The M949_1556 gene plays a role on the bacterial antigenicity and pathogenicity of Riemerella anatipestifer

doi:10.1016/j.vetmic.2015.03.003

Veterinary Microbiology

Volume 177, Issues 1–2, 15 May 2015, Pages 193-200

https://doi.org/10.1016/j.vetmic.2015.03.003 Get rights and content

Highlights

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M949_1556 gene inactivated RA mutant CH3ΔM949_1556 was identified.
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The mutant lost reactivity to the anti-LPS MAb in an indirect ELISA.
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The virulence of the mutant was attenuated more than 50 times.
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The mutant showed much higher susceptibility to complement-dependent killing.
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The mutant provided a broad cross-protection among RA serotypes 1, 2 and 10 strains.

Abstract

Riemerella anatipestifer is one of the most economically important pathogens of farm ducks worldwide. However, the molecular mechanisms regarding its antigenicity and pathogenicity are poorly understood. We previously constructed a library of random Tn4351 transposon mutants using R. anatipestifer strain CH3. In this study, M949_1556 gene inactivated mutant strain CH3ΔM949_1556 was identified by screening of the library using monoclonal antibody against R. anatipestifer serotype 1 lipopolysaccharide (LPS) (anti-LPS MAb) followed by sequence analysis. The mutant strain presented no reactivity to the anti-LPS MAb in an indirect ELISA. Animal studies showed that the median lethal dose (LD₅₀) of CH3ΔM949_1556 was >10¹⁰ colony forming units (CFU), which was attenuated more than 50 times, compared with that of wild-type strain CH3 (LD₅₀ = 2 × 10⁸ CFU). The bacterial loads in the blood of CH3ΔM949_1556 infected ducks were significantly decreased, compared with those of CH3-infected ducks. In addition, CH3ΔM949_1556 presented significant, higher susceptibility to complement-dependent killing than CH3 did in vitro. Furthermore, CH3ΔM949_1556 showed increased bacterial adhesion and invasion capacities to Vero cells. Immunization with CH3ΔM949_1556-inactived vaccine was effective in protecting the ducks from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2 and serotype 10 strain HXb2, suggesting that the mutant strain CH3ΔM949_1556 could provide a broad cross-protection among R. anatipestifer serotypes 1, 2 and 10 strains. Our results demonstrated that the M949_1556 gene plays a role on the bacterial antigenicity and pathogenicity of R. anatipestifer.

Introduction

Riemerella anatipestifer is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, and it belongs to the Flavobacteriaceae family in rRNA superfamily V (Segers et al., 1993). R. anatipestifer infection primarily causes disease in domestic ducks, and accounts for major economic losses to the industry (Sandhu and Rimler, 1997). A total of 21 serotypes of R. anatipestifer have been identified with no significant cross-protection reported (Pathanasophon et al., 1995, Pathanasophon et al., 2002). Serotypes 1, 2, and 10 are responsible for most of the major R. anatipestifer outbreaks in China (Hu et al., 2010). The chaperonin GroEL was the first reported protein providing a cross-protection among R. anatipestifer serotypes 1, 2 and 10 (Han et al., 2012), although the protection rate is not as high as that of inactivated bacterin (Liu et al., 2013). The mixture of recombinant Riean_1750 and Riean_1752, two proteins identified from R. anatipestifer cultured in iron restricted medium, provided 100% protection against challenge with R. anatipestifer, but no cross-protection was shown (Yang et al., 2013). On the other hand, several genes have been identified as virulence factors of R. anatipestifer strains, including VapD, CAMP cohemolysin and outer membrane protein A (OmpA) (Chang et al., 1998).

Our previous study has constructed a random Tn4351 transposon insertion library using R. anatipestifer strain CH3 and identified 33 biofilm-forming associated genes from the library in the genome wide (Hu et al., 2010). In this study, we screened the library using monoclonal antibody against R. anatipestifer serotype 1 lipopolysaccharide (LPS) (anti-LPS MAb) and one mutant which lost the reactivity with anti-LPS MAb was obtained. Sequence analysis showed that the M949_1556 gene was inactivated in the mutant strain, which was named as CH3ΔM949_1556. Furthermore, the bacterial antigenicity and pathogenicity of CH3ΔM949_1556 were characterized.

