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Barua A, Masum MHU, Mahdeen AA. A Reverse Vaccinology and Immunoinformatic Approach for the Designing of a Novel mRNA Vaccine Against Stomach Cancer Targeting the Potent Pathogenic Proteins of Helicobacter pylori. Bioinform Biol Insights 2025; 19:11779322251331104. [PMID: 40290636 PMCID: PMC12033411 DOI: 10.1177/11779322251331104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Accepted: 03/14/2025] [Indexed: 04/30/2025] Open
Abstract
Helicobacter pylori infection of the stomach's epithelial cells is a significant risk factor for stomach cancer. Various H pylori proteins (CagA, GGT, NapA, PatA, urease, and VacA) were targeted to design 2 messenger RNA (mRNA) vaccines, V1 and V2, using bioinformatics tools. Physicochemical parameters, secondary and tertiary structure, molecular docking and dynamic simulation, codon optimization, and RNA structure prediction have also been estimated for these developed vaccines. Physicochemical analyses revealed that these developed vaccines are soluble (GRAVY < 0), basic (pI < 7), and stable (aliphatic index < 80). The secondary and tertiary structure of the vaccines demonstrated robustness. The docking with toll-like receptors (TLRs) revealed that the vaccines have a potential affinity for TLR-2 (V1: -1132.3 kJ/mol, V2: -1093.6 kJ/mol) and TLR-4 (V1: -1042.7 kJ/mol, V2: -1201.2 kJ/mol), and molecular dynamics simulations confirmed their dynamic stability. Structural analyses of V1 (-505.96 kcal/mol) and V2 (-634.92 kcal/mol) mRNA vaccines underscored their stability. In addition, the vaccine showed a considerable rise in the counts of B cells and extended activation of both T cells was also observed for the vaccines, suggesting the potential for long-lasting immunity, and offering enhanced protection against H pylori. These findings not only suggest potential long-lasting immunity against H pylori but also offer hope for the future of stomach cancer prevention. Notably, the study emphasizes the need for subsequent animal and human-based studies to confirm these promising results.
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Affiliation(s)
- Abanti Barua
- Department of Microbiology, Noakhali Science and Technology University, Noakhali, Bangladesh
| | - Md. Habib Ullah Masum
- Department of Genomics and Bioinformatics, Faculty of Biotechnology and Genetic Engineering, Chattogram Veterinary and Animal Sciences University, Khulshi, Chattogram, Bangladesh
| | - Ahmad Abdullah Mahdeen
- Department of Microbiology, Noakhali Science and Technology University, Noakhali, Bangladesh
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2
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Reyes VE. Helicobacter pylori and Its Role in Gastric Cancer. Microorganisms 2023; 11:1312. [PMID: 37317287 PMCID: PMC10220541 DOI: 10.3390/microorganisms11051312] [Citation(s) in RCA: 37] [Impact Index Per Article: 18.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 05/08/2023] [Accepted: 05/15/2023] [Indexed: 06/16/2023] Open
Abstract
Gastric cancer is a challenging public health concern worldwide and remains a leading cause of cancer-related mortality. The primary risk factor implicated in gastric cancer development is infection with Helicobacter pylori. H. pylori induces chronic inflammation affecting the gastric epithelium, which can lead to DNA damage and the promotion of precancerous lesions. Disease manifestations associated with H. pylori are attributed to virulence factors with multiple activities, and its capacity to subvert host immunity. One of the most significant H. pylori virulence determinants is the cagPAI gene cluster, which encodes a type IV secretion system and the CagA toxin. This secretion system allows H. pylori to inject the CagA oncoprotein into host cells, causing multiple cellular perturbations. Despite the high prevalence of H. pylori infection, only a small percentage of affected individuals develop significant clinical outcomes, while most remain asymptomatic. Therefore, understanding how H. pylori triggers carcinogenesis and its immune evasion mechanisms is critical in preventing gastric cancer and mitigating the burden of this life-threatening disease. This review aims to provide an overview of our current understanding of H. pylori infection, its association with gastric cancer and other gastric diseases, and how it subverts the host immune system to establish persistent infection.
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Affiliation(s)
- Victor E Reyes
- Department of Pediatrics and Microbiology & Immunology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0372, USA
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Rajapaksha N, Soldano A, Yao H, Donnarumma F, Kashipathy MM, Seibold S, Battaile KP, Lovell S, Rivera M. Pseudomonas aeruginosa Dps (PA0962) Functions in H 2O 2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity. Int J Mol Sci 2023; 24:4669. [PMID: 36902100 PMCID: PMC10002758 DOI: 10.3390/ijms24054669] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 02/16/2023] [Accepted: 02/25/2023] [Indexed: 03/04/2023] Open
Abstract
We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps.
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Affiliation(s)
- Nimesha Rajapaksha
- Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803, USA
| | - Anabel Soldano
- Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803, USA
| | - Huili Yao
- Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803, USA
| | - Fabrizio Donnarumma
- Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803, USA
| | - Maithri M. Kashipathy
- Protein Structure and X-ray Crystallography Laboratory, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047, USA
| | - Steve Seibold
- Protein Structure and X-ray Crystallography Laboratory, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047, USA
| | | | - Scott Lovell
- Protein Structure and X-ray Crystallography Laboratory, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047, USA
| | - Mario Rivera
- Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803, USA
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Fu HW, Lai YC. The Role of Helicobacter pylori Neutrophil-Activating Protein in the Pathogenesis of H. pylori and Beyond: From a Virulence Factor to Therapeutic Targets and Therapeutic Agents. Int J Mol Sci 2022; 24:ijms24010091. [PMID: 36613542 PMCID: PMC9820732 DOI: 10.3390/ijms24010091] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Revised: 12/12/2022] [Accepted: 12/14/2022] [Indexed: 12/24/2022] Open
Abstract
Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of H. pylori, plays a role in bacterial protection and host inflammation. HP-NAP activates a variety of innate immune cells, including neutrophils, monocytes, and mast cells, to induce their pro-oxidant and pro-inflammatory activities. This protein also induces T-helper type 1 (Th1) immune response and cytotoxic T lymphocyte (CTL) activity, supporting that HP-NAP is able to promote gastric inflammation by activation of adaptive immune responses. Thus, HP-NAP is a potential therapeutic target for the treatment of H. pylori-induced gastric inflammation. The inflammatory responses triggered by HP-NAP are mediated by a PTX-sensitive G protein-coupled receptor and Toll-like receptor 2. Drugs designed to block the interactions between HP-NAP and its receptors could alleviate the inflammation in gastric mucosa caused by H. pylori infection. In addition, HP-NAP acts as a promising therapeutic agent for vaccine development, allergy treatment, and cancer immunotherapy. The high antigenicity of HP-NAP makes this protein a component of vaccines against H. pylori infection. Due to its immunomodulatory activity to stimulate the Th1-inducing ability of dendritic cells, enhance Th1 immune response and CTL activity, and suppress Th2-mediated allergic responses, HP-NAP could also act as an adjuvant in vaccines, a drug candidate against allergic diseases, and an immunotherapeutic agent for cancer. This review highlights the role of HP-NAP in the pathogenesis of H. pylori and the potential for this protein to be a therapeutic target in the treatment of H. pylori infection and therapeutic agents against H. pylori-associated diseases, allergies, and cancer.
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Affiliation(s)
- Hua-Wen Fu
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan
- Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan
- Correspondence: ; Tel.: +886-3-574-2485
| | - Yu-Chang Lai
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan
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Muñoz-Alía MÁ, Nace RA, Balakrishnan B, Zhang L, Packiriswamy N, Singh G, Warang P, Mena I, Narjari R, Vandergaast R, García-Sastre A, Schotsaert M, Russell SJ. Surface-modified measles vaccines encoding oligomeric, fusion-stabilized SARS-CoV-2 spike glycoproteins bypass measles seropositivity, boosting neutralizing antibody responses to omicron and historical variants. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2022:2022.12.16.520799. [PMID: 36561187 PMCID: PMC9774211 DOI: 10.1101/2022.12.16.520799] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/11/2023]
Abstract
Serum titers of SARS-CoV-2 neutralizing antibodies (nAb) correlate well with protection from symptomatic COVID-19, but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are life-long after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We therefore sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated potent induction of high titer nAb in measles-immune mice and confirmed the significant incremental contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin-display of the SARS-CoV-2 spike glycoprotein, and vaccine resurfacing. In animals primed and boosted with a MeV vaccine encoding the ancestral SARS-CoV-2 spike, high titer nAb responses against ancestral virus strains were only weakly cross-reactive with the omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the omicron BA.1 spike, antibody titers to both ancestral and omicron strains were robustly elevated and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that antigen engineering can enable the development of potent measles-based SARS-CoV-2 vaccine candidates.
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Affiliation(s)
- Miguel Á. Muñoz-Alía
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA
- Vyriad Inc, Rochester, MN, USA
| | - Rebecca A. Nace
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA
| | | | - Lianwen Zhang
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA
| | | | - Gagandeep Singh
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Prajakta Warang
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Ignacio Mena
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | | | | | - Adolfo García-Sastre
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Michael Schotsaert
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Stephen J. Russell
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA
- Vyriad Inc, Rochester, MN, USA
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Imanis Life Sciences, Rochester, MN, USA
- Division of Hematology, Mayo Clinic, Rochester, MN, USA
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6
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Deng R, Zheng H, Cai H, Li M, Shi Y, Ding S. Effects of helicobacter pylori on tumor microenvironment and immunotherapy responses. Front Immunol 2022; 13:923477. [PMID: 35967444 PMCID: PMC9371381 DOI: 10.3389/fimmu.2022.923477] [Citation(s) in RCA: 35] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Accepted: 07/04/2022] [Indexed: 12/13/2022] Open
Abstract
Helicobacter pylori is closely associated with gastric cancer. During persistent infection, Helicobacter pylori can form a microenvironment in gastric mucosa which facilitates the survival and colony formation of Helicobacter pylori. Tumor stromal cells are involved in this process, including tumor-associated macrophages, mesenchymal stem cells, cancer-associated fibroblasts, and myeloid-derived suppressor cells, and so on. The immune checkpoints are also regulated by Helicobacter pylori infection. Helicobacter pylori virulence factors can also act as immunogens or adjuvants to elicit or enhance immune responses, indicating their potential applications in vaccine development and tumor immunotherapy. This review highlights the effects of Helicobacter pylori on the immune microenvironment and its potential roles in tumor immunotherapy responses.
