stomach and for successful colonization. Adherence of H. pylori is achieved through several related families of outer membrane proteins and proteins of a type IV secretion system (T4SS), which bridge H. pylori to host cells through protein-protein and other protein-ligand interactions. Local environmental conditions such as cell type, available host cell surface proteins and/or ligands, as well as responses by the host immune system force H. pylori to alter expression of these proteins to adapt quickly to the local environment in order to colonize and survive. Some of these host-pathogen interactions appear to function in a "catch-and-release" manner, regulated by reversible binding at varying pH and allowing H. pylori to detach itself from cells or debris sloughed off the gastric epithelial lining in order to return for subsequent productive interactions. Other interactions between bacterial adhesin proteins and host adhesion molecules, however, appear to function as a committed step in certain pathogenic processes, such as translocation of the CagA oncoprotein through the H. pylori T4SS and into host gastric epithelial cells. Understanding these adhesion interactions is critical for devising new therapeutic strategies, as they are responsible for the earliest stage of infection and its maintenance. This review will discuss the expression and regulation of several outer membrane proteins and CagL, how they engage their known host cell protein/ligand targets, and their effects on clinical outcome.
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Affiliation(s)
- Daniel A Bonsor
- Institute of Human Virology, University of Maryland School of Medicine, University of Maryland, Baltimore, MD, USA
| | - Eric J Sundberg
- Institute of Human Virology, University of Maryland School of Medicine, University of Maryland, Baltimore, MD, USA. .,Department of Medicine, University of Maryland School of Medicine, University of Maryland, Baltimore, MD, USA. .,Department of Microbiology and Immunology, University of Maryland School of Medicine, University of Maryland, Baltimore, MD, USA.
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17
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Tegtmeyer N, Harrer A, Schmitt V, Singer BB, Backert S. Expression of CEACAM1 or CEACAM5 in AZ-521 cells restores the type IV secretion deficiency for translocation of CagA byHelicobacter pylori. Cell Microbiol 2018; 21:e12965. [DOI: 10.1111/cmi.12965] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2018] [Revised: 10/04/2018] [Accepted: 10/08/2018] [Indexed: 02/06/2023]
Affiliation(s)
- Nicole Tegtmeyer
- Department of Biology, Division of Microbiology; Friedrich Alexander University Erlangen; Erlangen Germany
| | - Aileen Harrer
- Department of Biology, Division of Microbiology; Friedrich Alexander University Erlangen; Erlangen Germany
| | - Verena Schmitt
- Medical Faculty, Institute of Anatomy; University of Duisburg-Essen; Essen Germany
| | - Bernhard B. Singer
- Medical Faculty, Institute of Anatomy; University of Duisburg-Essen; Essen Germany
| | - Steffen Backert
- Department of Biology, Division of Microbiology; Friedrich Alexander University Erlangen; Erlangen Germany
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18
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Kim SH. Comparative Proteome Analysis of Zerumbone-treated Helicobacter pylori. KOREAN JOURNAL OF CLINICAL LABORATORY SCIENCE 2018. [DOI: 10.15324/kjcls.2018.50.3.275] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Affiliation(s)
- Sa-Hyun Kim
- Department of Clinical Laboratory Science, Semyung University, Jecheon, Korea
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19
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Nell S, Estibariz I, Krebes J, Bunk B, Graham DY, Overmann J, Song Y, Spröer C, Yang I, Wex T, Korlach J, Malfertheiner P, Suerbaum S. Genome and Methylome Variation in Helicobacter pylori With a cag Pathogenicity Island During Early Stages of Human Infection. Gastroenterology 2018; 154:612-623.e7. [PMID: 29066327 DOI: 10.1053/j.gastro.2017.10.014] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Revised: 09/22/2017] [Accepted: 10/02/2017] [Indexed: 12/21/2022]
Abstract
BACKGROUND & AIMS Helicobacter pylori is remarkable for its genetic variation; yet, little is known about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori during experimental infection. METHODS We performed a randomized Phase I/II, observer-blind, placebo-controlled study of 12 healthy, H pylori-negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n = 7) or placebo (n = 5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 reisolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag pathogenicity island function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays. RESULTS The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss of cag pathogenicity island function was observed in 3 reisolates. In addition, 3 reisolates from the vaccine group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of vacuolization activity. We observed interstrain variation in methylomes due to phase variation in genes encoding methyltransferases. CONCLUSIONS We analyzed adaptation of a fully virulent strain of H pylori to 12 different volunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes in the absence of interstrain recombination. Our findings indicate that the large amount of genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476.
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Affiliation(s)
- Sandra Nell
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany
| | - Iratxe Estibariz
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany; Medical Microbiology and Hospital Epidemiology, Max von Pettenkofer Institute, Faculty of Medicine, LMU Munich, München, Germany
| | - Juliane Krebes
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany
| | - Boyke Bunk
- German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
| | - David Y Graham
- Baylor College of Medicine, Michael E. DeBakey VAMC, Houston, Texas
| | - Jörg Overmann
- German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
| | - Yi Song
- Pacific Biosciences, Menlo Park, California
| | - Cathrin Spröer
- German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
| | - Ines Yang
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany
| | - Thomas Wex
- Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany
| | | | - Peter Malfertheiner
- Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany
| | - Sebastian Suerbaum
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Site, Hannover, Germany; Medical Microbiology and Hospital Epidemiology, Max von Pettenkofer Institute, Faculty of Medicine, LMU Munich, München, Germany; National Reference Center for Helicobacter pylori, München, Germany.
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Preliminary study and bioinformatics analysis on the potential role of CagQ in type IV secretion system of H.pylori. Microb Pathog 2018; 116:1-7. [PMID: 29306012 DOI: 10.1016/j.micpath.2017.12.076] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2017] [Revised: 12/25/2017] [Accepted: 12/30/2017] [Indexed: 12/13/2022]
Abstract
Helicobacter pylori (H.pylori), is a major causative agent of chronic gastritis, gastric carcinoma and duodenal ulcer. Remarkably, H.pylori carries cytotoxin-associated gene pathogenicity island (CagPAI) which encodes a type IV secretion system (T4SS). T4SS is capable of forming a syringe-like structure to deliver oncoprotein cytotoxin-associated Antigen (CagA) into gastric epithelial cells and resulting in a cascade of events in host cells, such as induction of pro-inflammatory cytokines, alteration of cellular gene expression and cytoskeletal rearrangements. Among of those proteins in T4SS, CagQ still remains unknown functions. In this study, we performed analysis of protein-protein interaction and revealed that CagQ correlated with the most virulence factor CagA in T4SS. Interestingly, our data demonstrated that CagQ-deficient mutant strain had significantly lower expression in both mRNA and protein levels of CagA compared with H.pylori wild-type strain 26695. Moreover, we demonstrated that CagQ deletion also played a vital role in suppressing CagA-induced apoptosis of host gastric epithelial cells. To further investigate the role of CagQ in T4SS, we used bioinformatics analysis to provide a preliminary insight into CagQ. These results showed that CagQ possessed a transmembrane region from amino acid 50-68 which is also consistent with the prediction of hydrophobic scale and structure modeling. Thus, we conclude that CagQ is a membrane protein in T4SS and is crucial for maintaining CagA expression and CagA-induced apoptotic effects. This provides a novel specific therapeutic target for H.pylori CagA-induced gastroduodenal diseases.
