Role of Catabolite Control Protein A in the Regulation of Intermedilysin Production byStreptococcus intermedius

Abstract

Streptococcus intermedius is an opportunistic pathogen of humans that causes purulent infections, including brain and liver abscesses. This pathogen secretes a human-specific cytolysin, intermedilysin, which has been recognized as a major virulence factor. However, most of the expressional control mechanisms of ily are still unknown. To determine these mechanisms, we analyzed the nucleotide sequence of the ily promoter region. We found a highly homologous region to the catabolite-repressible element (cre) in the ily promoter region and observed a considerable decrease in the amount of secreted intermedilysin when cells were grown in a culture medium containing high concentrations of glucose/utilizable carbohydrates. Disruption of the ccpA gene, which encodes catabolite control protein A, did not induce catabolite repression of ily by glucose/utilizable carbohydrates. In cre mutants, catabolite repression of ily was partially restored, and purified catabolite control protein A bound to an oligonucleotide containing the cre consensus sequence in the ily promoter region. In addition, a prolonged lag phase and slower doubling time of the ccpA mutant cells were observed. Our data show that S. intermedius can modulate ily expression and growth rate through catabolite control protein A-mediated monitoring of the extracellular glucose/utilizable carbohydrate concentration.

Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

“…S. intermedius PC574 isolated from dental plaque (9) and the derivative strains were cultured at 37°C under anaerobic conditions. Brain heart infusion (BHI) broth (Becton, Dickinson and Company, Palo Alto, CA) and 3-(N-morpholino)propanesulfonic acidbuffered BHI (MOPS-BHI) broth were used as the culture media (17). Antibiotics were added at the following concentrations: chloramphenicol (Cm), 2 g/ml; erythromycin (Erm), 1 g/ml; and spectinomycin (Spc), 50 g/ml.…”

“…The amplified fragment was used to construct the ⌬nanA mutant. The ⌬msgA and ⌬nanA mutants were produced via transformation of competence-stimulating peptide (CSP)-treated PC574 cells with each PCR amplicon according to the method described previously (17). Colonies were selected on BHI agar containing 50 g/ml Spc.…”

“…PC574, the PC574 ⌬lacR mutant containing control vector pSETN1, and the PC574 ⌬lacR mutant complemented with the LacR-producing plasmid placR (Table 1) were cultured in MOPS-BHI medium at 37°C for 24 h under anaerobic conditions, and the cells were subsequently separated by centrifugation (5,000 ϫ g). Isolation of total RNA from the cells and quantitative RT-PCR (qRT-PCR) analysis were performed as described previously (17). The primer set qRT-msgA F and qRT-msgA R (Table 2) was used for quantification of msgA mRNA.…”

“…Previously, we characterized catabolite control protein A (CcpA), which can regulate ily expression and growth rate, depending on the extracellular glucose or utilizable carbohydrate concentration (17). We also showed that the lactose phospho-transferase system repressor (LacR) regulates transcription of ily by binding to its promoter region (18).…”

LacR Mutations Are Frequently Observed in Streptococcus intermedius and Are Responsible for Increased Intermedilysin Production and Virulence

“…The first is autoinducer 2 (AI-2) (a LuxS product used by several bacteria in quorum-sensing signaling), which is reported to be an exponential-growth-phase-specific activator of ily transcription (12). In addition, we recently revealed that ily expression and the growth rate of the bacteria are modulated through catabolite control protein A (CcpA), which is a LacI/GalR-type repressor that monitors the extracellular glucose/utilizable carbohydrate concentration (13).…”