LuxS/AI-2 Quorum Sensing System in Edwardsiella piscicida Promotes Biofilm Formation and Pathogenicity.

LuxS/AI-2 is an important quorum sensing system which affects the growth, biofilm formation, virulence, and metabolism of bacteria. LuxS is encoded by the luxS gene, but how this gene is associated with a diverse array of physiological activities in Edwardsiella piscicida (E. piscicida) is not known. Here, we constructed an luxS gene mutant strain, the △luxS strain, to identify how LuxS/AI-2 affects pathogenicity. The results showed that LuxS was not found in the luxS gene mutant strain, and this gene deletion decreased E. piscicida growth compared to that of the wild-type strain. Meanwhile, the wild-type strain significantly increased penetration and motility in mucin compared to levels with the △luxS strain. The 50% lethal dose (LD50) of the E. piscicida △luxS strain for zebrafish was significantly higher than that of the wild-type strain, which suggested that the luxS gene deletion could attenuate the strain's virulence. The AI-2 activities of EIB202 were 56-fold higher than those in the △luxS strain, suggesting that the luxS gene promotes AI-2 production. Transcriptome results demonstrated that between cells infected with the △luxS strain and those infected with the wild-type strain 46 genes were significantly differentially regulated, which included 34 upregulated genes and 12 downregulated genes. Among these genes, the largest number were closely related to cell immunity and signaling systems. In addition, the biofilm formation ability of EIB202 was significantly higher than that of the △luxS strain. The supernatant of EIB202 increased the biofilm formation ability of the △luxS strain, which suggested that the luxS gene and its product LuxS enhanced biofilm formation in E. piscicida All results indicate that the LuxS/AI-2 quorum sensing system in E. piscicida promotes its pathogenicity through increasing a diverse array of physiological activities.

The role of uhpA in Edwardsiella piscicida and the inflammatory cytokine response in tilapia.

Edwardsiella piscicida (E. piscicida) is an important zoonotic pathogen that infects fish by colonizing the intestines. The intestine provides nutrition including Glucose 6-phosphate (Glu6P) and a competitive environment for the microbiota. Although the transport system regulatory protein gene uhpA has been reported in E. piscicida genomes, whether the uhpA gene is involved in the pathogenicity of E. piscicida remains largely unknown. Therefore, the uhpA gene mutants strain E. piscicida ΔuhpA was constructed to elucidate the functions of Glu6P and the uhpA gene in E. piscicida. The results demonstrated that Glu6P significantly increased the gene expression of uhpC/uhpB/uhpA than without adding Glu6P in the culture. The gene expression of uhpC and uhpB was down regulated in the mutant strain than that of in the wild type strain. E. piscicida ΔuhpA exhibited an increase in virulence compared to that of E. piscicida EIB202 [LD50 value: (3.98 × 106 CFU/fish) and LD50 value: (1.45 × 107 CFU/fish) respectively]. Besides, although TNF-α did not show significant differences (p > 0.05) in the spleen of tilapia infected with ΔuhpA and EIB202 in the whole observed period, the gene expression of IL-1ß and TGF-ß in the spleen of tilapia infected with ΔuhpA showed significantly higher (p < 0.05) than that of in tilapia infected with EIB202. Meanwhile, the gene expression of IL-1ß and TGF-ß in spleen of tilapia infected with ΔuhpA showed significantly higher (p < 0.05) than that of in fish infected with EIB202 when zebrafish used as the control in the whole observed period. All these results suggested that Glu6P up-regulated the gene expression of uhpC/uhpB/uhpA; most important, the uhpA gene deletion in E. piscicida down-regulated the gene expression of uhpC and uhpB, enhanced its pathogenicity and its role in inducing the inflammatory cytokine responses in tilapia.

The UhpA mutant of Edwardsiella piscicida enhanced its motility and the colonization in the intestine of tilapia.

Edwardsiella piscicida (E. piscicida) is a significant bacterial pathogen of cultured fish, which infected fish meanly through the intestine. Glucose 6-phosphate (Glu6P) in the intestine is nutritious to the pathogen, Meanwhile, Glu6P was found using as a virulent regulating signal for bacteria. The UhpA, one of the Glu6P transport system regulatory proteins could down-regulate the uhpC/uhpB/uhpA system and decrease its pathogenicity. However, the motility and the colonization of E. piscicida affected by UhpA were still unclear. In this study, the motility and the colonization of E. piscicida were monitored. The result demonstrated that the motility of EIB202 was significantly stronger than that of in ΔuhpA according to fractions 4, 8 and 9. However, the motility of ΔuhpA was significantly stronger than that of EIB202 according to the total number at the whole experiment. Although, there was no difference in the number of bacteria in the posterior intestine of tilapia after infected with E. piscicida EIB202 and ΔuhpA. The number of bacteria in the anterior and the middle intestine of fish infected with ΔuhpA were significantly higher than that of in fish infected with EIB202 at the whole experiment (P < 0.05). Interestingly, both E. piscicida strains colonized in the anterior intestine than that of in the middle and posterior intestines of tilapia. Besides, the gene expression of IL-1ß and TNF-α in the head-kidney of fish infected with ΔuhpA showed significantly higher (p < 0.05) than fish infected with EIB202 during the whole experimental period. Most importantly, the survival rate of E. piscicida EIB202 and ΔuhpA were 57% and 37% respectively. All results indicate that the uhpA gene mutant in E. piscicida could enhance its motility and the colonization in the intestine of tilapia, this illustrates the mechanism of UhpA decreases the pathogenesis of E. piscicida in fish.

Cross-immunity in Nile tilapia vaccinated with Streptococcus agalactiae and Streptococcus iniae vaccines.