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21
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Functional Cytotoxin Associated Gene A in Helicobacter pylori Strains and Its Association with Integrity of Cag-pathogenicity Island and Histopathological Changes of Gastric Tissue. ARCHIVES OF CLINICAL INFECTIOUS DISEASES 2017. [DOI: 10.5812/archcid.62955] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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22
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Abstract
Helicobacter pylori is the most common bacterial infection worldwide, and virtually all infected persons develop co-existing gastritis. H. pylori is able to send and receive signals from the gastric mucosa, which enables both host and microbe to engage in a dynamic equilibrium. In order to persist within the human host, H. pylori has adopted dichotomous strategies to both induce inflammation as a means of liberating nutrients while simultaneously tempering the immune response to augment its survival. Toll-like receptors (TLRs) and Nod proteins are innate immune receptors that are present in epithelial cells and represent the first line of defense against pathogens. To ensure persistence, H. pylori manipulates TLR-mediated defenses using strategies that include rendering its LPS and flagellin to be non-stimulatory to TLR4 and TLR5, respectively; translocating peptidoglycan into host cells to induce NOD1-mediated anti-inflammatory responses; and translocating DNA into host cells to induce TLR9 activation.
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23
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Merino E, Flores-Encarnación M, Aguilar-Gutiérrez GR. Functional interaction and structural characteristics of unique components of Helicobacter pylori T4SS. FEBS J 2017; 284:3540-3549. [PMID: 28470874 DOI: 10.1111/febs.14092] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2016] [Revised: 03/14/2017] [Accepted: 04/26/2017] [Indexed: 12/25/2022]
Abstract
The Helicobacter pylori infection of the human gastric mucosa causes chronic active gastritis and peptic ulcers and is associated with the development of gastric cancer. Epidemiological studies show that these gastric diseases are related to virulent H. pylori strains that harbor the cytotoxin-associated gene pathogenicity island (cag PAI). The cag PAI is a DNA insertion in the H. pylori chromosome that encodes ~ 27 proteins, including the oncoprotein CagA. Approximately 20 of these proteins have been designated as cag type IV secretion system (T4SS) components. However, only 11 of these proteins share function, structure, and/or sequence similarities with the prototypical VirB/VirD4 T4SS of Agrobacterium tumefaciens. The VirB/VirD4 orthologs of the cag T4SS of H. pylori are required for CagA translocation and stimulate the gastric epithelial cells to produce and secrete interleukin-8 (IL-8). The cag PAI encodes eight additional proteins, such as Cag3 (Cagδ/HP0522), CagM (Cag16/HP0537), CagU (Cag11/HP0531), CagI (Cag19/HP0540), and CagH (Cag20/HP0541), which are also required for the translocation of CagA and IL-8 secretion, meanwhile CagF (Cag22/HP0543), CagG (Cag21/HP0542), and CagZ (Cag6/HP0526) are just required for the translocation of CagA. However, relatively little is known about their functions and structural organization because they exhibit a nondetectable sequence similarity with T4SS components in the current databases. In this review, we conducted an exhaustive analysis of the literature to present the biochemistry, putative role, localization, and interactions of each of these eight additional cag T4SS components.
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Affiliation(s)
- Enrique Merino
- Enrique Merino, Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México
| | - Marcos Flores-Encarnación
- Marcos Flores-Encarnación, Laboratorio de Microbiología Molecular y Celular, Facultad de Medicina, Benemérita Universidad Autónoma de Puebla, Puebla, México
| | - Germán Rubén Aguilar-Gutiérrez
- Germán Rubén Aguilar-Gutiérrez, Centro de Investigación Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, México
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Ogawa H, Iwamoto A, Tanahashi T, Okada R, Yamamoto K, Nishiumi S, Yoshida M, Azuma T. Genetic variants of Helicobacter pylori type IV secretion system components CagL and CagI and their association with clinical outcomes. Gut Pathog 2017; 9:21. [PMID: 28439300 PMCID: PMC5399799 DOI: 10.1186/s13099-017-0165-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2016] [Accepted: 03/25/2017] [Indexed: 12/20/2022] Open
Abstract
Background Helicobacter pylori infection is associated with risk for chronic gastritis (CG), gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC). The H. pylori Cag type IV secretion system (TFSS) translocates the virulence factor cytotoxin-associated gene A protein into host cells and plays an important role in initiating gastric carcinogenesis. The CagL and CagI proteins are components of the TFSS. The Arg-Gly-Asp (RGD) motif of CagL, and the six most distal C-terminal amino acids (Ser-Lys-Ile-Ile-Val-Lys, and Ser-Lys-Val-Ile-Val-Lys) of CagL and CagI are essential for TFSS adhesion to host cells. Additionally, the CagL variant Tyr58Glu59 was previously shown to be associated with GC patients. Results We isolated 43 H. pylori isolates from 17 CG, 8 GU, 8 DU, and 10 GC patients in Southeast Asia. Total DNAs were extracted and sequenced with MiSeq. H. pylori strain ATCC 26695, which was isolated from CG patients, was used as a reference. We examined the full sequences of H. pylori cagL and cagI using whole-genome sequencing (WGS), and analyzed whether single nucleotide variants and amino acid changes (AACs) correlated with adverse clinical outcomes. Three isolates were excluded from the analysis due to cagPAI rearrangements. CagL RGD motifs were conserved in 39 isolates (97.5%). CagL-Glu59 and Ile234 in the C-terminal motif were more common in 10 H. pylori isolates from GC patients (p < 0.001 and p < 0.05, respectively). When 5 Vietnamese isolates from GC patients were excluded, CagL-Glu59 still remains significant (p < 0.05), but not Ile234. CagL-Tyr58 was seen in only one isolate. The CagI C-terminal motif was completely conserved across all 40 isolates, and there were no significant AACs in CagI. Conclusions Using WGS, we analyzed genetic variants in clinical H. pylori isolates and identified putative novel and candidate variants in uncharacterized CagL and CagI sequences that are related to gastric carcinogenesis. In particular, CagL-Glu59 has the possible association with GC. Electronic supplementary material The online version of this article (doi:10.1186/s13099-017-0165-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Hirofumi Ogawa
- Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan
| | - Akira Iwamoto
- Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan
| | - Toshihito Tanahashi
- Local Incorporated Administrative Agency, Tokushima Prefecture Naruto Hospital, 32 Muya-cho, Kurosaki Aza Kotani, Naruto, Tokushima 772-0001 Japan
| | - Rina Okada
- Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan
| | - Koji Yamamoto
- Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan
| | - Shin Nishiumi
- Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan
| | - Masaru Yoshida
- Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan.,Division of Metabolomics Research, Department of Internal Related, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan.,AMED-CREST, AMED, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan
| | - Takeshi Azuma
- Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 Japan
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25
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Jang S, Kim J, Cha JH. Cot kinase plays a critical role in Helicobacter pylori-induced IL-8 expression. J Microbiol 2017; 55:311-317. [PMID: 28361341 DOI: 10.1007/s12275-017-7052-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Revised: 03/07/2017] [Accepted: 03/08/2017] [Indexed: 12/14/2022]
Abstract
Helicobacter pylori is a major pathogen causing various gastric diseases including gastric cancer. Infection of H. pylori induces pro-inflammatory cytokine IL-8 expression in gastric epithelial cells in the initial inflammatory process. It has been known that H. pylori can modulate Ras-Raf-Mek-Erk signal pathway for IL-8 induction. Recently, it has been shown that another signal molecule, cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, activates Mek and Erk and plays a role in the Erk pathway, similar to MAP3K signal molecule Raf kinase. Therefore, the objective of this study was to determine whether Cot kinase might be involved in IL-8 induction caused by H. pylori infection. AGS gastric epithelial cells were infected by H. pylori strain G27 or its isogenic mutants lacking cagA or type IV secretion system followed by treatment with Cot kinase inhibitor (KI) or siRNA specific for Cot kinase. Activation of Erk was assessed by Western blot analysis and expression of IL-8 was measured by ELISA. Treatment with Cot KI reduced both transient and sustained Erk activation. It also reduced early and late IL-8 secretion in the gastric epithelial cell line. Furthermore, siRNA knockdown of Cot inhibited early and late IL-8 secretion induced by H. pylori infection. Taken together, these results suggest that Cot kinase might play a critical role in H. pylori type IV secretion apparatus-dependent early IL-8 secretion and CagA-dependent late IL-8 secretion as an alternative signaling molecule in the Erk pathway.
