Detection of Quorum Sensing Signal Molecules in Edwardsiella ictaluri Ei-151.
ABSTRACT: Edwardsiella ictaluri is a Gram-negative pathogenic bacterium in the family Enterobacteriaceae that causes enteric septicemia of catfish, which has become a significant problem in the aquaculture of striped catfish (Pangasianodon hypophthalmus) in Vietnam. In this study, a bacterium designated as Ei-151 was isolated from diseased striped catfish and proved to be virulent. Based on 16S rDNA sequencing and phenotypic tests, the pathogenic bacterium was identified as Edw. ictaluri. The presence of quorum sensing signal molecules in Edw. ictaluri Ei-151 was detected with different biosensor strains. The results showed that Ei-151 produced at least three kinds of acylated homoserine lactone (AHL) signal molecules as detected with the biosensor Agrobacterium tumefaciens KYC55, and the AHLs fingerprint was similar to that of Edw. tarda. During its entire growth, the levels of AHLs and autoinducer-2 produced by Ei-151 peaked at the stationary phase (OD600 1.8), which suggested that both of them may function at the stationary phase. No Cholerae autoinducer-1-like activity (including Edw. ictaluri LMG7860(T)) was detected.
Project description:Edwardsiella ictaluri is an intracellular Gram-negative facultative pathogen causing enteric septicemia of catfish (ESC), a common disease resulting in substantial economic losses in the U.S. catfish industry. Previously, we demonstrated that several universal stress proteins (USPs) are highly expressed under in vitro and in vivo stress conditions, indicating their importance for E. ictaluri survival. However, the roles of these USPs in E. ictaluri virulence is not known yet. In this work, 10 usp genes of E. ictaluri were in-frame deleted and characterized in vitro and in vivo. Results show that all USP mutants were sensitive to acidic condition (pH 5.5), and Ei?usp05 and Ei?usp08 were very sensitive to oxidative stress (0.1% H2O2). Virulence studies indicated that Ei?usp05, Ei?usp07, Ei?usp08, Ei?usp09, Ei?usp10, and Ei?usp13 were attenuated significantly compared to E. ictaluri wild-type (EiWT; 20, 45, 20, 20, 55, and 10% vs. 74.1% mortality, respectively). Efficacy experiments showed that vaccination of catfish fingerlings with Ei?usp05, Ei?usp07, Ei?usp08, Ei?usp09, Ei?usp10, and Ei?usp13 provided complete protection against EiWT compared to sham-vaccinated fish (0% vs. 58.33% mortality). Our results support that USPs contribute E. ictaluri virulence in catfish.
Project description:<i>Edwardsiella ictaluri</i> is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. <i>E. ictaluri</i> strain 93-146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double <i>E. ictaluri</i> mutants (<i>Ei</i>Δ<i>evpC, Ei</i>Δ<i>hcp2</i>, and <i>Ei</i>Δ<i>evpC</i>Δ<i>hcp2</i>) were generated and characterized. Catfish peritoneal macrophages were able to kill <i>Ei</i>Δ<i>hcp2</i> better than <i>Ei</i>Δ<i>evpC, Ei</i>Δ<i>evpC</i>Δ<i>hcp2</i>, and <i>E. ictaluri</i> wild-type (<i>Ei</i>WT). The attachment of <i>Ei</i>Δ<i>hcp2</i> and <i>Ei</i>Δ<i>evpC</i>Δ<i>hcp2</i> to ovary cells significantly decreased compared to <i>Ei</i>WT whereas the cell invasion rates of these mutants were the same as that of <i>Ei</i>WT. Mutants exposed to normal catfish serum <i>in vitro</i> showed serum resistance. The fish challenges demonstrated that <i>Ei</i>Δ<i>evpC</i> and <i>Ei</i>Δ<i>evpC</i>Δ<i>hcp2</i> were attenuated completely and provided excellent protection against <i>Ei</i>WT infection in catfish fingerlings. Interestingly, <i>Ei</i>Δ<i>hcp2</i> caused higher mortality than that of <i>Ei</i>WT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with <i>Ei</i>Δ<i>evpC</i> and <i>Ei</i>Δ<i>evpC</i>Δ<i>hcp2</i>, their attenuation and protection were significantly higher compared to <i>Ei</i>WT and sham groups, respectively. Taken together, our data indicated that <i>evpC</i> (<i>hcp1</i>) is involved in <i>E. ictaluri</i> virulence in catfish while <i>hcp2</i> is involved in adhesion to epithelial cells and survival inside catfish macrophages.
Project description:Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen that causes enteric septicemia in catfish (ESC). Stress factors including poor water quality, poor diet, rough handling, overcrowding, and water temperature fluctuations increase fish susceptibility to ESC. The TonB energy transducing system (TonB-ExbB-ExbD) and TonB-dependent transporters of Gram-negative bacteria support active transport of scarce resources including iron, an essential micronutrient for bacterial virulence. Deletion of the tonB gene attenuates virulence in several pathogenic bacteria. In the current study, the role of TonB (NT01EI_RS07425) in iron acquisition and E. ictaluri virulence were investigated. To accomplish this, the E. ictaluri tonB gene was in-frame deleted. Growth kinetics, iron utilization, and virulence of the Ei?tonB mutant were determined. Loss of TonB caused a significant reduction in bacterial growth in iron-depleted medium (p > 0.05). The Ei?tonB mutant grew similarly to wild-type E. ictaluri when ferric iron was added to the iron-depleted medium. The Ei?tonB mutant was significantly attenuated in catfish compared with the parent strain (21.69 vs. 46.91% mortality). Catfish surviving infection with Ei?tonB had significant protection against ESC compared with naïve fish (100 vs. 40.47% survival). These findings indicate that TonB participates in pathogenesis of ESC and is an important E. ictaluri virulence factor.
