The Streptococcus mutans Cid and Lrg systems modulate virulence traits in response to multiple environmental signals

Abstract

The tight control of autolysis by Streptococcus mutans is critical for proper virulence gene expression and biofilm formation. A pair of dicistronic operons, SMU.575/574 (lrgAB) and SMU.1701/1700 (designated cidAB), encode putative membrane proteins that share structural features with the bacteriophage-encoded holin family of proteins, which modulate host cell lysis during lytic infection. Analysis of S. mutans lrg and cid mutants revealed a role for these operons in autolysis, biofilm formation, glucosyltransferase expression and oxidative stress tolerance. Expression of lrgAB was repressed during early exponential phase and was induced over 1000-fold as cells entered late exponential phase, whereas cidAB expression declined from early to late exponential phase. A two-component system encoded immediately upstream of lrgAB (LytST) was required for activation of lrgAB expression, but not for cid expression. In addition to availability of oxygen, glucose levels were revealed to affect lrg and cid transcription differentially and significantly, probably through CcpA (carbon catabolite protein A). Collectively, these findings demonstrate that the Cid/Lrg system can affect several virulence traits of S. mutans, and its expression is controlled by two major environmental signals, oxygen and glucose. Moreover, cid/lrg expression is tightly regulated by LytST and CcpA.

Fig. 1.

Schematic diagram of the lrg and cid loci in the S. mutans UA159 genome. Gene assignments and gene numbers above the schematic diagram are based on the S. mutans UA159 genome (GenBank accession no. AE014133). Numbers inside the schematic diagram and between ORFs indicate the nucleotides (bp) in the ORFs and intergenic regions, respectively. Arrows indicate the direction of transcription. A dicistronic operon, lytST, is located immediately upstream of lrgAB. The lytS operon encodes the putative sensor kinase and lytT encodes the putative response regulator of a TCS. The lrgA and lrgB operons are annotated as a regulator and effector of murein hydroase, respectively. The SMU.1699 and SMU.1703 genes are annotated as conserved hypothetical proteins, and SMU.1697 and SMU.1702 are predicted to encode a possible rRNA methylase and uncharacterized phosphatase, respectively.

Fig. 2.

Autolytic potential and biofilm-forming activity of S. mutans wild-type and its derivatives. (a) Autolysis assay. The autolytic activities of strains were monitored at 44 °C in a Bioscreen C system that was set to shake for 15 s before measurement every 30 min. Data are representative of three independent experiments and are presented as an average of triplicate samples. (b) Biofilm formation. Cultures were grown in BM medium supplemented with glucose or sucrose for 24 h. Data are representative of at least two separate experiments performed at least in triplicate. Error bars represent sd. *P<0.05; Student's t-test. (c) Production of GtfB and GtfC. Western blot analysis was performed by using bead-beaten, SDS-boiled extracts from S. mutans wild-type and its derivatives. Following SDS-PAGE, proteins were blotted onto an Immobilon P membrane and subjected to Western blotting with an anti-GtfB antiserum at a dilution of 1 : 500. Data are representative of at least three independent experiments. See text for details.

Fig. 3.

Growth curves of S. mutans wild-type and its lrg (a) and cid (b) derivatives under oxidative stress. Strains were grown in BHI medium containing 10 mM paraquat under anaerobic conditions. Growth was monitored in a Bioscreen C system that was set to shake for 15 s every 30 min. For anaerobic growth, sterile mineral oil (50 μl) was placed on top of the broth cultures. The results are representative of two independent experiments.

Fig. 4.

Expression of lrgA and cidA with growth phase. The expression of lrgA (a) and cidA (b) genes was measured in UA159 (▪) and its isogenic lytST mutant (▵) in the early (OD₆₀₀=0.2), mid (OD₆₀₀=0.5), late (OD₆₀₀=0.9) exponential and stationary phases of growth, using real-time RT-PCR. Results are the average of triplicate samples from three independent experiments.

Fig. 5.

Analysis of the effect of glucose concentration (a) and CcpA (b) on expression of the S. mutans cid and lrg operons by Northern blot. Total RNA was isolated from S. mutans UA159 cultures grown for 6 h in the presence of increasing concentrations of glucose (a), and UA159 and ccpA⁻ (TW1) cultures grown in the presence of either 11 or 45 mM glucose, at 2 h (early exponential growth), 6 h (late exponential phase) or 12 h (late stationary phase) (b). RNA (5 μg) from each sample was subjected to Northern blotting with DIG-labelled DNA probes corresponding to either lrgA or cidB. The size of each transcript is indicated to the left of each blot, and is also shown in a schematic diagram of the cid locus by the dotted arrows. The 1.1 kb cidAB transcript is indicated by an asterisk. The corresponding ethidium-bromide-stained gel is presented beneath the blots with arrows highlighting the 23S and 16S RNA gene bands.

Fig. 6.

Identification of cre sequences upstream of lrgAB. The B. subtilis cre consensus sequence derived by Miwa et al. (2000) is shown at the top, where W=A or T, and N=any nucleotide. The nucleotide sequence corresponding to 114 bp upstream of the ATG start codon (identified in bold italics) of the S. mutans lrgA gene is shown at the bottom. The putative cre elements are underlined and conserved nucleotides are shown in bold.

Fig. 7.

Working model for glucose and oxygen-dependent regulation of the S. mutans Cid/Lrg system. Lrg and Cid activities are regulated in a growth-phase-dependent fashion by oxygen and glucose levels, which would change dramatically as cells progress from early exponential to stationary phase. As cells actively grow (high levels of oxygen and glucose available), cid expression is dominant but lrg is minimal and probably under the control of CcpA. As cells enter stationary phase (oxygen/glucose-limited cells), lrg expression is dramatically increased by the LytST TCS. In contrast, cid expression is gradually diminished, in part due to CcpA-dependent regulation. The expression level of lrg and cid seems to be tightly balanced throughout growth, and direct interactions between Lrg and Cid proteins may be involved in achieving this balance. Therefore, by sensing multiple environmental inputs and modulating the balance between Lrg and Cid, S. mutans is able to modulate growth and biofilm formation, which are critical for virulence expression by this pathogen.