Yersinia ruckeri Infection and Enteric Redmouth Disease among Endangered Chinese Sturgeons, China, 2022.

During October 2022, enteric redmouth disease (ERM) affected Chinese sturgeons at a farm in Hubei, China, causing mass mortality. Affected fish exhibited characteristic red mouth and intestinal inflammation. Investigation led to isolation of a prominent bacterial strain, zhx1, from the internal organs and intestines of affected fish. Artificial infection experiments confirmed the role of zhx1 as the pathogen responsible for the deaths. The primary pathologic manifestations consisted of degeneration, necrosis, and inflammatory reactions, resulting in multiple organ dysfunction and death. Whole-genome sequencing of the bacteria identified zhx1 as Yersinia ruckeri, which possesses 135 drug-resistance genes and 443 virulence factor-related genes. Drug-susceptibility testing of zhx1 demonstrated high sensitivity to chloramphenicol and florfenicol but varying degrees of resistance to 18 other antimicrobial drugs. Identifying the pathogenic bacteria associated with ERM in Chinese sturgeons establishes a theoretical foundation for the effective prevention and control of this disease.

Mechanism of sturgeon intestinal inflammation induced by Yersinia ruckeri and the effect of florfenicol intervention.

The mechanism by which Y. ruckeri infection induces enteritis in Chinese sturgeon remains unclear, and the efficacy of drug prevention and control measures is not only poor but also plagued with numerous issues. We conducted transcriptomic and 16 S rRNA sequencing analyses to examine the differences in the intestinal tract of hybrid sturgeon before and after Y. ruckeri infection and florfenicol intervention. Our findings revealed that Y. ruckeri induced the expression of multiple inflammatory factors, including il1ß, il6, and various chemokines, as well as casp3, casp8, and multiple tumor necrosis factor family members, resulting in pathological injury to the body. Additionally, at the phylum level, the relative abundance of Firmicutes and Bacteroidota increased, while the abundance of Plesiomonas and Cetobacterium decreased at the genus level, altering the composition of the intestinal flora. Following florfenicol intervention, the expression of multiple apoptosis and inflammation-related genes was down-regulated, promoting tissue repair. However, the flora became further dysregulated, increasing the risk of infection. In conclusion, our analysis of the transcriptome and intestinal microbial composition demonstrated that Y. ruckeri induces intestinal pathological damage by triggering apoptosis and altering the composition of the intestinal microbiota. Florfenicol intervention can repair pathological damage, but it also exacerbates flora imbalance, leading to a higher risk of infection. These findings help elucidate the molecular mechanism of Y. ruckeri-induced enteritis in sturgeon and evaluate the therapeutic effect of drugs on intestinal inflammation in sturgeon.

Pan-genome survey of the fish pathogen Yersinia ruckeri links accessory- and amplified genes to virulence.

While both virulent and putatively avirulent Yersinia ruckeri strains exist in aquaculture environments, the relationship between the distribution of virulence-associated factors and de facto pathogenicity in fish remains poorly understood. Pan-genome analysis of 18 complete genomes, representing established virulent and putatively avirulent lineages of Y. ruckeri, revealed the presence of a number of accessory genetic determinants. Further investigation of 68 draft genome assemblies revealed that the distribution of certain putative virulence factors correlated well with virulence and host-specificity. The inverse-autotransporter invasin locus yrIlm was, however, the only gene present in all virulent strains, while absent in lineages regarded as avirulent. Strains known to be associated with significant mortalities in salmonid aquaculture display a combination of serotype O1-LPS and yrIlm, with the well-documented highly virulent lineages, represented by MLVA clonal complexes 1 and 2, displaying duplication of the yrIlm locus. Duplication of the yrIlm locus was further found to have evolved over time in clonal complex 1, where some modern, highly virulent isolates display up to three copies.

Screening and activity of potential gastrointestinal probiotic lactic acid bacteria against Yersinia ruckeri O1b.

