Edwardsiella ictaluri T3SS Effector EseN Modulates Expression of Host Genes Involved in the Immune Response.

The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, including NF-κß (c-rel and relB), creb3L4, socs6 and foxo3a. Regulation of transcription factors leads to regulation of proinflammatory interleukins (IL-8, IL-12a, IL-15, IL-6) and cyclooxygenase-2 (COX-2) expression. Inhibition of COX-2 mRNA by WT E. ictaluri leads to decreased production of prostaglandin E2 (PGE2), which is the product of COX-2 activity. Collectively, our results indicate that E. ictaluri EseN is an important player in the modulation of host immune responses to E.ictaluri infection.

Early Intracellular Trafficking and Subsequent Activity of Programmed Cell Death in Channel Catfish Macrophages Infected with Edwardsiella ictaluri.

The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 65ST and was significantly lower in WT-infected cells. Executioner caspase-3/7 activity also increased significantly in 65ST-infected cells compared to WT-infected cells. Repression of apoptosis was further demonstrated with flow cytometry using Alexa Fluor 647-labeled Annexin V and propidium iodide. Results indicate that WT ECV fused with early and late endosomes but that phagosomal/lysosomal fusion did not occur. Additionally, WT-infected cells recruited Golgi vesicles for vacuolar size increase and bacterial growth material, and both autophagy and apoptosis were repressed in the WT. This activity was all based on the function of the E. ictaluri T3SS.

Edwardsiella ictaluri type III secretion system (T3SS) effector EseN is a phosphothreonine lyase that inactivates ERK1/2.

EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase (PTL) activity, including OspF from Shigella and SpvC from Salmonella. A yeast-2-hybrid system was used to identify the major vault protein (MVP) as a specific host-cell binding partner for EseN, and the proximity ligation assay (PLA) confirmed the interaction. Similar to other pathogens, E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Structurally, EseN contains a highly conserved docking motif that is required for specific binding to mitogen-activated protein kinases, such as ERK1/2, and a motif that is essential for PTL activity. Immunoblotting and immunofluorescence analyses indicate that EseN inactivates ERK1/2 by dephosphorylation in vivo in the head kidney of infected fish and ex vivo in head kidney derived macrophages. Interaction of EseN with phosphorylated ERK1/2 (pERK1/2) was also confirmed using PLA, suggesting that MVP serves as a signaling scaffold for ERK1/2 and EseN. Channel catfish Ictalurus punctatus infected with E. ictaluri strains lacking the eseN gene had reduced numbers of E. ictaluri in the tissues following infection and reduced mortality compared to fish infected with the wild-type. Our results indicate that eseN encodes a PTL domain that interacts with MVP as a possible scaffold protein and inactivates pERK1/2 to ERK1/2, resulting in increased proliferation of E. ictaluri and, ultimately, death of the host.

Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System.

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.