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Hemolysin Co-regulated Family Proteins Hcp1 and Hcp2 Contribute to Edwardsiella ictaluri Pathogenesis.

ABSTRACT: Edwardsiella ictaluri 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. E. ictaluri 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 E. ictaluri mutants (EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2) were generated and characterized. Catfish peritoneal macrophages were able to kill EiΔhcp2 better than EiΔevpC, EiΔevpCΔhcp2, and E. ictaluri wild-type (EiWT). The attachment of EiΔhcp2 and EiΔevpCΔhcp2 to ovary cells significantly decreased compared to EiWT whereas the cell invasion rates of these mutants were the same as that of EiWT. Mutants exposed to normal catfish serum in vitro showed serum resistance. The fish challenges demonstrated that EiΔevpC and EiΔevpCΔhcp2 were attenuated completely and provided excellent protection against EiWT infection in catfish fingerlings. Interestingly, EiΔhcp2 caused higher mortality than that of EiWT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with EiΔevpC and EiΔevpCΔhcp2, their attenuation and protection were significantly higher compared to EiWT and sham groups, respectively. Taken together, our data indicated that evpC (hcp1) is involved in E. ictaluri virulence in catfish while hcp2 is involved in adhesion to epithelial cells and survival inside catfish macrophages.

SUBMITTER: Kalindamar S

PROVIDER: S-EPMC8207204 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:Edwardsiella ictaluri and Edwardsiella piscicida are important fish pathogens affecting cultured and wild fish worldwide. To investigate the genome-level differences and similarities between catfish-adapted strains in these two species, the complete E. ictaluri 93-146 and E. piscicida C07-087 genomes were evaluated by applying comparative genomics analysis. All available complete (10) and non-complete (19) genomes from five Edwardsiella species were also included in a systematic analysis. Average nucleotide identity and core-genome phylogenetic tree analyses indicated that the five Edwardsiella species were separated from each other. Pan-/core-genome analyses for the 29 strains from the five species showed that genus Edwardsiella members have 9474 genes in their pan genome, while the core genome consists of 1421 genes. Orthology cluster analysis showed that E. ictaluri and E. piscicida genomes have the greatest number of shared clusters. However, E. ictaluri and E. piscicida also have unique features; for example, the E. ictaluri genome encodes urease enzymes and cytochrome o ubiquinol oxidase subunits, whereas E. piscicida genomes encode tetrathionate reductase operons, capsular polysaccharide synthesis enzymes and vibrioferrin-related genes. Additionally, we report for what is believed to be the first time that E. ictaluri 93-146 and three other E. ictaluri genomes encode a type IV secretion system (T4SS), whereas none of the E. piscicida genomes encode this system. Additionally, the E. piscicida C07-087 genome encodes two different type VI secretion systems. E. ictaluri genomes tend to encode more insertion elements, phage regions and genomic islands than E. piscicida. We speculate that the T4SS could contribute to the increased number of mobilome elements in E. ictaluri compared to E. piscicida. Two of the E. piscicida genomes encode full CRISPR-Cas regions, whereas none of the E. ictaluri genomes encode Cas proteins. Overall, comparison of the E. ictaluri and E. piscicida genomes reveals unique features and provides new insights on pathogenicity that may reflect the host adaptation of the two species.

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Hemolysin Co-regulated Family Proteins Hcp1 and Hcp2 Contribute to Edwardsiella ictaluri Pathogenesis.

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