Yersiniosis of cultured Atlantic salmon is a recurrent fish health management challenge in many continents. The causative organism, Yersinia ruckeri, can reside latently in the gut and lead to acute infection and disease during hatchery and sea-transfer stages. One potential prevention approach is the administration of probiotic bacteria to suppress gut colonization of Y. ruckeri. Our study aimed to isolate and identify anti-Yersinia activity among lactic acid bacteria (LAB) isolated from the gastrointestinal tract (GIT) of aquatic animals. Of the 186 aquatic GIT isolates examined, three strains showed diffusible antimicrobial activity towards Y. ruckeri O1b. Analysis of 16 s rRNA gene sequences indicated the three bacterial strains were Enterococci, related to Enterococcus sp. (99%), Enterococcus thailandicus (99%), and Enterococcus durans (99%). Anti-Yersinia activity was maintained at neutral pH (~6.5-7.0), and in-vitro environmental tolerance assays showed the three strains could withstand simulated salmonids gastrointestinal tract conditions of: low pH (3.4) and 3% bile salt content. All three Enterococci strains showed higher adhesion to the intestinal mucus of Atlantic salmon than Y. ruckeri O1b (E. durans 24%, E. enterococcus sp. 25% and E. thailandicus 98%, compared to Y. ruckeri O1b 5%). However, only Enterococcus sp. and E. thailandicus were able to grow in the salmon intestinal mucus broth while E. durans showed no growth. Anti-Yersinia activity was completely inactivated by proteinase-K treatment, suggesting that the active compound/s are proteinaceous and may be bacteriocin-like inhibitory substances (BLIS). Our data indicate that Enterococcus sp. MA176 and E. thailandicus MA122 are potential probionts for the prevention of yersiniosis in salmonids. Further in-vivo studies are required to determine whether these bacteria reduce the incidence of yersiniosis in Atlantic salmon.

Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru.

Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.

qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture.

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.

Functional and Molecular Immune Response of Rainbow Trout (Oncorhynchus mykiss) Following Challenge with Yersinia ruckeri.

Currently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30-70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout's short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immune-related genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1ß, il-8, il-10, tnf-α1, tnf-α2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM.

Biotyping reveals loss of motility in two distinct Yersinia ruckeri lineages exclusive to Norwegian aquaculture.

Non-motile strains of Yersinia ruckeri, known as Y. ruckeri biotype 2, now dominate amongst clinical isolates retrieved from rainbow trout internationally. Due to an acute increase in the number of yersiniosis cases in Norway in recent years, followed by introduction of widespread intraperitoneal vaccination against the disease, an investigation on the prevalence of Y. ruckeri biotype 2 in Norwegian aquaculture was conducted. We biotyped 263 Y. ruckeri isolates recovered from diseased salmonids in Norway between 1985 and 2020. A total of seven biotype 2 isolates were identified, four of which were collected between 1985 and 1987, and three of which belong to the current epizootic clone, isolated from two different sea-farms in 2017. Whole-genome sequencing revealed single non-synonymous nucleotide polymorphisms in the flagellar genes flhC in isolates from the 1980s, and in fliP in isolates from 2017. In both variants, motility was restored both by complementation with wild-type alleles in trans and via spontaneous mutation-driven reversion following prolonged incubation on motility agar. While biotype 2 strains do not yet seem to have become broadly established in Norwegian aquaculture, the seven isolates described here serve to document a further two independent cases of Y. ruckeri biotype 2 emergence in salmonid aquaculture.

Tissue-specific differences in detection of Yersinia ruckeri carrier status in rainbow trout (Oncorhynchus mykiss).