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Affiliation(s)
- Ruiyi Deng
- Peking University Third Hospital, Research Center of Clinical Epidemiology, Beijing, China
- Peking University Health Science Center, Peking University First Medical School, Beijing, China
| | - Huiling Zheng
- Peking University Third Hospital, Department of Gastroenterology, Beijing, China
| | - Hongzhen Cai
- Peking University Third Hospital, Research Center of Clinical Epidemiology, Beijing, China
- Peking University Health Science Center, Peking University First Medical School, Beijing, China
| | - Man Li
- Peking University Third Hospital, Research Center of Clinical Epidemiology, Beijing, China
- Peking University Health Science Center, Peking University Third Medical School, Beijing, China
| | - Yanyan Shi
- Peking University Third Hospital, Research Center of Clinical Epidemiology, Beijing, China
| | - Shigang Ding
- Peking University Third Hospital, Department of Gastroenterology, Beijing, China
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Abstract
The DNA-binding protein from starved cells, Dps, is a universally conserved prokaryotic ferritin that, in many species, also binds DNA. Dps homologs have been identified in the vast majority of bacterial species and several archaea. Dps also may play a role in the global regulation of gene expression, likely through chromatin reorganization. Dps has been shown to use both its ferritin and DNA-binding functions to respond to a variety of environmental pressures, including oxidative stress. One mechanism that allows Dps to achieve this is through a global nucleoid restructuring event during stationary phase, resulting in a compact, hexacrystalline nucleoprotein complex called the biocrystal that occludes damaging agents from DNA. Due to its small size, hollow spherical structure, and high stability, Dps is being developed for applications in biotechnology.
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Tuan VP, Yahara K, Dung HDQ, Binh TT, Huu Tung P, Tri TD, Thuan NPM, Khien VV, Trang TTH, Phuc BH, Tshibangu-Kabamba E, Matsumoto T, Akada J, Suzuki R, Okimoto T, Kodama M, Murakami K, Yano H, Fukuyo M, Takahashi N, Kato M, Nishiumi S, Azuma T, Ogura Y, Hayashi T, Toyoda A, Kobayashi I, Yamaoka Y. Genome-wide association study of gastric cancer- and duodenal ulcer-derived Helicobacter pylori strains reveals discriminatory genetic variations and novel oncoprotein candidates. Microb Genom 2021; 7. [PMID: 34846284 PMCID: PMC8743543 DOI: 10.1099/mgen.0.000680] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Genome-wide association studies (GWASs) can reveal genetic variations associated with a phenotype in the absence of any hypothesis of candidate genes. The problem of false-positive sites linked with the responsible site might be bypassed in bacteria with a high homologous recombination rate, such as Helicobacter pylori, which causes gastric cancer. We conducted a small-sample GWAS (125 gastric cancer cases and 115 controls) followed by prediction of gastric cancer and control (duodenal ulcer) H. pylori strains. We identified 11 single nucleotide polymorphisms (eight amino acid changes) and three DNA motifs that, combined, allowed effective disease discrimination. They were often informative of the underlying molecular mechanisms, such as electric charge alteration at the ligand-binding pocket, alteration in subunit interaction, and mode-switching of DNA methylation. We also identified three novel virulence factors/oncoprotein candidates. These results provide both defined targets for further informatic and experimental analyses to gain insights into gastric cancer pathogenesis and a basis for identifying a set of biomarkers for distinguishing these H. pylori-related diseases.
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Affiliation(s)
- Vo Phuoc Tuan
- Department of Endoscopy, Cho Ray Hospital, Ho Chi Minh, Vietnam
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan
| | - Koji Yahara
- Antimicrobial Resistance ResearchCenter, National Institute of Infectious Diseases, Tokyo, Japan
- *Correspondence: Koji Yahara,
| | | | - Tran Thanh Binh
- Department of Endoscopy, Cho Ray Hospital, Ho Chi Minh, Vietnam
| | - Pham Huu Tung
- Department of Endoscopy, Cho Ray Hospital, Ho Chi Minh, Vietnam
| | - Tran Dinh Tri
- Department of Endoscopy, Cho Ray Hospital, Ho Chi Minh, Vietnam
| | | | - Vu Van Khien
- Department of GI Endoscopy, 108 Central Hospital, Hanoi, Vietnam
| | | | - Bui Hoang Phuc
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan
- Department of Microbiology, Cho Ray Hospital, Ho Chi Minh, Vietnam
| | | | - Takashi Matsumoto
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan
| | - Junko Akada
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan
| | - Rumiko Suzuki
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan
| | - Tadayoshi Okimoto
- Department of Gastroenterology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Masaaki Kodama
- Department of Gastroenterology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Kazunari Murakami
- Department of Gastroenterology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Hirokazu Yano
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo, Japan
- Institute of Medical Science, University of Tokyo, Tokyo, Japan
- Graduate School of Life Sciences, Tohoku University, Sendai, Japan
| | - Masaki Fukuyo
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo, Japan
- Institute of Medical Science, University of Tokyo, Tokyo, Japan
- Department of Molecular Oncology, Chiba University, Chiba, Japan
| | - Noriko Takahashi
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo, Japan
- Institute of Medical Science, University of Tokyo, Tokyo, Japan
- Department of Infectious Diseases, Kyorin University School of Medicine, Mitaka City, Tokyo, Japan
| | - Mototsugu Kato
- Division of Endoscopy, Hokkaido University Hospital, Sapporo, Hokkaido, Japan
- Department of Gastroenterology, National Hospital Organization Hakodate Hospital, Hakodate, Hokkaido, Japan
| | - Shin Nishiumi
- Department of Gastroenterology, Graduate School of Medicine, Kobe University, Chuou-ku, Kobe, Hyogo, Japan
- Department of Omics Medicine, Hyogo College of Medicine, Hyogo, Japan
| | - Takashi Azuma
- Department of Gastroenterology, Graduate School of Medicine, Kobe University, Chuou-ku, Kobe, Hyogo, Japan
| | - Yoshitoshi Ogura
- Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan
- Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine, Kurume, Fukuoka, Japan
| | - Tetsuya Hayashi
- Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Atsushi Toyoda
- Advanced GenomicsCenter, National Institute of Genetics, Shizuoka, Japan
| | - Ichizo Kobayashi
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo, Japan
- Institute of Medical Science, University of Tokyo, Tokyo, Japan
- Department of Infectious Diseases, Kyorin University School of Medicine, Mitaka City, Tokyo, Japan
- Research Center for Micro-Nano Technology, Hosei University, Tokyo, Japan
- *Correspondence: Ichizo Kobayashi, ;
| | - Yoshio Yamaoka
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan
- Department of Medicine, gastroenterology section, Baylor College of Medicine, Houston TX, USA
- *Correspondence: Yoshio Yamaoka,
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Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells. Int J Mol Sci 2021; 22:ijms222111560. [PMID: 34768994 PMCID: PMC8584237 DOI: 10.3390/ijms222111560] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 10/22/2021] [Accepted: 10/22/2021] [Indexed: 12/14/2022] Open
Abstract
Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam3CSK4, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.
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Dadinova L, Kamyshinsky R, Chesnokov Y, Mozhaev A, Matveev V, Gruzinov A, Vasiliev A, Shtykova E. Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations. Int J Mol Sci 2021; 22:ijms22116056. [PMID: 34205216 PMCID: PMC8199988 DOI: 10.3390/ijms22116056] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2021] [Revised: 05/29/2021] [Accepted: 05/31/2021] [Indexed: 12/12/2022] Open
Abstract
Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.
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Affiliation(s)
- Liubov Dadinova
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences”, Leninskiy Prospect, 59, 119333 Moscow, Russia; (R.K.); (Y.C.); (A.M.); (A.V.); (E.S.)
- Correspondence: ; Tel.: +7-(499)-135-62-00
| | - Roman Kamyshinsky
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences”, Leninskiy Prospect, 59, 119333 Moscow, Russia; (R.K.); (Y.C.); (A.M.); (A.V.); (E.S.)
- National Research Center “Kurchatov Institute”, Akademika Kurchatova, 1, 123182 Moscow, Russia
- Moscow Institute of Physics and Technology, Institutsky Lane 9, 141700 Dolgoprudny, Russia
| | - Yury Chesnokov
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences”, Leninskiy Prospect, 59, 119333 Moscow, Russia; (R.K.); (Y.C.); (A.M.); (A.V.); (E.S.)
- National Research Center “Kurchatov Institute”, Akademika Kurchatova, 1, 123182 Moscow, Russia
| | - Andrey Mozhaev
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences”, Leninskiy Prospect, 59, 119333 Moscow, Russia; (R.K.); (Y.C.); (A.M.); (A.V.); (E.S.)
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya, 16/10, 117997 Moscow, Russia
| | - Vladimir Matveev
- Physics Department, Lomonosov Moscow State University, 119991 Moscow, Russia;
| | - Andrey Gruzinov
- EMBL, Hamburg Outstation, c/o DESY, Notkestr. 85, Geb. 25a, 22607 Hamburg, Germany;
| | - Alexander Vasiliev
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences”, Leninskiy Prospect, 59, 119333 Moscow, Russia; (R.K.); (Y.C.); (A.M.); (A.V.); (E.S.)
- National Research Center “Kurchatov Institute”, Akademika Kurchatova, 1, 123182 Moscow, Russia
- Moscow Institute of Physics and Technology, Institutsky Lane 9, 141700 Dolgoprudny, Russia
| | - Eleonora Shtykova
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences”, Leninskiy Prospect, 59, 119333 Moscow, Russia; (R.K.); (Y.C.); (A.M.); (A.V.); (E.S.)
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12
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An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines. mBio 2021; 12:mBio.00930-21. [PMID: 33975938 PMCID: PMC8262850 DOI: 10.1128/mbio.00930-21] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.
