Requirement and Synergistic Contribution of Platelet-Activating Factor Acetylhydrolase Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and Systemic Infection by Hypervirulent emm3 Group A Streptococcus in Subcutaneous Infection of Mice Authors: Wenchao Feng, Dylan Minor, Mengyao Liu, & Benfang Lei This is a postprint of an article that originally appeared in Infection and Immunity on December 2017. The final version can be found at https://dx.doi.org/10.1128/IAI.00530-17. Feng, Wenchao, Dylan Minor, Mengyao Liu, and Benfang Lei. "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...." Infection and Immunity (September 2017). DOI: 10.1128/IAI.00530-17. Made available through Montana State University’s ScholarWorks scholarworks.montana.edu http://scholarworks.montana.edu/ http://scholarworks.montana.edu/ https://dx.doi.org/10.1128/IAI.00530-17 1 Requirement and Synergistic Contribution of PAF acetylhydrolase 1 Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and 2 Systemic Infection by Hypervirulent emm3 Group A Streptococcus 3 in Subcutaneous Infection of Mice 4 5 Wenchao Feng, Dylan Minor, Mengyao Liu, and Benfang Lei* 6 7 Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59718 8 9 Running Title: Synergistic Effects of Sse and SLS on GAS Immune Evasion 10 *Correspondence to Benfang Lei, blei@montana.edu11 12 mailto:blei@montana.edu 2 (ABSTRACT) 13 14 Hypervirulent Group A Streptococcus (GAS) can inhibit neutrophil recruitment and cause 15 systemic infection in mouse model of skin infection. The purpose of this study was to 16 determine whether PAF acetylhydrolase Sse and streptolysin S (SLS) have synergistic 17 contributions to inhibition of neutrophil recruitment and systemic infection in 18 subcutaneous infection of mice by MGAS315, a hypervirulent genotype emm3 GAS strain. 19 Deletions of sse and sagA in MGAS315 synergistically reduced skin lesion size and GAS 20 burden in the liver and spleen. However, the mutants were persistent at skin sites and had 21 similar growth factors in nonimmune blood. Thus, the low ssesagA numbers in the 22 liver/spleen is likely due to its reduction in systemic dissemination. Few intact and necrotic 23 neutrophils were detected at MGAS315 infection sites. In contrast, many neutrophils and 24 necrotic cells were present at the edge of sse sites on day 1 and at the edge and inside of 25 sse sites on day 2. sagA sites had massive and few intact neutrophils at the edge and 26 center of infection sites, respectively, on day 1 and were full of intact neutrophils or 27 necrotic cells on Day 2. ssesagA sites had massive intact neutrophils in the whole 28 infection sites. These sse and sagA deletion-caused changes in the histological pattern at 29 skin infection sites can be complemented. Thus, sse and sagA deletions synergistically 30 enhance neutrophil recruitment. These findings indicate that Sse and SLS are both 31 required but neither is sufficient for inhibition of neutrophil recruitment and systemic 32 infection by hypervirulent GAS. 33 34 3 (INTRODUCTION) 35 Group A streptococcus (GAS) is a major human pathogen that commonly causes 36 relatively mild pharyngitis and superficial skin infections (1). GAS can also cause potentially 37 lethal severe invasive infections with about 12,000 cases annually in the United States, and these 38 severe infections include bacteremic skin and soft-tissue infections, pneumonia, necrotizing 39 fasciitis, and bacteremia without focus (2). The severe invasive infections in 2005 to 2012 in the 40 United States are most frequently associated with GAS of the M protein gene-based genotypes 41 emm1, emm12, emm3, emm28, and emm89 (2). Invasive emm3 GAS causes a higher mortality 42 rate than invasive strains of other serotypes (3). 43 Necrotizing fasciitis (NF) is a rapidly progressive infection of the skin, subcutaneous and 44 deep soft tissue, and muscle and leads to systemic dissemination (4). Some NF patients have 45 numerous bacteria but few or no neutrophilic responses at infection sites (5, 6), which is 46 classified as stage III NF (6), and other histopathologic types include a moderate-to-severe 47 neutrophilic response (stage II) and positive Gram stain and an intense neutrophilic response and 48 an absence of bacteria (stage I) in infected tissues (6). Patients with the stage III NF have higher 49 mortality rate than patients with stages I and II NF (5, 6). 