Date of Award

2009

Degree Name

Biomedical Sciences

College

College of Science

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Hongwei Yu

Second Advisor

Philippe Georgel

Third Advisor

Todd Green

Fourth Advisor

Vernon Reichenbecher

Fifth Advisor

Donald Primerano

Abstract

Pseudomonas aeruginosa (P.a.) is a major threat to the health and well being of a person living with cystic fibrosis (CF). P.a. is capable of conversion to mucoidy or alginate overproducing phenotype. Mucoidy facilitates the chronic infection of P.a. infection in the CF lung. Mutations in the anti-sigma factor mucA, result in activation of the sigma factor AlgU which promotes expression of alginate through the alginate biosynthetic operon. However, P.a. can produce alginate independent of mucA mutation via a mechanism known as regulated proteolysis. Proteases AlgW and MucP can degrade MucA to liberate and activate AlgU. In this dissertation, two pathways to alginate production will be characterized through investigation of two negative regulators of alginate production: KinB and MucD. KinB is a histidine kinase which controls alginate production through two transcription factors: AlgB and RpoN along with the MucA protease AlgW. In the absence of KinB, the degradation rate of MucA is elevated which results in alginate production. MucD is a periplasmic protease that is presumed to degrade protein signals in the periplasm. It is hypothesized that if MucD does not degrade certain protein signals then they will accumulate and activate alginate production. Here, it is presented that in the absence of MucD, the intramembrane protease MucP activates degradation of MucA. To further characterize MucD, the roles of the PDZ domains in suppression of alginate production were probed. PDZ domains are conserved protein domains that function in protein to protein interaction. The PDZ domains of MucD were found to be dispensable for suppression of alginate production but required for the stability of MucD. When regulated proteolysis is activated, AlgU promotes expression of many genes. One such gene is LptF or Lipotoxin F. Here it is shown that in the absence of kinB, LptF is highly expressed. LptF was shown to be an outer membrane protein, but it is not involved in production of alginate. However, LptF mediates adhesion to lung epithelia and resistance to reactive oxygen species. P.a. is capable of activating AlgU through MucA proteolysis by several mechanisms that are characterized in this dissertation. Through elucidation of these pathways, we will be more prepared to combat alginate production and P.a. infection of the CF lung.

Subject(s)

Pseudomonas aeruginosa.

Cystic fibrosis.

Pseudomonas aeruginosa infections - Research.

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