2014. to reduce the cytotoxicity of an ILY-high-producing strain than an ILY-low-producing strain. Overall, our data show that blood contains factors that stimulate and inhibit ILY expression and activity, which may impact pathogenicity of is usually a facultative anerobic bacterium belonging to the anginosus group of streptococci (AGS), which also includes and (1). consists of two subspecies, subsp. and subsp. contains three subspecies, subsp. (2, 3). is usually associated with oral infections including periodontal disease and implantitis, respiratory infections including recurrent tonsillitis and pneumonia, and deep-seated purulent infections such as brain, lung, and liver abscesses (1, 4,C13). Among AGS species, only produces the cytotoxin intermedilysin (ILY) of the cholesterol-dependent cytolysin (CDC) family, encoded by the gene (14,C17). It has been shown that a knockout mutation of or inactivation of ILY using an anti-ILY antibody results in greatly decreased cytotoxicity of toward C-75 Trans the human hepatoma cell collection HepG2 (18), providing strong evidence that ILY is usually a crucial virulence factor of for infectivity and toxicity to human cells. In contrast to other CDC family members, ILY does not use cholesterol as a binding receptor and can specifically identify a glycosylphosphatidylinositol-linked human cell membrane protein, (h)CD59, which is a regulator of the terminal pathway of match (19). Therefore, is usually believed to be a purely human-specific pathogen. Two transcriptional repressors that can regulate expression, i.e., catabolite control protein (CcpA) and lactose phosphotransferase system repressor (LacR), have been reported thus far (20, 21). CcpA can regulate expression by binding to the catabolite-repressible element (promoter region as a part of the mechanism that monitors the extracellular concentration of utilizable carbohydrates such as glucose, fructose, and mannose (20). Therefore, high concentrations of these sugars in the environment may repress expression. It was shown that LacR is usually a member of the GntR family of transcriptional regulators (22) and can repress transcription of the operon by binding to the LacR acknowledgement element, which consists of direct repeats of the sequence TGTTTNWTTT (N = any base; W = A or T) in the promoter under galactose-limited conditions (22, 23). A pulldown assay C-75 Trans with a biotinylated DNA probe showed that LacR also binds to the promoter; however, the exact location of the binding site in this region is still unknown (21). Therefore, it is probable that for this reason can overproduce ILY when cultured in a galactose-containing medium. Disruption of in also causes constitutive overproduction of ILY and consequently increases toxicity to the human hepatoma cell collection HepG2 (21). Because a loss-of-function mutation in LacR is usually observed in almost all ILY-high-producing strains isolated from deep-seated abscesses, higher production of ILY seems to be necessary for increased virulence of this bacterium. can control the expression levels of by means of mechanisms monitoring the concentrations of sugars, such as galactose and glucose, that are present in the habitat, thereby affecting the pathogenicity of has a relatively wide range of glycosidase activities. It is known that this pathogen is the only AGS species that can secrete NanA that has neuraminidase (Neu) activity, encoded by (1, 3, 24, 25). In addition, and subsp. are C-75 Trans the only two AGS taxa with -d-galactosidase (-Gal), -d-fucosidase, cytotoxicity are IL18R1 antibody immunoglobulins that neutralize ILY, MsgA, and NanA activities. RESULTS ILY production by FBS-cultured cells. Recently, we exhibited that MsgA and NanA can degrade glycans on human serum glycoprotein 1-antitrypsin (26), suggesting C-75 Trans that galactose liberated from glycoproteins by these glycosidases in serum can activate ILY production through inactivation of LacR. To confirm this possibility, an ILY-low-producing strain, PC574, was cultured in FBS. ILY is usually a primary hemolytic factor; in FBS- and MOPS-BHI-cultured cells were also compared using PC574 luciferase gene with under the regulation of the promoter in an artificial operon. FBS-cultured cells showed much higher luciferase activity than MOPS-BHI-cultured cells (Fig. 1B), and together these data show that the increased hemolytic activity in the culture supernatant of FBS-cultured cells is due to the activation of expression. Open in a separate windows FIG 1 Hemolysis.