Mouse infection model using nga knockout mutant and complemented strain To investigate the extent with which NADase contributes to GAS virulence in the mouse model, nga gene encoding NADase of strain GT01 was replaced with an antibiotics marker. The resulting GT01Δnga did not show any detectable NADase activity and mortality in the invasive soft-tissue mouse-infection test

(Table 2). Therefore, we tried to complement the phenotype using a plasmid pLZN2 in which only the coding region of nga is cloned. However, the complementation study using GT01Δnga (pLZN2) strain was not successful in restoring survival times (Table 3). Unsuccessful complementation might be due to insufficient NADase activity in the GT01Δnga (pLZN2) strain (NADase activity: 1.28 ± 0.12 U). Therefore, two find more additional plasmids (pLZN-RBS

and pLZN-RBSII2) were constructed containing 16 and 26 base pair upstream DNA sequences encoding the potential ribosome-binding site, which is lacking in pLZN2 respectively (see Materials and Methods in detail). The resultant GT01Δnga (pLZN-RBSII2), but not GT01Δnga (pLZN-RBS), strain enhanced virulence compared to the mutant RG7420 in the mouse model (P = 0.019 for comparison of survival times). The result of the GT01Δnga (pLZN-RBS) strain may also be due to the same reason that the strain was non-functional, since it contained only slightly improved levels of NADase activity (1.78 ± 0.03 U). Table

3 Virulence (Mortality) to mouse of GT01Δnga with or without cloned nga gene Strain Mortalitya NADaseb GT01 (pLZ12-Km2, vector) 73% (8/11) 14.12 ± 1.30 GT01Δnga (pLZ12-Km2, vector) 0% (0/17) 0.04 ± 0.06 GT01Δnga (pLZN2, nga) 0% (0/11) 1.28 ± 0.12 GT01Δnga (pLZN-RBS, nga) 0% (0/10) 1.78 ± 0.03 GT01Δnga (EVP4593 concentration pLZN-RBSII2, nga) 29% (4/14) 4.57 ± 0.17 Bacteria were cultured in BHI-Y broth supplemented with kanamycin (100 μg/ml). a, Mice were observed for 8 days, because no mouse died after day 8 on previous study (see Figure 1). b, NADase activity was determined as described in Table 2. Furthermore those results encouraged us to construct plasmids almost containing longer upstream DNA sequences than what is present in pLZN-RBS and pLZN-RBSII2. However these plasmids were not successfully constructed (data not shown, see Discussion in detail). Assessment of body weight change in mouse infection model experiment First, we judged the virulence based only on the mortality rate. Although GT01Δnga (pLZN2) and GT01Δnga (pLZN-RBS) did not kill the injected mice (Table 3), possibly due to insufficient NADase activity, we found that there were some mice which exhibited a poor health condition but eventually survived. Hence, we also evaluated virulence of GAS infection in mice by monitoring body weight. In this method, lower body weight implies a more severe form of disease.