Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Dr. Miguel A. Valvano

Abstract

Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes chronic respiratory infection in cystic fibrosis (CF) patients, leading to rapid decline of lung function. CF patients harbour mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride (Cl) transport channel. B. cenocepacia survive in macrophages by delaying phagosome maturation and exaggerate this strategy in CFTR-defective macrophages. Neutrophils of CF patients display defective Cl transport resulting in impaired chlorination of bacteria via hypochlorous acid (HOCl), resulting in reduced bacterial clearance. I hypothesize that macrophages with dysfunctional CFTR lack sufficient Cl ion levels in their phagosomes for the chlorination of engulfed B. cenocepacia and subsequent clearance of the infection. B. cenocepacia were labelled with Cl-sensitive and -insensitive fluorophores to measure the intraphagosomal Cl ion concentration in infected CFTR-normal and -defective murine macrophages and no significant difference in the Cl ion level was found at 1, 4 and 6 hours post-infection. At 6, 8 and 10 hours post-infection, there was also no significant difference in the survival rate of phagocytosed B. cenocepacia in the two cell types exposed to Cl. HOCl production was not detected in the lysates of the two cell types suggesting that this bactericidal mechanism employed by neutrophils is absent or undetectably low. Our results, showing no significant difference in bacterial survival are consistent with the lack of HOCl production. A role for Cl ions in connection with bacterial clearance from the macrophages examined remains to be elucidated.

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