Section snippets

Animals

One-day old Cherry Valley ducks were obtained from ZhuangHang Duck Farm (Shanghai, China) and kept under controlled temperature (28–30 °C). The ducks were housed in cages and a 12-h light/dark cycle with free access to food and water was provided during the study. Animal experiments were carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines set by Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS). This animal

Identification of the mutant strain CH3ΔM949_1556

A stable hybridoma cell clone 8A9 producing monoclonal antibody against R. anatipestifer serotype 1 strain (anti-LPS MAb) was established. MAb titer in the ascite was assessed over 1:102,400 by ELISA. The mutant strain CH3ΔM949_1556 was obtained by screening the transposon library for the defective reactivity with anti-LPS MAb using an indirect ELISA. Single Tn4351 insertion in the CH3ΔM949_1556 chromosome was confirmed by Southern blot (Fig. 1A). Sequencing of the cloned DNA fragment revealed

Discussion

In this study, CH3ΔM949_1556 mutant was identified by screening the library of random Tn4351 transposon mutants using anti-LPS MAb. The mutant does not bind to anti-LPS MAb in an indirect ELISA. Further studies indicated that the mutant strain showed no significant difference in growth rate but obvious deficient in ladder-like binding pattern of CH3 LPS to anti-CH3 rabbit serum and significant attenuated virulence in duck model, compared to its wild-type strain CH3, indicating deletion of

Acknowledgement

This work was supported by the National Natural Science Foundation of China (31272591).