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Affiliation(s)
- Sungil Jang
- Department of Oral Biology, Oral Science Research Center, BK21 Plus Project, Yonsei University College of Dentistry, Seoul, 03722, Republic of Korea
| | - Jinmoon Kim
- Department of Oral Biology, Oral Science Research Center, BK21 Plus Project, Yonsei University College of Dentistry, Seoul, 03722, Republic of Korea.
| | - Jeong-Heon Cha
- Department of Oral Biology, Oral Science Research Center, BK21 Plus Project, Yonsei University College of Dentistry, Seoul, 03722, Republic of Korea.
- Microbiology and Molecular Biology, Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Stomatology Hospital of Guangzhou Medical University, Guangzhou, 510170, P. R. China.
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Jiang X, Yang Y, Zhou J, Zhu L, Gu Y, Zhang X, Li X, Fang W. Roles of the Putative Type IV-like Secretion System Key Component VirD4 and PrsA in Pathogenesis of Streptococcus suis Type 2. Front Cell Infect Microbiol 2016; 6:172. [PMID: 27995095 PMCID: PMC5133265 DOI: 10.3389/fcimb.2016.00172] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2016] [Accepted: 11/17/2016] [Indexed: 12/19/2022] Open
Abstract
Streptococcus suis type 2 (SS2) is a zoonotic pathogen causing septic infection, meningitis and pneumonia in pigs and humans. SS2 may cause streptococcal toxic shock syndrome (STSS) probably due to excessive release of inflammatory cytokines. A previous study indicated that the virD4 gene in the putative type IV-like secretion system (T4SS) within the 89K pathogenicity island specific for recent epidemic strains contributed to the development of STSS. However, the functional basis of VirD4 in STSS remains unclear. Here we show that deletion of virD4 led to reduced virulence as shown by about 65% higher LD50, lower bacterial load in liver and brain, and lower level of expression of inflammatory cytokines in mice and cell lines than its parent strain. The ΔVirD4 mutant was more easily phagocytosed, suggesting its role as an anti-phagocytic factor. Oxidative stress that mimic bacterial exposure to respiratory burst of phagocytes upregulated expression of virD4. Proteomic analysis identified 10 secreted proteins of significant differences between the parent and mutant strains under oxidative stress, including PrsA, a peptidyl-prolyl isomerase. The SS2 PrsA expressed in E. coli caused a dose-dependent cell death and increased expression of proinflammatory IL-1β, IL-6 and TNF-α in murine macrophage cells. Our data provide novel insights into the contribution of the VirD4 factor to STSS pathogenesis, possibly via its anti-phagocytic activity, upregulation of its expression upon oxidative stress and its involvement in increased secretion of PrsA as a cell death inducer and proinflammatory effector.
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Affiliation(s)
- Xiaowu Jiang
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
| | - Yunkai Yang
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
| | - Jingjing Zhou
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
| | - Lexin Zhu
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
| | - Yuanxing Gu
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
| | - Xiaoyan Zhang
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
| | - Xiaoliang Li
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
| | - Weihuan Fang
- Zhejiang University Institute of Preventive Veterinary Medicine, and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Zhejiang, China
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Fazeli Z, Alebouyeh M, Rezaei Tavirani M, Azimirad M, Yadegar A. Helicobacter pylori CagA induced interleukin-8 secretion in gastric epithelial cells. GASTROENTEROLOGY AND HEPATOLOGY FROM BED TO BENCH 2016; 9:S42-S46. [PMID: 28224027 PMCID: PMC5310799] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
AIM Since, contradictory data have been reported about the effect of diverse variants of H. pylori virulence factors on IL-8 induction, we aimed to analyze the effect of this diversity on levels of IL-8 secretion in AGS cell line. BACKGROUND Helicobacter pylori colonizes the human stomach and induces the activation of inflammatory cytokines, including interleukin (IL)-8, in the gastric mucosa. This induction promotes neutrophil and monocyte recruitment that causes gastric tissue damage. METHODS To determine whether different strains of H. pylori and their CagA variants have possible roles on IL-8 induction, polarized AGS cell line was infected with CagA+ H. pylori strains carrying different EPIYA motifs (ABCCC and ABC) and CagA- strain for 24 hours. Difference in stimulation of IL-8 was measured by ELISA. RESULTS IL-8 secretion was elevated in the treated cells with CagA encoding strains compared with the negative one. Furthermore, a noticeably increased level of IL-8 induction was measured by the CagA-EPIYA type ABCCC encoding strain in compare to that carried EPIYA type ABC. CONCLUSION Results of this study provide new evidence about different effects of H. pylori strains and possible roles of their CagA variants on IL-8 induction. It seems that not only carriage of cagA and its expression, but also diversity in EPIYA motif be involved in IL-8 induction in the gastric epithelial cells.
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Affiliation(s)
- Zeinab Fazeli
- Faculty of Paramedical Sciences, Department of Basic Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Masoud Alebouyeh
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran,Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | | | - Masoumeh Azimirad
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Abbas Yadegar
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Wiedemann T, Hofbaur S, Loell E, Rieder G. A C-Terminal Coiled-Coil Region of CagL is Responsible for Helicobacter Pylori-Induced Il-8 Expression. Eur J Microbiol Immunol (Bp) 2016; 6:186-196. [PMID: 27766167 PMCID: PMC5063011 DOI: 10.1556/1886.2016.00020] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2016] [Accepted: 06/07/2016] [Indexed: 02/07/2023] Open
Abstract
Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine which triggers the infiltration and migration of neutrophils into areas of bacterial infection. Helicobacter pylori-infected patient studies as well as animal models have revealed that H. pylori type I strains carrying an intact cytotoxin-associated gene pathogenicity island (cag-PAI) with a functional type IV secretion system (T4SS) induce IL-8 expression and secretion in gastric mucosa. This gastric mucosal IL-8 expression correlates with severe histological changes due to H. pylori infection. In the present study, we explored a new recognition pattern on the bacterial adhesion protein CagL inducing IL-8 expression in H. pylori-infected host cells. To analyze the secreted IL-8 concentration, we performed IL-8 enzyme-linked immunosorbent assay (ELISA). To investigate the H. pylori-induced IL-8 expression on the transcriptional level, we transiently transfected gastric epithelial cells (AGS) with a human IL-8 luciferase reporter construct. The results of this study demonstrate that specifically the C-terminal coiled-coil region of the H. pylori CagL protein, a protein described to be located on the tip of the T4SS-pilus, is responsible for several in vitro observations: 1) H. pylori-induced IL-8 secretion via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway; 2) H. pylori-induced elongation of the cells, a typical CagA-induced phenotype; and 3) the bridging of the T4SS to its human target cells. This novel bacterial-host recognition sequence allows a new insight into how H. pylori induces the inflammatory response in gastric epithelial cells and facilitates the development of precancerous conditions.
