The contribution of EndoS to GAS virulence was also studied in the less virulent AZD3965 cost strain NZ131 (serotype M49) in gain-of-function analysis. The results reveal that heterologous overexpression check details of EndoS in M49, NZ131[pNdoS] increased GAS resistance to killing by human neutrophils (Figure 1E). Monocyte killing assay As with neutrophil killing assays, no significant difference in bacterial survival was detected in the monocytic killing assays when comparing M1T1 GAS strain
5448 to the isogenic ndoS knockout strain (Figure 2A). Pretreatment of plasma with exogenous rEndoS resulted in a significant increase in GAS resistance to killing by monocytes (Figure 2B), as did heterologous expression of EndoS in the less virulent strain NZ131 (Figure 2C). Figure 2 Opsonized bacterial survival in U937 monocytic cell killing assays. (A) M1T1 GAS strain 5448 and isogenic ndoS knockout, 5448ΔndoS. (B) Exogenous pretreatment of plasma with rEndoS prior opsonization FDA-approved Drug Library of GAS. (C) Heterologous expression of EndoS in NZ131 (serotype M49). Error bars indicate standard deviation from the mean. *
indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, ns indicates no significant difference. In vivo mouse model Many major GAS virulence factors have been shown to decrease overall virulence when knocked out and studied in murine infection models [13–16]. It has also been shown pentoxifylline that EndoS has activity on all subclasses of murine IgG [17]. Taken together, this led us to believe that the contribution of EndoS to GAS virulence could be studied in vivo. However, in this murine model of infection GAS strain 5448ΔndoS showed no significant difference in virulence compared to wild-type 5448 (Figure 3A). Figure
3 Survival curves of female CD-1 mice following intraperitoneal challenge with GAS. (A) M1T1 GAS strain 5448 and isogenic ndoS knockout, 5448ΔndoS, at 2 × 107 cfu with 5% mucin (n = 6). (B) Heterologous expression of EndoS in NZ131 (serotype M49) at 5 × 108 cfu with 5% mucin (n = 10). However, when we studied the less virulent GAS strain NZ131 (serotype M49) overexpressing EndoS, it was found that strain NZ131[pNdoS] showed increased virulence in vivo (Figure 3B) compared to wild-type NZ131[empty vector]. This may be a function of the relatively high level of expression of EndoS in NZ131[pNdoS] compared to 5448 (Figure 1A). Discussion A single clone of the M1T1 serotype has disseminated globally during the last few decades to represent the leading cause of severe, invasive GAS infections [18].