50 Murine NF model with hypervirulent, invasive M1T1 GAS displays the stage III 51 histopathologic features, few or no neutrophils at bacterial sites (7, 8). Hypervirulent M1T1 52 GAS isolates are usually natural CovRS mutants (8-10). CovRS (also known as CsrRS) is the 53 two-component regulatory system that negatively regulates multiple virulence factors (11-14), 54 including those involved in innate immune evasion, such as the capsule synthase HasA (11), IL-55 8/CXC chemokine peptidase SpyCEP (15), platelet-activating factor acetylhydrolase SsE (7, 16), 56 opsonophagocytosis-inhibiting Mac (17), NAD+-glycohydrolase (18), and streptolysin O (19). 57 Natural CovRS mutations of a M1T1 clone of serotype M1 GAS enhance the expression of 58 4 CovRS-controlled virulence factors and down-regulates the expression of the protease SpeB, 59 resulting in a high capacity to cause skin invasion, innate immune evasion, systemic 60 dissemination, and hypervirulence (8, 14, 20, 21). The selection of CovRS M1T1 mutations 61 during infection has been readily demonstrated in invasive M1T1 GAS during experimental 62 mouse infections (10, 20-26). Neutrophils are required for in vivo selection of covRS mutants, 63 and M1T1 CovRS mutants exhibit greater resistance to clearance by neutrophils in vivo (27). 64 The severe inhibition of neutrophil responses by M1T1 GAS requires CovRS mutations, 65 and the PAF acetylhydrolase Sse, but not IL-8 peptidase SpyCEP and C5a peptidase ScpA, plays 66 a critical role in inhibition of neutrophil recruitment by hypervirulent M1T1 GAS CovS mutants 67 (7, 8). Streptolysin S (SLS) has been shown to inhibit neutrophil recruitment in a zebrafish 68 model of GAS infection and delays the exodus of the neutrophils from the vessel lumen into the 69 tissue at the early stage of GAS skin infection in mice (28). The purpose of this manuscript was 70 to determine whether Sse and SLS have synergistic effect on the capacity of MGAS315, a 71 hypervirulent emm3 strain (29, 30), to inhibit neutrophil recruitment and to cause systemic 72 infection in subcutaneous infection of mice. We generated single and double deletion mutants of 73 MGAS315 for sagA, which encodes the peptide component of SLS, and sse. We found that sse 74 and sagA deletions synergistically reduce skin invasion and GAS load in the liver and spleen and 75 that both sse and sagA are both required for inhibition of neutrophil recruitment by MGAS315. 76 RESULTS 77 Synergistic effects of Sse and SLS to skin invasion, systemic infection, and 78 neutrophil recruitment in murine model of subcutaneous MGAS315 infection. MGAS315 79 caused large lesions in female CD-1 mice at 48 h after subcutaneous inoculation (Lesion area ± 80 SD: 934 ± 165 mm2) whereas MGAS315 sse, sagA, and ssesagA caused lesion sizes of 277 81 5 ± 64, 380 ± 126, and 188 ± 58 mm2, respectively (Fig. 1A and 1B). There were (4.0 ± 2.6) x 104 82 neutrophils at the MGAS315 skin infection site as estimated by the myeloperoxidase (MPO) 83 assay, and sse, sagA, and ssesagA recruited (2.8 + 1.5) x 105, (2.5 + 0.8) x 105, and (5.1 + 84 1.3) x 105 neutrophils at the infection sites, respectively. There were (2.5 + 3.6) x 108 and 2.0 + 85 1.1 x 107 GAS bacteria in the spleen and in per gram liver tissue in the MGAS315 infection, 86 respectively, and deletion of sse or sagA in MGAS315 reduced GAS loads in the spleen by the 87 magnitude of ≥4 orders and in the liver by the magnitude of 2 orders (Fig. 1). More importantly, 88 there were no GAS in the liver and spleen of the majority of mice infected with ssesagA on 89 day 2 after inoculation (Fig. 1C and 1D). The data indicate that Sse and SLS significantly 90 contribute to skin invasion, inhibition of neutrophil recruitment, and systemic infection in 91 subcutaneous infection of mice and that Sse and SLS have synergistic contribution to skin 92 invasion, inhibition of neutrophil recruitment, and systemic infection in skin infections of CD-1 93 mice. 94 These strains were also compared in subcutaneous infection of C57BL/6J mice on day 1 95 after inoculation. The effects of sse and/or sagA deletions in MGAS315 on skin invasion, 96 neutrophil recruitment, and bacterial loads in the liver and spleen in C57BL/6J mice were similar 97 to those in CD-1 mice (Fig. 2). Particularly, the sse and sagA deletions also showed the 98 synergistic contribution to the enhancement in neutrophil recruitment and reduction in GAS 99 loads in the liver and spleen. We also compared sse infection in female and male C57BL/6J 100 mice and observed no gender difference in lesion size, neutrophil levels, and GAS loads in the 101 liver and spleen (Fig. 2). 