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      Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni

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          Abstract

          The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.

          Author Summary

          Bacterial cell shape is dictated by the composition of the cell envelope component peptidoglycan. Some important pathogens have a characteristic helical corkscrew morphology that may help them burrow into mucus overlaying cells to initiate colonization and pathogenicity. One example is Campylobacter jejuni, the leading cause of bacterial-induced diarrheal disease in the developed world. Direct evidence supporting the hypothesis that C. jejuni shape is related to its pathogenicity traits has not previously been provided. We identified a gene encoding a peptidase modifying peptidoglycan that is essential for maintaining the C. jejuni corkscrew shape. We can now connect a C. jejuni gene with morphology and peptidoglycan biosynthesis. Loss of this gene was also found to affect pathogenic attributes such as chicken colonization, biofilms, motility, and activation of host inflammatory mediators. In addition, this is the first study to thoroughly characterize C. jejuni peptidoglycan structure and to identify a gene involved in peptidoglycan maintenance. Our findings highlight an emerging theme in bacterial pathogenesis research: the connection between bacterial cell biology and pathogenesis. Finally, our characterization of C. jejuni cell shape and peptidoglycan provides a starting point for further work in this area in C. jejuni and other bacteria with curved and helical morphologies.

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          Most cited references68

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          Peptidoglycan structure and architecture.

          The peptidoglycan (murein) sacculus is a unique and essential structural element in the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the cytoplasmic membrane. There is a high diversity in the composition and sequence of the peptides in the peptidoglycan from different species. Furthermore, in several species examined, the fine structure of the peptidoglycan significantly varies with the growth conditions. Limited number of biophysical data on the thickness, elasticity and porosity of peptidoglycan are available. The different models for the architecture of peptidoglycan are discussed with respect to structural and physical parameters.
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            The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences.

            Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium-properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain-Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.
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              Experimental Campylobacter jejuni infection in humans.

              Two strains of Campylobacter jejuni ingested by 111 adult volunteers, in doses ranging from 8 x 10(2) to 2 x 10(9) organisms, caused diarrheal illnesses. Rates of infection increased with dose, but development of illness did not show a clear dose relation. Resulting illnesses with strain A3249 ranged from a few loose stools to dysentery, with an average of five diarrheal stools and a volume of 509 mL. Infection with strain 81-176 was more likely to cause illness, and these illnesses were more severe, with an average of 15 stools and 1484 mL of total stool volume. All patients had fecal leukocytes. The dysenteric nature of the illness indicates that the pathogenesis of C. jejuni infection includes tissue inflammation. Ill volunteers developed a serum antibody response to the C. jejuni group antigen and were protected from subsequent illness but not infection with the same strain.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2012
                March 2012
                22 March 2012
                23 March 2012
                : 8
                : 3
                : e1002602
                Affiliations
                [1 ]Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
                [2 ]The Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom
                [3 ]Wine Research Centre, Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada
                [4 ]Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
                [5 ]Department of Microbiology and Immunology & Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
                University of Illinois, United States of America
                Author notes

                Conceived and designed the experiments: EF JB CA JL JE LDG VJD SEG WV ECG. Performed the experiments: EF JB CA JL JE LDG. Analyzed the data: EF JB CA JL JE LDG VJD SEG WV ECG. Contributed reagents/materials/analysis tools: VJD SEG WV ECG. Wrote the paper: EF JB CA JL JE LDG VJD SEG WV ECG.

                Article
                PPATHOGENS-D-11-01940
                10.1371/journal.ppat.1002602
                3310789
                22457624
                176dca62-920f-4d28-b9b5-ce9fceb9292d
                Frirdich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 27 August 2011
                : 8 February 2012
                Page count
                Pages: 14
                Categories
                Research Article
                Biology
                Biochemistry
                Glycobiology
                Genetics
                Microbiology
                Bacterial Pathogens
                Bacteriology
                Medicine
                Infectious Diseases
                Bacterial Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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