DOI
https://doi.org/10.25772/PB85-BE11
Defense Date
2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Oral Health Research
First Advisor
Dr. Chunhao Li
Abstract
Sialic acids (SA) are complex sugars critical for self-recognition, a feature exploited by pathogens by deploying sialidases, a class of enzymes that cleave SA from host glycoconjugates. Previously, we found that Porphyromonas gingivalis, an agent of periodontal disease, produces a conserved sialidase (PG0352) that was critical for its pathogenicity. The current studies expand on previous findings with a newly constructed PG0352-complemented strain (CΔPG0352) using a multi-disciplinary approach involving cryo-electron microscopy (cryo-EM), glycobiology, eukaryotic cell culture, enzyme kinetics, and x-ray crystallography. Cryo-EM revealed ΔPG0352 had defective capsule formation which was restored in CΔPG0352. Serum killing assays revealed complementation of PG0352 restored serum resistance. Mechanistically, PG0352 disarms the host complement system by desialylating key factors which attenuated its bactericidal activity. Neutrophil killing assays revealed ΔPG0352 was more sensitive to neutrophil killing and had impaired intracellular survival. PG0352 harbors two functional units: an N-terminal domain (PGD) of unknown function and a conserved C-terminal sialidase domain. We present a 1.84Å crystal structure of PG0352 without its signal peptide. Biochemical analysis suggest PGD is a carbohydrate binding module (CBM) not required for sialidase activity. Functional analysis reveals PG0352 has a broad pH range for activity, with optimal activity at pH 5. Kinetic studies indicate PG0352 demonstrates higher affinity towards N-acetylneuraminic acid than N-glycolylneuraminic acid. Parallel studies with a sialidase-deficient mutant (ΔPGN1608) and complemented strain (CΔPGN1608) in ATCC33277 revealed ΔPGN1608 produces a thinner non-capsule electron dense surface layer, has impaired biofilm formation, increased susceptibility to serum killing, and diminished neutrophil resistance which was rescued in CΔPGN1608.
Rights
© Christopher Khoa Pham
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
7-17-2023