Identifier

etd-10132011-154436

Degree

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

Document Type

Dissertation

Abstract

Edwardsiella ictaluri encodes a urease operon and an arginine decarboxylase (AdiA) that are required for virulence in head kidney derived macrophages (HKDM). The urease produces ammonia in amounts sufficient to alter environmental pH from acid to neutral. A hypothetical model was proposed, involving arginine metabolism in E. ictaluri infected HKDM, focusing on bacterial urease, AdiA, a second arginine decarboxylase (SpeA), and agmatinase (SpeB). Using fluorescence based ratiometric pH determination of E. ictaluri in live HKDM, it was shown that E. ictaluri modulates HKDM phagosome pH to above six. Urease and AdiA mutants failed to up-regulate vacuole pH, while vacuole pH for the SpeA and SpeB mutants was similar to the wild-type. These mutants could also replicate in HKDM similar to wild type E. ictaluri. These data show that urease and AdiA are required for phagosome pH neutralization. To determine the source of urea for E. ictaluri’s urease, an arginase inhibitor, L-norvaline, was used to partially block HKDM urea production. In arginase inhibited HKDM, E. ictaluri could not neutralize phagosome pH, nor could it replicate. Nitric oxide production in HKDM was not significantly different between controls and experimental groups. This indicates that HKDM have limited capacity to produce NO. Levels of urea produced in infected and control HKDM were at the lowest limit of assay detection and were not significantly different from one another. Together, these data show that E. ictaluri uses its urease and AdiA to neutralize phagosome pH, and that it uses urea derived from HKDM arginase to do so.

Date

2011

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Thune, Ronald

Share

COinS