|Beekman, J -|
|Thune, R -|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 1, 2009
Publication Date: November 1, 2009
Citation: Booth, N.J., Beekman, J.B., Thune, R.L. 2009. Edwardsiella ictaluri Encodes an Acid Activated Urease that is Required for Intracellular Replication in Channel Catfish Ictalurus punctatus Macrophages. Applied and Environmental Microbiology. 75: 6712-6720. Interpretive Summary: Edwardsiella ictaluri has a urease enzyme that contributes to pathogenesis by altering environmental pH through a mechanism that involves excretion of ammonium produced by the breakdown of urea. Disruption of this enzyme renders the bacteria incapable of producing disease in channel catfish and inhibits replication of the bacterium in macrophages, the natural defence cells of the host. The urease enzyme is always expressed, but is activated by low pH. This enzyme probably enables transversal of the low pH digestive tract of the catfish host, and enables survival and replication of the bacteria inside macrophages by neutralizing the acidified phagocytic compartment in these cells where bacteria are normally destroyed.
Technical Abstract: Genomic analysis indicated that Edwardsiella ictaluri encodes a putative ureasepathogenicity island containing 9 open reading frames, including urea and ammonium transporters. In vitro studies with the wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly resistant to acid conditions, but that urease activity is not required for acid resistance. Growth studies, however, demonstrated that E. ictaluri is unable to grow at pH5 in the absence of urea, but is able to elevate the environmental pH from pH5 to pH7 and grow when exogenous urea is available. Additional data demonstrates substantial production of ammonia by wild-type E. ictaluri in the presence of urea at low pH, but none by the urease mutant. Results also indicate that the E. ictaluri urease has an optimal activity at pH2 to 3. Proteomic analysis with 2-D gel electrophoresis indicated that urease proteins are expressed at both pH5 and pH7, although urease activity is only detectable at pH5. In vivo data shows that urease was not required for initial invasion of catfish, but was required for subsequent proliferation and virulence. Ex vivo studies also indicated that urease was not required for initial uptake or survival in head kidney derived macrophages (HKDM), but was required for intracellular replication. Intracellular replication of the wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.