Detection of Edwardsiella ictaluri virulence genes required to prevent killing by catfish neutrophils

NRI competitive grant no: 2004-35204-14211

The disease caused by Edwardsiella ictaluri, enteric septicemia of catfish (ESC), is the most economically important disease of farm-raised channel catfish, which is the largest aquaculture industry in the United States. The mechanisms used by E. ictaluri to establish infection in catfish and avoid the catfish's early defense systems are not well understood. The purpose of this project is to determine the mechanism used by E. ictaluri to avoid killing by catfish neutrophils, which are immune cells responsible for engulfing and killing bacteria.

The long-range goal of our laboratory is to determine the strategy and mechanisms used by E. ictaluri in the early pathogenesis of enteric septicemia of catfish (ESC), and to use this knowledge for the rational development of novel strategies for blocking the penetration or dissemination of E. ictaluri in channel catfish. The immediate objective of this project is to identify E. ictaluri genes that are required to prevent killing by channel catfish neutrophils and to determine their effects on pathogenesis.

Our strategy will be to construct a bank of random transposon-generated insertion mutations in the chromosome of E. ictaluri. Each mutant will then be individually transferred into microplates with and without neutrophils, and bacterial viability will be monitored over a 90 minute period. Mutants with increased susceptibility to killing by catfish neutrophils compared to wild type E. ictaluri will be identified; these mutants will contain transposon insertions in genes required for survival against killing by neutrophils. Following confirmation of the selected mutants, the affected genes in the susceptible mutants will be determined. After eliminating any mutations that may have occurred in housekeeping genes, we will choose a subset of mutants with insertions in virulence genes for subsequent cloning and further characterization.

We have constructed new stable plasmid vectors carrying bacterial luciferase and green fluorescent protein under the control of both constitutive and inducible promoters, and we have transferred them into E. ictaluri and tested their function.  An inducible luciferase and gfp labeled E. ictaluri random mutant library using a mariner-based transposon has been constructed in collaboration with the USDA-ARS Catfish Genetics Laboratory in Stoneville, MS. Mutants were initially screened with channel catfish neutrophils. Then, mutants that have increased susceptibility to killing by catfish neutrophils were screened for attenuation in channel catfish. Gene identifications and further characterization of these mutants are pending.

The construction of luciferase and gfp-labeled E. ictaluri from this project has enabled pathogenesis research in which we are able to track the progress of E. ictaluri infection in living catfish using bioluminescence imaging. The methods developed in this project should be applicable to investigate pathogenesis of disease caused by other bacterial pathogens.

Project director: Mark Lawrence
Project co-director: Attila Karsi
Project personnel: Simon Menanteau, Pradeep Dumpala, Joey Greene, Erin Corb

Publications:

Karsi, A. and M. L. Lawrence.  2007.  Broad host range fluorescence and bioluminescence expression vectors for gram negative bacteria.  Plasmid 57:286-295.

Karsi, A., S. Menanteau-Ledouble, and M. L. Lawrence.  2006.  Development of bioluminescent Edwardsiella ictaluri for non-invasive disease monitoring.  FEMS Microbiol. Lett. 260:216-223.

Karsi, A. and M. L. Lawrence.  2006.  Development of fluorescent Edwardsiella ictaluri and visualization of the pathogen in channel catfish neutrophils.  106th General Meeting of the American Society for Microbiology, Orlando, Florida.

Karsi, A., S. Menanteau, and M. L. Lawrence.  2005.  Real-time monitoring of bioluminescent Edwardsiella ictaluri infections in living catfish.  105th General Meeting of the American Society for Microbiology, Atlanta, Georgia.

Karsi, A., S. Menanteau, and M. L. Lawrence.  2005.  Use of bioluminescent Edwardsiella ictaluri for real-time monitoring of enteric septicemia of catfish.  Fish Health Newsletter 33:10-11.