Date of Award

2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Molecular Genetics

First Advisor

Keith P. Mintz

Abstract

The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host'pathogen interactions. Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function. The MorC sequence was determined to be conserved in Gammaproteobacteria. Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A. actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. MorC from all organisms restored at least one of the A. actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria. Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane. The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant. Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains. A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools. All proteins, except MorC, were detected in the mutant strain. However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain. These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems.

One protein found to be reduced was a component of the fimbrial secretion system of A. actinomycetemcomitans. The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study. Therefore, the defect in fimbriation was identified and complemented in trans. The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy. The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes. The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate. The mutant strain displayed phenotypes typically associated with inactivation of morC. However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent. Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy.

MorC plays a critical role in maintaining secretion of major virulence determinants of A. actinomycetemcomitans. Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology. The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria. This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion.

Language

en

Number of Pages

225 p.

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Microbiology Commons

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