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Affiliation(s)
- Tobias Wiedemann
- Helmholtz Zentrum München, German Research Center for Environmental Health, Institute for Diabetes and Cancer , Neuherberg, Germany
| | - Stefan Hofbaur
- Max-von-Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University , Munich, Germany
| | - Eva Loell
- Max-von-Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University , Munich, Germany
| | - Gabriele Rieder
- Bavarian Health and Food Safety Authority, Veterinaerstrasse 2 , D-85764 Oberschleissheim, Germany
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29
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Lang BJ, Gorrell RJ, Tafreshi M, Hatakeyama M, Kwok T, Price JT. The Helicobacter pylori cytotoxin CagA is essential for suppressing host heat shock protein expression. Cell Stress Chaperones 2016; 21:523-33. [PMID: 26928021 PMCID: PMC4837183 DOI: 10.1007/s12192-016-0680-x] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Revised: 01/11/2016] [Accepted: 02/10/2016] [Indexed: 02/07/2023] Open
Abstract
Bacterial infections typically elicit a strong Heat Shock Response (HSR) in host cells. However, the gastric pathogen Helicobacter pylori has the unique ability to repress this response, the mechanism of which has yet to be elucidated. This study sought to characterize the underlying mechanisms by which H. pylori down-modulates host HSP expression upon infection. Examination of isogenic mutant strains of H. pylori defective in components of the type IV secretion system (T4SS), identified the secretion substrate, CagA, to be essential for down-modulation of the HSPs HSPH1 (HSP105), HSPA1A (HSP72), and HSPD1 (HSP60) upon infection of the AGS gastric adenocarcinoma cell line. Ectopic expression of CagA by transient transfection was insufficient to repress HSP expression in AGS or HEK293T cells, suggesting that additional H. pylori factors are required for HSP repression. RT-qPCR analysis of HSP gene expression in AGS cells infected with wild-type H. pylori or isogenic cagA-deletion mutant found no significant change to account for reduced HSP levels. In summary, this study identified CagA to be an essential bacterial factor for H. pylori-mediated suppression of host HSP expression. The novel finding that HSPH1 is down-modulated by H. pylori further highlights the unique ability of H. pylori to repress the HSR within host cells. Elucidation of the mechanism by which H. pylori achieves HSP repression may prove to be beneficial in the identification of novel mechanisms to inhibit the HSR pathway and provide further insight into the interactions between H. pylori and the host gastric epithelium.
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Affiliation(s)
- Ben J Lang
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Victoria, Australia
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Rebecca J Gorrell
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Victoria, Australia
- Infection and Immunity, and Cancer Programs, Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, 3800, Victoria, Australia
| | - Mona Tafreshi
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Victoria, Australia
| | - Masanori Hatakeyama
- Division of Microbiology, Graduate School of Medicine, University of Tokyo, Tokyo, 113-0033, Japan
| | - Terry Kwok
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Victoria, Australia.
- Infection and Immunity, and Cancer Programs, Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, 3800, Victoria, Australia.
| | - John T Price
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Victoria, Australia.
- Centre for Chronic Disease, College of Health and Biomedicine, Victoria University, St Albans, Melbourne, VIC, Australia.
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Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens. mBio 2016; 7:e00221-16. [PMID: 27118587 PMCID: PMC4850256 DOI: 10.1128/mbio.00221-16] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.
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31
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Biological function of hpsh4590 localized in the plasticity zone of Helicobacter pylori. Microb Pathog 2016; 93:63-9. [DOI: 10.1016/j.micpath.2016.01.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2015] [Revised: 01/07/2016] [Accepted: 01/07/2016] [Indexed: 01/01/2023]
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Tohidpour A. CagA-mediated pathogenesis of Helicobacter pylori. Microb Pathog 2016; 93:44-55. [DOI: 10.1016/j.micpath.2016.01.005] [Citation(s) in RCA: 75] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Revised: 11/14/2015] [Accepted: 01/07/2016] [Indexed: 12/20/2022]
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Szkaradkiewicz A, Karpiński TM, Linke K, Majewski P, Rożkiewicz D, Goślińska-Kuźniarek O. Expression of cagA, virB/D Complex and/or vacA Genes in Helicobacter pylori Strains Originating from Patients with Gastric Diseases. PLoS One 2016; 11:e0148936. [PMID: 26866365 PMCID: PMC4750868 DOI: 10.1371/journal.pone.0148936] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2015] [Accepted: 01/24/2016] [Indexed: 12/17/2022] Open
Abstract
In order to better understand pathogenicity of Helicobacter pylori, particularly in the context of its carcinogenic activity, we analysed expression of virulence genes: cagA, virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, virD4) and vacA in strains of the pathogen originating from persons with gastric diseases. The studies were conducted on 42 strains of H. pylori isolated from patients with histological diagnosis of non-atrophic gastritis-NAG (group 1, including subgroup 1 containing cagA+ isolates and subgroup 2 containing cagA- strains), multifocal atrophic gastritis-MAG (group 2) and gastric adenocarcinoma-GC (group 3). Expression of H. pylori genes was studied using microarray technology. In group 1, in all strains of H. pylori cagA+ (subgroup 1) high expression of the gene as well as of virB/D was disclosed, accompanied by moderate expression of vacA. In strains of subgroup 2 a moderate expression of vacA was detected. All strains in groups 2 and 3 carried cagA gene but they differed in its expression: a high expression was detected in isolates of group 2 and its hyperexpression in strains of group 3 (hypervirulent strains). In both groups high expression of virB/D and vacA was disclosed. Our results indicate that chronic active gastritis may be induced by both cagA+ strains of H. pylori, manifesting high expression of virB/D complex but moderate activity of vacA, and cagA- strains with moderate expression of vacA gene. On the other hand, in progression of gastric pathology and carcinogenesis linked to H. pylori a significant role was played by hypervirulent strains, manifesting a very high expression of cagA and high activity of virB/D and vacA genes.
