Whole Genome Sequencing of an Invasive Cbm⁺ Streptococcus mutans strain

Objectives: Streptococcus mutans, a well-known dental pathogen, has been consistently associated with extra-oral infections, such as infective endocarditis. To colonize oral and extra-oral tissues, S. mutans utilizes surface-associated proteins that tightly interact with host extracellular matrix (ECM) (e.g. collagen and laminin). Cnm and Cbm are examples of collagen-binding proteins (CBPs) which mediate adhesion to, and invasion of, host cells. However, while the whole genome sequence (WGS) of 8 Cnm⁺ strains is available, no Cbm⁺ strain has been sequenced to date. Here, we report the WGS and phenotypic characterization of the serotype e Cbm⁺ S. mutans LAR01 strain.

Methods: LAR01 chromosomal DNA was obtained and fully sequenced with the Real-Time Sequencing (SMRT) system. The genome was assembled, annotated and deposited in the NCBI/GenBank. Mutational analysis as well as heterologous expression of Cbm in Lactococcus lactis were performed, and the strains were tested for biofilm formation, stress tolerance, ECM binding, endothelial cell invasion, and virulence in Galleria mellonella.

Results: The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. It contains 15 PTS gene clusters, 7 surface-anchored (LPxTG) proteins, and genes encoding mutacins VI and K8. Like in Cnm⁺ strains, the cbm gene was found to be genetically linked to the pgf glycosylation machinery. When compared to UA159 strain, phenotypic characterization of LAR01 revealed increased biofilm formation and similar ability to withstand acid and oxidative stresses. Deletion of cbm significantly impaired the ability to bind to collagen and laminin, invade human coronary endothelial cells, and attenuated virulence in G. mellonella. Heterologous expression of Cbm in L. lactis revealed that Cbm is indeed a virulence factor.

Conclusions: This is the first study reporting the WGS of an invasive Cbm⁺ S. mutans strain. Collectively, our findings provide new insights on both the virulence properties and genetic variability present in S. mutans.