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13
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Guo Y, He W, Mou H, Zhang L, Chang J, Peng S, Ojha A, Tavora R, Parcells MS, Luo G, Li W, Zhong G, Choe H, Farzan M, Quinlan BD. An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines. mBio 2021; 12:mBio.00930-21. [PMID: 33975938 DOI: 10.1101/2020.04.10.036418] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023] Open
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.
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MESH Headings
- Angiotensin-Converting Enzyme 2/genetics
- Angiotensin-Converting Enzyme 2/immunology
- Animals
- Binding Sites
- COVID-19 Vaccines/chemistry
- COVID-19 Vaccines/immunology
- Female
- Genetic Engineering
- Glycosylation
- HEK293 Cells
- Humans
- Immunogenicity, Vaccine
- Mice
- Mice, Inbred BALB C
- Models, Molecular
- Protein Domains
- Rats
- Rats, Sprague-Dawley
- Receptors, Coronavirus/genetics
- Receptors, Coronavirus/immunology
- Spike Glycoprotein, Coronavirus/genetics
- Spike Glycoprotein, Coronavirus/immunology
- Vaccines, Conjugate/genetics
- Vaccines, Conjugate/immunology
- Vaccines, Synthetic/chemistry
- Vaccines, Synthetic/immunology
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Affiliation(s)
- Yan Guo
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Wenhui He
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Huihui Mou
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Lizhou Zhang
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Jing Chang
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Shoujiao Peng
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Amrita Ojha
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Rubens Tavora
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Mark S Parcells
- Department of Animal and Food Sciences, University of Delaware, Newark, Delaware, USA
| | - Guangxiang Luo
- Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA
| | - Wenhui Li
- National Institute of Biological Sciences, Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China
| | - Guocai Zhong
- Scripps Research SZBL Chemical Biology Institute, Shenzhen Bay Laboratory, Shenzhen, China
- School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Hyeryun Choe
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Michael Farzan
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
| | - Brian D Quinlan
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA
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14
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Lv C, Zhang X, Liu Y, Zhang T, Chen H, Zang J, Zheng B, Zhao G. Redesign of protein nanocages: the way from 0D, 1D, 2D to 3D assembly. Chem Soc Rev 2021; 50:3957-3989. [PMID: 33587075 DOI: 10.1039/d0cs01349h] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Compartmentalization is a hallmark of living systems. Through compartmentalization, ubiquitous protein nanocages such as viral capsids, ferritin, small heat shock proteins, and DNA-binding proteins from starved cells fulfill a variety of functions, while their shell-like structures hold great promise for various applications in the field of nanomedicine and nanotechnology. However, the number and structure of natural protein nanocages are limited, and these natural protein nanocages may not be suited for a given application, which might impede their further application as nanovehicles, biotemplates or building blocks. To overcome these shortcomings, different strategies have been developed by scientists to construct artificial protein nanocages, and 1D, 2D and 3D protein arrays with protein nanocages as building blocks through genetic and chemical modification to rival the size and functionality of natural protein nanocages. This review outlines the recent advances in the field of the design and construction of artificial protein nanocages and their assemblies with higher order, summarizes the strategies for creating the assembly of protein nanocages from zero-dimension to three dimensions, and introduces their corresponding applications in the preparation of nanomaterials, electrochemistry, and drug delivery. The review will highlight the roles of both the inter-subunit/intermolecular interactions at the key interface and the protein symmetry in constructing and controlling protein nanocage assemblies with different dimensions.
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Affiliation(s)
- Chenyan Lv
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing Key Laboratory of Functional Food from Plant Resources, Beijing 100083, China.
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15
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Dubrovin EV, Dadinova LA, Petoukhov MV, Soshinskaya EY, Mozhaev AA, Klinov DV, Schäffer TE, Shtykova EV, Batishchev OV. Spatial organization of Dps and DNA-Dps complexes. J Mol Biol 2021; 433:166930. [PMID: 33713674 DOI: 10.1016/j.jmb.2021.166930] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2020] [Revised: 02/24/2021] [Accepted: 03/05/2021] [Indexed: 02/04/2023]
Abstract
DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA-Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA-Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA-Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA-Dps complexes. Angle analysis has demonstrated that in DNA-Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet.
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Affiliation(s)
- Evgeniy V Dubrovin
- A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, 31/4 Leninskiy prospekt, Moscow 119071, Russia; Lomonosov Moscow State University, Faculty of Physics, Leninskie Gory 1 bld 2, 119991 Moscow, Russia.
| | - Liubov A Dadinova
- Shubnikov Institute of Crystallography, Federal Scientific Research Centre "Crystallography and Photonics," Russian Academy of Sciences, 119333 Moscow, Russia
| | - Maxim V Petoukhov
- Shubnikov Institute of Crystallography, Federal Scientific Research Centre "Crystallography and Photonics," Russian Academy of Sciences, 119333 Moscow, Russia
| | - Ekaterina Yu Soshinskaya
- Shubnikov Institute of Crystallography, Federal Scientific Research Centre "Crystallography and Photonics," Russian Academy of Sciences, 119333 Moscow, Russia
| | - Andrey A Mozhaev
- Shubnikov Institute of Crystallography, Federal Scientific Research Centre "Crystallography and Photonics," Russian Academy of Sciences, 119333 Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
| | - Dmitry V Klinov
- Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya 1a, 119435 Moscow, Russia
| | - Tilman E Schäffer
- University of Tübingen, Institute of Applied Physics, Auf der Morgenstelle 10, 72076 Tübingen, Germany
| | - Eleonora V Shtykova
- Shubnikov Institute of Crystallography, Federal Scientific Research Centre "Crystallography and Photonics," Russian Academy of Sciences, 119333 Moscow, Russia
| | - Oleg V Batishchev
- A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, 31/4 Leninskiy prospekt, Moscow 119071, Russia
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16
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Wu H, Gu L, Ma X, Tian X, Fan S, Qin M, Lu J, Lyu M, Wang S. Rapid Detection of Helicobacter pylori by the Naked Eye Using DNA Aptamers. ACS OMEGA 2021; 6:3771-3779. [PMID: 33585756 PMCID: PMC7876845 DOI: 10.1021/acsomega.0c05374] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Accepted: 01/12/2021] [Indexed: 05/02/2023]
Abstract
Helicobacter pylori was first isolated from gastritis patients by Barry J. Marshall and J. Robin Warren in 1982, and more than 90% of duodenal ulcers and about 80% of gastric ulcers are caused by H. pylori infection. Most detection methods require sophisticated instruments and professional operators, making detection slow and expensive. Therefore, it is critical to develop a simple, fast, highly specific, and practical strategy for the detection of H. pylori. In this study, we used H. pylori as a target to select unique aptamers that can be used for the detection of H. pylori. In our study, we used random ssDNA as an initial library to screen nucleic acid aptamers for H. pylori. We used binding rate and the fluorescence intensity to identify candidate aptamers. One DNA aptamer, named HPA-2, was discovered through six rounds of positive selection and three rounds of negative selection, and it had the highest affinity constant of all aptamers tested (K d = 19.3 ± 3.2 nM). This aptamer could be used to detect H. pylori and showed no specificity for other bacteria. Moreover, we developed a new sensor to detect H. pylori with the naked eye for 5 min using illumination from a hand-held flashlight. Our study provides a framework for the development of other aptamer-based methods for the rapid detection of pathogenic bacteria.
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Affiliation(s)
- Hangjie Wu
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Lide Gu
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Xiaoyi Ma
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Xueqing Tian
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Shihui Fan
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Mingcan Qin
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Jing Lu
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Mingsheng Lyu
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
| | - Shujun Wang
- Jiangsu Key Laboratory
of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine
Biotechnology, Jiangsu Ocean University, Lianyungang 222005, PR China
- Co-Innovation Center of Jiangsu Marine
Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, PR China
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17
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Soshinskaya EY, Dadinova LA, Mozhaev AA, Shtykova EV. Effect of Buffer Composition on Conformational Flexibility of N-Terminal Fragments of Dps and the Nature of Interactions with DNA. Small-Angle X-Ray Scattering Study. CRYSTALLOGR REP+ 2020. [DOI: 10.1134/s1063774520060334] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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18
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Quinlan BD, He W, Mou H, Zhang L, Guo Y, Chang J, Peng S, Ojha A, Tavora R, Parcells MS, Luo G, Li W, Zhong G, Choe H, Farzan M. An engineered receptor-binding domain improves the immunogenicity of multivalent SARS-CoV-2 vaccines. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2020:2020.11.18.388934. [PMID: 33236008 PMCID: PMC7685318 DOI: 10.1101/2020.11.18.388934] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/22/2023]
Abstract
The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.
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Affiliation(s)
- Brian D. Quinlan
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
- These authors contributed equally
| | - Wenhui He
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
- These authors contributed equally
| | - Huihui Mou
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
- These authors contributed equally
| | - Lizhou Zhang
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
- These authors contributed equally
| | - Yan Guo
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
- These authors contributed equally
| | - Jing Chang
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Shoujiao Peng
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Amrita Ojha
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Rubens Tavora
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Mark S. Parcells
- Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USA
| | - Guangxiang Luo
- Department of Microbiology, University of Alabama at Birmingham School Of Medicine, Birmingham, AL 35294, USA
| | - Wenhui Li
- National Institute of Biological Sciences, Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China
| | - Guocai Zhong
- Scripps Research | SZBL Chemical Biology Institute, Shenzhen Bay Laboratory (SZBL), Shenzhen, China
- School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Hyeryun Choe
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Michael Farzan
- Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
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19
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Amano KI, Sawazumi R, Imamura H, Sumi T, Hashimoto K, Fukami K, Kitaoka H, Nishi N, Sakka T. An Improved Model-potential-free Analysis of the Structure Factor Obtained from a Small-angle Scattering: Acquisitions of the Pair Distribution Function and the Pair Potential. CHEM LETT 2020. [DOI: 10.1246/cl.200292] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Ken-ichi Amano
- Faculty of Agriculture, Meijo University, 1-501 Shiogamaguchi, Tempaku-ku, Nagoya, Aichi 468-8502, Japan
- Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
| | - Ryosuke Sawazumi
- Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
| | - Hiroshi Imamura
- Department of Applied Chemistry, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
| | - Tomonari Sumi
- Research Institute for Interdisciplinary Science, Okayama University, Okayama 700-8530, Japan
| | - Kota Hashimoto
- Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
| | - Kazuhiro Fukami
- Department of Materials Science and Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan
| | - Haru Kitaoka
- Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
| | - Naoya Nishi
- Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
| | - Tetsuo Sakka
- Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
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20
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Kamyshinsky R, Chesnokov Y, Dadinova L, Mozhaev A, Orlov I, Petoukhov M, Orekhov A, Shtykova E, Vasiliev A. Polymorphic Protective Dps-DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering. Biomolecules 2019; 10:biom10010039. [PMID: 31888079 PMCID: PMC7023142 DOI: 10.3390/biom10010039] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2019] [Revised: 12/19/2019] [Accepted: 12/22/2019] [Indexed: 12/01/2022] Open
Abstract
Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.