102 Resistance of MGAS315 and its sse and sagA deletion mutants to clearance at skin 103 infection sites and in nonimmune human and mouse blood. Bacterial loads of ssesagA in 104 6 the liver and spleen at days 1 and 2 after inoculation were >1000-fold lower than those of 105 MGAS315, sse, and sagA. This difference could be due to effective clearance of ssesagA 106 at skin infection sites because of the more robust neutrophil recruitment at ssesagA sites. 107 Alternatively, the double mutant might be compromised in growth and survival in blood. These 108 possibilities were examined. The numbers of viable GAS at skin infection sites at 24 h after 109 inoculation were 286%, 156%, 55%, and 85% of those at 1 h after inoculation in MGAS315, 110 sse, sagA, and ssesagA infections, respectively (Fig. 3A). The numbers of wt strain and 111 sse at skin infection sites slightly increased whereas the numbers of sagA and ssesagA 112 slightly decreased with time. The data indicate that the mutants with enhanced neutrophil 113 responses, especially ssesagA, were not efficiently cleared at 24 h after inoculation. To 114 determine whether the mutants were compromised in growth and survival in blood, the apparent 115 growth factors of MGAS315 and its sse, sagA, ssesagA mutants, and their complement 116 strains in heparinized blood and serum from two persons and in pooled heparinized mouse blood 117 were measured. The two persons were healthy and lacked anti-SLO and anti-NADase antibodies 118 in their serum (data not shown). The numbers of MGAS315, the mutants, and complement 119 strains of the mutants after 3-h incubation in blood and sera were more than 40-fold higher than 120 inoculum (Fig. 3B). The apparent growth factors of the test strains in each test had no significant 121 difference as the 1 way ANOVA multiple comparison analyses of the data using the GraphPad 122 Prism software (version 7.03) resulted in P values of ≥0.1184. Thus, the sse and/or sagA 123 deletions did not alter growth and survival of MGAS315 in human and mouse blood. These data 124 suggest that the lowered loads of the sse and sagA deletion mutants in the liver and spleen 125 appears to at least partially be due to reduced systemic dissemination. 126 7 Inhibition of neutrophil recruitment inside skin infection sites by MGAS315. The 127 MPO assay data in Figs. 1 and 2 indicate that there was no robust neutrophil response at 128 MGAS315 infection sites in the skin. The Gram and H&E stain patterns were consistent with 129 this measurement. Presentative Gram and H&E stain images of a section covering the center of 130 the whole subcutaneous infection site at days 1 and 2 after GAS inoculation are shown in Figs. 131 S1 and S2, respectively. Bacterial sites are referred as GAS zone, and a thin layer of neutrophils 132 referred as neutrophil zone was present at the edge of GAS zone. Images at higher magnification 133 show that bacterial site had very few neutrophils or necrotic materials, which is indicated by the 134 dashed, red box on both days 1 and 2 after inoculation of MGAS315 (Figs. 4 and 5). There was 135 a thin layer of neutrophils on day 1 and more neutrophils on day 2, which are indicated by the 136 dashed, yellow box. The yellow box of the neutrophil zone was at the edge of the GAS site, and 137 the red and yellow boxes were not overlapped with each other. These results indicate that 138 MGAS315 can inhibit neutrophil recruitment within GAS sites. 139 Synergistic effect of sse and sagA deletions on neutrophil recruitment inside GAS 140 sites. Skin infection sites were analyzed by Gram and H&E stains for subcutaneous infections of 141 C57BL/6J mice with sse, sagA, and ssesagA at 24 h (5 mice) and 48 h (5 mice) after 142 inoculation. A series of sections through each skin infection site were analyzed by Gram and 143 H&E stains, and representative stain images of whole skin infection sites at days 1 and 2 are 144 presented as Figs. S3-S8. The stain patterns of sse site at day 1 (Fig. S3) was largely similar to 145 that of MGAS315 site (Fig. S1), having neutrophils mainly at the edge of bacterial sites. 