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INVESTIGATOR: Rogge, M. L.

PERFORMING INSTITUTION:
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE, LOUISIANA 70803-0100

THE ROLE OF TYPE VI SECRETION IN EDWARDSIELLA ICTALURI VIRULENCE

NON-TECHNICAL SUMMARY: The gram-negative pathogen Edwardsiella ictaluri is the etiological agent of enteric septicemia of catfish (ESC), an economically-important disease of channel catfish (Ictalurus punctatus). Channel catfish and trout are the major aquaculture species of the United States, with catfish farming accounting for approximately 90% of the total finfish aquaculture production in the United States in 2009. Enteric septicemia of catfish is reported in all catfish production regions of the US, and losses due to ESC were reported on over 60% of production facilities. Recent research has identified the importance of bacterial secretion systems in E. ictaluri virulence. Edwardsiella ictaluri encodes a complete type VI secretion system (T6SS), which are recently identified bacterial secretion systems commonly associated with pathogenic organisms. Relatively little is known about how T6SS are involved in bacterial pathogenesis, but preliminary data indicates that the E. ictaluri T6SS is required for virulence. Further understanding of the role of the T6SS in E. ictaluri pathogenesis will provide information about the host-pathogen interaction, leading to improved knowledge of pathogenesis, as well as provide information for vaccine development.

OBJECTIVES: The goal is to determine the role of the E. ictaluri type VI secretion system (T6SS) in virulence associated with enteric septicemia of catfish (ESC). Based on preliminary data, the T6SS is an important virulence factor of E. ictaluri. Further understanding of how this system is involved in pathogenesis will lead to effective treatments against this deadly fish pathogen. Furthermore, this information will be useful to advance the understanding of the role of the T6SS in pathogens of higher vertebrates, including Salmonella, Yersinia, Burkholderia, Vibrio, and others. Using ESC to study T6SSs provides a significant advantage in that test subjects are readily available for in vivo studies, and the host/pathogen relationship represents a natural disease system and not a model. Our lab is capable of screening virulence mutants in a number of in vitro, ex vivo, and in vivo assays, including gene expression analyses by quantitative real-time PCR, western blot, phagocytic and non-phagocytic cell culture assays, and in vivo invasion, persistence, and mortality assays to evaluate virulence. This research puts forth two specific objectives that will confirm the role of the T6SS in pathogenesis of E. ictaluri, identify proteins secreted by the E. ictaluri T6SS, and identify how the T6SS is involved in pathogenesis. The first objective of this research is to identify the conditions involved in the expression of the E. ictaluri T6SS by evaluating expression of T6SS genes in varying culture conditions. Furthermore, proteins secreted by the T6SS will be identified and evaluated for their role in E. ictaluri pathogenesis. The second objective is to determine the role of the E. ictaluri T6SS in pathogenesis. The effects of T6SS secretion on intracellular replication within channel catfish cells will be evaluated, as well as effects in vivo within the channel catfish. Expected outputs from this research include the results of the studies being presented at national and international meetings in addition to peer-reviewed publications.

APPROACH: Work to identify the role of the E. ictaluri type III secretion system (T3SS) led to the discovery that a type VI secretion system (T6SS) is regulated by the T3SS. Type VI secretion systems are recently discovered systems that are present in many Gram-negative pathogens. The role of the E. ictaluri T6SS in pathogenesis within channel catfish is unknown, but preliminary data indicates it is required for virulence. The goal of this research is to characterize the role of the T6SS in E. ictaluri pathogenesis in channel catfish. Expression of the T6SS will be analyzed by quantitative real-time PCR (qRT-PCR) to determine conditions in which T6SS genes are expressed in vitro. Expression of T6SS genes will be compared to expression of the 16s region and gyrB. Protein expression will be verified by Western blot. Furthermore, substrates secreted by the T6SS will be identified and analyzed to determine their role in virulence. Isogenic mutations will be constructed in specific T6SS-related genes, and their phenotypes will be evaluated in ex vivo and in vivo assays to determine the role of the specific T6SS genes in pathogenesis. Intracellular replication within channel catfish ovary cells and channel catfish head kidney-derived macrophages will be evaluated using gentamicin exclusion assays. The ability of wild type and mutant strains of E. ictaluri to induce cytotoxicity or apoptosis will be evaluated using commercially available kits. Quantitative data will be evaluated by analysis of variance by using Statistical Analysis Systems v9.1 (SAS Institute, Cary, NC). Where applicable, when the overall model indicates a significant difference (P < 0.05), pairwise comparisons will be conducted using Scheffe's test.

PROGRESS: 2011/09 TO 2012/08
OUTPUTS: Data were presented at the Postdoctoral Works-in-Progress Seminar Series at the Louisiana State University School of Veterinary Medicine in Baton Rouge, LA, December 2011, the American Fisheries Society Fish Health Section Annual Meeting in La Crosse, WI, August 2012, and the USDA AFRI NIFA Investigator's Meeting in Washington, D.C., August 2012. A manuscript is currently being prepared for submission. PARTICIPANTS: Dr. Matthew Rogge, project director. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2011/09 TO 2012/08
The findings of this research have contributed to the understanding of the importance of the type VI secretion system to E. ictaluri pathogenesis. The reverse-transcriptase PCR results indicate that the T6SS is expressed as one large operon including the genes evpABCDEFGHIJKLMNO, presumably from a promoter and regulatory region upstream of evpA. Transcription of evpP was also detected, but not in conjunction with the larger transcript of evpA-O. None of the mutations in the T6SS-related genes resulted in attenuation of intracellular replication in either naive macrophages or channel catfish ovary cells, indicating the T6SS does not have a role in intracellular replication. None of the mutations affected the ability of E. ictaluri to invade the channel catfish head kidney. All strains were detected at similar densities 30 min post-immersion. Persistence within the head kidney, however, was affected by mutations in some T6SS genes. Mutation of hcp1 had no effect on persistence. Mutation of evpD and evpJ did not significantly reduce bacterial loads in the head kidney, but did result in reduced mortality during the challenge. The evpJ and evpD double mutant significantly decreased in density 3 days post-immersion, however, and persisted only 9 days. The evpI mutant also significantly decreased in density 3 days post-immersion and was only sporadically found in the head kidney after day 8. Results of the mortality challenge demonstrate that the evpI and the evpJ/evpD double mutant are fully attenuated, as no fish died in either challenge. The evpD and evpJ single mutants were still virulent, but caused significantly less mortality than the wild type. The hcp1 mutant caused similar mortality as the wild type. These results demonstrate that the T6SS is required for persistence and virulence within the channel catfish host. In vitro expression analyses using the 6xHis-tagged proteins demonstrates that the T6SS is not expressed in rich media (BHI). Growth in neutral minimal media induces low level expression of the T6SS, and a shift to acidic minimal media (pH 5.5) greatly increases expression of the T6SS. Expression of the T6SS does not appear to be dependent on the iron concentration of the media. Analysis of the extracellular protein during culture in acidic minimal media indicates that EvpC and EvpD are secreted. Secretion of EvpD is dependent on EvpC and the type III secretion system (T3SS) regulatory protein EsrB, indicating that secretion of T6SS proteins is regulated by the T3SS.

PUBLICATIONS (not previously reported): 2011/09 TO 2012/08
Rogge, M.L. and R. L. Thune. 08/01/2012. Mutagenic Analysis of the Edwardsiella ictaluri Type VI Secretion System. 2012 AFS-FHS Annual Meeting Program, La Crosse, WI.