Modification of Streptococcus mutans Cnm by PgfS contributes to adhesion, endothelial cell invasion, and virulence.
Expression of the surface protein Cnm has been directly implicated in the ability of certain strains of Streptococcus mutans to bind to collagen and to invade human coronary artery endothelial cells (HCAEC) and in the killing of Galleria mellonella. Sequencing analysis of Cnm(+) strains revealed that cnm is located between the core genes SMU.2067 and SMU.2069. Reverse transcription-PCR (RT-PCR) analysis showed that cnm is cotranscribed with SMU.2067, encoding a putative glycosyltransferase referred to here as PgfS (protein glycosyltransferase of streptococci). Notably, Cnm contains a threonine-rich domain predicted to undergo O-linked glycosylation. The previously shown abnormal migration pattern of Cnm, the presence of the threonine-rich domain, and the molecular linkage of cnm with pgfS lead us to hypothesize that PgfS modifies Cnm. A ΔpgfS strain showed defects in several traits associated with Cnm expression, including collagen binding, HCAEC invasion, and killing of G. mellonella. Western blot analysis revealed that Cnm from the ΔpgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ΔpgfS was highly susceptible to proteinase K degradation, in contrast to the high-molecular-weight Cnm version found in the parent strain. Lectin-binding analyses confirmed the glycosylated nature of Cnm and strongly suggested the presence of N-acetylglucosamine residues attached to Cnm. Based on these findings, the phenotypes observed in ΔpgfS are most likely associated with defects in Cnm glycosylation that affects protein function, stability, or both. In conclusion, this study demonstrates that Cnm is a glycoprotein and that posttranslational modification mediated by PgfS contributes to the virulence-associated phenotypes linked to Cnm.
Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Alejandro
A
Avilés-Reyes
Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
James H
JH
Miller
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA Department of Medicine/Hematology-Oncology Division, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Patricia J
PJ
Simpson-Haidaris
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Fred K
FK
Hagen
Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA jacqueline_abranches@urmc.rochester.edu jose_lemos@urmc.rochester.edu.
Jacqueline
J
Abranches
Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA jacqueline_abranches@urmc.rochester.edu jose_lemos@urmc.rochester.edu.
José A
JA
Lemos
D018829:Adhesins, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism; D003331:Coronary Vessels; D042783:Endothelial Cells / Q000382:microbiology; D008040:Genetic Linkage; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism; D009036:Moths; D010641:Phenotype; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D059748:Proteolysis; D017384:Sequence Deletion; D013290:Streptococcal Infections / Q000382:microbiology; D013295:Streptococcus mutans / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014774:Virulence
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; C488758:Cnm protein, Streptococcus mutans; D037102:Lectins; D003094:Collagen; D016695:Glycosyltransferases