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

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 four 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 to characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only two 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, NMR, 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.

O-glycosylation of intrinsically disordered regions regulates homeostasis of membrane proteins in streptococci

“…In S. pyogenes and S. pneumoniae , we elucidated a novel Glc-type O -linked glycosylation mechanism in which glycosyltransferase GtrB initiates the synthesis of a Glc-modified bactoprenol-phosphate lipid molecule for the transfer of Glc to extracytoplasmic proteins harboring the Ser/Thr-rich IDRs. Previous studies have reported that S. mutans possesses the O -linked protein glycosylation system encoded by the Pgf gene cluster which covalently links N-acetylhexosamine sugars, presumably GalNAc and GlcNAc, with the cell wall-associated adhesins Cnm and WapA 32, 33 . Like GtrB in S. pyogenes and S. pneumoniae , the Pgf-encoded glycosyltransferase, PgfS, initiates the protein glycosylation pathway 27, 28 .…”

“…Pgf pathway glycosylates PrsA, PBP1a, FtsQ and PknB with GalNAc in S. mutans Contrary to S. pyogenes and S. pneumoniae, no ConA-bound proteins were recovered from solubilized extracts of the S. mutans membranes. However, S. mutans possesses a protein glycosylation machinery that decorates the cell wall-associated collagenbinding adhesins WapA and Cnm with O-N-acetylhexosamines, presumably Nacetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) 32,33 . O-Glycosylation of WapA and Cnm requires the participation of the Pgf gene cluster encoded enzymes: the GtrB-type glycosyltransferase PgfS, an epimerase PgfE, and two GT-C fold glycosyltransferases PgfM1 and PgfM2.…”

Galleria mellonella—A Model for the Study of aPDT—Prospects and Drawbacks

“…In summary, G. mellonella has been used for testing many infections caused by different gram-positive and gram-negative bacteria. Among gram-positive microorganisms, Staphylococcus aureus [ 71 , 72 , 73 , 74 , 75 , 76 , 77 ], Streptococcus pyogenes [ 78 , 79 , 80 ], Streptococcus pneumoniae [ 81 , 82 , 83 ], Streptococcus mutans [ 19 , 20 , 84 , 85 , 86 ], L. monocytogenes [ 4 , 44 , 65 , 66 , 87 , 88 ], Enterococus faecalis [ 89 , 90 , 91 , 92 ], Enterococcus faecium [ 93 , 94 , 95 , 96 ], L. rhamnosus GG [ 63 , 64 ], C. butyricum Miyairi 588 [ 63 ], C. perfringens [ 67 , 68 ], Mycobacterium bovis [ 23 ], Mycobacterium abscessus [ 97 , 98 ], and Mycobacterium tuberculosis [ 99 , 100 , 101 , 102 ] were mentioned. Among gram-neg...…”

Post‐translational modification by the Pgf glycosylation machinery modulates Streptococcus mutans OMZ175 physiology and virulence

“…Our group demonstrated that the Pgf machinery is required for the post-translational glycosylation of both core (WapA) and non-core (Cnm) LPXTG-anchored surface proteins (Avilés-Reyes et al, 2018). In addition to the LPXTG anchoring motif, Cnm possesses an amyloidogenic β-sheet-rich Collagen-Binding Domain (CBD) (Cologna et al, 2021) and a Threonine-Rich Repeat Region (TRRR) where O-glycosylations with HexNAc 2 moieties (presumably GalNAc and GlcNAc) have been identified (Andresen et al, 2023). Deletion of the pgf genes in OMZ175 results in a reduction of both Cnm and WapA molecular weights and stability.…”

“…Moreover, these deletions significantly hinder Cnmassociated virulence traits such as binding to collagenous surfaces, invasion of endothelial and epithelial cells, and killing of Galleria mellonella, an invertebrate model of systemic infection (Avilés-Reyes, Miller, Simpson-Haidaris, Hagen, et al, 2014;Avilés-Reyes et al, 2018). Furthermore, apart from Cnm and WapA, we have recently identified two other potential targets of Pgf glycosylation: the surface-anchored SpaP (also known as Antigen I/II or P1) and the glucosyltransferase GtfC, which is involved in glucan biosynthesis as part of sucrose-dependent biofilm formation (Andresen et al, 2023).…”