Indeed, the virulence between the two strains also appears to be slightly different from each other, although we were unable to explain the reason. Although the plasmid pLZN-RBSII2 conferred significant virulence to the nga strain when compared

to a control vector (Table 3 and Figure 2), we found that the strain nga (pLZN-RBSII2) produced only 8% of the NADase activity found in the wild type strain. In order to restore NADase levels to near normal, we attempted to construct plasmids containing longer upstream DNA sequences than what is present in pLZN-RBS and pLZN-RBSII2. However these plasmids were not successfully constructed, possibly due to the potential check details toxicity of over produced NADase to bacterial cell. As shown in Figure 4, injection of NADase inhibitor (His-IFS) significantly OICR-9429 molecular weight rescued mice from strains GT01. To further investigate the potential of the His-IFS solution, we tested strain CR01, which showed the highest virulence in the mouse-infection model among our collected strains (see Table 2). Although His-IFS alone was not sufficient to significantly rescue mice from the strain CR01, a combination of His-IFS solution and ampicillin was able to significantly decrease GAS virulence in mice

compared with ampicillin alone (unpublished data). These results also show that NADase activity occurs in vivo and can be inhibited. Using western blot analysis, we detected two bands from pHis-IFS using anti-RGS-HIS antibody (Figure 3). Based on the specificity of this antibody, we attributed Oxymatrine the smaller band to degradation of the His-IFS protein. The higher virulence of strain CR01 when compared to the other isolates belonging to high activity group (Table 2) may not only be due to higher level of NADase activity, but also due to additional unknown factors. For example,

two-dimensional gel electrophoresis Cell Cycle inhibitor demonstrates that CR01 presents a different pattern of secreted extracellular proteins compared to the other isolates belonging to high activity group, including markedly lower level of the SpeB protein (unpublished results). Further analysis of the strain CR01, although the less representative strain among the high activity isolates had not been focused on very much in this study, would be a very interesting advance for the field. Finally, we should discuss the discrepancy between NADase activity being important to the virulence of S. pyogenes during in vivo mouse models and our epidemiological data showing that low and high levels of NADase activity do not correlate with the severity of the S. pyogenes isolates in human infection. One possibility is that there is no statistical difference due to low sample number which is a result of a very small number of cases of the STSS disease. There is another possibility. After human passage, the isolated S. pyogenes could be different from the original strain which caused the infection due to getting genetic mutations.