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      Disruption of the M949_RS01915 gene changed the bacterial lipopolysaccharide pattern, pathogenicity and gene expression of Riemerella anatipestifer

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          Abstract

          Riemerella anatipestifer is an important pathogen that causes septicemia anserum exsudativa in ducks. Lipopolysaccharide (LPS) is considered to be a major virulence factor of R. anatipestifer. To identify genes involved in LPS biosynthesis, we screened a library of random Tn 4351 transposon mutants using a monoclonal antibody against R. anatipestifer serotype 1 LPS (anti-LPS MAb). A mutant strain RA1067 which lost the reactivity in an indirect ELISA was obtained. Southern blot and sequencing analyses indicated a single Tn 4351 was inserted at 116 bp in the M949_RS01915 gene in the RA1067 chromosomal DNA. Silver staining and Western blot analyses indicated that the RA1067 LPS was defected compared to the wild-type strain CH3 LPS. The RA1067 displayed a significant decreased growth rate at the late stage of growth in TSB in comparison with CH3. In addition, RA1067 showed higher susceptibility to complement-dependent killing, more than 360-fold attenuated virulence based on the median lethal dose determination, increased bacterial adhesion and invasion capacities to Vero cells and significantly decreased blood bacterial loads in RA1067 infected ducks, when compared to the CH3. An animal experiment indicated that inactivated RA1067 cells was effective in cross-protecting of the ducks from challenging with R. anatipestifer strains WJ4 (serotype 1), Yb2 (serotype 2) and HXb2 (serotype 10), further confirming the alteration of the RA1067 antigenicity. Moreover, RNA-Seq analysis and real-time PCR verified two up-regulated and three down-regulated genes in RA1067. Our findings demonstrate that the M949_RS01915 gene is associated to bacterial antigenicity, pathogenicity and gene regulation of R. anatipestifer.

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          Most cited references38

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          A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels.

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            Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly.

            The O-antigen is an important component of the outer membrane of Gram-negative bacteria. It is a repeat unit polysaccharide and consists of a number of repeats of an oligosaccharide, the O-unit, which generally has between two and six sugar residues. O-Antigens are extremely variable, the variation lying in the nature, order and linkage of the different sugars within the polysaccharide. The genes involved in O-antigen biosynthesis are generally found on the chromosome as an O-antigen gene cluster, and the structural variation of O-antigens is mirrored by genetic variation seen in these clusters. The genes within the cluster fall into three major groups. The first group is involved in nucleotide sugar biosynthesis. These genes are often found together in the cluster and have a high level of identity. The genes coding for a significant number of nucleotide sugar biosynthesis pathways have been identified and these pathways seem to be conserved in different O-antigen clusters and across a wide range of species. The second group, the glycosyl transferases, is involved in sugar transfer. They are often dispersed throughout the cluster and have low levels of similarity. The third group is the O-antigen processing genes. This review is a summary of the current knowledge on these three groups of genes that comprise the O-antigen gene clusters, focusing on the most extensively studied E. coli and S. enterica gene clusters.
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              Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz.

              Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16-35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random-length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.
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                Author and article information

                Contributors
                douyafeng@qq.com
                xlwang99@shvri.ac.cn
                403319195@qq.com
                shwang@shvri.ac.cn
                mxtian@shvri.ac.cn
                qijingjing@shvri.ac.cn
                litao@shvri.ac.cn
                shoveldeen@shvri.ac.cn
                yus@shvri.ac.cn
                Journal
                Vet Res
                Vet. Res
                Veterinary Research
                BioMed Central (London )
                0928-4249
                1297-9716
                6 February 2017
                6 February 2017
                2018
                : 48
                : 6
                Affiliations
                ISNI 0000 0001 0526 1937, GRID grid.410727.7, Shanghai Veterinary Research Institute, , Chinese Academy of Agricultural Sciences, ; Shanghai, 200241 China
                Article
                409
                10.1186/s13567-017-0409-6
                5294843
                28166822
                adb2d17c-dc3c-4f87-9f13-40f654a1f790
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 29 September 2016
                : 5 December 2016
                Funding
                Funded by: the National Natural Science Foundation of China
                Award ID: 31272591
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2017

                Veterinary medicine
                Veterinary medicine

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