Role of RegM, a homologue of the catabolite repressor protein CcpA, in the virulence of Streptococcus pneumoniae

Abstract

As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both alpha-glucosidase and beta-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The regM mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a lacZ transcriptional fusion construct indicated that mutation of regM resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (cps). Thus, regM is the first gene outside of the cps locus to be implicated in regulation of capsular gene expression.

FIG. 1.

(A) Genetic organization of the regM region in S. pneumoniae. The relative positions of the oligonucleotides used to create the knockout mutant (a and b) and the deletion mutant (c, d, e, and f) are indicated by the arrows. (B) Sequence of the promoter region of regM. The potential ribosome binding sites (RBS), as well as −35 and −10 promoter elements, are indicated. A 14-bp sequence with homology to the cre consensus sequence is underlined. (C) Comparison of the cre-like sequence in the regM promoter region with the consensus cre sequence. The sequence differs from the consensus cre sequence defined by Weickert and Chambliss (34) at one position, indicated in boldface type.

FIG. 2.

Comparative virulence of D39 and the D39 regM knockout mutant (D39regM⁻). Groups of six mice were challenged intraperitoneally with either 4 × 10³ CFU (A) or 1 × 10⁵ CFU (B). Survival times of individual mice are indicated.

FIG. 3.

Comparative virulence of D39 and D39 carrying ΔregM. Groups of 10 mice were challenged intraperitoneally with approximately 500 CFU (A) or 4 × 10⁴ CFU (B). Survival times of individual mice are indicated.

FIG. 4.

Catabolite repression of β-galactosidase in D39 and D39 carrying ΔregM. Cells were grown at 37°C in CH medium supplemented with lactose or lactose plus glucose. Specific activity of β-galactosidase in exponentially grown cells was measured using O-nitrophenol β-d-galactopyranoside as the substrate. The data represent the means + standard errors (error bars) of three independent experiments.

FIG. 5.

Catabolite repression of α-glucosidase in D39 and D39 carrying ΔregM. Cells were grown at 37°C in CH medium supplemented with maltose or maltose plus glucose. Specific activity of α-glucosidase in exponentially grown cells was measured using p-nitrophenyl α-d-glucopyranoside as the substrate. The data represent the means + standard errors (error bars) of three independent experiments.

FIG. 6.

Expression of the cps operon. D39 lacking ebg and carrying cps::lacZ and D39 lacking ebg and carrying ΔregM cps::lacZ were grown in CH supplemented with glucose, sucrose, or lactose, and expression of capsule genes was monitored as expression of lacZ in fusion. The data represent the means + standard errors (error bars) of three independent experiments.

FIG. 7.

Colony size of D39 and D39 carrying ΔregM. Cells were grown on CH agar plates with 5% defibrinated blood and incubated for 16 h at 37°C in a CO₂ incubator. (A) D39 carrying ΔregM; (B) D39. (C) Mean colony diameter + standard error (error bars) (50 colonies measured).