146 However, the higher magnification images show a couple of differences between MGAS315 and 147 sse sites. More neutrophils were present at the edge of sse site and were engaged with 148 bacteria (pink box), and an intense stain zone of bacteria and necrotic materials (green box) was 149 8 between the intense neutrophil/GAS zone (pink box) and sites with free bacteria and few 150 neutrophils (red box) (Fig. 4). While the stain patterns of MGAS315 site on day 2 were very 151 similar to that of MGAS315 site on day 1, the stain patterns of sse site on day2 were totally 152 different from those of sse site on day 1 and MGAS315 site on day 2, showing robust 153 neutrophil recruitment throughout the whole infection site (Fig. S4). The higher magnification 154 images of sse site on day 2 in Fig. 5 show intense staining of bacteria and necrotic neutrophils 155 (green box) and staining of intact neutrophils and bacteria at the edges of infection site (yellow 156 box). Thus, deletion of sse abolishes MGAS315 inhibition of neutrophil recruitment inside GAS 157 site. 158 The deletion of sagA had more profound effect on neutrophil recruitment than sse 159 deletion on day 1 (Fig. S5). sagA site on day 1 showed intense neutrophil stains at the edge 160 (pink box), and free bacteria and fewer neutrophils were observed at the center of sagA sites , 161 which contained bacteria/necrotic material regions (green box), and region containing mainly 162 free bacteria (red box) (Fig. 4). Compared with sse site, sagA site on day 2 showed more 163 intense staining of intact neutrophils (pink box) and had necrotic materials only at the center of 164 infection site (green box) (Figs. 5 and S6). Thus, deletion of sagA also abolishes MGAS315 165 inhibition of neutrophil recruitment inside GAS site. 166 In contrast to the sse and sagA infection in which the center of sse and sagA sites 167 had sparsely scattered neutrophils at day 1 after inoculation, ssesagA site was almost full of 168 neutrophils at day 1 after inoculation (Figs. 4 and S7). Stain patterns of ssesagA on day 2 169 were similar to those on day 1 (Figs. 5 and S8). These results indicate that the sse and sagA 170 deletions have synergistic promotion of neutrophil recruitment inside GAS sites in the skin. 171 9 Thus, neither of Sse or SLS is sufficient to cause the inhibition of neutrophil infiltration by 172 MGAS315, and both Sse and SLS are required for and synergistically contribute to inhibition of 173 neutrophil recruitment by MGAS315. In addition, ssesagA sites on both days did not have 174 many necrotic neutrophils, suggesting that Sse and SLS can both contribute to apoptosis or 175 necrosis of recruited neutrophils. 176 Complementation of sse, sagA, and ssesagA. To confirm the phenotype of sse, 177 sagA, and ssesagA was due to the sse and sagA deletions but not a spurious mutation, the sse 178 gene was put back into sse and ssesagA, and the sagA gene was put back to sagA and 179 ssesagA, yielding sse-sse, sagA-sagA, ssesagA-sse and ssesagA-sagA. These 180 complementation strains of the sse and sagA deletion mutants restored Sse production and SLS, 181 respectively, according to the PAF acetylhydrolase activity in the culture supernatant of sse-sse 182 and ssesagA-sse and β-hemolytic activity of sagA-sagA and ssesagA-sagA on blood agar 183 plates (data not shown). The histological patterns of these complementation strains in 184 subcutaneous infection of C57BL/6J mice were determined on days 1 and 2 after inoculation. 185 The Gram and H&E stain pattern of sse-sse (Fig. S9) and sagA-sagA (Fig. S10) were similar 186 to that of MGAS315 (Figs. S1 and S2), and the pattern of ssesagA-sse (Fig. S11) and 187 ssesagA-sagA (Fig. S12) were similar to that of sagA (Figs. S5 and S6) and sse (Figs. S3 188 and S4), respectively. In addition, the levels of emm3, hasA, spyCEP, and scpA transcripts in 189 sse, sagA, ssesagA at the exponential growth phase in THY were similar to those of 190 MGAS315 (data not shown). Thus, the phenotype of sse, sagA, and ssesagA is caused by 191 the sse and/or sagA deletions. 192 10 For better understanding of the histopathological features of the skin infections in the 193 histological figures, Gram and H&E stain images of the skin of C57BL/6J without GAS 194 infection is presented in Fig. S13 for comparison. 