Supplemental materials Requirement and Synergistic Contribution of PAF acetylhydrolase Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and Systemic Infection by Hypervirulent emm3 Group A Streptococcus in Subcutaneous Infection of Mice Wenchao Feng, Dylan Minor, Mengyao Liu, and Benfang Lei Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59718 Figure S1. Histological analyses of MGAS315 skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 1.7 x 108 cfu MGAS315. Skin infection sites were collected on day 1 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and neutrophil (PMN) zones. The boxes indicate the areas that are shown in Fig. 4 at higher magnification. Figure S2. Histological analyses of MGAS315 skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 1.7 x 108 cfu MGAS315. Skin infection sites were collected on day 2 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and PMN zones. The boxes indicate the areas that are shown in Fig. 5 at higher magnification. Figure S3. Histological analyses of MGAS315 sse skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 2.0 x 108 cfu sse. Skin infection sites were collected on day 1 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and neutrophil (PMN) zones. The boxes indicate the areas that are shown in Fig. 4 at higher magnification. Figure S4. Histological analyses of MGAS315 sse skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 2.0 x 108 cfu sse. Skin infection sites were collected on day 2 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and neutrophil (PMN) zones. The boxes indicate the areas that are shown in Fig. 5 at higher magnification. Figure S5. Histological analyses of MGAS315 sagA skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 1.9 x 108 cfu sagA. Skin infection sites were collected on day 1 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and neutrophil (PMN) zones. The boxes indicate the areas that are shown in Fig. 4 at higher magnification. Figure S6. Histological analyses of MGAS315 sagA skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 1.9 x 108 cfu sagA. Skin infection sites were collected on day 2 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and neutrophil (PMN) zones. The boxes indicate the areas that are shown in Fig. 5 at higher magnification. Figure S7. Histological analyses of MGAS315 ssesagA skin infection sites in mice. Five 6- week old female C57BL/6J mice were subcutaneously inoculated with 2.0 x 108 cfu ssesagA. Skin infection sites were collected on day 1 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and neutrophil (PMN) zones. The boxes indicate the areas that are shown in Fig. 4 at higher magnification. Figure S8. Histological analyses of MGAS315 ssesagA skin infection sites in mice. Five 6- week old female C57BL/6J mice were subcutaneously inoculated with 2.0 x 108 cfu ssesagA. Skin infection sites were collected on day 2 after inoculation, fixed, and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images taken at 2X magnification with the bar represents 200 µm. The half curly parentheses indicate the bacterial and neutrophil (PMN) zones. The boxes indicate the areas that are shown in Fig. 5 at higher magnification. Figure S9. Histological analyses of sse-sse skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 1.8 x 108 cfu sse-sse. Two and three mice were sacrificed on days 1 and 2 after inoculation, respectively, to collect skin infection sites. The skin infection sites were fixed and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images on days 1 and 2 after inoculation. The bar represents 200 µm. Figure S10. Histological analyses of sagA-sagA skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 1.4 x 108 cfu sagA-sagA. Two and three mice were sacrificed on days 1 and 2 after inoculation, respectively, to collect skin infection sites. The skin infection sites were fixed and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images on days 1 and 2 after inoculation. The bar represents 200 µm. Figure S11. Histological analyses of ssesagA-sse skin infection sites in mice. Five 8-week old female C57BL/6J mice were subcutaneously inoculated with 1.6 x 108 cfu ssesagA-sse. Two and three mice were sacrificed on days 1 and 2 after inoculation, respectively, to collect skin infection sites. The skin infection sites were fixed and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images on days 1 and 2 after inoculation. The bar represents 200 µm. Figure S12. Histological analyses of ssesagA-sagA skin infection sites in mice. Five 6-week old female C57BL/6J mice were subcutaneously inoculated with 1.5 x 108 cfu ssesagA-sagA. Two and three mice were sacrificed on days 1 and 2 after inoculation, respectively, to collect skin infection sites. The skin infection sites were fixed and analyzed with Gram and H&E stains, as described in the Materials and Methods section. Presented are representative Gram and H&E stain images on days 1 and 2 after inoculation. The bar represents 200 µm. Figure S13. Gram and H&E stain images of the skin of a C57BL/6J mouse. eof ssesagA- sagA skin infection sites in mice. The skin infection sites were fixed and analyzed with Gram and H&E stains, as described in the Materials and Methods section. The bar represents 20 µm.