The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells

Abstract

Streptococcus mutans is considered the primary etiologic agent of dental caries, a global health problem that affects 60 to 90% of the population, and a leading causative agent of infective endocarditis. It can be divided into four different serotypes (c, e, f, and k), with serotype c strains being the most common in the oral cavity. In this study, we demonstrate that in addition to OMZ175 and B14, three other strains (NCTC11060, LM7, and OM50E) of the less prevalent serotypes e and f are able to invade primary human coronary artery endothelial cells (HCAEC). Invasive strains were also significantly more virulent than noninvasive strains in the Galleria mellonella (greater wax worm) model of systemic disease. Interestingly, the invasive strains carried an additional gene, cnm, which was previously shown to bind to collagen and laminin in vitro. Inactivation of cnm rendered the organisms unable to invade HCAEC and attenuated their virulence in G. mellonella. Notably, the cnm knockout strains did not adhere to HCAEC as efficiently as the parental strains did, indicating that the loss of the invasion phenotype observed for the mutants was linked to an adhesion defect. Comparisons of the invasive strains and their respective cnm mutants did not support a correlation between biofilm formation and invasion. Thus, Cnm is required for S. mutans invasion of endothelial cells and possibly represents an important virulence factor of S. mutans that may contribute to cardiovascular infections and pathologies.

Fig. 1.

Invasive properties of Streptococcus mutans strains isolated from dental plaque (A) and from blood of patients with bacteremia and/or endocarditis (B). The number of S. mutans CFU recovered from the intracellular compartment of HCAEC after 5 h of infection is shown. The serotypes of the strains are indicated (c, e, or f). The data represent the averages and standard deviations (SD) for at least three independent experiments.

Fig. 2.

Killing of G. mellonella larvae infected with the noninvasive strain UA159 and the invasive strains OMZ175, B14, OM50E, LM7, and NCTC11060 of S. mutans. The experiments were repeated three times, and the results are representative of a typical experiment.

Fig. 3.

(A) Detection of the cnm gene by PCR reveals that cnm is found only in invasive strains. (B) Invasion of HCAEC by cnm knockout strains. The number of S. mutans CFU recovered from the intracellular compartment of HCAEC after 5 h of infection is shown. The data represent the averages and SD for at least three independent experiments.

Fig. 4.

Adherence of S. mutans to HCAEC. Data shown represent percentages of adherent cells in relation to the initial bacterial inoculum. #, statistically significant difference between the noninvasive strains UA159, 19, and 1237-00 and the invasive strain OMZ175 (P < 0.01); *, statistically significant difference (P < 0.01) between the cnm knockout strains and their respective parental strains. The data represent the averages and SD for at least three independent experiments.

Fig. 5.

(A) Killing of G. mellonella larvae infected with the noninvasive strain UA159 (□), the invasive strain OMZ175 (⧫), and the cnm knockout strain OMZ175-cnm (•). The experiments were repeated three times, and the results are representative of a typical experiment. (B) Wax worms infected with UA159, OMZ175, and OMZ175-cnm in a typical experiment at 24 h postinjection.

Fig. 6.

Biofilm assays of cells grown in LMW supplemented with 1% sucrose (A) and 1% glucose (B) on the surfaces of 96-well microtiter plates. Results shown are averages for three separate experiments plus SD. Statistically significant differences between strains were assessed by Student's t test and are indicated by asterisks.

Fig. 7.

Expression of cnm in trans partially restored the invasive phenotype (A) and virulence (B) of OMZ175-cnm.