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      Helicobacter pylori Exploits a Unique Repertoire of Type IV Secretion System Components for Pilus Assembly at the Bacteria-Host Cell Interface

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          Abstract

          Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island ( cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. Δ cagI and Δ cagL mutant strains fail to form pili, whereas a Δ cagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.

          Author Summary

          Helicobacter pylori persistently colonizes the stomach in approximately half of the human population. People who are infected with H. pylori strains harboring the cag pathogenicity island (PAI) have an increased risk of developing gastric cancer. The cag PAI encodes a type IV secretion system (T4SS) that is utilized by the bacteria to inject the bacterial oncoprotein CagA into gastric epithelial cells. Related T4SSs found in several other bacteria have been studied in detail, but thus far there has been very little study of the H. pylori cag T4SS. Here, we utilized a mass spectrometry-based approach to reveal co-purification of three constituents of the H. pylori T4SS (CagH, CagI, and CagL) that lack homology to components of T4SSs in other bacterial species. These proteins are essential for CagA translocation into host cells, and scanning electron microscope studies reveal that the proteins are involved in the formation of pili at the bacterial-host cell interface. A conserved C-terminal motif present in CagH, CagI, and CagL is essential for functionality of the T4SS. This study highlights the important role played by unique constituents of the H. pylori cag T4SS, and illustrates the marked variation that exists among bacterial T4SSs.

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          Most cited references 86

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          WebLogo generates sequence logos, graphical representations of the patterns within a multiple sequence alignment. Sequence logos provide a richer and more precise description of sequence similarity than consensus sequences and can rapidly reveal significant features of the alignment otherwise difficult to perceive. Each logo consists of stacks of letters, one stack for each position in the sequence. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of symbols within the stack reflects the relative frequency of the corresponding amino or nucleic acid at that position. WebLogo has been enhanced recently with additional features and options, to provide a convenient and highly configurable sequence logo generator. A command line interface and the complete, open WebLogo source code are available for local installation and customization. Copyright 2004 Cold Spring Harbor Laboratory Press
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              A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.

              A rapid method based on a defined methanol-chloroform-water mixture for the quantitative precipitation of soluble as well as hydrophobic proteins from dilute solutions (e.g., column chromatography effluents) has been developed. The effectiveness of this method is not affected by the presence of detergents, lipids, salt, buffers, and beta-mercaptoethanol.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                September 2011
                September 2011
                1 September 2011
                : 7
                : 9
                Affiliations
                [1 ]Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [2 ]Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [3 ]Proteomics Laboratory, Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [4 ]Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, and Department of Oncological Genetics, Cancer Center Institute, Warsaw, Poland
                [5 ]Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, United States of America
                Fred Hutchinson Cancer Research Center, United States of America
                Author notes

                Conceived and designed the experiments: CLS WHM TLC. Performed the experiments: CLS JAG JTL EMJ MSM. Analyzed the data: CLS WHM JAG JTL EMJ SH MSM TLC. Contributed reagents/materials/analysis tools: WHM SH EEH. Wrote the paper: CLS TLC.

                PPATHOGENS-D-11-00533
                10.1371/journal.ppat.1002237
                3164655
                21909278
                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
                Counts
                Pages: 20
                Categories
                Research Article
                Biology
                Microbiology
                Host-Pathogen Interaction
                Medicine
                Gastroenterology and Hepatology
                Gastrointestinal Cancers
                Gastrointestinal Infections
                Infectious Diseases
                Bacterial Diseases
                Helicobacter Pylori

                Infectious disease & Microbiology

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