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The PEP Phosphotransferase System (PTS) in the Group A Streptococcus 1 Acts to Reduce SLS activity and Lesion Severity During Soft Tissue Infection 2 3 4 Kanika Gera 1 , Tuquynh Le 1 , Rebecca Jamin 2 , Zehava Eichenbaum 2 and Kevin S. McIver 1 * 5 6 7 1 Department of Cell Biology & Molecular Genetics and Maryland Pathogen Research Institute, 8 University of Maryland, College Park, MD 20742 9 2 Biology Department, Georgia State University, Atlanta, GA 30302 10 11 12 Running Title: PTS and virulence in GAS 13 14 *Address Correspondence To: 15 Kevin S. McIver, University of Maryland, 3124 Bioscience Research Bldg (413), College Park, 16 MD 20742-4451; Tel: (301) 405-4136; Fax: (301) 314-9489 17 Email: [email protected] 18 IAI Accepts, published online ahead of print on 30 December 2013 Infect. Immun. doi:10.1128/IAI.01271-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved. on November 28, 2018 by guest http://iai.asm.org/ Downloaded from

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Page 1: The PEP Phosphotransferase System (PTS) in the Group A ... · yz P~Hpr-Ser interacts with the catabolite control protein CcpA, ... sur Summerson colorimeter (A filter). Alternatively,

The PEP Phosphotransferase System (PTS) in the Group A Streptococcus 1 Acts to Reduce SLS activity and Lesion Severity During Soft Tissue Infection 2

3 4

Kanika Gera1, Tuquynh Le1, Rebecca Jamin2, Zehava Eichenbaum2 and Kevin S. McIver1* 5 6 7

1Department of Cell Biology & Molecular Genetics and Maryland Pathogen Research Institute, 8 University of Maryland, College Park, MD 20742 9

2Biology Department, Georgia State University, Atlanta, GA 30302 10 11 12 Running Title: PTS and virulence in GAS 13 14 *Address Correspondence To: 15 Kevin S. McIver, University of Maryland, 3124 Bioscience Research Bldg (413), College Park, 16 MD 20742-4451; Tel: (301) 405-4136; Fax: (301) 314-9489 17 Email: [email protected] 18

IAI Accepts, published online ahead of print on 30 December 2013Infect. Immun. doi:10.1128/IAI.01271-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 19 Obtaining essential nutrients, such as carbohydrates, is an important process for bacterial 20

pathogens to successfully colonize host tissues. The Phosphoenolpyruvate Phosphotransferase 21 System (PTS) is a primary mechanism by which bacteria transport sugars and sense the carbon 22 state of the cell. The Group A Streptococcus (GAS) is a fastidious microorganism that has 23 adapted to a variety of niches in the human body to elicit a wide array of diseases. A ∆ptsI 24 mutant (EI deficient) generated in three different strains of M1T1 GAS was unable to grow on 25 multiple carbon sources (PTS and non-PTS). Complementation with ptsI expressed under its 26 native promoter in single copy was able to rescue the growth defect of the mutant. In a mouse 27 model of GAS soft tissue infection, all ∆ptsI mutants exhibited a significantly larger and more 28 severe ulcerative lesion than mice infected with their respective wild type. Increased transcript 29 levels of sagA and Streptolysin S (SLS) activity during exponential phase growth was observed. 30 We hypothesized that early onset of SLS activity would correlate with the severity of the lesions 31 induced by the ΔptsI mutant. In fact, infection of mice with a ∆ptsI sagB double mutant resulted 32 in a lesion comparable to either a wild type or a sagB mutant alone. Therefore, a functional PTS 33 is not required for subcutaneous skin infection in mice; however, it does play a role in 34 coordinating virulence factor expression and disease progression. 35 36 37

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INTRODUCTION 38 The ability to obtain essential nutrients during an infection is critical for bacterial 39 pathogens to successfully colonize and proliferate within host tissues. One such key process 40 involves the ability to import and catabolize optimal carbon sources such as carbohydrates. 41 Bacteria have evolved elegant regulatory pathways that detect the presence of preferred 42 carbohydrates, repress the utilization of non-preferred sugars, and regulate their metabolism 43 based on this information flow (1, 2). In fact, many pathogens tightly control the genes involved 44 in carbohydrate utilization and regulation in response to in vivo growth and these same genes 45 have been shown to be important to the disease process (3-8). Therefore, it is apparent that 46 bacterial pathogens have closely linked their sugar metabolic sensing networks to virulence gene 47 expression during infection. 48 The primary bacterial system coupling the transport of carbohydrates across the 49 cytoplasmic membrane with their phosphorylation is a multiprotein phosphorelay called the 50 phosphoenolpyruvate (PEP)-dependent phosphotransferase system or PTS (9). The PTS is 51 composed of at least three distinct proteins: the cytosolic proteins enzyme 1 (EI) and Hpr, 52 encoded by ptsI and ptsH, respectively, and the membrane-bound sugar-specific enzyme II (EII) 53 proteins. Each EII consists of one or two integral membrane-bound domains (EIIC/EIID) that are 54 necessary for sugar translocation and these domains form complexes with two hydrophilic 55 components (EIIA & EIIB) that are required for substrate phosphorylation (1, 10). EI initiates 56 the phosphorelay after autophosphorylation by PEP, followed by transfer of the phosphoryl 57 group to the histidine at position 15 of HPr. P~HPr–His then donates the phosphoryl group to 58 one of several sugar-specific EIIA proteins, which in turn transfers it to its cognate EIIB, and 59 finally onto the incoming cognate sugar transported by EIIC/D. 60

