STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF CAZYME AND CAZYME-RELATED PROTEINS FROM: BACTEROIDES THETAIOTAOMICRON AND PORPHYROMONAS GINGIVALIS: TWO ABUNDANT COLONIZERS OF THE HUMAN MICROBIOME
Master of Science (MSc)
Biological and Chemical Sciences
Faculty of Science
Dr. Michael Suits
Provided project guidance, training and education, funding
The human body consists of approximately 30 trillion cells, while non-human microbes that reside on and within the body outnumber human somatic cells by a factor of 1.3 – 2.3. The interplay between our cells and those of the colonizing microorganisms affect physiology in a multitude of ways, both beneficial and detrimental. Microbes found in the oral cavity, such as the Red Complex member Porphyromonas gingivalis, are associated with pathology, namely periodontal diseases including gum deterioration, tooth decay, and loss of underlying alveolar bone. At the other end of the gastrointestinal tract, microbes such as Bacteroides thetaiotaomicron are found in abundance in the distal intestine and form symbiotic relationships that are critical for the proper function of the mammalian colon. Carbohydrates are integral for the survival of both of these organisms; as sources of energy, physical anchors, and barriers against environmental stress. As such, these microbes present interesting models for the study of extremely important host-microbial relationships involving carbohydrates and pathologic or beneficial mechanisms in carbohydrate metabolism.
The successful expression and purification of four homologous putative galactosidases from B. thetaiotaomicron, BT2109, BT2918, BT2966, and BT3158 are reported, along with BT2857, which has been expressed and purified previously. Crystallization and X-ray diffraction analyses of three of these full-length galactosidases was achieved, along with two truncations of BT2857 and one of BT3158. Functional kinetic characterization and generation of Michaelis-Menten constants was conducted using the galactose analogs para-nitrophenyl-β-galactopyranoside and para-nitrophenyl-α-galactopyranoside. Thin layer-chromatographic analysis using a variety of galactose-containing oligo/poly-saccharides was used to analyze degradative capability of these enzymes, along with nuclear magnetic resonance and mass spectrometric analysis to characterize the products of these degradative reactions.
The expression and purification of three proteins from P. gingivalis, PGN1176 (a short-chain fatty-acid dehydrogenase), PGN1459 (a putative DNA-defense and mutagenic protein), and PGN1461 (a putative spore-maturation protein, researched in collaboration with Katarina Mandić) are reported. Crystallization and preliminary X-ray diffraction of all three proteins was completed. Preliminary functional characterization of two proteins, PGN1459 and PGN1461, was conducted using a crude DNA-shift assay and thin-layer chromatographic analysis against a diverse array of (poly)saccharides, respectively.
Crystallization, diffraction, structural determination, and functional definition of each of these proteins is of critical importance in gaining insight into the roles these proteins play in either the symbiotic or pathogenic relationships between the human body and the resident organisms of the microbiome.
Stevenson, James, "STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF CAZYME AND CAZYME-RELATED PROTEINS FROM: BACTEROIDES THETAIOTAOMICRON AND PORPHYROMONAS GINGIVALIS: TWO ABUNDANT COLONIZERS OF THE HUMAN MICROBIOME" (2020). Theses and Dissertations (Comprehensive). 2260.