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Applied and Environmental Microbiology, November 2009, p. 6712-6720, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01670-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (Ictalurus punctatus) Macrophages{triangledown}

Natha J. Booth,1,{dagger} Judith B. Beekman,1,2 and Ronald L. Thune1,2*

Department of Veterinary Science, Louisiana State University Agricultural Center, Louisiana State University, Baton Rouge, Louisiana 70803,1 Department of Pathobiological Sciences, School of Veterinary Medicine, Skip Bertman Drive and River Road, Louisiana State University, Baton Rouge, Louisiana 708032

Received 14 July 2009/ Accepted 4 September 2009

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of 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.

* Corresponding author. Mailing address: LSU School of Veterinary Medicine, Skip Bertman Drive and River Rd., Baton Rouge, LA 70803. Phone: (225) 578-9680. Fax: (225) 578-9701. E-mail: thune{at}vetmed.lsu.edu

{triangledown} Published ahead of print on 11 September 2009.

{dagger} Present address: USDA-ARS Catfish Genetics Research Unit, P.O. Box 38, Stoneville, MS 38776.

Applied and Environmental Microbiology, November 2009, p. 6712-6720, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01670-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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