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Affiliation(s)
- Andrzej Szkaradkiewicz
- Department of Medical Microbiology, University of Medical Sciences in Poznań, Wieniawskiego 3, Str., 61–712, Poznań, Poland
| | - Tomasz M. Karpiński
- Department of Medical Microbiology, University of Medical Sciences in Poznań, Wieniawskiego 3, Str., 61–712, Poznań, Poland
| | - Krzysztof Linke
- Department of Gastroenterology, Human Nutrition and Internal Diseases, University of Medical Sciences in Poznań, Przybyszewskiego 49, Str., 60–355, Poznań, Poland
| | - Przemysław Majewski
- Department of Clinical Pathomorphology, University of Medical Sciences in Poznań, Przybyszewskiego 49, Str., 60–355, Poznań, Poland
| | - Dorota Rożkiewicz
- Department of Paediatric Infectious Diseases, Medical University of Białystok, University Children’s Hospital, Waszyngtona 17, Str., 15–274, Białystok, Poland
| | - Olga Goślińska-Kuźniarek
- Department of Medical Microbiology, University of Medical Sciences in Poznań, Wieniawskiego 3, Str., 61–712, Poznań, Poland
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Pérez-Figueroa E, Torres J, Sánchez-Zauco N, Contreras-Ramos A, Alvarez-Arellano L, Maldonado-Bernal C. Activation of NLRP3 inflammasome in human neutrophils by Helicobacter pylori infection. Innate Immun 2016; 22:103-112. [DOI: 10.1177/1753425915619475] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023] Open
Abstract
TLRs and NLRs participate in the immune system recognition of Helicobacter pylori. However, little is known about the mechanisms leading to inflammasome activation by H. pylori and if NLRs in neutrophils are involved in the process. We studied how NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome components are involved in IL-1β maturation in human neutrophils in response to the infection and if they are dependent on T4SS (type IV secretion system) and TLRs. Human neutrophils were cultured and infected with the 26695 or the VirD4− H. pylori strains; the IL-1β concentration was analyzed by ELISA, and we also evaluated the activation of TLRs 2 and 4. The infection of neutrophils with both strains of H. pylori induced production of IL-1β and expression of the NLRP3 inflammasome components such as apoptosis-associated speck-like protein with CARD domain and NLRP3 protein. The infection also increased the activity of caspase-1, which is required for the maturation of IL-1β. Our study shows, for the first time, that H. pylori infection induces the expression and activation of components of NLRP3 inflammasomes in human neutrophils and that the activation is independent of a functional T4SS and TLR2 and TLR4.
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Affiliation(s)
- Erandi Pérez-Figueroa
- Laboratorio de Investigación en Inmunología y Proteómica, Hospital Infantil de México Federico Gómez, México City, México
- Unidad de Investigación Médica en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, IMSS, México City, México
| | - Javier Torres
- Unidad de Investigación Médica en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, IMSS, México City, México
| | - Norma Sánchez-Zauco
- Laboratorio de Investigación en Inmunología y Proteómica, Hospital Infantil de México Federico Gómez, México City, México
- Unidad de Investigación Médica en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, IMSS, México City, México
| | - Alejandra Contreras-Ramos
- Laboratorio de Biología del Desarrollo, Hospital Infantil de México Federico Gómez, México City, México
| | | | - Carmen Maldonado-Bernal
- Laboratorio de Investigación en Inmunología y Proteómica, Hospital Infantil de México Federico Gómez, México City, México
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35
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Kim SH, Kim JB. Comparative Proteome Analysis of Cyanidin 3-O-glucoside Treated Helicobacter pylori. ACTA ACUST UNITED AC 2015. [DOI: 10.15616/bsl.2015.21.4.233] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Affiliation(s)
- Sa-Hyun Kim
- Department of Clinical Laboratory Science, Semyung University, Jecheon 27136, Korea
| | - Jong-Bae Kim
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea
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Analysis of the intactness of Helicobacter pylori cag pathogenicity island in Iranian strains by a new PCR-based strategy and its relationship with virulence genotypes and EPIYA motifs. INFECTION GENETICS AND EVOLUTION 2015. [PMID: 26205689 DOI: 10.1016/j.meegid.2015.07.026] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Variants of the Helicobacter pylori cag pathogenicity island (cagPAI) and certain virulence genotypes have been proposed to be associated with different gastric disorders. In the present study, we designed a new PCR-based strategy to investigate the intactness of cagPAI in Iranian patients using highly specific primer sets spanning the cagPAI region. The possible relationship between the cagPAI status of the strains and clinical outcomes was also determined. We also characterized virulence genotypes (cagL, cagA, vacA, babA2 and sabA) and variants of CagA EPIYA motifs in these strains. H. pylori was detected in 61 out of 126 patients with various gastroduodenal diseases. The cagL, cagA, vacA s1m1, vacA s1m2, vacA s2m2, babA2, and sabA genotypes were detected in 96.7%, 85.2%, 29.5%, 45.9%, 24.6%, 96.7%, and 83.6% of the strains, respectively. Among the 52 cagA-positive strains, EPIYA motifs ABC, ABCC, ABCCC, and mixed types were orderly detected in the 39, 7, 1, and 5 strains. The cagPAI positivity included both intact and partially deleted, with the overall frequencies of 70.5% and 26.2%, respectively. The majority of the strains from patients with PUD (87.5%), gastric erosion (83.3%) and cancer (80%) presented an intact cagPAI, while a lower frequency of cagPAI intactness was detected in gastritis patients (61.1%). However, no significant relationship was found between the possession of intact cagPAI and clinical outcomes. Furthermore, we found that cagA and vacA s1m1 genotypes were significantly correlated with intact cagPAI (P=0.015 and P=0.012). A significant correlation was also found between EPIYA-ABC and intact cagPAI (P=0.010). The proposed PCR-based scheme was found to be useful for determining the intactness of cagPAI. Our findings also indicate that the cagPAI appears to be intact and rather conserved in majority of Iranian strains. Finally, our study proposed that H. pylori strains with partially deleted cagPAI were less likely to cause severe diseases in comparison with those carrying intact cagPAI.
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Gopal GJ, Pal J, Kumar A, Mukhopadhyay G. C-terminal domain of CagX is responsible for its interaction with CagT protein of Helicobacter pylori type IV secretion system. Biochem Biophys Res Commun 2014; 456:98-103. [PMID: 25446105 DOI: 10.1016/j.bbrc.2014.11.041] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Accepted: 11/13/2014] [Indexed: 12/23/2022]
Abstract
Helicobacter pylori are the well known human pathogen associated with gastric cancer and peptic ulcer. Pathogenesis is mainly due to the presence of 40 kb cagPAI (cag Pathogenicity Island) region that encodes the type IV secretion system (TFSS) consisting of a cytoplasmic part, a middle part/core complex (spans from inner membrane to outer membrane), and an outer membrane associated part. CagX and CagT are two important proteins of TFSS that have homology with virB9 and virB7 of Agrobacterium tumefaciens TFSS. In this study, we have shown that the CagX and CagT interact directly by using co-immunoprecipitation of endogenous CagX and CagT and MBP pull down assay. We further authenticate this observation using yeast two-hybrid assay and co-expression of both the protein coding gene in Escherichia coli. We also observed that the C-terminal region of CagX is important for CagT interaction. We reconfirm that CagT depends on CagX for its stabilization. These observations could contribute in overall visualization of assembly and architecture of TFSS because protein-protein interactions among Cag proteins are likely to have an important role in assembly. Thorough understanding about architecture and mechanism of action of cag-TFSS may lead to design controlled drug delivery system.