195 196 DISCUSSION 197 This study was designed to examine the role of Sse and SLS in the inhibition of 198 neutrophil recruitment by hypervirulent emm3 GAS. The findings are as follows: 1) deletion of 199 sse or sagA enhances neutrophil recruitment and reduces skin invasion and GAS loads in the 200 liver and spleen in subcutaneous infection of mice with MGAS315; 2) MGAS315 inhibits 201 neutrophil recruitment inside GAS sites, and both sse and sagA are required for the inhibition of 202 neutrophil recruitment; and 3) sse and sagA deletions have synergistic effects on skin invasion, 203 neutrophil recruitment, and systemic infection. These findings indicate that Sse and SLS are 204 required but either of them is not sufficient for the inhibition of neutrophil recruitment by 205 hypervirulent emm3 GAS and synergistically contribute to GAS skin invasion and systemic GAS 206 infections. 207 Bakleh et al. has classified necrotizing fasciitis caused by GAS into 3 stages according to 208 histopathologic features (6). Stage I NF has intense neutrophilic response and an absence of 209 bacteria in Gram stain; stage II NF has a moderate-to-severe neutrophilic response and positive 210 Gram stain; and stage III NF is characterized by the presence of few or no neutrophils and a 211 Gram stain results positive for bacteria. Patients with stage III NF have a higher mortality rate 212 than patients with stage I and II NF. The subcutaneous MGAS315 infection site in mice displays 213 the stage III histopathologic features. Skin infection sites of MGAS5005 in mice also have few 214 11 neutrophils at sites where Gram stain is positive for GAS (7). MGAS5005, a M1T1 isolate, and 215 MGAS315 are both hypervirulent CovS mutants, and the correction of their covS mutation 216 enhances neutrophil recruitment by more than 10 fold (8, 30). Thus, the findings in our current 217 and previous studies indicate that hypervirulent M1T1 and M3 GAS CovRS mutants can cause 218 the stage III histopathologic pattern in skin infections by inhibiting neutrophil recruitment. 219 The enhancement in neutrophil recruitment by sse deletion in MGAS315 confirms our 220 previous findings on the function of Sse in inhibition of neutrophil responses. Deletion of sse in 221 M1T1 strain MGAS5005 enhances neutrophil recruitment (7). Sse is a potent PAF 222 acetylhydrolase, and the function of Sse is medicated by targeting PAF (7, 31). Passive 223 immunization with an enzymatic activity-neutralizing monoclonal antibody of Sse enhances 224 neutrophil recruitment (32). Sse, but not CXC chemokine peptidase SpyCEP and C5a peptidase 225 ScpA, is critical for MGAS5005 inhibition of neutrophil recruitment in skin infection of mice (8). 226 Our results indicate that Sse also plays a critical role in the inhibition of neutrophil recruitment 227 by hypervirulent emm3 GAS. 228 SLS is another critical factor for the inhibition of neutrophil responses by MGAS315 in 229 skin infection of mice. SLS is a cytolytic toxin, and its peptide component is encoded by sagA 230 (33). GAS sagA deletion mutant is attenuated in virulence and skin invasion (34). SLS-negative 231 mutant is associated with the robust recruitment of neutrophils and significantly reduces lethal 232 myositis in zebrafish, and the extravasation of neutrophils is quicker in the early stage of 233 subcutaneous infection of mice with SLS- mutant than wild-type GAS (28). In this study, we 234 show that SLS is required for the inhibition of neutrophil recruitment by hypervirulent M3 GAS. 235 Thus, both sse and sagA are essential but not sufficient for the inhibition of neutrophil 236 recruitment by hypervirulent M3 GAS CovS mutants in subcutaneous infections of mice. 237 12 The essential but insufficient role of each of Sse and SLS in MGAS315 inhibition of 238 neutrophil recruitment implies that Sse and SLS evade neutrophil responses through different 239 mechanisms. The synergistic effects of sse and sagA deletions on neutrophil recruitment support 240 this implication. Sse has the potent PAF acetylhydrolase activity, and neutrophil recruitment is 241 lowered in infection of PAF receptor KO mice with MGAS5005 sse than that of wild-type 242 control mice (7, 31). PAF is an important lipid mediator in inflammation and chemoattractant 243 for neutrophils (35, 36). Deletion of sse leads to the reduction of the capacity of MGAS315 to 244 inhibit neutrophil recruitment inside GAS sites, suggesting that PAF may play a critical role in 245 neutrophils recruitment in the presence of SLS. Whether PAF mainly functions as 246 chemoattractant for neutrophils in GAS infections is currently investigated in our laboratory. 