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Precise prevention and control of RA may be achieved through the development of live bacterial vaccines. In recent years, a large number of virulence factors of RA have been confirmed (Chang, et al., 1998; Crasta, et al., 2002; Hu, et al., 2018; Wang, et al., 2017; Zhao, et al., 2016; Zou, et al., 2015), and as such, an attenuated strain has the potential to be a vaccine. Furthermore, the mutation of global regulators may be preferable over single virulence factors for targeting live attenuated vaccines because global regulators control a cascade of genes that are related to bacterial virulence, which may improve safety and immunogenicity (Asensio, et al., 2008; Miller, et al., 1993; Zhao, et al., 2016).
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Characterization of the Riemerella anatipestifer M949_RS00050 gene
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Interestingly, the result showed that LPS purified from the mutant strain CH3ΔM949_RS00050 had the same ladder-like pattern compared to the wild-type strain CH3. Furthermore, in our previous work, we demonstrate that several genes, including M949_RS07405, M949_RS01915, M949_RS07580 and M949_RS07630 were involved in regulating the genes mainly responsible for bacterial polysaccharide biosynthesis in R. anatipestifer CH3 (Dou et al., 2017; Zou et al., 2015a, b). Consequently, we investigated the variability of these gene expressions between the wild-type strain CH3 and the mutant strain CH3ΔM949_RS00050.
Based on its causing ever-increasing heavy economic losses, Riemerella anatipestifer has been viewed as an important bacterial pathogen in the duck industry worldwide. However, the molecular mechanisms regarding its pathogenicity are poorly understood. In our previous study, we have built a random mutagenesis library of Riemerella anatipestifer CH3 using transposon Tn4351. In this study, we screened the library by determining bacterial median lethal dose in ducklings. A mutant strain showed about 376-fold attenuated virulence in comparison with the wild-type strain CH3 was obtained. Subsequently, the Tn4351 inserted gene was identified as M949_RS00050, which encodes a putative protein containing an outer membrane protein beta-barrel domain by genome walking and sequence analyses. Southern blot analysis indicated a single Tn4351 insertion in the CH3 chromosomal DNA. Inactivation of M949_RS00050 gene did not affect bacterial metabolic activity and the silver stained lipopolysaccharide pattern. However, the bacterial sensitivity to normal duck sera killing and bacterial hydrophobicity were dramatically enhanced in the M949_RS00050 gene inactivated mutant strain, compared to its wild-type strain CH3. Moreover, bacterial adherence and invasion abilities, bacterial capsular polysaccharide quantity, biofilm formation capacity and the bacterial virulence of the mutant strain were obviously decreased, compared to the wild-type strain CH3. Thus, our finding demonstrates that the M949_RS00050 gene functions on multiple bacterial biological properties and virulence in Riemerella anatipestifer.
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Riemerellosis, a Riemerella anatipestifer infection, can cause meningitis, pericarditis, parahepatitis, and airsacculitis in ducks, leading to serious economic losses in the duck meat industry. However, the molecular mechanism of the pathogenesis and virulence factors of this infection are poorly understood. In the present study, we created a mutant strain RA-YMΔCas9 using trans-conjugation. Bacterial virulence tests indicated that the median lethal dose (LD₅₀) of RA-YMΔCas9 was 5.01 × 10⁷ CFU, significantly lower than that of the RA-YM strain, which was 1.58 × 10⁵ CFU. The distribution and blood bacterial load from the infection groups showed no significant difference in the brain between the RA-YMΔCas9 mutant and the wild-type RA-YM strains, however, the number of mutant strains were significantly reduced in the liver, heart, and blood. Animal immunization experiments demonstrated that the intranasal administration of RA-YMΔCas9 in ducklings provided 80% protection after challenge with the wild-type strain, showing potential use as a live mucosal vaccine. RNAseq analysis indicated that Cas9 protein played a regulatory role in gene expression. This study is the first to report on the involvement of Cas9 in the regulation and pathogenesis of R. anatipestifer, and provides a theoretical basis for the development of relevant genetic engineering vaccines.
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Riemerella anatipestifer (R. anatipestifer) causes severe perihepatitis, pericarditis, airsacculitis and meningitis in the duck, leading to great economic losses worldwide. Given the increased prevalence of drug-resistance strains, vaccination is the best strategy to prevent R. anatipestifer infection in ducklings. In this study, we identified a gene in R. anatipestifer (B739-2187) that can restore the resistance of the Salmonella phoP mutant to polymyxin B using genetic complementation. Furthermore, the deletion of B739-2187 in R. anatipestifer resulted in a mutant exhibiting increased sensitivity to polymyxin B. The R. anatipestifer B739-2187 mutant did not exhibit phenotypic defects, as indicated by its growth curve, lipopolysaccharide and outer membrane protein profiles, and attachment and invasion of duck embryo fibroblast cells. The duck animal experiments demonstrated that the deletion of B739-2187 significantly decreased the virulence of R. anatipestifer, and the B739-2187 mutant provided 100% protection against challenge with wild-type R. anatipestifer, exhibiting the characteristics of an ideal live vaccine.
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The M949_1556 gene plays a role on the bacterial antigenicity and pathogenicity of Riemerella anatipestifer

Highlights

Abstract

Introduction

Section snippets

Animals

Identification of the mutant strain CH3ΔM949_1556

Discussion

Acknowledgement

J. Mol. Biol.

Microb. Pathog.

Vet. Microbiol.

Vet. Microbiol.

Trends Microbiol.

FEMS Microbiol. Lett.

Methods Enzymol.

Methods Enzymol.

FEMS Microbiol. Lett.

Anal. Biochem.

Carbohydr. Res.

Carbohydr. Res.

Molecular characterization of a plasmid isolated from Riemerella anatipestifer

Avian Pathol.

Surface proteins that promote adherence of Staphylococcus aureus to human desquamated nasal epithelial cells

BMC Microbiol.

Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels

J. Clin. Microbiol.

Synthesis and characterization of lipooligosaccharide-based conjugates as vaccine candidates for Moraxella (Branhamella) catarrhalis

Infect. Immun.

Mutation of the maturase lipoprotein attenuates the virulence of Streptococcus equi to a greater extent than does loss of general lipoprotein lipidation

Infect. Immun.

Identification and immunological characteristics of chaperonin GroEL in Riemerella anatipestifer

Appl. Microbiol. Biotechnol.

Maturation of Streptococcus pneumoniae lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence

Mol. Microbiol.