Adherence and Antibiotic Protection Assay: Human cells were infected with wildtype strains of S.mutans and their corresponding cnm mutants: OMZ175, OM50E, LM7, NCTC11060. S.mutans UA159, a natural cnm negative strain, was used as a negative control. For adherence, human cells were infected at MOI 100 and incubated for 30 min at 4oC. For invasion, human cells were infected at MOI 100 for 2 hours, washed, antibiotic treated for 3 hours to kill extracellular bacteria. Cells were lysed, serially diluted and plated on BHI, and CFUs determined.
Quantitative PCR: Antibiotic protection assay with strain OMZ175 was completed, except cells were incubated for 5, 10, 24, and 48 hours and treated with antibiotic containing media continuously or pulsed with antibiotic for three hours only. Genomic DNA was extracted using QIAamp DNA Mini Kit. qPCR was used to measure relative quantities of 18S and 16S with iQ SYBR-green supermix.
Result: All S.mutans strains were able to adhere to CASMC. Wildtype S.mutans strains expressing cnm were more successful at invading CASMC by ~2 logs than the cnm mutants and UA159. qPCR results suggest that OMZ175 exits the host by 24 hours. There was no evidence of OMZ175 degradation or loss after 48 hours, and intracellular replication could not be confirmed.
Conclusion: Collagen binding protein, Cnm, facilitates S.mutans invasion of human cardiovascular smooth muscle cells and may be a contributing factor to cardiovascular disease. Future experiments will investigate intracellular bacterial replication and host cell exit strategies.
Keywords: Adherence and colonization, Bacterial, Cardiovascular disease, Invasion and Oral biology