Identifier

etd-01252006-184722

Degree

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

Document Type

Dissertation

Abstract

An Edwardsiella ictaluri strain with disruption of ureG was identified through the use of signature tagged insertion mutagenesis as being attenuated for virulence in the channel catfish host. Sequencing of the flanking regions surrounding the insert showed that the gene was part of a urease gene complex that included ureE, ureF, ureG, ureD, ureI, and an ammonium transporter homologue. The ureG gene encodes a GTP-binding accessory protein which is thought to function in energy-dependent urease assembly. The ureG mutant strain was found to be attenuated for mortality, persistence, and for the ability to establish infection in a competition challenge during co-infection with the wild type (WT) strain. In an experimental infection of channel catfish macrophages, the ureG mutant strain was attenuated for intracellular replication while the WT strain showed more than a ten-fold increase in numbers of viable organisms recovered at 12 hours post infection (PI), even though there were no significant differences in initial uptake of either strain. Light microscopy of prepared cell culture slides at 8 and 12 hours PI showed macrophages containing large numbers of WT bacteria, confirming the replication of the WT strain. A gentamicin exclusion assay performed with macrophages treated with 6 mM urea revealed that numbers of WT recovered from macrophages treated with urea was more than twice that recovered from macrophages without urea. Survival in macrophages requires the ability to tolerate or alter the acidic environment of the phagolysosome. Growth curves performed at acidic pH and survival assays following extreme acid shock indicate that E. ictaluri is naturally acid resistant and is able to utilize urea to enhance growth and replication at acidic pH by the neutralization of environmental pH. Two-dimensional gel analysis of WT cell lysates, prepared following growth at neutral and acidic pH, identified three urease proteins, UreA, UreC, and UreG, that were uniquely expressed during growth at acidic pH, indicating that expression of these proteins is acid inducible. The results presented here confirm the importance of the urease enzyme complex in virulence, intracellular survival, and acid resistance of E. ictaluri despite its "urease negative" characterization in traditional biochemical tests.

Date

2005

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Ronald Thune

DOI

10.31390/gradschool_dissertations.706

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