247 The difference in transepithelial migration of neutrophils between the treatments of the HaCaT 248 human keratinocyte cells with wild-type and SLS-negative GAS mutant suggests that SLS 249 inhibits the host cells’ production of signals chemotactic for neutrophils (28). SLS can promotes 250 programmed cell death of epithelial keratinocytes during GAS infection (37). In the MGAS315 251 sse infections, necrotic cells were present at the edge of bacteria sites on day 1 and inside GAS 252 sites on day 2 after inoculation, suggesting that SLS may kill neutrophils when they encounter 253 GAS bacteria and affect neutrophil recruitment at early stage of infection and result in the 254 histological pattern of sse infection at day 1 after inoculation. 255 There is a synergistic effect of sse and sagA deletions on GAS loads in the liver and 256 spleen in subcutaneous infection of mice. The sse and sagA deletion mutants were not efficiently 257 cleared at skin infection sites, and the growth factors of the mutants in nonimmune human blood 258 and serum are similar to those of the parent strain. These observations suggest that Sse and SLS 259 synergistically contribute to GAS dissemination and systemic infections by hypervirulent GAS. 260 13 261 MATERIALS AND METHODS 262 Declaration of ethical approval. All animal procedures were carried out in strict 263 accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals 264 of the National Institutes of Health (38). The protocols for the experiments were approved by 265 the Institutional Animal Care and Use Committee at Montana State University (Permit number: 266 2014-45). Blood was collected from healthy donors in accordance with a protocol approved by 267 the Institutional Review Board at MSU (Protocol No.: BL120513). Written informed consent 268 was provided by study participants. 269 Bacterial strains and growth. Genotype emm3 strain MGAS315 has been described 270 (29), and this hypervirulent clinical isolate has function-losing CovS G457V mutation (30). 271 MGAS315 and its derivative strains were grown in Todd-Hewitt broth supplemented with 0.2% 272 yeast extract (THY). 273 Generation of MGAS315 sse, sagA, and ssesagA mutants. MGAS315 sse was 274 generated using the suicide plasmid pGRV-Δsse (16), as described (16). The mutant lacked a 275 621-bp fragment that encodes amino acids 55 to 261 of Sse and had no foreign DNA including 276 antibiotic selection marker. 277 In the deletion of the sagA gene, a DNA fragment containing the whole sagA gene and its 278 23-bp upstream and 164-bp downstream was deleted whereas the downstream sagB was intact. 279 The 5′and 3′ ∼1,000-bp flanking fragments of the sagA fragment to be deleted were amplified 280 from MGAS315 chromosomal DNA and using high fidelity Phusion DNA polymerase and 281 primer pairs 5’-AGATCTGCAAACGTGTTCAATTGGTTG-3’/5’-282 CTCGAGTAACTGATAAGAACACGAG-3’ and 5’-283 14 TTCTCGAGCTAGATAGTACCTGCTAATTAC-3’/5’-284 TTGGATCCGAAATGTCACGACACGAAC-3’. The PCR products of the upstream and 285 downstream flanking fragments were sequentially cloned into pGRV (39) at the BglII/XhoI and 286 XhoI/BamHI sites, respectively, yielding the suicide plasmid pGRV-sagA. The plasmid was 287 introduced into MGAS315 using electroporation and inserted into the sagA locus of the 288 MGAS315 chromosome through a homologous recombination event at one flanking fragment. 289 Bacteria with this first crossover were selected on THY agar plates with 10 mg/l 290 chloramphenicol. One strain with the first crossover was grown on THY agar plate. At every 12 291 h after plating, bacteria were recovered from a whole plate, resuspended in 10 ml THY, and 292 vortexed for 2 min, and 10 µl of the vortexed GAS suspension was streaked on THY agar plate, 293 which was regarded as one passage. Deletion mutants were identified after >12 passages. 294 Bacteria were plated on THY agar plates after the last passage, and resultant colonies were 295 spotted in parallel on THY agar with and without chloramphenicol to identify potential double-296 crossover strains that were chloramphenicol-sensitive. Chloramphenicol-sensitive strains were 297 analyzed to identify deletion mutants by PCR using primers 5’-298 GTGATAAGAACTAGATAGTTG-3’ and 5’-GGCATTAAATGTTGAGCAAC-3’. The sagA 299 gene in MGAS315 sse was deleted similarly to yield ssesagA. 300 The sse and sagA deletions in the mutants were confirmed by DNA sequencing of the sse 301 and sagA loci and by the loss of the Sse activity in culture supernatant of sse mutants using the 302 PAF acetylhydrolase activity assay (31) and by the loss of β-hemolytic activity of sagA mutants 303 on blood agar plates. 