Date of Award

2011

Degree Type

Thesis

Degree Name

Master of Science

Program

Microbiology and Immunology

Supervisor

Dr. Miguel Valvano

Abstract

Burkholderia cepacia complex bacteria are important opportunistic pathogens in cystic fibrosis patients and are associated in some cases with acute necrotizing pneumonia, sepsis, and increased risk of death. B. cenocepacia and other Burkholderia are commonly found in the environment, particularly in the rhizosphere. It is not surprising that they have evolved high resistance against several antimicrobial agents, as well as the ability to interact with multiple hosts, including the ability to survive intracellularly in amoebae, macrophages, and epithelial cells. Like most opportunistic pathogens, B. cenocepacia must adapt to environmental stresses and have the ability to persist in the infected host. Persistence is in part associated to the bacterial ability to control cell division. We have identified two cell wall hydrolases, BCAL1938 and BCAL2128, with high sequence similarity to a Mycobacterium tuberculosis protein known to be essential for later stages of cell division. These proteins belong to the NlpC/P60 family of endopeptidases that chiefly function to modify peptidoglycan during growth, but are often required for cell wall turnover, separation of daughter cells during cell division, biofilm formation, bacterial adhesion, and motility. These proteins could contribute directly to B. cenocepacia pathogenicity and may also be required for revival from a resting state during infection. In other bacteria, including M. tuberculosis,

NlpC/P60 proteins interact with resuscitation promoting factors (RPFs) that mediate resuscitation from a dormant state. B. cenocepacia mutant strains not expressing BCAL1938 and BCAL2128 show growth defects characterized by aggregation of cells and the formation of long chains. BCAL2128 mutant strains are sensitive to high osmotic pressure, oxidative stress, detergents, and cell-wall acting antibiotics. BCAL1938 mutant

iii

strains have a defect in O antigen production and are sensitive to polymyxin. We also have evidence that these predicted NlpC/P60 proteins are able to degrade peptidoglycan in vitro and thus might be involved in cell wall remodeling. Therefore, the potential necessity and non-redundancy of these proteins in B. cenocepacia during adaptive responses make them novel putative targets for antimicrobial drug development.

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