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Affiliation(s)
- Roman Kamyshinsky
- National Research Center “Kurchatov Institute”, Akademika Kurchatova pl., 1, 123182 Moscow, Russia; (Y.C.); (A.O.); (A.V.)
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
- Moscow Institute of Physics and Technology, Institutsky lane 9, 141700 Dolgoprudny, Moscow Region, Russia
- Correspondence: ; Tel.: +7-916-356-3963
| | - Yury Chesnokov
- National Research Center “Kurchatov Institute”, Akademika Kurchatova pl., 1, 123182 Moscow, Russia; (Y.C.); (A.O.); (A.V.)
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
| | - Liubov Dadinova
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
| | - Andrey Mozhaev
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
- Shemyakin-Ovchinnikov Institute of bioorganic chemistry of Russian Academy of Sciences, Miklukho-Maklaya, 16/10, 117997 Moscow, Russia
| | - Ivan Orlov
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
| | - Maxim Petoukhov
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
- Frumkin Institute of Physical Chemistry and Electrochemistry of Russian Academy of Sciences, Leninsky prospect, 31, 119071 Moscow, Russia
| | - Anton Orekhov
- National Research Center “Kurchatov Institute”, Akademika Kurchatova pl., 1, 123182 Moscow, Russia; (Y.C.); (A.O.); (A.V.)
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
- Moscow Institute of Physics and Technology, Institutsky lane 9, 141700 Dolgoprudny, Moscow Region, Russia
| | - Eleonora Shtykova
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
| | - Alexander Vasiliev
- National Research Center “Kurchatov Institute”, Akademika Kurchatova pl., 1, 123182 Moscow, Russia; (Y.C.); (A.O.); (A.V.)
- Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia; (L.D.); (A.M.); (I.O.); (M.P.); (E.S.)
- Moscow Institute of Physics and Technology, Institutsky lane 9, 141700 Dolgoprudny, Moscow Region, Russia
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21
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Blaser N, Backert S, Pachathundikandi SK. Immune Cell Signaling by Helicobacter pylori: Impact on Gastric Pathology. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1149:77-106. [PMID: 31049845 DOI: 10.1007/5584_2019_360] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Helicobacter pylori represents a highly successful colonizer of the human stomach. Infections with this Gram-negative bacterium can persist lifelong, and although in the majority of cases colonization is asymptomatic, it can trigger pathologies ranging from chronic gastritis and peptic ulceration to gastric cancer. The interaction of the bacteria with the human host modulates immune responses in different ways to enable bacterial survival and persistence. H. pylori uses various pathogenicity-associated factors such as VacA, NapA, CGT, GGT, lipopolysaccharide, peptidoglycan, heptose 1,7-bisphosphate, ADP-heptose, cholesterol glucosides, urease and a type IV secretion system for controlling immune signaling and cellular functions. It appears that H. pylori manipulates multiple extracellular immune receptors such as integrin-β2 (CD18), EGFR, CD74, CD300E, DC-SIGN, MINCLE, TRPM2, T-cell and Toll-like receptors as well as a number of intracellular receptors including NLRP3, NOD1, NOD2, TIFA and ALPK1. Consequently, downstream signaling pathways are hijacked, inducing tolerogenic dendritic cells, inhibiting effector T cell responses and changing the gastrointestinal microbiota. Here, we discuss in detail the interplay of bacterial factors with multiple immuno-regulatory cells and summarize the main immune evasion and persistence strategies employed by H. pylori.
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Affiliation(s)
- Nicole Blaser
- Department of Biology, Institute for Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany
| | - Steffen Backert
- Department of Biology, Institute for Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany
| | - Suneesh Kumar Pachathundikandi
- Department of Biology, Institute for Microbiology, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany.
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22
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Howe C, Moparthi VK, Ho FM, Persson K, Stensjö K. The Dps4 from Nostoc punctiforme ATCC 29133 is a member of His-type FOC containing Dps protein class that can be broadly found among cyanobacteria. PLoS One 2019; 14:e0218300. [PMID: 31369577 PMCID: PMC6675082 DOI: 10.1371/journal.pone.0218300] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Accepted: 07/18/2019] [Indexed: 11/18/2022] Open
Abstract
Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe2+ to reduce H2O2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe2+ utilizing O2, but no evidence for its use of the oxidant H2O2 could be found. We identified Zn2+ to be an effective inhibitor of the O2-mediated Fe2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins.
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Affiliation(s)
- Christoph Howe
- Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, Sweden
| | - Vamsi K. Moparthi
- Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, Sweden
| | - Felix M. Ho
- Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, Sweden
| | - Karina Persson
- Department of Chemistry, Umeå University, Umeå, Sweden
- * E-mail: (KS); (KP)
| | - Karin Stensjö
- Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, Sweden
- * E-mail: (KS); (KP)
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23
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Matsuzaki J, Tsugawa H, Kashiwazaki Y, Mori H, Yamamoto Y, Kameyama H, Masaoka T, Kanai T, Suzuki H. Neutrophil-activating Protein Polymorphism of Helicobacter pylori Determines the Host Risk of Dyspepsia. Cell Mol Gastroenterol Hepatol 2019; 8:295-297.e6. [PMID: 31108232 PMCID: PMC6718361 DOI: 10.1016/j.jcmgh.2019.05.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/03/2018] [Revised: 05/05/2019] [Accepted: 05/06/2019] [Indexed: 12/11/2022]
Affiliation(s)
- Juntaro Matsuzaki
- Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Hitoshi Tsugawa
- Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Yuki Kashiwazaki
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Hideki Mori
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan; Department of Gastroenterology, National Hospital Organization Tokyo Medical Center, Tokyo, Japan
| | - Yuta Yamamoto
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Hisako Kameyama
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Tatsuhiro Masaoka
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Takanori Kanai
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
| | - Hidekazu Suzuki
- Department of Gastroenterology and Hepatology, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
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24
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Kira JI, Isobe N. Helicobacter pylori infection and demyelinating disease of the central nervous system. J Neuroimmunol 2019; 329:14-19. [DOI: 10.1016/j.jneuroim.2018.06.017] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2018] [Accepted: 06/28/2018] [Indexed: 12/29/2022]
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25
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Devaraj A, Buzzo J, Rocco CJ, Bakaletz LO, Goodman SD. The DNABII family of proteins is comprised of the only nucleoid associated proteins required for nontypeable Haemophilus influenzae biofilm structure. Microbiologyopen 2017; 7:e00563. [PMID: 29230970 PMCID: PMC6011942 DOI: 10.1002/mbo3.563] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2017] [Revised: 10/23/2017] [Accepted: 10/30/2017] [Indexed: 11/20/2022] Open
Abstract
Biofilms play a central role in the pathobiology of otitis media (OM), bronchitis, sinusitis, conjunctivitis, and pneumonia caused by nontypeable Haemophilus influenzae (NTHI). Our previous studies show that extracellular DNA (eDNA) and DNABII proteins are essential components of biofilms formed by NTHI. The DNABII protein family includes integration host factor (IHF) and the histone‐like protein HU and plays a central role in NTHI biofilm structural integrity. We demonstrated that immunological targeting of these proteins during NTHI‐induced experimental OM in a chinchilla model caused rapid clearance of biofilms from the middle ear. Given the essential role of DNABII proteins in maintaining the structure of an NTHI biofilm, we investigated whether any of the other nucleoid associated proteins (NAPs) expressed by NTHI might play a similar role, thereby serving as additional target(s) for intervention. We demonstrated that although several NAPs including H‐NS, CbpA, HfQ and Dps are present within the biofilm extracellular matrix, only the DNABII family of proteins is critical for the structural integrity of the biofilms formed by NTHI. We have also demonstrated that IHF and HU are located at distinct regions within the extracellular matrix of NTHI biofilms formed in vitro, indicative of independent functions of these two proteins.
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Affiliation(s)
- Aishwarya Devaraj
- Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, OH, USA
| | - John Buzzo
- Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, OH, USA
| | - Christopher J Rocco
- Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, OH, USA
| | - Lauren O Bakaletz
- Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, OH, USA
| | - Steven D Goodman
- Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, OH, USA
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26
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Helicobacter pylori antigenic Lpp20 is a structural homologue of Tipα and promotes epithelial-mesenchymal transition. Biochim Biophys Acta Gen Subj 2017; 1861:3263-3271. [PMID: 28947343 DOI: 10.1016/j.bbagen.2017.09.017] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2017] [Revised: 09/07/2017] [Accepted: 09/21/2017] [Indexed: 02/07/2023]
Abstract
BACKGROUND Helicobacter pylori is a bacterium that affects about 50% of the world population and, despite being often asymptomatic, it is responsible of several gastric diseases, from gastritis to gastric cancer. The protein Lpp20 (HP1456) plays an important role in bacterium survival and host colonization, but the possibility that it might be involved in the etiology of H. pylori-related disorders is an unexplored issue. Lpp20 is a lipoprotein bound to the external membrane of the bacterium, but it is also secreted inside vesicles along with other two proteins of the same operon, i.e. HP1454 and HP1457. RESULTS In this study we determined the crystal structure of Lpp20 and we found that it has a fold similar to a carcinogenic factor released by H. pylori, namely Tipα. We demonstrate that Lpp20 promotes cell migration and E-cadherin down-regulation in gastric cancer cells, two events recalling the epithelial-mesenchymal transition (EMT) process. Differently from Tipα, Lpp20 also stimulates cell proliferation. CONCLUSIONS This identifies Lpp20 as a new pathogenic factor produced by H. pylori that promotes EMT and thereby the progression of cancer to the metastatic state.
