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.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-01252006-184722 |
| Date | 26 January 2006 |
| Creators | Booth, Natha Joy |
| Contributors | John Hawke, Ronald Thune, Richard Cooper, Dean Shi, Philip Elzer |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-01252006-184722/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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