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Affiliation(s)
- Gopal Jee Gopal
- Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India; Department of Biochemistry, M.S. University of Baroda, Vadodara, Gujarat, India.
| | - Jagannath Pal
- Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India; Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA
| | - Awanish Kumar
- Department of Biotechnology, National Institute of Technology, Raipur, Chhattisgarh, India
| | - Gauranga Mukhopadhyay
- Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
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Devi S, Ansari SA, Vadivelu J, Mégraud F, Tenguria S, Ahmed N. Helicobacter pylori antigen HP0986 (TieA) interacts with cultured gastric epithelial cells and induces IL8 secretion via NF-κB mediated pathway. Helicobacter 2014; 19:26-36. [PMID: 24205801 DOI: 10.1111/hel.12100] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND The envisaged roles and partly understood functional properties of Helicobacter pylori protein HP0986 are significant in the context of proinflammatory and or proapoptotic activities, the two important facilitators of pathogen survival and persistence. In addition, sequence analysis of this gene predicts a restriction endonuclease function which remained unknown thus far. To evaluate the role of HP0986 in gastric inflammation, we studied its expression profile using a large number of clinical isolates but a limited number of biopsies and patient sera. Also, we studied antigenic role of HP0986 in altering cytokine responses of human gastric epithelial (AGS) cells including its interaction with and localization within the AGS cells. MATERIALS AND METHODS For in vitro expression study of HP0986, 110 H. pylori clinical isolates were cultured from patients with functional dyspepsia. For expression analysis by qRT PCR of HP0986, 10 gastric biopsy specimens were studied. HP0986 was also used to detect antibodies in patient sera. AGS cells were incubated with recombinant HP0986 to determine cytokine response and NF-κB activation. Transient transfection with HP0986 cloned in pEGFPN1 was used to study its subcellular localization or homing in AGS cells. RESULTS Out of 110 cultured H. pylori strains, 34 (31%) were positive for HP0986 and this observation was correlated with in vitro expression profiles. HP0986 mRNA was detected in 7 of the 10 biopsy specimens. Further, HP0986 induced IL-8 secretion in gastric epithelial cells in a dose and time-dependent manner via NF-κB pathway. Serum antibodies against HP0986 were positively associated with H. pylori positive patients. Transient transfection of AGS cells revealed both cytoplasmic and nuclear localization of HP0986. CONCLUSION HP0986 was moderately prevalent in clinical isolates and its expression profile in cultures and gastric biopsies points to its being naturally expressed. Collective observations including the induction of IL-8 via TNFR1 and NF-κB, subcellular localization, and seropositivity data point to a significant role of HP0986 in gastroduodenal inflammation. We propose to name the HP0986 gene/protein as 'TNFR1 interacting endonuclease A (TieA or tieA)'.
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Affiliation(s)
- Savita Devi
- Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046, India
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Copper promotes TFF1-mediated Helicobacter pylori colonization. PLoS One 2013; 8:e79455. [PMID: 24236136 PMCID: PMC3827375 DOI: 10.1371/journal.pone.0079455] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2013] [Accepted: 10/01/2013] [Indexed: 02/08/2023] Open
Abstract
The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.
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Salih BA, Guner A, Karademir A, Uslu M, Ovali MA, Yazici D, Bolek BK, Arikan S. Evaluation of the effect of cagPAI genes of Helicobacter pylori on AGS epithelial cell morphology and IL-8 secretion. Antonie van Leeuwenhoek 2013; 105:179-89. [PMID: 24170115 DOI: 10.1007/s10482-013-0064-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/18/2013] [Accepted: 10/23/2013] [Indexed: 02/07/2023]
Abstract
Helicobacter pylori cagPAI genes play an important role in pathogenesis, however little is known about their functions in isolates from Turkish patients. We aimed to evaluate the intactness and the effect of the cagPAI genes (cagT, cagM, cagE, cagA) and cagA EPIYA motifs on the AGS morphological changes and IL-8 induction. Of 53 patients 38 were found infected with H. pylori. PCR amplification of the cagPAI genes showed 42.1 % intact, 39.5 % partially deleted and 18.4 % with complete deletions. Isolates from gastritis, duodenal and gastric ulcer patients with intact and partially deleted cagPAI genes induced higher IL-8 secretion than those with complete deletions. Isolates from gastritis patients had higher deletion frequencies of the cagT and cagM genes than the other two genes. Infection of AGS cells with isolates that possess intact cagPAI and EPIYA-ABC resulted in the formation of the hummingbird phenotype. The cagA positive isolates induced higher IL-8 secretion than cagA negative isolates. Isolates from DU patients with more than one EPIYA-C motif induced higher concentrations of IL-8 than those with EPIYA-ABC. In conclusion, the intactness of the cagPAI in our isolates from different patients was not conserved. An intact cagPAI was found to play an important role in the pathogenesis of DU but not GU or gastritis. The cagA gene, but not other cagPAI genes, was associated with the induction of IL-8 and the morphological changes of the AGS cells. An increase in the number of EPIYA-C motifs had noticeable effect on the formation of the hummingbird phenotype.
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Affiliation(s)
- Barik A Salih
- Department of Biology, Faculty of Science and Literature, Fatih University, B. Cekmece, Istanbul, Turkey,
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Zhu ZH, Tang XD, Wang FY, Guo P. Immune factors affecting eradication of Helicobacter pylori: Implications for immunotherapy. Shijie Huaren Xiaohua Zazhi 2013; 21:2674-2678. [DOI: 10.11569/wcjd.v21.i26.2674] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
In this paper, we discuss immune factors affecting eradication of Helicobacter pylori (H. pylori) and analyze their implications for immunotherapy. By reviewing related literature and comparing with conventional pure acid suppression, we analyze factors related to the eradication of H. pylori from an immunological point of view and put forward new hypotheses concerning the development of oral H. pylori vaccines.
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Bullman S, Lucid A, Corcoran D, Sleator RD, Lucey B. Genomic investigation into strain heterogeneity and pathogenic potential of the emerging gastrointestinal pathogen Campylobacter ureolyticus. PLoS One 2013; 8:e71515. [PMID: 24023611 PMCID: PMC3758288 DOI: 10.1371/journal.pone.0071515] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2013] [Accepted: 07/01/2013] [Indexed: 12/29/2022] Open
Abstract
The recent detection and isolation of C. ureolyticus from patients with diarrhoeal illness and inflammatory bowel diseases warrants further investigation into its role as an emerging pathogen of the human gastrointestinal tract. Regarding the pathogenic mechanisms employed by this species we provide the first whole genome analysis of two C. ureolyticus isolates including the type strain. Comparative analysis, subtractive hybridisation and gene ontology searches against other Campylobacter species identifies the high degree of heterogenicity between C. ureolyticus isolates, in addition to the identification of 106 putative virulence associated factors, 52 of which are predicted to be secreted. Such factors encompass each of the known virulence tactics of pathogenic Campylobacter spp. including adhesion and colonisation (CadF, PEB1, IcmF and FlpA), invasion (ciaB and 16 virB-virD4 genes) and toxin production (S-layer RTX and ZOT). Herein, we provide the first virulence catalogue for C. ureolyticus, the components of which theoretically provide this emerging species with sufficient arsenal to establish pathology.
