Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans

Abstract

We report the whole genome sequence of the serotype e Cbm⁺ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra-oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall-anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram-positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.

Keywords: Streptococcus mutans; Adhesins; collagen-binding proteins; dental caries; genome; intracellular invasion.

Figure 1

Circular representation of the genome of Streptococcus mutans LAR01. Outermost circle indicates the distances from the putative origin of replication. Blue circle represent genes in the forward (+) and red circle represent genes in the reverse strand (−). Secreted genes are represented in green whereas orange indicated genes that are unique to LAR01 in relation to UA159. Innermost circle represents the % G + C with more and less than average shown as light blue and purple, respectively.

Figure 2

Genetic arrangement of cbm locus in LAR01 compared to the cbm⁻/cnm⁻ strain UA159 and the cnm⁺ OMZ175. Like cnm in OMZ175, cbm is flanked by the pgf glycosylation machinery in LAR01. Interestingly, the last two genes of the pgf system (pgfE-pgfM2) are separated by an insertion of 29.1 Kb that contain genes coding for a putative type VII secretion system (T7SS).

Figure 3

Comparative analysis of the expression of virulence-related traits in LAR01, OMZ175 and UA159. (A) 24h biofilm formation of strains UA159, OMZ175 and LAR01 on the surface of microtiter plates in the presence of glucose or sucrose. (B) Percent survival of unadapted (open symbols) and acid-adapted (closed symbols) cells of UA159, OMZ175 and LAR01 after exposure to pH 3.0. (C) Percent survival of UA159, OMZ175 and LAR01 exposed to 0.2% H₂O₂. (D) Growth competition between S. mutans strains. Absence of growth inhibition after addition of aminopeptidase confirmed that inhibition zones were due to mutacin activity. (E–F) Deferred antagonism assays. Cultures of S. mutans were grown on BHI agar plates for 24 h incubation and a soft BHI agar overlay containing S. gordonii (E) or L. lactis (F) cells was incubated for additional 48 h before zone of inhibition was recorded. All experiments were performed at least in triplicate and asterisks indicate statistical significance (p ≤ 0.05).

Figure 4

Phenotypic characterization of the Δcbm mutant in LAR01. (A) Western blot analysis using anti-Cnm antibody. (B) Binding to collagen type I and laminin in vitro. (C) Invasion of HCAECs and (D) killing of G. mellonella. All experiments were performed at least in triplicate and asterisks indicate statistical significance (p ≤ 0.05) between Δcbm and the wild-type LAR01 or between Δcbm and the genetically complemented Δcbm (CΔcbm).

Figure 5

Heterologous expression of Cbm in L. lactis NZ9800. (A) Immunofluorescence microscopy analysis demonstrated that Cbm was effectively expressed and translocated to the cell surface of L. lactis. (B) Expression of Cbm in L. lactis enables binding to collagen type I. (C) Expression of Cbm in NZ9800 contributes to systemic virulence in G. mellonella. (*) p ≤ 0.05.