Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division

Author:

Andresen Silke12,de Mojana di Cologna Nicholas3,Archer-Hartmann Stephanie1,Rogers Ashley M12,Samaddar Sandip3,Ganguly Tridib3,Black Ian M1,Glushka John1,Ng Kenneth K S4,Azadi Parastoo1,Lemos José A3,Abranches Jacqueline3,Szymanski Christine M12

Affiliation:

1. University of Georgia Complex Carbohydrate Research Center, , Athens, GA 30602 , USA

2. University of Georgia Department of Microbiology and Complex Carbohydrate Research Center, , Athens, GA 30602 , USA

3. University of Florida Department of Oral Biology, College of Dentistry, , Gainesville, FL 32603 , USA

4. University of Windsor Department of Chemistry and Biochemistry, , Windsor, ON N9B 3P4 , Canada

Abstract

Abstract Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

Funder

NIH

Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, U.S. Department of Energy

NIDCR

Publisher

Oxford University Press (OUP)

Subject

Biochemistry

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