Effective monitoring for subclinical infections is a cornerstone of proactive disease management in aquaculture. Salmonid fish that survive enteric redmouth disease (ERM) can carry Yersinia ruckeri as a latent infection for several months, potentially facilitating cryptic spread between facilities that exchange fish. In this study, fingerling rainbow trout (Oncorhynchus mykiss) were infected by immersion and sampled for up to 14 weeks post-infection. Yersinia ruckeri was cultured from the posterior kidney of more than 89% of fish up to 4 weeks post-infection, but from 2% or fewer of fish sampled at later time points. In contrast, qPCR-based detection of the Y. ruckeri 16s rRNA gene in intestine and spleen extracts revealed a much higher rate of infection: at 14 weeks post-infection Y. ruckeri was detected in nearly 50% of spleens and 15% of intestines. The difference between spleen and intestine is likely due at least in part to technical limitations of qPCR on intestinal DNA extracts; accordingly, we propose that qPCR of spleen DNA ought to be considered the preferred standard for detection of carriers of Y. ruckeri.

Absolute quantification of priority bacteria in aquaculture using digital PCR.

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans.

Participation of two sRNA RyhB homologs from the fish pathogen Yersinia ruckeri in bacterial physiology.

Small noncoding RNAs (sRNAs) are important regulators of gene expression and physiology in bacteria. RyhB is an iron-responsive sRNA well characterized in Escherichia coli and conserved in other Enterobacteriaceae. In this study, we identified and characterized two RyhB homologs (named RyhB-1 and RyhB-2) in the fish pathogen Yersinia ruckeri. We found that, as in other Enterobacteriaceae, both RyhB-1 and RyhB-2 are induced under iron starvation, repressed by the Fur regulator, and depend on Hfq for stability. Despite these similarities in expression, the mutant strains of Y. ruckeri lacking RyhB-1 (ΔryhB-1) or RyhB-2 (ΔryhB-2) exhibited differential phenotypes. In comparison with the wild type, the ΔryhB-1 strain showed a hypermotile phenotype, reduced biofilm formation, increased replication rate, faster growth, and increased ATP levels in bacterial cultures. By contrast, in salmon cell cultures, the ΔryhB-1 strain exhibited an increased survival. On the other hand, the ΔryhB-2 strain was non-motile and showed augmented biofilm formation as compared to the wild type. The expression of a subset of RyhB conserved targets, selected from different bacterial species, was analyzed by quantitative RT-PCR in wild type, ΔryhB-1, ΔryhB-2, and ΔryhB-1 ΔryhB-2 strains cultured in iron-depleted media. RyhB-1 negatively affected the expression of most analyzed genes (sodB, acnA, sdhC, bfr, fliF, among others), whose functions are related to metabolism and motility, involving iron-containing proteins. Among the genes analyzed, only sdhC and bfr appeared as targets for RyhB-2. Taken together, these results indicate that Y. ruckeri RyhB homologs participate in the modulation of the bacterial physiology with non-redundant roles.

Investigation of the effects of some phytochemicals on Yersinia ruckeri and antimicrobial resistance / Investigação dos efeitos de alguns fitoquímicos em Yersinia ruckeri e resistência antimicrobiana

Abstract In this study, it is aimed to investigate the effects of Moringa oleifera and Sorbus domestica plant extracts on bacterial disease agents Yersinia ruckeri in aquaculture. Morphological and biochemical properties of 2 different Y. ruckeri isolates were determined. Then, Real-Time PCR analysis and gene sequencing of the isolates were identified. Phytochemicals (M. oleifera and S. domestica) and antibiotics (Oxytetracycline (OX) and Enrofloxacin (ENR)) were used together in the antibiogram test of antibiotics compared to the effect status of antibiotics. Also, the effects of phytochemicals on Y. ruckeri growth was examined comparatively by spectrophotometrically measuring at 600 nm wavelength every 2 hours according to bacterial growth densities with 10 different groups formed on TSB medium. As a result of the study, it was observed that the isolates formed Gram negative, catalase positive, oxidase negative, mobile and typical Y. ruckeri colonies. After the biochemical tests performed with Microgen ID panel, 99.85% similarity was determined. The isolates overlap with the 16S rRNA gene region after sequence analysis, and 99% of the isolates were similar in phylogenetic analysis. After the antibiogram test, Oxytetracycline and Enrofloxacin antibiotics were resistant to Y. ruckeri but the effects of phytochemicals were less on solid medium (MHA). As a result of the measurements carried out in liquid medium (TSB), it was observed that phytochemicals such as M. oliefera and S. domestica inhibit the growth of bacteria by 40-50%. As the importance of antibiotic resistance is increasing day by day, we believe that these phytochemicals will give positive results in treatment instead of using antibiotics.