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27
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Hong ZW, Yang YC, Pan T, Tzeng HF, Fu HW. Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein. PLoS One 2017; 12:e0173632. [PMID: 28328957 PMCID: PMC5362085 DOI: 10.1371/journal.pone.0173632] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2016] [Accepted: 02/22/2017] [Indexed: 01/19/2023] Open
Abstract
Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification.
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Affiliation(s)
- Zhi-Wei Hong
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
| | - Yu-Chi Yang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
| | - Timothy Pan
- Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
| | - Huey-Fen Tzeng
- Department of Applied Chemistry, National Chi Nan University, Puli, Nantou, Taiwan, Republic of China
- * E-mail: (HWF); (HFT)
| | - Hua-Wen Fu
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
- Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
- * E-mail: (HWF); (HFT)
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28
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Tavares R, Pathak SK. Helicobacter pylori Secreted Protein HP1286 Triggers Apoptosis in Macrophages via TNF-Independent and ERK MAPK-Dependent Pathways. Front Cell Infect Microbiol 2017; 7:58. [PMID: 28293545 PMCID: PMC5329642 DOI: 10.3389/fcimb.2017.00058] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2016] [Accepted: 02/13/2017] [Indexed: 01/10/2023] Open
Abstract
Macrophages constitute a powerful line of defense against H. pylori. The final disease outcome is highly dependent on the bacterial ability to modulate the effector functions of activated macrophages. Here, we report that H. pylori secreted protein HP1286 is a novel regulator of macrophage responses. Differential expression and release of HP1286 homologues were observed among H. pylori strains. Recombinant purified HP1286 (rHP1286) had the ability to bind to primary human monocyte-derived macrophages (MDM) and macrophage cell lines. Exposure to rHP1286 induced apoptosis in macrophages in a dose- and time-dependent manner. Although interaction of rHP1286 was observed for several other cell types, such as human monocytes, differentiated neutrophil-like HL60 cells, and the T lymphocyte Jurkat cell line, rHP1286 failed to induce apoptosis under similar conditions, indicating a macrophage-specific effect of the protein. A mutant strain of H. pylori lacking HP1286 protein expression was significantly impaired in its ability to induce apoptosis in macrophages. Significantly higher caspase 3 activity was detected in rHP1286-challenged macrophages. Furthermore, rHP1286-induced macrophages apoptosis was not inhibited in the presence of neutralizing antibodies against TNF. These observations indicate that rHP1286 induced a caspase-dependent and TNF-independent macrophage apoptosis. Pre-treatment of macrophages with U0126, an inhibitor of the ERK MAPK signaling pathway significantly reduced rHP1286-induced apoptosis. Furthermore, nuclear translocation of ERK and phosphorylation of c-Fos was detected in rHP1286-treated macrophages. These results provide functional insight into the potential role of HP1286 during H. pylori infection. Considering the ability of HP1286 to induce macrophage apoptosis, the protein could possibly help in the bacterial escape from the activated macrophages and persistence in the stomach.
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Affiliation(s)
- Raquel Tavares
- Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Stockholm, Sweden
| | - Sushil Kumar Pathak
- Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Stockholm, Sweden
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29
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Zhou S, Huang Y, Liang B, Dong H, Yao S, Chen Y, Xie Y, Long Y, Gong S, Zhou Z. Systemic and mucosal pre-administration of recombinant Helicobacter pylori neutrophil-activating protein prevents ovalbumin-induced allergic asthma in mice. FEMS Microbiol Lett 2017; 364:fnw288. [PMID: 28087613 DOI: 10.1093/femsle/fnw288] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 09/18/2016] [Accepted: 01/11/2017] [Indexed: 12/28/2022] Open
Abstract
PURPOSE Previous epidemiologic studies have demonstrated an inverse association between Helicobacter pylori infection and the frequency of allergic asthma. The neutrophil-activating protein (NAP) of H. pylori has been identified as a modulator possessing anti-Th2 inflammation activity. Here, we sought to determine whether systemic or mucosal pre-administration of recombinant H. pylori NAP (rNAP) could prevent ovalbumin (OVA)-induced allergic asthma in mice. METHODS Mice were exposed to purified rNAP through intraperitoneal injection or inhalation and then sensitized with OVA. Following a challenge with aerosolized OVA, the bronchoalveolar lavage fluid (BALF) cell count, lung tissue histology, BALF cytokines and serum IgE were evaluated. RESULTS Both intraperitoneal injection and inhalation of rNAP prior to OVA sensitization significantly reduced eosinophil accumulation and inflammatory infiltration in lung tissue in OVA-induced asthma mice; eosinophils were reduced in the BALF of rNAP-treated mice. In addition, IL-4 and IL-13 levels were lower (P < 0.01), IL-10 and IFN-γ levels were higher (P < 0.01) and IgE serum levels were lower (P < 0.01) in the treated groups compared to the control group. CONCLUSIONS Systemic and mucosal pre-administration of rNAP could suppress the development of OVA-induced asthma in mice; rNAP may be utilized as part of novel strategies for the prevention or treatment of allergic diseases.
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Affiliation(s)
- Shuai Zhou
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China.,Translational Medicine Center, Guangdong Women and Children Hospital, No. 521 Xingnan Avenue, Panyu district, Guangzhou, Guangdong 511400, People's Republic of China
| | - Yanmei Huang
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Bingshao Liang
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Hui Dong
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Shuwen Yao
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Yinshuang Chen
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Yongqiang Xie
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Yan Long
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Sitang Gong
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
| | - Zhenwen Zhou
- Guangzhou Women and Children's Medical Center, Guangzhou Medical University, No. 318 Renminzhong Road, Yuexiu, Guangzhou, Guangdong 510120, People's Republic of China
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30
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D'Elios MM, Vallese F, Capitani N, Benagiano M, Bernardini ML, Rossi M, Rossi GP, Ferrari M, Baldari CT, Zanotti G, de Bernard M, Codolo G. The Helicobacter cinaedi antigen CAIP participates in atherosclerotic inflammation by promoting the differentiation of macrophages in foam cells. Sci Rep 2017; 7:40515. [PMID: 28074932 PMCID: PMC5225449 DOI: 10.1038/srep40515] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2016] [Accepted: 12/06/2016] [Indexed: 01/09/2023] Open
Abstract
Recent studies have shown that certain specific microbial infections participate in atherosclerosis by inducing inflammation and immune reactions, but how the pathogens implicated in this pathology trigger the host responses remains unknown. In this study we show that Helicobacter cinaedi (Hc) is a human pathogen linked to atherosclerosis development since at least 27% of sera from atherosclerotic patients specifically recognize a protein of the Hc proteome, that we named Cinaedi Atherosclerosis Inflammatory Protein (CAIP) (n = 71). CAIP appears to be implicated in this pathology because atheromatous plaques isolated from atherosclerotic patients are enriched in CAIP-specific T cells (10%) which, in turn, we show to drive a Th1 inflammation, an immunopathological response typically associated to atherosclerosis. Recombinant CAIP promotes the differentiation and maintenance of the pro-inflammatory profile of human macrophages and triggers the formation of foam cells, which are a hallmark of atherosclerosis. This study identifies CAIP as a relevant factor in atherosclerosis inflammation linked to Hc infection and suggests that preventing and eradicating Hc infection could reduce the incidence of atherosclerosis.
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Affiliation(s)
- Mario Milco D'Elios
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - Francesca Vallese
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | - Nagaja Capitani
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.,Department of Life Sciences, University of Siena, Siena, Italy
| | - Marisa Benagiano
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - Maria Lina Bernardini
- Department of Biology and Biotechnology, "C. Darwin", Sapienza University of Rome, Rome, Italy.,Institute Pasteur Italy - Fondazione Cenci Bolognetti, Rome, Italy
| | - Mirko Rossi
- Department of Food Hygiene and Environmental Health, University of Helsinki, Helsinki, Finland
| | - Gian Paolo Rossi
- Internal Medicine, Department of Medicine-DIMED, University of Padua, Italy
| | - Mauro Ferrari
- Vascular Surgery Unit, Cisanello University Hospital AOUP, Pisa, Italy
| | | | - Giuseppe Zanotti
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | | | - Gaia Codolo
- Department of Biology, University of Padua, Padua, Italy
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31
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Xia W. Competition for Iron Between Host and Pathogen: A Structural Case Study on Helicobacter pylori. Methods Mol Biol 2017; 1535:65-75. [PMID: 27914073 DOI: 10.1007/978-1-4939-6673-8_5] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Helicobacter pylori (H. pylori) is a highly successful bacterial pathogen, which colonizes the stomach of more than half of the world's population. To colonize and survive in such an acidic and inhospitable niche, H. pylori cells have evolved complex mechanisms to acquire nutrients from human hosts, including iron, an essential nutrient for both the pathogens and host cells. However, human cells also utilize diverse strategies in withholding of irons to prevent the bacterial outgrowth. The competition for iron is the central battlefield between pathogen and host. This mini-review summarizes the updated scenarios of the battle for iron between H. pylori and human host from a structural biology perspective.
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Affiliation(s)
- Wei Xia
- MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, 135 West Xingang Road, Guangzhou 510275, China.