Streptococcus agalactiae and Streptococcus iniae are major bacterial pathogens of tilapia that can cause high mortality concomitant with large economic losses to aquaculture. Although development of vaccines using formalin-killed bacteria to control these diseases has been attempted, the mechanism of immunity against streptococcal infections and the cross-protective ability of these two bacteria remains unclear. To explore the immunological role of these vaccines, we compared the immune responses of tilapia after immunization with both vaccines and compared the relative percent survival (RPS) and cross-immunization protection of tilapia after separate infection with S. agalactiae and S. iniae. All results revealed that vaccinated fish had significantly higher (P < 0.05) levels of specific antibodies than control fish 14 days post secondary vaccination (PSV) and 7 days post challenge. In vaccinated fish, the mRNA expression of interleukin-8 (IL-8), interleukin-12 (IL-12), caspase-3 (C-3), tumour necrosis factor (TNF), and interferon (IFN) was significantly up regulated (P < 0.05) in the head kidney after immunized; similar results were found for IL-8, TNF and IFN in the posterior kidney, meanwhile the expression levels of C-3 and IFN were significantly increased (P < 0.05) in the spleen of vaccinated fish. Additionally, the levels of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in vaccinated fish were improved at different degree when compared to the control fish. These results showed that vaccination with formalin-killed cells (FKCs) of either S. agalactiae or S. iniae conferred protection against infection by the corresponding pathogen in Nile tilapia, resulting in RPS values of 92.3% and 91.7%, respectively. Furthermore, cross-protection was observed, as the S. agalactiae FKC vaccine protected fish from S. iniae infection, and vice versa. These results suggested that the S. agalactiae and S. iniae FKC vaccines can induce immune responses and generate excellent protective effects in Nile tilapia.

The role of regulator FucP in Edwardsiella tarda pathogenesis and the inflammatory cytokine response in tilapia.

The animal intestine provides a competitive environment for the microbiota. Successful colonization by pathogens requires sensing chemical signals to regulate the expression of virulence genes. Some bacteria rely on a two-component chemical signal transduction system, named FusKR, to regulate virulence genes expression by intestinal fucose. Here we construct the fucP gene deletion strain prove FucP regulation of the T3SS in E. tarda. The result showed that the mutant strain had down-regulated significantly the gene expression of FusKR and T3SS compared to the wild-type strain (P < 0.05). This mutant strain significantly increased LD50 in zebrafish compared to the wild-type strain (P < 0.05), and significantly decreased penetration and motility in mucin than the wild-type strain (P < 0.05). Meanwhile, tilapia infected with mutant strain show significantly reduced E. tarda adherence and colonization than those infected with the wild-type strain (P < 0.05). Fish infected with EIB202 and ΔfucP showed significantly higher (P < 0.05) gene expression of IL-1ß, TNF-α, IFN-γ, TGF-ß and HSP-70 in head kidney than fish treated with PBS in the whole observed period; however CPP-3 did not show significant differences (P > 0.05) in all groups. Fish infected with EIB202 showed significantly higher (P < 0.05) gene expression of TGF-ß in head kidney than fish treated with ΔfucP in the whole observed period; however other cytokines did not show significant differences (P > 0.05) in the whole observed period. In addition, Fish infected with EIB202 showed significantly higher (P < 0.05) gene expression of IL-1ß, TNF-α and TGF-ß in spleen than fish treated with ΔfucP in the whole observed period, however IFN-γ, CPP-3, and HSP-70 did not show significant differences (P > 0.05) in the whole observed period. Although the gene expression of cytokines was induced similarly by both strains, all results indicate that the fucP gene deletion down-regulates the key gene expression of FucKR and T3SS, reduces the pathogenicity of E. tarda in fish, particularly decreases inducing the gene expression of TGF-ß in the head kidney and IL-1ß, TNF-α and TGF-ß in the spleen.

Different concentrations of Edwardsiella tarda ghost vaccine induces immune responses in vivo and protects Sparus macrocephalus against a homologous challenge.

Bacterial ghosts (BGs) can be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria. They are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. Here we prepared Edwardsiella tarda ghost (ETG) and tested different concentrations in vaccination trials. The results showed that serum IgM antibody titers were significantly higher in three different concentration immunization groups than control group (P < 0.05), However, there was no significant (P > 0.05) difference between the immunized groups. The phagocytic percentage (PP) was significantly higher (P < 0.05) in ETG immunized groups than in the control group from 3 days post-treatment. The PP continued to rise with time until day 21, when the values of three ETG immunized groups were 45.7%,51.2% and 50.7%, respectively. In addition, phagocytic index (PI) was significantly higher (P < 0.05) in ETG immunized groups than in the control group after 7 days post-treatment. However, there was no significant (P > 0.05) difference of PP or PI between immunized groups. In addition, non-specific immune immunity, such as acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme activities displayed a similar pattern in all immunized groups, all immunized fish showed significantly higher activities than control group fish (P < 0.05). Most importantly three ETG immunized groups were all significantly more protected against the E. tarda challenge (19/25, 76% survival), (21/25, 84% survival) and (20/25, 80% survival) respectively, compared to (9/25, 36% survival) survival in the control group, but there was no significant (P > 0.05) difference of survival rate (SR) or relative percent survival (RPS) between immunized groups. All these results suggest that an ETG could stimulate cellular and humoral immunity, and could be used as a vaccine candidate in S.m. In summary, ETG can protect fish from Edwardsiellosis, and there is no significant difference in SR and RPS when three different concentrations of ETG are used, so it can easily be developed as a vaccine for mechanical and artificial operations.