Resumo Neste estudo, objetiva-se investigar os efeitos dos extratos de plantas de Moringa oleifera e Sorbus domestica sobre agentes bacterianos Yersinia ruckeri na aquicultura. Foram determinadas as propriedades morfológicas e bioquímicas de 2 isolados diferentes de Y. ruckeri. Em seguida, a análise de PCR em tempo real e o seqüenciamento genético dos isolados foram identificados. Fitoquímicos (M. oleifera e S. domestica) e antibióticos (Oxitetraciclina e Enrofloxacina) foram usados ​​juntos no teste de antibiograma dos antibióticos em comparação com o status de efeito dos antibióticos. Além disso, os efeitos dos fitoquímicos no crescimento de Y. ruckeri foram examinados comparativamente por medição espectrofotométrica no comprimento de onda de 600 nm a cada 2 horas de acordo com as densidades de crescimento bacteriano com 10 grupos diferentes formados no meio TSB. Como resultado do estudo, observou-se que os isolados formaram colônias Gram-negativas, catalase-positivas, oxidase-negativas, móveis e típicas de Y. ruckeri. Após os testes bioquímicos realizados com o painel Microgen ID, foi determinada uma similaridade de 99,85%. Os isolados se sobrepõem à região do gene 16S rRNA após a análise da sequência e 99% dos isolados foram semelhantes na análise filogenética. Após o teste do antibiograma, os antibióticos Oxitetraciclina e Enrofloxacina foram resistentes a Y. ruckeri, mas os efeitos dos fitoquímicos foram menores no meio sólido (MHA). Como resultado das medições realizadas em meio líquido (TSB), observou-se que os fitoquímicos inibem o crescimento de bactérias em 40-50%. Como a importância da resistência aos antibióticos está aumentando dia a dia, acreditamos que as plantas que são mais alternativas e mais adequadas para o uso de antibióticos hoje em dia darão resultados positivos no tratamento.

Investigation of the effects of some phytochemicals on Yersinia ruckeri and antimicrobial resistance.

In this study, it is aimed to investigate the effects of Moringa oleifera and Sorbus domestica plant extracts on bacterial disease agents Yersinia ruckeri in aquaculture. Morphological and biochemical properties of 2 different Y. ruckeri isolates were determined. Then, Real-Time PCR analysis and gene sequencing of the isolates were identified. Phytochemicals (M. oleifera and S. domestica) and antibiotics (Oxytetracycline (OX) and Enrofloxacin (ENR)) were used together in the antibiogram test of antibiotics compared to the effect status of antibiotics. Also, the effects of phytochemicals on Y. ruckeri growth was examined comparatively by spectrophotometrically measuring at 600 nm wavelength every 2 hours according to bacterial growth densities with 10 different groups formed on TSB medium. As a result of the study, it was observed that the isolates formed Gram negative, catalase positive, oxidase negative, mobile and typical Y. ruckeri colonies. After the biochemical tests performed with Microgen ID panel, 99.85% similarity was determined. The isolates overlap with the 16S rRNA gene region after sequence analysis, and 99% of the isolates were similar in phylogenetic analysis. After the antibiogram test, Oxytetracycline and Enrofloxacin antibiotics were resistant to Y. ruckeri but the effects of phytochemicals were less on solid medium (MHA). As a result of the measurements carried out in liquid medium (TSB), it was observed that phytochemicals such as M. oliefera and S. domestica inhibit the growth of bacteria by 40-50%. As the importance of antibiotic resistance is increasing day by day, we believe that these phytochemicals will give positive results in treatment instead of using antibiotics.

Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture.

We report the development of ddPCR assays for single and simultaneous detection of the bacterial pathogens Flavobacterium psychrophilum and Yersinia ruckeri in water from land-based recirculation aquaculture systems (RAS), producing Atlantic salmon (Salmo salar) smolt. The method was tested and verified for use in water analyses from RAS production sites, and proved to be specific and with sensitivity 0.0011 ng DNA for F. psychrophilum and 1.24 ng for Y. ruckeri. These bacteria are important fish pathogens that have caused reoccurring salmonid infection disease in RAS. Monitoring pathogen levels in water samples could be a useful alternative surveillance strategy to evaluate operational risk assessment connected to stress factors. Water quality is essential for fish health and growth in RAS production in general, and high or increasing levels of these pathogens in the RAS water may generate an early indication of unfavourable conditions in the RAS environment, and give directions to operational actions. This approach may reduce fish mortality, reduce production loss, and offer more effective and targeted preventive measures within RAS production.

The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence.

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface-exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin-like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM.

Changes in physiology and virulence during the selection of resistant Yersinia ruckeri mutants under subinhibitory cefotaxime concentrations.

Bacterial antibiotic resistance is one of the main healthcare problems currently. Apart from reducing antibiotic efficacy, it has awakened the interest of scientists due to its association with bacterial fitness and virulence. Interestingly, antibiotic resistance can be a source of both increased fitness and decreased fitness, even though the molecular basis of these relationships remains unknown. The aim of this work is to define the effects of sub-MIC concentrations of cefotaxime, an antibiotic extensively used in clinical practice, on the physiology and virulence of Yersinia ruckeri and to determine the importance of these sub-MIC concentrations for the selection of antibiotic-resistant mutants in the aquatic environment. Results indicated that exposure to sub-MIC concentrations of cefotaxime selected Y. ruckeri populations with irreversible alterations in the physiology, such as slow growth, aggregation in liquid cultures and modification of the colony morphology. These bacteria also displayed changes in the OMPs and LPS profiles and a full attenuation of virulence. An overexpression of the envelope stress regulator RpoE was also detected after exposure to the antibiotic. In conclusion, exposure to cefotaxime selected, at high frequency, Y. ruckeri strains that survive the antibiotic stress at the expense of a fitness cost and the loss of virulence.

Overcoming Fish Defences: The Virulence Factors of Yersinia ruckeri.

Yersinia ruckeri is the causative agent of enteric redmouth disease, a bacterial infection of marine and freshwater fish. The disease mainly affects salmonids, and outbreaks have significant economic impact on fish farms all over the world. Vaccination routines are in place against the major serotypes of Y. ruckeri but are not effective in all cases. Despite the economic importance of enteric redmouth disease, a detailed molecular understanding of the disease is lacking. A considerable number of mostly omics-based studies have been performed in recent years to identify genes related to Y. ruckeri virulence. This review summarizes the knowledge on Y. ruckeri virulence factors. Understanding the molecular pathogenicity of Y. ruckeri will aid in developing more efficient vaccines and antimicrobial compounds directed against enteric redmouth disease.

A Yersinia ruckeri TIR Domain-Containing Protein (STIR-2) Mediates Immune Evasion by Targeting the MyD88 Adaptor.

