Essential to oral biofilm development is the initial colonization by oral streptococci. The abundant oral streptococci prevent pathogens from arising in numbers that tips the balance to a pathogenic state. We have used an oral streptococcus Streptococcus parasanguinis as a model to study bacterial adhesion and colonization and identified a new family of bacterial serine-rich repeat protein (SRRP) named a fimbriae- associated protein-Fap1. Previous funding support has led to the discovery of a new glycosylation and secretion pathway responsible for biogenesis of Fap1. Biogenesis of Fap1 is controlled by a gene cluster encoding glycosyltransferases and accessory secretion proteins. Fap1-like SRRPs are highly conserved in Gram-positive bacteria and play an important role in bacterial colonization and virulence. We have generated genetic and biochemical tools and established Fap1 as a model system to investigate this important family of SRRP adhesins. In our study of Fap1 glycosylation, we have identified two putative glycosyltransferases (Gtf), Gtf1 and Gtf2 that are required for the first step of Fap1 glycosylation, as well as additional glycosyltransferases GalT1, GalT2 and Gly that are responsible for the subsequent Fap1 glycosylation steps. Gtf2 interacts with Gtf1 and functions as a molecular chaperone to enhance glycosyltransferase activity of Gtf1. GalT1 contains a novel domain of unknown function (DUF1792) that possesses glycosyltransferase activity. However the molecular events underlying how they contribute to Fap1 glycosylation is unknown. In our study of Fap1 secretion, we identified a protein complex consisting of two distinct glycosylation associated proteins, Gap1 and Gap3, which regulate Fap1 biogenesis. Gap3 is a lectin that organizes the accessory secretion protein complex. Gap1 is a putative glycosyltransferase that interacts with Gap3, and also stabilizes Gap3. However the biochemical function of the complex is unknown.
Two specific aims are proposed:
Aim 1 Dissect Fap1 glycosylation steps and determine the contribution of glycosyltransferases. We will determine A) how Gtf2 modulates Gtf1 by solving 3D structure of Gtf2 and define the underlying mechanism how Gtf2 modulates Gtf1;B) how DUF1972 functions as a new glycosyltransferase by solving its 3-D structure;C) how GalT2 and Gly contribute to Fap1 glycosylation.
Aim 2 Define the role of the glycosylation-associated protein complex in Fap1 biogenesis. We will determine A) the role of Gap3 in Fap1 biogenesis as a lectin;B) the engagement of Gap1 in Fap1 biogenesis via three interactive mechanisms, 1) glycosylation of Gap3;2) interaction with Gap3;and 3) prevention of Gap3 from proteolytic degradation since the presence of Gap1 stabilizes Gap3. As biogenesis of SRRPs is highly conserved, deciphering the molecular details that modulate bacterial adhesion by the Fap1 biogenesis will provide new information useful for the design of strategies to maintain the oral cavity healthy, as well as aid in the design of novel therapeutics that control pathogens that use SRRPs.

Public Health Relevance

Dental plaque formation is fundamental for oral health and disease. Oral microbial homeostasis is not only important for oral health but also affect systemic health. Our proposed studies will investigate streptococcal glycosylation and secretion mechanisms that are crucial for streptococcal biofilm formation and colonization. The information generated from the studies will help us to device new strategies to maintain healthy oral environment, thereby inhibiting pathogenic biofilm formation and preventing bacterial infections.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Oral, Dental and Craniofacial Sciences Study Section (ODCS)
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Lunsford, Dwayne
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University of Alabama Birmingham
Schools of Dentistry
United States
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Zhang, Hua; Zhu, Fan; Yang, Tiandi et al. (2014) The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold. Nat Commun 5:4339
Shi, Wei-Wei; Jiang, Yong-Liang; Zhu, Fan et al. (2014) Structure of a novel O-linked N-acetyl-D-glucosamine (O-GlcNAc) transferase, GtfA, reveals insights into the glycosylation of pneumococcal serine-rich repeat adhesins. J Biol Chem 289:20898-907
Zhu, Fan; Wu, Ren; Zhang, Hua et al. (2013) Structural and biochemical analysis of a bacterial glycosyltransferase. Methods Mol Biol 1022:29-39
Echlin, Haley; Zhu, Fan; Li, Yirong et al. (2013) Gap2 promotes the formation of a stable protein complex required for mature Fap1 biogenesis. J Bacteriol 195:2166-76
Zhang, Hua; Zhu, Fan; Ding, Lei et al. (2013) Preliminary X-ray crystallographic studies of an N-terminal domain of unknown function from a putative glycosyltransferase from Streptococcus parasanguinis. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:520-3
Zhou, Meixian; Zhu, Fan; Li, Yirong et al. (2012) Gap1 functions as a molecular chaperone to stabilize its interactive partner Gap3 during biogenesis of serine-rich repeat bacterial adhesin. Mol Microbiol 83:866-78
Zhu, Fan; Erlandsen, Heidi; Ding, Lei et al. (2011) Structural and functional analysis of a new subfamily of glycosyltransferases required for glycosylation of serine-rich streptococcal adhesins. J Biol Chem 286:27048-57
Liang, Xiaobo; Chen, Yi-Ywan M; Ruiz, Teresa et al. (2011) New cell surface protein involved in biofilm formation by Streptococcus parasanguinis. Infect Immun 79:3239-48
Wu, Ren; Wu, Hui (2011) A molecular chaperone mediates a two-protein enzyme complex and glycosylation of serine-rich streptococcal adhesins. J Biol Chem 286:34923-31
Zhou, Meixian; Zhang, Hua; Zhu, Fan et al. (2011) Canonical SecA associates with an accessory secretory protein complex involved in biogenesis of a streptococcal serine-rich repeat glycoprotein. J Bacteriol 193:6560-6

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