Enhanced expression of the slo and the scpC genes in severe invasive GAS is attributed to mutation of a transcriptional regulator CsrS 
Although sequences of the slo gene and the scpC gene were identical among clinically isolated non-invasive and severe invasive GAS (data not shown), Figure 5A shows that the slo and the scpC genes were expressed in the severe invasive GAS greater in extent than those in the non-invasive GAS.
The expression of the other virulence-associated genes, such as IgG degrading protease of GAS, Mac-1-like protein (mac), nicotine adenine dinucleotide glycohydrolase (nga), polysaccharide capsule production (hasA), and C5a peptidase (scpA), was also upregulted in the severe invasive GAS, greater than that detected in the non-invasive GAS (Figure 5A).
Contrarily, the levels of streptococcal pyrogenic endotoxin (speB), SLS (sagA), and mitogenic factor (speF) genes were downregulated in the severe invasive GAS, compared to that found in the non-invasive GAS (Figure 5A and data not shown).
These results demonstrate the prominent changes in the transcriptional profile of several virulence-associated genes, including the slo and the scpC, in the all severe invasive GAS.
Mutation of csrR or csrS can cause significant alterations in virulence in mouse models of infection,either increasing lethality or the severity of localized soft tissue lesions [5], [6].
GAS isolates from mice with severe invasive disease had mutations in csrS, raising the notion that CsrR/S function is important in modulating gene expression during infection.
Therefore, we analyzed the linkage between the csrS and/or csrR genes and the property of invasive GAS infection by sequencing these genes in the emm49 strains used in this study.
The nucleotide sequence of the csrR gene was identical in all the isolates, and that of the csrS gene was identical among the all non-invasive GAS isolates (data not shown).
However, as shown in Figure 5B, the csrS genes of all clinically isolated severe invasive GAS had a deletion, a point mutation, or an insertion, thereby, resulting in the creation of translational stop codons (NIH147, NIH226, NIH230, and NIH269) or in a mutation in the presumed kinase domain (NIH200).
In order to clarify the role of CsrS regarding expression of the virulence-associated genes and resistance to PMN killing, we introduced the intact csrS gene of the 1566 strain into the severe invasive GAS (see Figure 5A).
The csrS-introduced severe invasive GAS reduced the expression levels of the slo and the scpC genes, comparable to those detected in the non-invasive GAS.
In contrast, the expression of speB was upregulated to the level observed in the non-invasive GAS (Figure 5A).
In parallel with the expression profile of slo and scpC in the severe invasive GAS, introduction of the intact csrS gene into the severe invasive GAS restored the susceptibility to the killing by PMN (p=0.015 compared with severe invasive isolates +CsrS) (Figure 6A), abrogated the inhibition of PMN migration by degradation of IL-8 (p=0.002 compared with invasive isolates +CsrS) (Figure 4A, 4B, and 6B), and diminished the killing activity for PMN by necrosis (p=0.00016 compared with invasive isolates +CsrS) (Figure 3A, 3C, and 6C).
These results strongly suggest that mutations in the csrS gene correspond to the immunocompromized activity in the severe invasive isolates, associated with inhibition of PMN recruitment and survival.
