Advisory Committee Chair
Advisory Committee Members
Carlos J Orihuela
Date of Award
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Serine - rich repeat glycoproteins (SRRPs) play important roles in mediating bac-terial attachment to a variety of host and microbial surfaces. They have been im-plicated in the development of tissue infections such as pneumonia, infective en-docarditis, meningitis and oral infectious diseases. Streptococcus parasanguinis, one of the commensal streptococci present in the oral cavity, utilizes its fimbriae to mediate the initial attachment to the tooth pellicle. Fap1 (Fimbriae-associated-protein 1), the first identified SRRP, is the subunit of the fimbriae which utilizes proteins encoded by an 11-gene cluster (gly-gtf3-dGT1-galT2 and secY2-gap1-gap2-gap3-secA2-gtf1-gtf2) to mediate its glycosylation and secretion. The key enzymes responsible for the initial steps of the Fap1 glycosylation are Gtf12, Gtf3 and dGT1. They catalyze the transfer of GlcNAc, Glc, and both Glc and GlcNAc residues to the Fap1 polypeptide sequentially. The role of other two pu-tative glycosyltransferases, GalT2 and Gly in the Fap1 glycosylation is still un-known. In this study, we established a recombinant in vivo glycosylation system in Escherichia coli and synthesized the fully modified Fap1 glycan by incorporat-ing the necessary glycosyltransferases. Our study reported the first reconstitu-tion of an exogenous stepwise O-linked protein glycosylation synthetic pathway in E. coli. In addition, we have determined that GalT2 mediates the fifth step of the Fap1 glycosylation by adding a rhamnose residue, and Gly mediates the final glycosylation step by transferring glucosyl residues. Since the gene cluster in-volved in the biogenesis of SRRPs is highly conserved, we additionally character-ized the glucosyltransferase GtfC which is important for the glycosylation of Srr2, a SRRP from Streptococcus agalactiae COH1. We further identified a conserved loop region that is crucial for acceptor substrate binding of GtfC homologs in streptococci through structural and biochemical analysis. Taken together, these findings shed new insight into the mechanism of SRRP biogenesis, which is vital in designing novel antibacterial agents.
Zhu, Fan, "Glycosyltransferases Involved In The Glycosylation Of Serine-Rich Glycoproteins From Streptococci" (2016). All ETDs from UAB. 3457.