TIR domain-containing proteins are essential for bacterial pathogens to subvert host defenses. This study describes a fish pathogen, Yersinia ruckeri SC09 strain, with a novel TIR domain-containing protein (STIR-2) that affects Toll-like receptor (TLR) function. STIR-2 was identified in Y. ruckeri by bioinformatics analysis. The toxic effects of this gene on fish were determined by in vivo challenge experiments in knockout mutants and complement mutants of the stir-2 gene. In vitro, STIR-2 downregulated the expression and secretion of IL-6, IL-1ß, and TNF-α. Furthermore, the results of NF-κB-dependent luciferase reporter system, co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid assay indicated that STIR-2 inhibited the TLR signaling pathway by interacting with myeloid differentiation factor 88 (MyD88). In addition, STIR-2 promoted the intracellular survival of pathogenic Yersinia ruckeri SC09 strain by binding to the TIR adaptor protein MyD88 and inhibiting the pre-inflammatory signal of immune cells. These results showed that STIR-2 increased virulence in Y. ruckeri and suppressed the innate immune response by inhibiting TLR and MyD88-mediated signaling, serving as a novel strategy for innate immune evasion.

TcpA, a novel Yersinia ruckeri TIR-containing virulent protein mediates immune evasion by targeting MyD88 adaptors.

TIR domain-containing protein is an important member for some bacterial pathogens to subvert host defenses. Here we described a fish virulent Yersinia ruckeri SC09 strain that interfered directly with Toll-like receptor (TLR) function by a TIR-containing protein. Firstly, the novel TIR-containing protein was identified by bioinformatics analysis and named as TcpA. Secondly, the toxic effects of TcpA in fish was demonstrated in vivo challenge experiments through knockout mutant and complement mutant of tcpA gene. Thirdly, The study in vitro revealed that TcpA could down-regulate the expression and secretion of IL-6, IL-1ß and TNF-α. Finally, we demonstrated that TcpA could inhibit the TLR signaling pathway through interaction with myeloid differentiation factor 88 (MyD88) in experiments such as NF-κB dependent luciferase reporter system, co-immunoprecipitation, GST pull-down and yeast two-hybrid. The study revealed that TcpA was essential for virulence and was able to interact with the TIR adaptor protein MyD88 and inhibit the pre-inflammatory signal of immune cells and promote the intracellular survival of pathogenic Yersinia ruckeri SC09 strain. In conclusion, our results showed that TcpA acted as a new virulence factor in Y. ruckeri could suppress innate immune response and increase virulence by inhibiting TLR and MyD88-mediated specific signaling, highlighting a novel strategy for innate immune evasion in bacteria.

Flagellar regulation mediated by the Rcs pathway is required for virulence in the fish pathogen Yersinia ruckeri.

The flagellum is a complex surface structure necessary for a number of activities including motility, chemotaxis, biofilm formation and host attachment. Flagellin, the primary structural protein making up the flagellum, is an abundant and potent activator of innate and adaptive immunity and therefore expression of flagellin during infection could be deleterious to the infection process due to flagellin-mediated host recognition. Here, we use quantitative RT-PCR to demonstrate that expression of the flagellin locus fliC is repressed during the course of infection and subsequently up-regulated upon host mortality in a motile strain of Yersinia ruckeri. The kinetics of fliC repression during the infection process is relatively slow as full repression occurs 7-days after the initiation of infection and after approximately 3-logs of bacterial growth in vivo. These results suggests that Y. ruckeri possesses a regulatory system capable of sensing host and modulating the expression of motility in response. Examination of the master flagellar operon (flhDC) promoter region for evidence of transcriptional regulation and regulatory binding sites revealed potential interaction with the Rcs pathway through an Rcs(A)B Box. Deletion of rcsB (ΔrcsB) by marker-exchange mutagenesis resulted in overproduction of flagellin and unregulated motility, showing that the Rcs pathway negatively regulates biosynthesis of the flagellar apparatus. Experimental challenge with ΔrcsB and ΔrcsBΔfliC1ΔfliC2 mutants revealed that mutation of the Rcs pathway results in virulence attenuation which is dependent on presence of the flagellin gene. These results suggest that the inappropriate expression of flagellin during infection triggers host recognition and thus immune stimulation resulting in attenuation of virulence. In addition, RNAseq analyses of the ΔrcsB mutant strain verified the role of this gene as a negative regulator of the flagellar motility system and identified several additional genes regulated by the Rcs pathway.