Identification and characterization of a novel secreted glycosidase with multiple glycosidase activities in Streptococcus intermedius.

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit ß-d-galactosidase and N-acetyl-ß-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated ß-d-galactosidase, ß-d-fucosidase, N-acetyl-ß-d-glucosaminidase, and N-acetyl-ß-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-ß-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked ß-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had ß-d-galactosidase and ß-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-ß-d-glucosaminidase and N-acetyl-ß-d-galactosaminidase activities. The ß-d-galactosidase and ß-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-ß-d-glucosaminidase and N-acetyl-ß-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.

LacR mutations are frequently observed in Streptococcus intermedius and are responsible for increased intermedilysin production and virulence.

Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.

Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity.

Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor σ(32) in E. coli, although S. intermedius does not utilize σ(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.

Role of catabolite control protein A in the regulation of intermedilysin production by Streptococcus intermedius.

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.