Full length articleTcpA, a novel Yersinia ruckeri TIR-containing virulent protein mediates immune evasion by targeting MyD88 adaptors
Introduction
Yersinia ruckeri is the etiological agent of enteric redmouth disease (ERM) in rainbow trout and has been reported worldwide from many different species [1]. Over the past few decades, ERM disease has induced substantial economic losses in fish due to the associated mortality and veterinary costs [2]. Despite the general administration of an effective vaccine in fish, outbreaks continue to occur, which are mainly attributed to specific strains [3,4]. However, very little is known about the virulence mechanisms of Y. ruckeri even ERM is serious in fish. Many potential virulence factors have been reported, such as extracellular toxins [5,6], a high affinity iron uptake system named ruckerbactin [5], flagellar motility genes [7], two-component system [8], etc. To further systematically understand Y. ruckeri and its virulence, we had reported the complete genome of Y.ruckeri SC09, a highly virulent strain isolated from diseased fish with severe septicaemia in China [9]. In the genome of SC09, we found a great deal of horizontal gene transfer (HGT) events in Y. ruckeri, and these HGT carried many genes related to immune evasion, which prompted us to further study the correlation between immune evasion and the virulence of Y. ruckeri.
Among the gene horizontal transfer elements in the genome of SC09, the most representative one is a class called Integrative and conjugative elements (ICEs). ICEs are a class of newly recognized mobile DNA elements in prokaryotes [10]. Many ICEs harbor a tyrosine recombinase gene and are flanked by direct repeats corresponding to the 3′ end of a conserved gene (e.g., a tRNA gene), which suggest integration happen via site-specific recombination in the 3′ end of this gene [11]. Furthermore, ICEs typically have a core of conserved modular structures that mediate their integration, excision, conjugation, and regulation, interspersed with “accessory regions” that are variably present across members of a species [12]. A mobile element named ICE(r2) in SC09 (NJ56_RS12425-NJ56_RS12600) was integrated between an intact or partial tRNA-Asn copy. Interestingly, a class of bacterial proteins which were homology to the Toll/IL-1 receptor (TIR) domain had been identified, it was encoded by the ICE(r2) and acted as “accessory genes”. One of these proteins had been named as TcpA, encoded by NJ56_RS12465, which was specific in SC09 and absent in other Y. ruckeri strains, such as RS41 (NCBI RefSeq: NZ_CQBN00000000.1), OMBL4 (NCBI RefSeq: NZ_CPUZ00000000.1) and CSF007-82 (NCBI RefSeq: NZ_CCYO00000000.1) strains. The TIR domain is essential for the interaction between the Toll-like receptor (TLR) and adaptor, and is onset of a signalling cascade resulting in nuclear translocation of the transcription factor, NF-κB, followed by the production of inflammatory cytokines and type I interferons [13]. TIR domain interactions play a key role in activating conserved cellular signal transduction pathways in response to pathogen signals [14]. Accordingly, SC09 may target TLR signaling to facilitate its escape from the host innate immune response and enhance virulence by TcpA. In fact, the intracellular survival of Y. ruckeri had been detected in previous studies [15].
In the present study, a novel Y. ruckeri TIR-containing virulent protein named as TcpA were identified through in vivo and in vitro experiments. The results showed that TcpA efficiently inhibited TLR signaling and contributed to toxicity in vitro and in vivo. Besides, we proposed TIR-containing protein TcpA represent a new class of virulence factors in Y. ruckeri and modulated host inflammatory responses during infection.
Section snippets
Bacterial strains
Wild-type Y. ruckeri SC09 was isolated from diseased fish in a commercial farm in Jianyang, Sichuan province of China, and was routinely cultured in Luria-Bertani (LB) medium at 28 °C. The virulent Y. ruckeri SC10 strain was isolated from the aquatic environment.
Construction of Y. ruckeri ΔtcpA mutant and complemented strains
Gene knockout was previously described by Luo et al. [16]. The tcpA gene sequence (gene accession number: NJ56_RS12465) of the Y. ruckeri SC09 strain (genome accession number: NZ_CP025800) is available in GenBank. The left and right
Genomic location of tcpA in Yersinia ruckeri
Integrative and conjugative elements (ICEs) are widespread mobile DNA in prokaryotes [19]. Numerous ICEs harbor a tyrosine recombinase gene and are flanked by direct repeats corresponding to a conserved gene [20]. ICEs typically have some modular structures that mediate their integration, excision, conjugation, and regulation, interspersed with “accessory regions” that are variably present across different species [21]. The mobile element ICE(r2) in SC09 (NJ56_RS12425-NJ56_RS12600) was
Discussion
In the current study, we found that fish pathogen Yersinia ruckeri SC09 produced TLR homologs to inhibit TLR signaling and promoted immune evasion. Previously we used whole-genome sequencing to obtain the genome-wide sequence of Y. ruckeri SC09 [9], and by comparative genomics we found specific ICE elements in the bacterial genome. ICE elements are currently thought to mediate the horizontal transfer of genes in bacteria [23]. This element can provide the host with some specific “cargo” genes
Acknowledgments
This work was supported by Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0848) and the Science and Technology Support Program by Science and Technology Department of Sichuan Province (No. 2014NZ0003).
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These authors contributed equally to this work.