Effects of oxygen on virulence traits of Streptococcus mutans

Abstract

Oxygen profoundly affects the composition of oral biofilms. Recently, we showed that exposure of Streptococcus mutans to oxygen strongly inhibits biofilm formation and alters cell surface biogenesis. To begin to dissect the underlying mechanisms by which oxygen affects known virulence traits of S. mutans, transcription profiling was used to show that roughly 5% of the genes of this organism are differentially expressed in response to aeration. Among the most profoundly upregulated genes were autolysis-related genes and those that encode bacteriocins, the ClpB protease chaperone subunit, pyruvate dehydrogenase, the tricarboxylic acid cycle enzymes, NADH oxidase enzymes, and certain carbohydrate transporters and catabolic pathways. Consistent with our observation that the ability of S. mutans to form biofilms was severely impaired by oxygen exposure, transcription of the gtfB gene, which encodes one of the primary enzymes involved in the production of water-insoluble, adhesive glucan exopolysaccharides, was down-regulated in cells growing aerobically. Further investigation revealed that transcription of gtfB, but not gtfC, was responsive to oxygen and that aeration causes major changes in the amount and degree of cell association of the Gtf enzymes. Moreover, inactivation of the VicK sensor kinase affected the expression and localization the GtfB and GtfC enzymes. This study provides novel insights into the complex transcriptional and posttranscriptional regulatory networks used by S. mutans to modulate virulence gene expression and exopolysaccharide production in response to changes in oxygen availability.

FIG. 1.

Distribution of functions of genes affected by oxygen availability. The 106 genes differentially expressed at P ≤ 0.001 were grouped by functional classification according to the Los Alamos S. mutans genome database (http://www.oralgen.lanl.gov/).

FIG. 2.

CAT assay. The gtfB and gtfC promoter fusions with the cat gene were inserted in a single copy into the chromosome of the wild type, and the resulting strains were designated TW54 (PgtfB) and TW55 (PgtfC), respectively. The strains were grown under aerobic or anaerobic conditions. See the text for more details. The data presented are means ± standard deviations (error bars) of at least three independent experiments. *, P < 0.01 (Student's t test).

FIG. 3.

Western blot analysis of different cell extracts from wild-type S. mutans grown under anaerobic (AN) or aerobic (AE) conditions, i.e., bead-beaten SDS boiling extract (whole-cell lysates, A), 4% SDS extracts (B), bead-beaten Tris extracts (soluble fraction, C), the insoluble fraction (D), and the supernatant fraction (E). See Materials and Methods for more details about the preparation of the cellular fractions. Following SDS-PAGE, proteins were blotted onto a nitrocellulose membrane and subjected to Western blotting with an anti-GtfB polyclonal antiserum at a 1:500 dilution.

FIG. 4.

The behaviors of GtfB and GtfC in the vicK-deficient mutant (vicK-NP). (A) Western blot analysis of different cell extracts from wild-type (UA159) and vicK-NP strains of S. mutans, i.e., bead-beaten Tris extract (soluble fraction), the insoluble fraction, and the supernatant fraction. Following SDS-PAGE, proteins were blotted onto a nitrocellulose membrane and subjected to Western blotting with an anti-GtfB polyclonal antiserum at a dilution of 1:500. See the text for more details. (B) Differential expression of gtfB and gtfC in the wild-type and vicK mutant strains by real-time PCR. The data shown are means ± standard deviations (error bars) from at least three independent experiments. *, P < 0.001 (Student's t test).