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32
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Flint A, Stintzi A, Saraiva LM. Oxidative and nitrosative stress defences of Helicobacter and Campylobacter species that counteract mammalian immunity. FEMS Microbiol Rev 2016; 40:938-960. [PMID: 28201757 PMCID: PMC5091033 DOI: 10.1093/femsre/fuw025] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 03/29/2016] [Accepted: 07/02/2016] [Indexed: 12/18/2022] Open
Abstract
Helicobacter and Campylobacter species are Gram-negative microaerophilic host-associated heterotrophic bacteria that invade the digestive tract of humans and animals. Campylobacter jejuni is the major worldwide cause of foodborne gastroenteritis in humans, while Helicobacter pylori is ubiquitous in over half of the world's population causing gastric and duodenal ulcers. The colonisation of the gastrointestinal system by Helicobacter and Campylobacter relies on numerous cellular defences to sense the host environment and respond to adverse conditions, including those imposed by the host immunity. An important antimicrobial tool of the mammalian innate immune system is the generation of harmful oxidative and nitrosative stresses to which pathogens are exposed during phagocytosis. This review summarises the regulators, detoxifying enzymes and subversion mechanisms of Helicobacter and Campylobacter that ultimately promote the successful infection of humans.
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Affiliation(s)
- Annika Flint
- Ottawa Institute of Systems Biology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada
| | - Alain Stintzi
- Ottawa Institute of Systems Biology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada
| | - Lígia M. Saraiva
- Instituto de Tecnologia Química e Biológica, NOVA, Av. da República, 2780-157 Oeiras, Portugal
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33
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Shan W, Kung HF, Ge R. Comparison of Iron-Binding Ability Between Thr70-NapA and Ser70-NapA of Helicobacter pylori. Helicobacter 2016; 21:192-200. [PMID: 26347349 DOI: 10.1111/hel.12266] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
BACKGROUND The neutrophil-activating protein (NapA) of Helicobacter pylori (H. pylori), with DNA-binding and iron seizing properties, is a fundamental virulence factor involved in H. pylori-related diseases. Compared with Ser70-NapA strain, Thr70-NapA strain is more intimately correlated with iron-deficiency anemia. METHODS To investigate whether two types of proteins differ in iron-binding ability, mutated Thr70-NapA and Ser70-NapA strains were established. Isothermal titration calorimetry (ITC) method was conducted to measure the binding between the NapA protein and Fe(2+) . The structural changes of NapA protein were also tested during iron interaction by fast protein liquid chromatography (FPLC) and circular dichroism (CD) methods. DNA-binding assay was performed for evaluate the affinity of both mutated and wild types of NapA with DNA. RESULTS Mutated Thr70-NapA had higher iron-binding ability than wild Ser70-NapA. The structural stability of Thr70-NapA was disrupted and became more active along with the rising concentration of Fe(2+) , whereas no similar association was observed between Ser70-NapA and Fe(2+) level. When the iron/protein molar ratio ranged from 10 to 20, both Ser70-NapA and Thr70-NapA displayed weaker DNA-binding ability. CONCLUSIONS Thr70-NapA has much stronger ability to sequester ferrous ion compared with Ser70-NapA in H. pylori. In addition, the DNA-binding property of NapA is dependent upon the Fe(2+) concentration.
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Affiliation(s)
- Weiran Shan
- Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou, China
| | - Hsiang-Fu Kung
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, China
| | - Ruiguang Ge
- Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou, China
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Yang YC, Kuo TY, Hong ZW, Chang HW, Chen CC, Tsai TL, Fu HW. High yield purification of Helicobacter pylori neutrophil-activating protein overexpressed in Escherichia coli. BMC Biotechnol 2015; 15:23. [PMID: 25880121 PMCID: PMC4425898 DOI: 10.1186/s12896-015-0136-x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Accepted: 03/18/2015] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-induced gastric inflammation. Due to its immunogenic and immunomodulatory properties, HP-NAP has been used for developing vaccines against H. pylori infection and new drugs for cancer therapy. RESULTS Here, we provide a simple process for high-yield production of HP-NAP by applying one-step negative chromatography to purify recombinant HP-NAP expressed in Escherichia coli (E. coli). In our E. coli expression system, recombinant HP-NAP constitutes nearly 70% of the total protein. Overexpressed recombinant HP-NAP is almost completely soluble upon cell lysis at pH 9.5. Under the optimal condition at pH 8.0, recombinant HP-NAP with purity higher than 95% can be obtained from E. coli by collecting the unbound fraction using diethylaminoethyl (DEAE) Sephadex resin in batch mode. The overall yield of HP-NAP from a 50-ml E. coli culture is ~19 mg. The purified HP-NAP folds into a multimer with a secondary structure of α-helix and is able to trigger the production of reactive oxygen species by neutrophils. CONCLUSIONS Purification of recombinant HP-NAP overexpressed in E. coli using DEAE Sephadex negative mode batch chromatography is an efficient method for high-yield production of highly pure HP-NAP in its native state. The purified HP-NAP is useful for various clinical applications including vaccine development, diagnosis, and new drug development.
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Affiliation(s)
- Yu-Chi Yang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China.
| | - Ting-Yu Kuo
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China.
| | - Zhi-Wei Hong
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China.
| | - Han-Wen Chang
- Department of Internal Medicine, Division of Hepatology and Gastroenterology, Mackay Memorial Hospital, Hsinchu, 30055, Taiwan, Republic of China.
| | - Chung-Chu Chen
- Department of Internal Medicine, Division of Hepatology and Gastroenterology, Mackay Memorial Hospital, Hsinchu, 30055, Taiwan, Republic of China.
| | - Te-Lung Tsai
- Department of Pathology and Laboratory Medicine, Mackay Memorial Hospital, Hsinchu, 30055, Taiwan, Republic of China.
| | - Hua-Wen Fu
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China. .,Department of Life Science, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China.
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Sanchuki HBS, Valdameri G, Moure VR, Oliveira MA, Pedrosa FO, Souza EM, Korolik V, Huergo LF. Purification of the Campylobacter jejuni Dps protein assisted by its high melting temperature. Protein Expr Purif 2015; 111:105-10. [PMID: 25707373 DOI: 10.1016/j.pep.2014.12.011] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2014] [Revised: 12/15/2014] [Accepted: 12/16/2014] [Indexed: 11/17/2022]
Abstract
Dps proteins (DNA binding protein from starved cell) form a distinct group within the ferritin superfamily. All Dps members are composed of 12 identical subunits that assemble into a conserved spherical protein shell. Dps oxidize Fe(2+) in a conserved ferroxidase center located at the interface between monomers, the product of the reaction Fe(3+), is then stored inside the protein shell in the form of non-reactive insoluble Fe2O3. The Campylobacter jejuni Dps (CjDps) has been reported to play a plethora of functions, such as DNA binding and protection, iron storage, survival in response to hydrogen peroxide and sulfatide binding. CjDps is also important during biofilm formation and caecal colonization in poultry. In order to facilitate in vitro characterisation of CjDps, it is important to have a simple and reproducible protocol for protein purification. Here we report an observation that CjDps has an unusual high melting temperature. We exploited this property for protein purification by introducing a thermal treatment step which allowed achieving homogeneity by using only two chromatographic steps. Gel filtration chromatography, circular dichroism, mass spectrometry, DNA-binding and iron oxidation analysis confirmed that the CjDps structure and function were unaffected.
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Affiliation(s)
- Heloisa B S Sanchuki
- Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Departamento de Bioquímica e Biologia Molecular, UFPR Curitiba, PR, Brazil
| | - Glaucio Valdameri
- Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Departamento de Bioquímica e Biologia Molecular, UFPR Curitiba, PR, Brazil
| | - Vivian R Moure
- Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Departamento de Bioquímica e Biologia Molecular, UFPR Curitiba, PR, Brazil
| | - Marco A Oliveira
- Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Departamento de Bioquímica e Biologia Molecular, UFPR Curitiba, PR, Brazil
| | - Fábio O Pedrosa
- Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Departamento de Bioquímica e Biologia Molecular, UFPR Curitiba, PR, Brazil
| | - Emanuel M Souza
- Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Departamento de Bioquímica e Biologia Molecular, UFPR Curitiba, PR, Brazil
| | - Victoria Korolik
- Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia
| | - Luciano F Huergo
- Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Departamento de Bioquímica e Biologia Molecular, UFPR Curitiba, PR, Brazil.
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Structures and metal-binding properties of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center. Biomolecules 2014; 4:600-15. [PMID: 24971723 PMCID: PMC4192664 DOI: 10.3390/biom4030600] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2014] [Revised: 06/03/2014] [Accepted: 06/04/2014] [Indexed: 01/24/2023] Open
Abstract
Helicobacter pylori causes severe diseases, such as chronic gastritis, peptic ulcers, and stomach cancers. H. pylori neutrophil-activating protein (HP-NAP) is an iron storage protein that forms a dodecameric shell, promotes the adhesion of neutrophils to endothelial cells, and induces the production of reactive oxygen radicals. HP-NAP belongs to the DNA-protecting proteins under starved conditions (Dps) family, which has significant structural similarities to the dodecameric ferritin family. The crystal structures of the apo form and metal-ion bound forms, such as iron, zinc, and cadmium, of HP-NAP have been determined. This review focused on the structures and metal-binding properties of HP-NAP. These metal ions bind at the di-nuclear ferroxidase center (FOC) by different coordinating patterns. In comparison with the apo structure, metal loading causes a series of conformational changes in conserved residues among HP-NAP and Dps proteins (Trp26, Asp52, and Glu56) at the FOC. HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of pores. Metal ions have been identified around one of the pores; therefore, the negatively-charged pore is suitable for the passage of metal ions.
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Fu HW. Helicobacter pylori neutrophil-activating protein: From molecular pathogenesis to clinical applications. World J Gastroenterol 2014; 20:5294-5301. [PMID: 24833859 PMCID: PMC4017044 DOI: 10.3748/wjg.v20.i18.5294] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/29/2013] [Revised: 12/02/2013] [Accepted: 01/06/2014] [Indexed: 02/06/2023] Open
Abstract
Helicobacter pylori (H. pylori) neutrophil-activating protein (HP-NAP) was originally identified as a virulence factor of H. pylori for its ability to activate neutrophils to generate respiratory burst by releasing reactive oxygen species. Later on, HP-NAP was also found to be involved in the protection of H. pylori from DNA damage, supporting the survival of H. pylori under oxidative stress. This protein is highly conserved and expressed by virtually all clinical isolates of H. pylori. The majority of patients infected with H. pylori produced antibodies specific for HP-NAP, suggesting its important role in immunity. In addition to acting as a pathogenic factor by activating the innate immunity through a wide range of human leukocytes, including neutrophils, monocytes, and mast cells, HP-NAP also mediates adaptive immunity through the induction of T helper cell type I responses. The pro-inflammatory and immunomodulatory properties of HP-NAP not only make it play an important role in disease pathogenesis but also make it a potential candidate for clinical use. Even though there is no convincing evidence to link HP-NAP to a disease outcome, recent findings supporting the pathogenic role of HP-NAP will be reviewed. In addition, the potential clinical applications of HP-NAP in vaccine development, clinical diagnosis, and drug development will be discussed.
