| The Gram-positive bacterium Streptococcus mutans is a human oral pathogen that is also associated with cardiovascular malignancies such as infective endocarditis and, possibly, atherosclerosis. Preliminary epidemiological investigations of primary human aortic valve replacement specimens detected S. mutans DNA in nearly 40% of examined samples suggesting a previous or current exposure of cardiac tissue to S. mutans. Adhesion to extracellular matrix (ECM) components (e.g. collagen and laminin) by S. mutans surface proteins is recognized to be an important colonization strategy in both oral and extra-oral infections. Specifically, S. mutans binding to collagen is mediated by Cnm, a bacterial surface collagen- and laminin-binding protein. The Cnm protein contains an N-terminal secretion signal, a collagen-binding domain, a unique threonine-rich B-domain and the C-terminal LPXTG cell wall anchoring motif. Previously, we showed that Cnm mediates invasion of various human cell types and enhances the virulence in the Galleria mellonella invertebrate model. The cnm gene is present in ~15% of all S. mutans isolates with an uneven distribution among the different serotypes. Analysis of the cnm genomic location in 6 cnm+ strains revealed that cnm is part of a conserved three-gene locus including two additional surface proteins (cnaB and cbpA) and is located immediately upstream pgfS, encoding a putative glycosyltransferase. In contrast to cnm, inactivation of cnaB, cbpA or both in the serotype f strain OMZ175 did not interfere with the ability of S. mutans to interact with various ECM proteins, invade human coronary artery endothelial cells (HCAECs), or kill G. mellonella larvae. Conversely, deletion of cnm in OMZ175, expression in the cnm- UA159 strain, or heterologous expression in Lactococcus lactis demonstrated that Cnm is responsible for robust ECM-binding, invasion of non-professional phagocytic cells and efficient killing of G. mellonella. Furthermore, we showed that ECM-binding, intracellular invasion and virulence of Cnm+ strains can be specifically blocked by anti-rCnmA antibodies. A closer analysis of the region downstream of cnm indicated that pgfS is the first gene of a four-gene operon (pgfS-pgfM1-pgfE-pgfM2) potentially associated with protein glycosylation. Lectin binding analysis of Cnm in the different pgf mutants confirmed that Cnm is a glycoprotein and that these genes are important for proper Cnm maturation. While Cnm glycosylation is predicted to occur at the threonine-rich domain, the extent of Cnm function and proteolytic stability was markedly affected by improper glycosylation. Our results indicate that Cnm, the first glycoprotein characterized in S. mutans, is a major virulence factor mediating important steps in colonization of oral and extra-oral infections. |