Project description:Edwardsiella ictaluri is a Gram-negative intracellular pathogen causing enteric septicemia of channel catfish (ESC). Type six secretion system (T6SS) is a sophisticated nanomachine that delivers effector proteins into eukaryotic host cells as well as other bacteria. In the current work, we in-frame deleted the E. ictalurievpB gene located in the T6SS operon by allelic exchange. The safety and efficacy of Ei?evpB as well as Aquavac-ESC, a commercial vaccine manufactured by Intervet/Merck Animal Health, were evaluated in channel catfish (Ictalurus punctatus) fingerlings and fry by immersion exposure. Our results showed that the Ei?evpB strain was avirulent and fully protective in catfish fingerlings. The Ei?evpB strain was also safe in catfish fry, and immersion vaccination with Ei?evpB at doses 106 and 107 CFU/ml in water resulted in 34.24 and 80.34% survival after wild-type immersion challenge compared to sham-vaccinated fry (1.79% survival). Catfish fry vaccinated with Ei?evpB at doses 106, 107, and 108 CFU/ml in water exhibited dose-dependent protection. When compared with Aquavac-ESC, Ei?evpB provided significantly higher protection in catfish fingerlings and fry (p < 0.05). Results indicate that the Ei?evpB strain is safe and can be used to protect catfish fingerlings and fry against E. ictaluri.
Project description:Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, Ei?evpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-? gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-? gene expression patterns in the AK resembled that of the patterns induced by Ei?evpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the Ei?evpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-? and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the Ei?evpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines.
Project description:We have investigated the quorum sensing control in Aeromonas veronii MTCC 3249, originally isolated as A. culicicola from the midgut of Culex quinquefasciatus. Based on biosensor assays, the bacterium showed constant production of multiple acyl-homoserine lactones (AHLs) with increasing cell-density. The luxRI gene homologs, acuR (A. culicicola transcriptional Regulator) and acuI (A. culicicola autoInducer) were successfully amplified by inverse-PCR. Sequence analysis indicated acuRI were divergent from all known quorum sensing gene homologs in Aeromonas. Two localized regions in the C-terminal autoinducer binding domain of acuR showed indels suggesting variations in autoinducer specificity. Further, only a single copy of the quorum sensing genes was detected, suggesting a tight regulation of mechanisms under its control. Chromatography and further chemical analysis identified two AHLs in the culture supernatant: 6-carboxy-HHL (homoadipyl homoserine lactone), a novel AHL, and N-tetradecanoylhomoserine lactone. The existence of a potentially variant quorum sensing system might therefore, reflect in some way the ecological strategies adopted by this bacterium in the mosquito midgut.
Project description:Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.
Project description:Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000 tons produced annually, and ESC is the leading cause of disease loss in the industry. We have demonstrated the survival and replication of E. ictaluri within channel catfish cells and identified a secretion system that is essential for E. ictaluri intracellular replication and virulence. We have also identified nine proteins encoded in the E. ictaluri genome that we believe are actively transferred from the bacterium to the cytoplasm of the host cell and act to manipulate host cell physiology to the advantage of the bacterium. The data presented here confirm that the proteins are actually transferred during an infection, which will lead to further work on approaches to preventing or controlling ESC.
Project description:asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ?asdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ?asdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ?asdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ?asdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.
Project description:<i>Edwardsiella ictaluri</i>, a Gram-negative facultative intracellular pathogen, is the causative agent of enteric septicemia of catfish (ESC). The innate functions of B cells have been demonstrated in several teleost fish, including zebrafish, rainbow trout, and channel catfish. Recently, our group has developed several protective <i>E. ictaluri</i> live attenuated vaccines (LAVs). However, the innate role of catfish B cells to phagocytose and destroy <i>E. ictaluri</i> wild-type (WT) and live attenuated vaccine (LAV) strains has not been evaluated. In this study, we assessed the efficacy of <i>E. ictaluri</i> WT and two LAVs on phagocytosis, microbial killing, and survival of catfish anterior kidney (AK) B cells. Initially, we documented active uptake of <i>E. ictaluri</i> WT and two LAVs in B cells by flow cytometry and light microscopy. Then, we observed the <i>E. ictaluri</i> strains-induced phagosome and/or phagolysosome formation in the cytoplasm of catfish magnetically sorted IgM<sup>+</sup> B cells. Furthermore, we demonstrated that AK B cells were able to destroy the internalized <i>E. ictaluri</i> WT and LAV strains efficiently. Finally, we documented early and late apoptotic/necrotic manifestations induced by <i>E. ictaluri</i> in catfish AK B cells. In conclusion, our results suggest that both LAVs and WT strain initiate similar innate immune responses such as active phagocytic uptake, induced bactericidal activity as well as promote early and late apoptotic changes in catfish B cells. Our data suggest that phagocytic and microbicidal B cells may serve as professional APCs in initiation of protective adaptive immune responses against ESC in channel catfish.