41 protein and ECM components. Therefore, a whole-cell binding assay (Fig. 2) was carried out using the wild-type MGAS 6183 strain, the scl1-inactivated isogenic mutant, and the mutant complemented with plasmid pSL230 expressing in trans the Scl1.41 protein (Caswell et al., 2007). All three strains were first transformed with the plasmid pSB027 to generate GFP-expressing cells (Fig. 2a, images at left). The stability of two plasmids pSL230 and pSB027 within the complemented mutant strain was confirmed by isolating total DNA from these cells (Fig. 2d). Fluorescent GAS strains were next tested for binding to ECM-coated glass cover slips (Fig. 2a, images at middle and right columns). More fluorescent wild-type

cells were seen attached to the cover Protease Inhibitor Library slips coated with cFn and Lm, as compared with scl1 mutant GAS. Furthermore, complementation of the scl1 mutant with pSL230 considerably increased cell binding

to both ECMs. Quantitative analysis by counting the numbers of GAS cells in random fields fully supported visual observations (Fig. 2b). The scl1-inactivated mutant bound 30% and 45% less to cFn and Lm, respectively, compared with the wild-type strain. Importantly, the complementation of the mutant for Scl1.41 expression restored the wild-type levels of binding to both cFn and Lm, indicating that this phenotype was due to the lack of Scl1 expression. Residual cFn binding by the Scl1 find more mutant could be attributed to the presence of the prtf2 gene in this strain (Caswell et al., 2007) encoding an additional Fn-binding protein, F2 (Jaffe et al., 1996). Similarly, the observed binding of the Scl1-deficient mutant to Lm could be attributed to Lbp and Shr expression; however, the M41-type GAS was not included in the studies that characterized these ECM-binding proteins (Terao et al., 2002; Fisher et al., 2008). Because lbp and shr genes are conserved among GAS strains of various M-types, we used PCR to demonstrate

the presence of both genes in 4��8C M41-type strain MGAS 6183 (Fig. 2c). Altogether, our results demonstrate that Scl1.41 protein is an important surface adhesin that selectively binds to human cFn and Lm and significantly contributes to ECM– GAS interactions. GAS interactions with ECM components have been exhaustively reported in the literature and considerable effort has been directed toward understanding its function in GAS adherence and internalization pertaining to human disease (Cue et al., 2000). The bulk of that work focuses on Fn, although the effect of exogenous cFn on GAS internalization was not specifically investigated. Far less is known about the contribution of Lm to GAS adherence and internalization. Recently, the Lbp of the M1-type strain was shown to facilitate the adherence to and internalization by HEp-2 cells; however, the observed decrease in internalization of the lbp mutant was not statistically significant compared with the wild-type strain (Terao et al., 2002).