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Affiliation(s)
- Susan Bullman
- Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland
| | - Alan Lucid
- Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland
| | - Daniel Corcoran
- Department of Medical Microbiology, Cork University Hospital, Cork, Ireland
| | - Roy D. Sleator
- Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland
- * E-mail:
| | - Brigid Lucey
- Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland
- Department of Medical Microbiology, Cork University Hospital, Cork, Ireland
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Gorrell RJ, Guan J, Xin Y, Tafreshi MA, Hutton ML, McGuckin MA, Ferrero RL, Kwok T. A novel NOD1- and CagA-independent pathway of interleukin-8 induction mediated by the Helicobacter pylori type IV secretion system. Cell Microbiol 2012; 15:554-70. [PMID: 23107019 DOI: 10.1111/cmi.12055] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2012] [Revised: 08/24/2012] [Accepted: 10/09/2012] [Indexed: 12/12/2022]
Abstract
The type IV secretion system (T4SS) of Helicobacter pylori triggers massive inflammatory responses during gastric infection by mechanisms that are poorly understood. Here we provide evidence for a novel pathway by which the T4SS structural component, CagL, induces secretion of interleukin-8 (IL-8) independently of CagA translocation and peptidoglycan-sensing nucleotide-binding oligomerization domain 1 (NOD1) signalling. Recombinant CagL was sufficient to trigger IL-8 secretion, requiring activation of α5 β1 integrin and the arginine-glycine-aspartate (RGD) motif in CagL. Mutation of the encoded RGD motif to arginine-glycine-alanine (RGA) in the cagL gene of H. pylori abrogated its ability to induce IL-8. Comparison of IL-8 induction between H. pylori ΔvirD4 strains bearing wild-type or mutant cagL indicates that CagL-dependent IL-8 induction can occur independently of CagA translocation. In line with this notion, exogenous CagL complemented H. pylori ΔcagL mutant in activating NF-κB and inducing IL-8 without restoring CagA translocation. The CagA translocation-independent, CagL-dependent IL-8 induction involved host signalling via integrin α5 β1 , Src kinase, the mitogen-activated protein kinase (MAPK) pathway and NF-κB but was independent of NOD1. Our findings reveal a novel pathway whereby CagL, via interaction with host integrins, can trigger pro-inflammatory responses independently of CagA translocation or NOD1 signalling.
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Affiliation(s)
- Rebecca J Gorrell
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia; Department of Microbiology, Monash University, Clayton, Vic., Australia
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Zechner EL, Lang S, Schildbach JF. Assembly and mechanisms of bacterial type IV secretion machines. Philos Trans R Soc Lond B Biol Sci 2012; 367:1073-87. [PMID: 22411979 PMCID: PMC3297438 DOI: 10.1098/rstb.2011.0207] [Citation(s) in RCA: 129] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Type IV secretion occurs across a wide range of prokaryotic cell envelopes: Gram-negative, Gram-positive, cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the cell surface as a pilus structure important for direct contact and penetration of the recipient cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.
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Affiliation(s)
- Ellen L Zechner
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/I, Graz 8010, Austria.
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Conradi J, Tegtmeyer N, Woźna M, Wissbrock M, Michalek C, Gagell C, Cover TL, Frank R, Sewald N, Backert S. An RGD helper sequence in CagL of Helicobacter pylori assists in interactions with integrins and injection of CagA. Front Cell Infect Microbiol 2012. [PMID: 22919661 DOI: 10.3389/fcimb.2012.00070.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Helicobacter pylori is a specific gastric pathogen that colonizes the stomach in more than 50% of the world's human population. Infection with this bacterium can induce several types of gastric pathology, ranging from chronic gastritis to peptic ulcers and even adenocarcinoma. Virulent H. pylori isolates encode components of a type IV secretion system (T4SS), which form a pilus for the injection of virulence proteins such as CagA into host target cells. This is accomplished by a specialized adhesin on the pilus surface, the protein CagL, a putative VirB5 ortholog, which binds to host cell β(1) integrin, triggering subsequent delivery of CagA across the host cell membrane. Like the human extracellular matrix protein fibronectin, CagL contains an RGD (Arg-Gly-Asp) motif and is able to trigger intracellular signaling pathways by RGD-dependent binding to integrins. While CagL binding to host cells is mediated primarily by the RGD motif, we identified an auxiliary binding motif for CagL-integrin interaction. Here, we report on a surface exposed FEANE (Phe-Glu-Ala-Asn-Glu) interaction motif in spatial proximity to the RGD sequence, which enhances the interactions of CagL with integrins. It will be referred to as RGD helper sequence (RHS). Competitive cell adhesion assays with recombinant wild type CagL and point mutants, competition experiments with synthetic cyclic and linear peptides, and peptide array experiments revealed amino acids essential for the interaction of the RHS motif with integrins. Infection experiments indicate that the RHS motif plays a role in the early interaction of H. pylori T4SS with integrin, to trigger signaling and to inject CagA into host cells. We thus postulate that CagL is a versatile T4SS surface protein equipped with at least two motifs to promote binding to integrins, thereby causing aberrant signaling within host cells and facilitating translocation of CagA into host cells, thus contributing directly to H. pylori pathogenesis.
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Affiliation(s)
- Jens Conradi
- Department of Chemistry, Bielefeld University Bielefeld, Germany
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46
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Conradi J, Tegtmeyer N, Woźna M, Wissbrock M, Michalek C, Gagell C, Cover TL, Frank R, Sewald N, Backert S. An RGD helper sequence in CagL of Helicobacter pylori assists in interactions with integrins and injection of CagA. Front Cell Infect Microbiol 2012; 2:70. [PMID: 22919661 PMCID: PMC3417467 DOI: 10.3389/fcimb.2012.00070] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2011] [Accepted: 05/02/2012] [Indexed: 12/12/2022] Open
Abstract
Helicobacter pylori is a specific gastric pathogen that colonizes the stomach in more than 50% of the world’s human population. Infection with this bacterium can induce several types of gastric pathology, ranging from chronic gastritis to peptic ulcers and even adenocarcinoma. Virulent H. pylori isolates encode components of a type IV secretion system (T4SS), which form a pilus for the injection of virulence proteins such as CagA into host target cells. This is accomplished by a specialized adhesin on the pilus surface, the protein CagL, a putative VirB5 ortholog, which binds to host cell β1 integrin, triggering subsequent delivery of CagA across the host cell membrane. Like the human extracellular matrix protein fibronectin, CagL contains an RGD (Arg-Gly-Asp) motif and is able to trigger intracellular signaling pathways by RGD-dependent binding to integrins. While CagL binding to host cells is mediated primarily by the RGD motif, we identified an auxiliary binding motif for CagL–integrin interaction. Here, we report on a surface exposed FEANE (Phe-Glu-Ala-Asn-Glu) interaction motif in spatial proximity to the RGD sequence, which enhances the interactions of CagL with integrins. It will be referred to as RGD helper sequence (RHS). Competitive cell adhesion assays with recombinant wild type CagL and point mutants, competition experiments with synthetic cyclic and linear peptides, and peptide array experiments revealed amino acids essential for the interaction of the RHS motif with integrins. Infection experiments indicate that the RHS motif plays a role in the early interaction of H. pylori T4SS with integrin, to trigger signaling and to inject CagA into host cells. We thus postulate that CagL is a versatile T4SS surface protein equipped with at least two motifs to promote binding to integrins, thereby causing aberrant signaling within host cells and facilitating translocation of CagA into host cells, thus contributing directly to H. pylori pathogenesis.
