Riemerella anatipestifer Type IX Secretion System Is Required for Virulence and Gelatinase Secretion.

Yunqing Guo,Di Hu,Zili Li,Dingren Bi,Yuncai Xiao,Xiliang Wang,Tao Wang,Shaowen Li,Mei Liu,Zutao Zhou,Jie Guo

doi:10.3389/fmicb.2017.02553

Abstract

Riemerella anatipestifer (RA), a major causative agent of septicemia anserum exsudativa in domesticated ducklings, has a protein secretion system known as the type IX secretion system (T9SS). It is unknown whether the T9SS contributes to the virulence of RA through secretion of factors associated with pathogenesis. To answer this question, we constructed an RA mutant deficient in sprT, which encodes a core protein of the T9SS. Deletion of sprT yielded cells that failed to digest gelatin, an effect that was rescued via complementation by a plasmid encoding wild-type sprT. Complement-mediated killing was significantly increased in the deletion mutant, suggesting that proteins secreted by the T9SS are necessary for complement evasion in RA. Liquid chromatography-tandem mass spectrometry analysis revealed that RAYM_01812 and RAYM_04099 proteins containing a subtilisin-like serine protease domain and exhibiting extracellular gelatinase activity were secreted by the T9SS. Animal experiments demonstrated that the virulence of mutant strain ΔsprT strain was attenuated by 42,000-fold relative to wild-type RA-YM. Immunization with the ΔsprT protected ducks from challenge with RA-YM, suggesting that the former can be used as a live attenuated vaccine. These results indicate that the T9SS is functional in RA and contributes to its virulence by exporting key proteins. In addition, subtilisin-like serine proteases which are important virulence factors that interact with complement proteins may enable RA to evade immune surveillance in the avian innate immune system.

Highlights

Riemerella anatipestifer (RA) is a Gram-negative, non-spore-forming, rod-shaped bacterium belonging to the genus Riemerella and family Flavobacteriaceae of the phylum Bacteroidetes (Segers et al, 1993)
The deletion of the sprT gene after allelic exchange was confirmed by PCR amplification of the smaller fragment (1752-bp) in RAYM as compared to the larger Leftarm-Spc-Rightarm fragment (2538-bp) in RA-YM sprT (Figure 1A)
There were no differences in biological characteristics between wild-type RA-YM, mutant sprT, and complemented C sprT strains except in the early logarithmic growth phase, with sprT exhibiting more rapid growth as compared to RA-YM, which had a similar growth rate as C sprT (Figure 1C)

Summary

Introduction

Riemerella anatipestifer (RA) is a Gram-negative, non-spore-forming, rod-shaped bacterium belonging to the genus Riemerella and family Flavobacteriaceae of the phylum Bacteroidetes (Segers et al, 1993). It usually causes septicemia and serum exudate in ducks, geese, turkeys, and various other domestic and wild birds, resulting in serious economic losses worldwide (Sandhu, 2008). 21 serotypes of RA have been identified and no significant cross-protection has been reported. Live avirulent strains induce longerlasting protection and are more convenient; there are few reports on attenuated live vaccines (Sandhu, 1991; Higgins et al, 2000; Liu et al, 2013). Chemotherapy is the most widely used approach for treating RA infection, but the increasing incidence of drug resistance (Yang et al, 2012; Li et al, 2016) compels the search for new strategies for controlling this disease

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