University of Western Ontario - Electronic Thesis and Dissertation Repository

Location of Thesis Examination

Room 6150 Support Services Building

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Dr. Miguel A. Valvano

Abstract

Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in cystic fibrosis and other immunocompromised patients. The bacterium survives within macrophages by interfering with typical endocytic trafficking, resulting in delayed maturation of a B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia alters gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation. Furthermore, we hypothesize that specialized bacterial secretion systems are involved in the interactions between intracellular bacteria and macrophages. In this work, we characterize later-stage infection of macrophages by B. cenocepacia, showing replication within an acidified endosomal compartment suggestive of a phagolysosome. We examine differential gene expression by intracellular B. cenocepacia using selective capture of transcribed sequences (SCOTS) with both competitive enrichment and microarray analysis. We identified 766 genes differentially regulated in intracellular bacteria, of which 329 were induced and 437 repressed. Affected genes are involved in all aspects of cellular life, including information storage and processing, cellular processes and signalling, and metabolism; in general, intracellular gene expression demonstrates a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. Deletion of various SCOTS-identified genes affects B. cenocepacia entry into macrophages and intracellular replication, as well as host-directed cytotoxicity and spread to neighbouring cells. Expression of secretion system genes is differentially-regulated by intracellular B. cenocepacia. Although none of the five major secretion systems are essential for growth in culture, we show that bacterial secretion systems are involved in macrophage entry, intracellular replication, and host-directed cytotoxicity. Type IV secretion systems play a role in early interactions with macrophages, while type II and IV secretion systems contribute to post-internalization intracellular replication and host-directed cytotoxicity. As a whole, secretion systems appear to increase pathogenicity in macrophages while limiting the spread of B. cenocepacia infection.

Together, these studies advance our understanding of the intracellular behaviour of B. cenocepacia in macrophages. Further investigation into the remaining SCOTS-identified genes, as well as putative secreted effectors, will provide a better understanding of the adaptive responses of intracellular B. cenocepacia, leading to life in a phagosomal niche and host cell cytotoxicity.