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Affiliation(s)
- Jens Conradi
- Department of Chemistry, Bielefeld University Bielefeld, Germany
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Ta LH, Hansen LM, Sause WE, Shiva O, Millstein A, Ottemann KM, Castillo AR, Solnick JV. Conserved transcriptional unit organization of the cag pathogenicity island among Helicobacter pylori strains. Front Cell Infect Microbiol 2012; 2:46. [PMID: 22919637 PMCID: PMC3417554 DOI: 10.3389/fcimb.2012.00046] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2011] [Accepted: 03/17/2012] [Indexed: 12/14/2022] Open
Abstract
The Helicobacter pyloricag pathogenicity island (cag PAI) encodes a type IV secretion system that is more commonly found in strains isolated from patients with gastroduodenal disease than from those with asymptomatic gastritis. Genome-wide organization of the transcriptional units in H. pylori strain 26695 was recently established using RNA sequence analysis (Sharma et al., 2010). Here we used quantitative reverse-transcription polymerase chain reaction of open reading frames and intergenic regions to identify putative cag PAI operons in H. pylori; these operons were analyzed further by transcript profiling after deletion of selected promoter regions. Additionally, we used a promoter-trap system to identify functional cag PAI promoters. The results demonstrated that expression of genes on the H. pyloricag PAI varies by nearly five orders of magnitude and that the organization of cag PAI genes into transcriptional units is conserved among several H. pylori strains, including, 26695, J99, G27, and J166. We found evidence for 20 transcripts within the cag PAI, many of which likely overlap. Our data suggests that there are at least 11 operons: cag1-4, cag3-4, cag10-9, cag8-7, cag6-5, cag11-12, cag16-17, cag19-18, cag21-20, cag23-22, and cag25-24, as well as five monocistronic genes (cag4, cag13, cag14, cag15, and cag26). Additionally, the location of four of our functionally identified promoters suggests they are directing expression of, in one case, a truncated version of cag26 and in the other three, transcripts that are antisense to cag7, cag17, and cag23. We verified expression of two of these antisense transcripts, those antisense to cag17 and cag23, by reverse-transcription polymerase chain reaction. Taken together, our results suggest that the cag PAI transcriptional profile is generally conserved among H. pylori strains, 26695, J99, G27, and J166, and is likely complex.
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Affiliation(s)
- Linda H Ta
- Departments of Medicine and Microbiology & Immunology, Center for Comparative Medicine, University of California Davis, Davis, CA, USA
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Pham KT, Weiss E, Jiménez Soto LF, Breithaupt U, Haas R, Fischer W. CagI is an essential component of the Helicobacter pylori Cag type IV secretion system and forms a complex with CagL. PLoS One 2012; 7:e35341. [PMID: 22493745 PMCID: PMC3320882 DOI: 10.1371/journal.pone.0035341] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2011] [Accepted: 03/15/2012] [Indexed: 12/11/2022] Open
Abstract
Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly.
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Affiliation(s)
- Kieu Thuy Pham
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
| | - Evelyn Weiss
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
| | - Luisa F. Jiménez Soto
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
| | - Ute Breithaupt
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
| | - Rainer Haas
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
| | - Wolfgang Fischer
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
- * E-mail:
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Mueller D, Tegtmeyer N, Brandt S, Yamaoka Y, De Poire E, Sgouras D, Wessler S, Torres J, Smolka A, Backert S. c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains. J Clin Invest 2012; 122:1553-66. [PMID: 22378042 PMCID: PMC3314471 DOI: 10.1172/jci61143] [Citation(s) in RCA: 194] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2011] [Accepted: 01/11/2012] [Indexed: 12/24/2022] Open
Abstract
Many bacterial pathogens inject into host cells effector proteins that are substrates for host tyrosine kinases such as Src and Abl family kinases. Phosphorylated effectors eventually subvert host cell signaling, aiding disease development. In the case of the gastric pathogen Helicobacter pylori, which is a major risk factor for the development of gastric cancer, the only known effector protein injected into host cells is the oncoprotein CagA. Here, we followed the hierarchic tyrosine phosphorylation of H. pylori CagA as a model system to study early effector phosphorylation processes. Translocated CagA is phosphorylated on Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs EPIYA-A, EPIYA-B, and EPIYA-C in Western strains of H. pylori and EPIYA-A, EPIYA-B, and EPIYA-D in East Asian strains. We found that c-Src only phosphorylated EPIYA-C and EPIYA-D, whereas c-Abl phosphorylated EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D. Further analysis revealed that CagA molecules were phosphorylated on 1 or 2 EPIYA motifs, but never simultaneously on 3 motifs. Furthermore, none of the phosphorylated EPIYA motifs alone was sufficient for inducing AGS cell scattering and elongation. The preferred combination of phosphorylated EPIYA motifs in Western strains was EPIYA-A and EPIYA-C, either across 2 CagA molecules or simultaneously on 1. Our study thus identifies a tightly regulated hierarchic phosphorylation model for CagA starting at EPIYA-C/D, followed by phosphorylation of EPIYA-A or EPIYA-B. These results provide insight for clinical H. pylori typing and clarify the role of phosphorylated bacterial effector proteins in pathogenesis.
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Affiliation(s)
- Doreen Mueller
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Nicole Tegtmeyer
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Sabine Brandt
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Yoshio Yamaoka
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Eimear De Poire
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Dionyssios Sgouras
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Silja Wessler
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Javier Torres
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Adam Smolka
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Steffen Backert
- University of Magdeburg, Department of Medical Microbiology, Magdeburg, Germany.
University College Dublin, School of Biomolecular and Biomedical Sciences, Dublin, Ireland.
Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Department Medicine-Gastroenterology, Houston, Texas, USA.
Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan.
Hellenic Pasteur Institute, Laboratory of Medical Microbiology, Athens, Greece.
Division of Microbiology, University Salzburg, Salzburg, Austria.
Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mexico.
Department of Medicine/Gastroenterology, Medical University of South Carolina, Charleston, South Carolina, USA
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Ooi Y, Daikoku E, Wu H, Aoki H, Morita C, Nakano T, Kohno T, Takasaki T, Sano K. Morphology and infectivity of virus that persistently caused infection in an AGS cell line. Med Mol Morphol 2011; 44:213-20. [PMID: 22179184 DOI: 10.1007/s00795-010-0530-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2010] [Accepted: 10/13/2010] [Indexed: 12/13/2022]
Abstract
A recent report has indicated that proteins and genes of simian virus 5 (SV5) are detected in a human gastric adenocarcinoma (AGS) cell line, which is widely provided for oncology, immunology, and microbiology research. However, the production of infective virions has not been determined in this cell line. In this study, the morphology and infectivity of the virus particles of the AGS cell line were studied by light and electron microscopy and virus transmission assay. The virus particles were approximately 176.0 ± 41.1 nm in diameter. The particles possessed projections 8-12 nm long on the surface and contained a nucleocapsid determined to be 13-18 nm in width and less than 1,000 nm in length. The virus was transmissible to the Vero cell line, induced multinuclear giant cell formation, and reproduced the same shape of antigenic virions. In this study, the persistently infected virus in the AGS cell line was determined to be infective and form reproducible virions, and a new morphological feature of SV5 was determined.
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Affiliation(s)
- Yukimasa Ooi
- Department of Microbiology and Infection Control, Osaka Medical College, Daigaku-machi, Takatsuki, Osaka, Japan
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