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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetics, Physiology, and Health Research to Improve Catfish Production

Location: Warmwater Aquaculture Research Unit

Title: A developing model for Edwardsiella ictaluri pathogenesis

Authors
item Thune, Ronald -
item Rogge, Matt -
item Booth, Natha
item Baumgartner, Wes -
item Beekman, Judith -

Submitted to: Catfish Farmers of America Research Symposium
Publication Type: Abstract Only
Publication Acceptance Date: December 1, 2011
Publication Date: N/A

Technical Abstract: Edwardsiella ictaluri, the causative agent of Enteric Septicemia of Catfish (ESC), belongs to a rather short list of bacteria known to survive and replicate in macrophages. Macrophages are ameboid-like cells that engulf and then digest cellular debris and pathogens, including bacteria. The process, known as phagocytosis, involves attaching to the bacterial cell, bringing it into the cell in a specialized vacuole called a phagosome, then fusing with another specialized vacuole called a lysosome, forming a phagolysosome. The lysosome releases acid-activated enzymes and toxic metabolites, the vacuole acidifies to pH 4-5, and the bacterial cell is killed and broken down by the enzymes and toxins. Edwardsiella ictaluri is able to avoid this process and replicate in these cells. The key to the ultimate control and prevention of ESC involves developing an intimate knowledge of the mechanisms that E. ictaluri uses to advance the disease process and avoid the host defense system. This means identifying the genes and their encoded proteins that are important to the virulence of the bacterium and determining the function of the proteins. As a first step in this process, a virulence factor detection assay was used in a waterborne infection model in specificpathogen- free channel catfish, which resulted in the identification of 5 genes encoding proteins similar to virulence factors in other pathogens, including three genes involved in type III secretion systems (T3SS) and two in genes involved in urease activity. Using the sequences obtained to probe the E. ictaluri genome, a 26,135 bp pathogenicity island encoding 33 genes of a T3SS with similarity to the Salmonella pathogenicity island-2 class of T3SS and a putative urease pathogenicity island containing 9 genes were identified. When the T3SS is knocked out, the mutant retains its ability to enter catfish cell lines and head-kidney-derived-macrophages (HKDM), but is defective in intracellular replication. The mutant also invades catfish tissues in equal numbers to the wild-type (WT) E. ictaluri, but replicates poorly and is slowly cleared from the tissues, while the wild-type increases in number. Finally, quantitative PCR and western blotting identified low pH and phosphate limitation as conducive to expression of the E. ictaluri T3SS, growth conditions that mimic the phagosomal environment. Initial studies indicated that the urease enzyme is activated by low pH. When urea is available in the media at pH 5, WT E. ictaluri produces substantial quantities of ammonia, but no ammonia is produced by the urease mutant in the same conditions. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in minimal media, but, when exogenous urea is available, the ammonia produced elevates the environmental pH from pH 5 to pH 7, which is conducive to replication. Urease is not required for uptake or survival in HKDM or for initial invasion of catfish, but it is required for intracellular replication and proliferation in catfish tissues. Urea in HKDM appears to be provided by the macrophage arginase enzyme. Based on the data presented, a model for the early pathogenesis of E. ictaluri in HKDM will be presented. In addition, virulence factors that are likely involved in later stages of infection will be discussed. Although some things about these proteins can be predicted from the protein sequence and structure, further experimentation is required to determine their function.

Last Modified: 10/23/2014
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