304 Construction of complement strains. The sse gene was put back into sse and 305 ΔssesagA using pGRV-sse (16), as previously described (16), yielding sse-sse and 306 15 ΔssesagA-sse for complementation of the sse deletion mutants. For the complementation of the 307 sagA deletion mutants in sagA and ΔssesagA, a fragment containing the full-length sagA gene 308 and its flanking regions was amplified with PCR using the Phusion High Fidelity PCR kit from 309 New England BioLabs, MGAS315 genomic DNA, and primers 5’-310 TTGGATCCGCAAACGTGTTCAATTGGTTG-3’ and 5’-311 TTGGATCCGAAATGTCACGACACGAAC-3’. The PCR product was cloned into pGRV at 312 BamHI site, yielding suicide plasmid pGRV-sagA. This plasmid was introduced into sagA and 313 ΔssesagA by electroporation. The procedures described above to generate the ΔsagA mutant 314 were followed to generate complement strains sagA-sagA and ΔssesagA-sagA. These 315 complement strains were confirmed by sequencing the sse or sagA loci. The sse-sse and 316 ΔssesagA-sse strains restored production of Sse as assayed for the Sse enzymatic activity in its 317 culture supernatant using the Sse PAF acetylhydrolase activity assay (31), and the sagA-sagA 318 and ΔssesagA-sagA strains restored SLS production as the strains displayed β-hemolytic 319 activity on blood agar plates. 320 Subcutaneous Mouse infections. Six-week old, female CD-1 and female and male 321 C57BL/6J mice were used in subcutaneous infections with MGAS315, sse, sagA, ssesagA, 322 and complement strains of the mutants. CD-1 mice were purchased from Charles River 323 Laboratories, and C57BL/6J mice were breed at the Animal Resources Center at Montana State 324 University using the breeding mice from The Jackson Laboratory. GAS bacteria grown in THY 325 were harvested at the exponential growth phase and washed with pyrogen-free Dulbecco's 326 phosphate-buffered saline (DPBS) three times, and resuspended in DPBS. Groups of mice were 327 subcutaneously inoculated with 0.2 ml of GAS in DPBS at OD600 of 1.0, and actual inoculum 328 was determined by plating and usually contained about 108 colony-forming units (cfu). Mice 329 16 were euthanized at indicated time after inoculation to collect skin samples for measurement of 330 lesion size, viable bacteria, and neutrophil recruitment and histological analyses, and the liver 331 and spleen were also harvested to measure numbers of viable GAS. The euthanasia of mice was 332 done with a gradual fill method at a displacement rate of 30% CO2 of the chamber volume per 333 minute, as recommended in The 2013 American Veterinary Medical Association Guidelines. 334 Measurement of skin lesion size, neutrophil recruitment, and viable GAS in tissues. 335 The skin around the infection site was peeled off, and the skin lesion was recognized by the 336 boundary of the inflammation area. The size of skin lesions was measured by analyzing the 337 lesion pictures using the area measurement tool of the Adobe Acrobat 9 software program. The 338 skin containing infection area was excised for neutrophil measurement. Numbers of recruited 339 neutrophils in the infected skin samples were determined by a myeloperoxidase assay, as 340 described previously (7). The numbers of viable GAS in skin infection sites, liver and spleen 341 were determined by homogenizing the tissues in DPBS using Kontes pestles and then plating at 342 appropriate dilutions. 343 Histological analyses. Skin GAS infection sites collected at 24 h and 48 h after 344 inoculation were fixed in 10% neutral buffered formalin for 24 h. The samples were dehydrated 345 with ethanol, cleared with xylene, and infiltrated with paraffin using a Tissue Embedding 346 Console System (Sakura Finetek, Inc.). The paraffin blocks were processed to obtain 4-μm 347 sections, which were stained with hematoxylin and eosin (H&E) or with a Gram stain kit from 348 Becton, Dickinson and Company. Stained slides were examined using a Nikon Eclipse 80i 349 microscope. 350 GAS growth in nonimmune human blood and pooled mouse blood. GAS growth in 351 nonimmune blood and serum from two persons and pooled blood of female C57BL/6J mice was 352 17 determined as previously described (7). Bacteria of MGAS315, Δsse, sagA, ΔssesagA, and 353 complement strains were harvested at the exponential growth phase in THY, washed three times 354 with DPBS, and inoculated in triplicates at ∼ 105 CFU/ml into 0.5 ml of heparinized nonimmune 355 human blood or serum and into 0.2 ml of heparinized mouse blood pool. The samples were 356 rotated end-to-end for 3 h at 37°C, and the numbers of viable GAS in the samples and inocula 357 were determined by plating. The growth factor was defined as the ratio of CFU for each sample 358 after a 3-h incubation over the CFU of the corresponding inoculum. 