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

Yafeng Dou,Chan Ding,Xiaolan Wang,Guijing Yu,Mingxing Tian,Shengqing Yu,Tao Li,Shaohui Wang,Jingjing Qi

doi:10.1186/s13567-017-0409-6

Yafeng Dou, Chan Ding + Show 7 more

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https://doi.org/10.1186/s13567-017-0409-6

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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 Tn4351 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 Tn4351 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.

Highlights

Riemerella anatipestifer is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, which causes epizootic infectious disease in poultry, especially in ducks [1,2,3]
Animal experiments were carried out in agreement with the Institutional Animal Care and Use Committee (IACUC) guidelines set by Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS)
Identification of the mutant strain RA1067 The mutant RA1067 that lacked reactivity with anti-LPS MAb was obtained by screening the transposon library using an indirect ELISA, and identified by Polymerase chain reaction (PCR) amplification using primers 16S rRNA F/16S rRNA R, Erm-F/ Erm-R and RA1067-F/RA1067-R

Summary

Introduction

Riemerella anatipestifer is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, which causes epizootic infectious disease in poultry, especially in ducks [1,2,3]. 21 serotypes of R. anatipestifer have been identified, there is poor cross-protection among them [5,6,7]. Several virulence-associated genes, including VapD, CAMP cohemolysis, outer membrane protein and TonB-dependent receptor tbdr have been identified in R. anatipestifer strains [3, 9, 10]. Lipopolysaccharide (LPS), the main component of the outer membrane of Gram-negative bacteria, is a potent stimulant of innate immune response [12]. The LPS is composed of three distinct components: lipid A, O-antigen and core oligosaccharide. The O-antigen consists of oligosaccharide repeating units (O units), which usually contain two to eight residues from a broad range of sugars, Dou et al Vet Res (2017) 48:6 both common and rare, and their derivatives. The O-antigen repeats are the most variable constituent of the LPS molecule, imparting the antigenic specificity

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