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Williams SM, Chandran AV, Vijayabaskar MS, Roy S, Balaram H, Vishveshwara S, Vijayan M, Chatterji D. A histidine aspartate ionic lock gates the iron passage in miniferritins from Mycobacterium smegmatis. J Biol Chem 2014; 289:11042-11058. [PMID: 24573673 PMCID: PMC4036245 DOI: 10.1074/jbc.m113.524421] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2013] [Revised: 02/24/2014] [Indexed: 11/06/2022] Open
Abstract
Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8-2.2 Å for the various mutants to compare structural alterations vis à vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.
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Affiliation(s)
| | - Anu V Chandran
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India
| | - Mahalingam S Vijayabaskar
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom, and
| | - Sourav Roy
- Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India
| | - Hemalatha Balaram
- Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India
| | | | - Mamannamana Vijayan
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India
| | - Dipankar Chatterji
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India,; Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India.
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Zanotti G, Cendron L. Structural and functional aspects of the Helicobacter pylori secretome. World J Gastroenterol 2014; 20:1402-1423. [PMID: 24587618 PMCID: PMC3925851 DOI: 10.3748/wjg.v20.i6.1402] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Accepted: 01/06/2014] [Indexed: 02/06/2023] Open
Abstract
Proteins secreted by Helicobacter pylori (H. pylori), an important human pathogen responsible for severe gastric diseases, are reviewed from the point of view of their biochemical characterization, both functional and structural. Despite the vast amount of experimental data available on the proteins secreted by this bacterium, the precise size of the secretome remains unknown. In this review, we consider as secreted both proteins that contain a secretion signal for the periplasm and proteins that have been detected in the external medium in in vitro experiments. In this way, H. pylori’s secretome appears to be composed of slightly more than 160 proteins, but this number must be considered very cautiously, not only because the definition of secretome itself is ambiguous but also because the included proteins were observed as secreted in in vitro experiments that were not representative of the environmental situation in vivo. The proteins that appear to be secreted can be grouped into different classes: enzymes (48 proteins), outer membrane proteins (43), components of flagella (11), members of the cytotoxic-associated genes pathogenicity island or other toxins (8 and 5, respectively), binding and transport proteins (9), and others (11). A final group, which includes 28 members, is represented by hypothetical uncharacterized proteins. Despite the large amount of data accumulated on the H. pylori secretome, a considerable amount of work remains to reach a full comprehension of the system at the molecular level.
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40
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Amedei A, Codolo G, Ozolins D, Ballerini C, Biagioli T, Jaunalksne I, Zilevica A, D Elios S, De Bernard M, D' Elios MM. Cerebrospinal fluid T-regulatory cells recognize Borrelia burgdorferi NAPA in chronic Lyme borreliosis. Int J Immunopathol Pharmacol 2013; 26:907-915. [PMID: 24355226 DOI: 10.1177/039463201302600409] [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] [Indexed: 01/07/2023] Open
Abstract
The NapA protein of B. burgdorferi is essential for the persistence of spirochetes in ticks. One of the most intriguing aspects of NapA is its potential to interfere with the host immune system. Here, we investigated the role of the acquired immune responses induced by NapA in the cerebrospinal fluids (CSF) of patients with chronic Lyme borreliosis. We evaluated the cytokine profile induced in microglia cells and CSF T cells following NapA stimulation. We report here that NapA induced a regulatory T (Treg) response in the CSF of patients with chronic Lyme borreliosis and it is able to expand this suppressive response by promoting the production of TGF-beta and IL-10 by microglia cells. Collectively, these data strongly support a central role of NapA in promoting both Treg response and immune suppression in the CSF of patients with chronic Lyme borreliosis and suggest that NapA and the Treg pathway may represent novel therapeutic targets for the prevention and treatment of the disease.
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Affiliation(s)
- A Amedei
- Department of Experimental and Clinical Medicine, University of Firenze, Italy
| | - G Codolo
- Department of Biology, University of Padova, Padova, Italy
| | - D Ozolins
- Microbiology, Faculty of Medicine, University of Latvia, Riga, Latvia
| | - C Ballerini
- Department of Neurological Sciences, University of Firenze, Firenze, Italy
| | - T Biagioli
- Department of Neurological Sciences, University of Firenze, Firenze, Italy
| | | | - A Zilevica
- Microbiology, Faculty of Medicine, University of Latvia, Riga, Latvia
| | - S D Elios
- Department of Experimental and Clinical Medicine, University of Firenze, Italy
| | - M De Bernard
- Department of Biology, University of Padova, Padova, Italy
| | - M M D' Elios
- Department of Experimental and Clinical Medicine, University of Firenze, Italy
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An infection-enhanced oncolytic adenovirus secreting H. pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors. Mol Ther 2013; 21:2008-18. [PMID: 23817216 PMCID: PMC3831034 DOI: 10.1038/mt.2013.153] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2013] [Accepted: 06/24/2013] [Indexed: 12/13/2022] Open
Abstract
Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. HP-NAP can mediate antitumor effects by recruiting neutrophils and inducing Th1-type differentiation in the tumor microenvironment. It therefore holds strong potential as a therapeutic gene. Here, we armed a replication-selective, infection-enhanced adenovirus with secretory HP-NAP, Ad5PTDf35-[Δ24-sNAP], and evaluated its therapeutic efficacy against neuroendocrine tumors. We observed that it could specifically infect and eradicate a wide range of tumor cells lines from different origin in vitro. Insertion of secretory HP-NAP did not affect the stability or replicative capacity of the virus and infected tumor cells could efficiently secrete HP-NAP. Intratumoral administration of the virus in nude mice xenografted with neuroendocrine tumors improved median survival. Evidence of biological HP-NAP activity was observed 24 hours after treatment with neutrophil infiltration in tumors and an increase of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and MIP2-α in the systemic circulation. Furthermore, evidence of Th1-type immune polarization was observed as a result of increase in IL-12/23 p40 cytokine concentrations 72 hours postvirus administration. Our observations suggest that HP-NAP can serve as a potent immunomodulator in promoting antitumor immune response in the tumor microenvironment and enhance the therapeutic effect of oncolytic adenovirus.
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Shih KS, Lin CC, Hung HF, Yang YC, Wang CA, Jeng KC, Fu HW. One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis. PLoS One 2013; 8:e60786. [PMID: 23577158 PMCID: PMC3620106 DOI: 10.1371/journal.pone.0060786] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2012] [Accepted: 03/02/2013] [Indexed: 12/12/2022] Open
Abstract
Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.
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Affiliation(s)
- Kuo-Shun Shih
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
| | - Chih-Chang Lin
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
| | - Hsiao-Fang Hung
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
- Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan, Republic of China
| | - Yu-Chi Yang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
| | - Chung-An Wang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
| | - Kee-Ching Jeng
- Departments of Research, Taichung Veterans General Hospital, Taiwan, Republic of China
| | - Hua-Wen Fu
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
- Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
- * E-mail:
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Yokota SI, Toita N, Yamamoto S, Fujii N, Konno M. Positive relationship between a polymorphism in Helicobacter pylori neutrophil-activating protein a gene and iron-deficiency anemia. Helicobacter 2013; 18:112-6. [PMID: 23067298 DOI: 10.1111/hel.12011] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND Numerous studies have suggested a link between iron-deficiency anemia (IDA) and Helicobacter pylori infection. Previously, we found that strains isolated from IDA patients showed higher levels of Fe ion uptake and Fe-ion-dependent rapid proliferation than those of strains derived from patients without IDA. MATERIALS AND METHODS Twenty-four H. pylori strains from IDA patients (IDA strains) and 25 strains from patients who had H. pylori gastritis without anemia (non-IDA strains) were examined. Their nucleotide sequences of napA, fur, and feoB, which contribute to Fe ion uptake, were determined. RESULTS Numerous polymorphisms of the three genes were found in both strains. Frequency of neutrophil-activating protein A (NapA), which encoded by napA, with threonine at amino acid residue No. 70 (Thr70-type NapA) was significantly higher in IDA strains than in non-IDA strains. Strains with Thr70-type NapA showed significantly higher levels of Fe(3+) and Fe(2+) uptake than did strains with other types, Ser70-type of NapA, which is found in standard strains. Other significantly different occurrences of polymorphisms between IDA and non-IDA groups were not observed in these genes. CONCLUSION The results suggest that H. pylori strains with Thr70-type NapA have enhanced Fe ion uptake ability and are associated with the pathogenesis of IDA.
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Affiliation(s)
- Shin-Ichi Yokota
- Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, 060-8556, Japan
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Uberti AF, Olivera-Severo D, Wassermann GE, Scopel-Guerra A, Moraes JA, Barcellos-de-Souza P, Barja-Fidalgo C, Carlini CR. Pro-inflammatory properties and neutrophil activation by Helicobacter pylori urease. Toxicon 2013; 69:240-9. [PMID: 23466444 DOI: 10.1016/j.toxicon.2013.02.009] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2012] [Revised: 12/25/2012] [Accepted: 02/05/2013] [Indexed: 01/25/2023]
Abstract
The gastric pathogen Helicobacter pylori produces large amounts of urease, whose enzyme activity enables the bacterium to survive in the stomach. We have previously shown that ureases display enzyme-independent effects in mammalian models, most through lipoxygenases-mediated pathways. Here, we evaluated potential pro-inflammatory properties of H. pylori urease (HPU). Mouse paw edema and activation of human neutrophils were tested using a purified, cell-free, recombinant HPU. rHPU induced paw edema with intense neutrophil infiltration. In vitro 100 nM rHPU was chemotactic to human neutrophils, inducing production of reactive oxygen species. rHPU-activated neutrophils showed increased lifespan, with inhibition of apoptosis accompanied by alterations of Bcl-XL and Bad contents. These effects of rHPU persisted in the absence of enzyme activity. rHPU-induced paw edema, neutrophil chemotaxis and apoptosis inhibition reverted in the presence of the lipoxygenase inhibitors esculetin or AA861. Neutrophils exposed to rHPU showed increased content of lipoxygenase(s) and no alteration of cyclooxygenase(s). Altogether, our data indicate that HPU, besides allowing the bacterial survival in the stomach, could play an important role in the pathogenesis of the gastrointestinal inflammatory disease caused by H. pylori.