359 Other analyses. DNA sequencing of the amplified PCR products was performed by 360 using the BigDye Terminator v3.1 cycle sequencing kit and an Applied Biosystems 3130 genetic 361 analyzer. Sequence data were analyzed by using Sequencer 5.1 software (Gene Codes 362 Corporation). The PAF acetylhydrolase activity in culture supernatant of MGAS315 and its 363 isogenic mutants was assayed using the 2-thio PAF-based colorimetric assay as described (31). 364 Statistical analyses. The statistical analyses were done using the GraphPad Prism 365 software (version 7.03). 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Effects of sse and sagA deletions on skin invasion, GAS loads in liver and spleen, 499 and neutrophil recruitment in subcutaneous MGAS315 infection of CD-1 mice. Six-week 500 old female CD-1 mice were subcutaneously inoculated with 1.7 x 108 CFU MGAS315, 2.0 x108 501 cfu sse, 2.1 x 108 cfu sagA, and 1.7 x 108 cfu ssesagA and euthanized at 48 h after 502 inoculation for analyses. Shown are representative inside-out images of the skin infection sites 503 (A), lesion size (B), neutrophil recruitment (C), GAS loads in spleen (D), and GAS load in liver 504 (E). The Mann-Whitney t Test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. 505 FIG. 2. Effects of sse and sagA deletions on skin invasion, GAS loads in liver and spleen, 506 and neutrophil recruitment in subcutaneous MGAS315 infection of C57BL/6J mice. six-507 week old C57BL/6J mice were subcutaneously inoculated with 1.5 x 108 CFU MGAS315, 1.9 508 x108 cfu sse, 1.8 x 108 cfu sagA, and 1.7 x 108 cfu ssesagA and euthanized at 24 h after 509 inoculation for analyses. Shown are lesion size (A), neutrophil recruitment (B), GAS loads in 510 24 liver (C), and GAS load in spleen (D). The Mann-Whitney t Test: *, P < 0.05; **, P < 0.01; ***, 511 P < 0.001; ****, and P < 0.0001. 512 FIG. 3. Lack of the detrimental effect of sse and sagA deletion on the clearance of 513 MGAS315 at skin infection sites and human blood. (A) Persistence of MGAS315, sse, 514 sagA, ssesagA at skin infection sites. About 108 cfu bacteria of each strain were 515 subcutaneously inoculated in groups of sixteen 6-week old female C57BL/6J mice. Eight mice 516 of each group were euthanized at 1 h and 24 h after inoculation. Presented are the numbers and 517 median values of viable GAS at the skin infection sites. The P values are for 1 h versus 24 h 518 comparison of each strain by the Mann-Whitney t Test. (B) Growth factors of MGAS315 and its 519 derivative strains in nonimmune blood and serum. The strains were inoculated (∼105 CFU) into 520 0.5 ml of nonimmune blood or serum in triplicates and incubated for 3 h at 37°C with end-to-end 521 rotation. Growth factor was defined as the ratio of viable CFU of GAS in each sample over the 522 CFU of the inoculum. The 1way ANOVA multiple comparison analyses: P ≥ 0.1184. 523 FIG. 4. Histological analyses of MGAS315, sse, sagA, and ssesagA skin infection sites 524 in mice at day 1 after inoculation. Groups of ten 6-week old female C57BL/6J mice were 525 subcutaneously inoculated with 1.5 x 108 cfu MGAS315, 1.8 x 108 cfu sse, 1.9 x 108 cfu sagA, 526 2.0 x 108 cfu ssesagA in 0.2 ml DPBS. Five mice of each group were sacrificed on days 1 and 527 2 after inoculation, respectively, to collect skin infection sites. The skin infection sites were 528 fixed and analyzed with Gram and H&E stains, as described in the Materials and Methods 529 section. Shown are representative Gram and H&E stain images of infection sites. Shown are the 530 Gram and H&E stain images for the regions in skin infection site that are indicated by the boxes 531 in Figs. S1, S3, S5, and S7. The bar represents 20 µm. 532 25 FIG. 5. Histological analyses of MGAS315, sse, sagA, and ssesagA skin infection sites 533 in mice at day 2 after inoculation. The infection conditions were described in Fig. 4. Shown 534 are representative Gram and H&E stain images of infection sites. Shown are the Gram and H&E 535 stain images for the regions in skin infection site that are indicated by the boxes in Figs. S2, S4, 536 S6, and S8. The bar represents 20 µm. 537 Manuscript Text File Figure 1 Figure 2 Figure 3 Figure 4 Figure 5