NADase has the ability to cleave β-NAD+, which is universally important in numerous essential redox and energy-producing biological reactions, depleting intracellular NAD pools [8, 9]. NADase is also toxic for bacterial cells themselves, therefore, GAS

encodes ifs gene whose product (IFS) is an endogenous inhibitor of NADase activity and localized in the bacterial cytoplasmic compartment [9, 10]. NADase precursor exists as an inactive complex with IFS [9, 10]. In vitro, intoxication of keratinocytes with NADase was associated with cytotoxic effects [11, 12]. Bricker et al. presented Proteases inhibitor that NADase enhances GAS virulence in vivo using mouse models [13]. These results enabled us to further study the NADase as a target molecule GW786034 molecular weight to reduce GAS virulence. However, another study of GAS infection among aboriginal people in Australia

found no relationship between NADase production and severity or outcome of GAS infection [14]. Furthermore, we recently reported that M-1 group A streptococcal isolates were divided into three groups based on NADase activity: high activity, low activity and no activity [15], whereas we did not find that low and high levels of the NADase activity correlated with severity of GAS human infection (data not shown). Meanwhile, Ajdic et al. reported Mirabegron that among 73 NCT-501 mw strains isolated from patients with mostly invasive GAS infections from a recent outbreak of streptococcal infection, 67 (92%) were NADase producer [16], although strains isolated from patients with non-invasive GAS infections were not assayed. It is unknown why the 8% strains isolated from patients with mostly invasive GAS infections were not NADase producer. Therefore, we thought that before taking up the study of our interest, it should be further determined how NADase is important as a virulence factor for severe invasive disease. We mainly focused on the following two points: (i) How do NADase activity levels correlate with virulence? (ii) If

NADase is important for severe invasive disease, and whether it is possible that IFS suppresses the severity. In this study, we present further evidences to prove the importance of NADase in severe invasive disease. Methods Bacterial strains Streptococcal strains were isolated as causative organisms from invasive diseases patients in Japan (Table 1). S. pyogenes (GAS) strain SF370, which is prevalent as the database reference isolate (accession NC_002737), was provided by the courtesy of J. J. Ferretti [17, 18]. Streptococcal strains were cultured in brain heart infusion (E-MC62, EIKEN Chemical Co., Tokyo, Japan) supplemented with 0.3% yeast extract (BD, Sparks, MD, USA) (BHI-Y) broth unless otherwise described.