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Affiliation(s)
- Augusto F Uberti
- Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
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Campylobacter jejuni Dps protein binds DNA in the presence of iron or hydrogen peroxide. J Bacteriol 2013; 195:1970-8. [PMID: 23435977 DOI: 10.1128/jb.00059-13] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Iron is an essential cofactor for many enzymes; however, this metal can lead to the formation of reactive oxygen species. Ferritin proteins bind and oxidize Fe(2+) to Fe(3+), storing this metal in a nonreactive form. In some organisms, a particular subfamily of ferritins, namely, Dps proteins, have the ability to bind DNA. Here we show that the Campylobacter jejuni Dps has DNA binding activity that is uniquely activated by Fe(2+) or H2O2 at below neutral pH. The Dps-DNA binding activity correlated with the ability of Dps to self-aggregate. The Dps-DNA interaction was inhibited by NaCl and Mg(2+), suggesting the formation of ionic interactions between Dps and DNA. Alkylation of cysteines affected DNA binding in the presence of H2O2 but not in the presence of Fe(2+). Replacement of all cysteines in C. jejuni Dps with serines did not affect DNA binding, excluding the participation of cysteine in H2O2 sensing. Dps was able to protect DNA in vitro from enzymatic cleavage and damage by hydroxyl radicals. A C. jejuni dps mutant was less resistant to H2O2 in vivo. The concerted activation of Dps-DNA binding in response to low pH, H2O2, and Fe(2+) may protect C. jejuni DNA during host colonization.
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Up-regulation of neutrophil activating protein in Helicobacter pylori under high-salt stress: structural and phylogenetic comparison with bacterial iron-binding ferritins. Biochimie 2013; 95:1136-45. [PMID: 23352965 DOI: 10.1016/j.biochi.2012.12.017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2012] [Accepted: 12/30/2012] [Indexed: 02/08/2023]
Abstract
It is generally accepted that most gastrointestinal diseases are probably caused by the bacterial pathogen Helicobacter pylori (H. pylori). In this study we have focused on the comparison of protein expression profiles of H. pylori grown under normal and high-salt conditions by a proteomics approach. We have identified about 190 proteins whose expression levels changed after growth at high salt concentration. Among these proteins, neutrophil-activating protein (NapA) was found to be consistently up-regulated under osmotic stress brought by high salts. We have investigated the effect of high salt on secondary and tertiary structures of NapA by circular dichroism spectroscopy followed by analytical ultracentrifugation to monitor the change of quaternary structure of recombinant NapA with increasing salt concentration. The loss of iron-binding activity of NapA coupled with noticeable energetic variation in protein association of NapA as revealed by isothermal titration calorimetry was found under high salt condition. The phylogenetic tree analysis based on sequence comparison of 16 protein sequences encompassing NapA proteins and ferritin of H. pylori and other prokaryotic organisms pointed to the fact that all H. pylori NapA proteins of human origin are more homologous to NapA of Helicobacter genus than to other bacterial NapA. Based on computer modeling, NapA proteins from H. pylori of human isolates are found more similar to ferritin from H. pylori than to NapA from other species of bacteria. Taken together, these results suggested that divergent evolution of NapA and ferritin possessing dissimilar and diverse sequences follows a path distinct from that of convergent evolution of NapA and ferritin with similar dual functionality of iron-binding and ferroxidase activities.
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Abstract
ABSTRACT Iron is an essential transition metal for mammalian cellular and tissue viability. It is critical to supplying oxygen through heme, the mitochondrial respiratory chain, and enzymes such as ribonucleotide reductase. Mammalian organisms have evolved with the means of regulating the metabolism of iron, because if left unregulated, the resulting excess amounts of iron may induce chronic toxicities affecting multiple organ systems. Several homeostatic mechanisms exist to control the amount of intestinal dietary iron uptake, cellular iron uptake, distribution, and export. Within these processes, numerous molecular participants have been identified because of advancements in basic cell biology and efforts in disease-based research of iron storage abnormalities. For example, dietary iron uptake across the intestinal duodenal mucosa is mediated by an intramembrane divalent metal transporter 1 (DMT1), and cellular iron efflux involves ferroportin, the only known iron exporter. In addition to duodenal enterocytes, ferroportin is present in other cell types, and exports iron into plasma. Ferroportin was recently discovered to be regulated by the expression of the circulating hormone hepcidin, a small peptide synthesized in hepatocytes. These recent studies on the role of hepcidin in the regulation of dietary, cellular, and extracellular iron have led to a better understanding of the pathways by which iron balance in humans is influenced, especially its involvement in human genetic diseases of iron overload. Other important molecular pathways include iron binding to transferrin in the bloodstream for cellular delivery through the plasma membrane transferrin receptor (TfR1). In the cytosol, iron regulatory proteins 1 and 2 (IRP1 and IRP2) play a prominent role in sensing the presence of iron in order to posttranscriptionally regulate the expression of TfR1 and ferritin, two important participants in iron metabolism. From a toxicological standpoint, posttranscriptional regulation of these genes aids in the sequestration, control, and hence prevention of cytotoxic effects from free-floating nontransferrin-bound iron. Given the importance of dietary iron in normal physiology, its potential to induce chronic toxicity, and recent discoveries in the regulation of human iron metabolism by hepcidin, this review will address the regulatory mechanisms of normal iron metabolism in mammals with emphasis on dietary exposure. It is the goal of this review that this information may provide in a concise format our current understanding of major pathways and mechanisms involved in mammalian iron metabolism, which is a basis for control of iron toxicity. Such a discussion is intended to facilitate the identification of deficiencies so that future metabolic or toxicological studies may be appropriately focused. A better knowledge of iron metabolism from normal to pathophysiological conditions will ultimately broaden the spectrum of the usefulness of this information in biomedical and toxicological sciences for improving and protecting human health.
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Affiliation(s)
- Luis G Valerio
- U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition,Office of Food Additive Safety, Division of Biotechnology and GRAS Notice Review, College Park, MD, 20470, USA
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Iankov ID, Penheiter AR, Carlson SK, Galanis E. Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein. J Immunol Methods 2012; 384:1-9. [PMID: 22750540 DOI: 10.1016/j.jim.2012.06.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2012] [Revised: 06/19/2012] [Accepted: 06/20/2012] [Indexed: 12/15/2022]
Abstract
Neutrophil-activating protein (NAP) is a major virulence factor expressed by Helicobacter pylori isolates associated with severe chronic gastroduodenal inflammation and peptic ulcers. NAP is one of the main protective antigens and a target for vaccine development against Helicobacter infection. In addition, NAP is a potent immune stimulator with potential application as a general vaccine adjuvant and in treatment of allergic diseases and cancer immunotherapy. NAP-specific immunoassays are needed for both H. pylori diagnostics and characterization of NAP-based vaccines and immunomodulatory preparations. We generated a panel of NAP-specific monoclonal antibodies (MAbs) by immunization of BALB/c mice with synthetic NAP peptides. The antibody reactivity against recombinant or native NAP antigen was characterized by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence. A sensitive capture ELISA was developed using MAbs 23C8 and 16F4 (directed against different NAP epitopes) for detection of native or measles virus (MV) vector-expressed recombinant NAP in a concentration range of 4 ng/ml to 2000 ng/ml. MAb 23C8 antigen-binding depends on Tyr101 in a variable amino acid sequence of the NAP molecule, indicating the existence of antigenic variants among H. pylori strains. MAb 16F4 reacted with NAP from different H. pylori strains and was a sensitive tool for detection of small amounts of isolated NAP antigen or whole bacteria by immunoblotting or immunofluorescence. In conclusion, MAb-based immunoassays are highly specific and sensitive for detection of native NAP antigen and recombinant NAP immunostimulatory transgenes expressed by replication competent virus vectors.
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Affiliation(s)
- Ianko D Iankov
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA
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Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form. Biochem Biophys Res Commun 2012; 422:745-50. [PMID: 22618234 DOI: 10.1016/j.bbrc.2012.05.073] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2012] [Accepted: 05/12/2012] [Indexed: 11/23/2022]
Abstract
Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn(2+)- or Cd(2+)-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.
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Tsuruta O, Yokoyama H, Fujii S. A new crystal lattice structure of Helicobacter pylori neutrophil-activating protein (HP-NAP). Acta Crystallogr Sect F Struct Biol Cryst Commun 2012; 68:134-40. [PMID: 22297984 PMCID: PMC3274388 DOI: 10.1107/s1744309111052675] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2011] [Accepted: 12/07/2011] [Indexed: 05/31/2023]
Abstract
A new crystal lattice structure of Helicobacter pylori neutrophil-activating protein (HP-NAP) has been determined in two forms: the native state (Apo) at 2.20 Å resolution and an iron-loaded form (Fe-load) at 2.50 Å resolution. The highly solvated packing of the dodecameric shell is suitable for crystallographic study of the metal ion-uptake pathway. Like other bacterioferritins, HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of channels. Iron loading causes a series of conformational changes of amino-acid residues (Trp26, Asp52 and Glu56) at the ferroxidase centre.
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Affiliation(s)
- Osamu Tsuruta
- School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan
| | - Hideshi Yokoyama
- School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan
| | - Satoshi Fujii
- School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan
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