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In addition to carbohydrate transport, the PTS in Gram-positive bacteria also plays an 61 important role in modulating the activity of transcriptional regulators (e.g., antiterminators, 62 activators) that control expression of sugar utilization genes. The PTS intermediates P~HPr–His 63 and P~EIIsugar can directly phosphorylate transcriptional regulators that contain either PTS 64 regulatory domains (PRD) and/or EII-like domains to effect their activity. In Bacillus subtilis, 65 the phosphorylation state of EI and Hpr regulates the activity of PRD-containing antiterminators 66 (e.g., LicT) and activators (e.g., LevR) in response to glucose availability, thus controlling their 67 ability to regulate the expression of alternative sugar operons (1, 11). PTS-mediated signaling 68 can also influence virulence gene expression in Gram-positive pathogens. The cellobiose PTS of 69 Streptococcus pneumonia is important for virulence in a murine pneumonia/sepsis model (12). 70 Inactivation of ptsI (EI) and ptsH (Hpr) in Clostridium difficile led to induction of toxin gene 71 expression in the presence of glucose (13). Activity of PrfA, the major virulence regulator in 72 Listeria monocytogenes, is controlled by the phosphorylation state of PTS components (14). The 73 Bacillus anthracis AtxA master virulence regulator contains both PRD and EIIB domains, and 74 requires an intact PTS pathway for full activity (15). 75 The PTS is also involved in carbon catabolite repression (CCR) via Hpr kinase (HprK)-76 mediated phosphorylation at Ser46 of Hpr in Gram-positive bacteria. In the presence of glucose, 77 P~Hpr-Ser interacts with the catabolite control protein CcpA, which then is able to bind to cre 78 sites found in promoters of sugar utilization and metabolism operons to mediate CCR (1, 16). In 79 addition to its central role in CCR, CcpA has been shown be important for the virulence of a 80 number of important Gram-positive pathogens (17-22). CcpA-independent pathways for CCR 81 also exist in Streptococcus mutans, which is exerted through a network of PTS permeases (23). 82

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Thus, the sugar status of the bacterial cell can have a profound impact on virulence and 83 expression of important virulence phenotypes in Gram-positive pathogens. 84 The group A streptococcus (GAS, S. pyogenes) is a Gram-positive human-adapted 85 pathogen with the ability to cause benign and life-threatening diseases in various host niches, 86 including the respiratory tract (pharyngitis), the skin (impetigo), deep tissues (necrotizing 87 fasciitis), and the bloodstream (streptococcal toxic shock syndrome). A conservative estimate 88 indicates that more than 745 million cases of GAS infection occur worldwide each year, leading 89 to over half a million deaths (24). The clinical impact of GAS is based largely on its ability to 90 produce a large array of surface exposed (e.g., M protein, capsule) and secreted (e.g., 91 Streptolysins SLS and SLO, SpeB protease) virulence factors that contribute significantly to its 92 pathogenesis. GAS exhibits coordinate regulation of its virulence genes in response to changing 93 host environments through global regulatory networks such as the stand-alone regulator Mga and 94 the two-component system (TCS) CovRS. Mutations in the histidine kinase CovS are associated 95 with an invasive GAS disease phenotype and the altered regulation of virulence genes, including 96 sagA (SLS), speB (cysteine protease), and hasA (capsule). 97 GAS is a fermentative lactic acid bacterium that relies heavily on sugar metabolism for 98 its energy production given that it lacks the necessary enzymes for a functional TCA cycle and 99 oxidative-cytochromes for electron transport. Early studies established that glucose, sucrose, 100 maltose, galactose, and mannose could support growth of an M3 GAS strain in chemically 101 defined medium (25). Addition of PTS-transported sugars such as glucose, sucrose or trehalose 102 to a semisynthetic growth medium stimulate the synthesis of M protein, an important GAS 103 virulence factor involved in resistance to phagocytosis and adherence to host tissues (26). 104 Furthermore, genes involved in carbohydrate uptake and utilization are up regulated in vivo and 105

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contribute to virulence in GAS models of infection (19, 21, 27, 28). CcpA-mediated CCR via 106 Hpr is also important for GAS virulence, regulating over 6% of the genome, including indirectly 107 repressing transcription of the sag operon encoding SLS (18, 19, 21). Finally, we have recently 108 shown that the Mga virulence regulator contains PRD domains that are phosphorylated by the 109 PTS to control Mga-regulated gene expression and virulence (3). Despite the increasing 110 evidence that carbohydrate utilization influences GAS pathogenesis, the role of PTS transport 111 and signaling on disease progression has not been investigated. 112 In this study, we characterize the PTS pathway in multiple strains of GAS by creating a 113 deletion mutant of ptsI (EI), the first protein of the phosphorelay. We show that a functional PTS 114 is not necessary for eliciting a GAS subcutaneous soft tissue infection. However, it does limit 115 lesion severity at the site of subcutaneous infection in mice by regulating the temporal expression 116 level of Streptolysin S. Thus, PTS transport and signaling contributes to GAS pathogenesis. 117 118 119 MATERIALS AND METHODS 120 Bacterial strains and media 121 MGAS5005 (covS) is an invasive M1T1 strain with an available genome sequence (29). GAS 122 strain 5448 is also a M1T1 strain isolated from an invasive infection and 5448AP is an isogenic 123 animal passaged covS variant. GAS was either cultured in complete Todd-Hewitt medium 124 (Alpha Biosciences) supplemented with 0.2% yeast extract (THY) or in 2x Chemically Defined 125 Medium (CDM) prepared by MP Biomedical as previously described (30, 31). Prior to use, 126 freshly prepared sodium bicarbonate (59.51 µM final) and L-cysteine (11.68 µM final) were 127

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added along with a carbohydrate source at a final concentration of either 0.5% (D-glucose) or 1% 128 (all other sources). Growth of GAS was assayed by measuring absorbance using a Klett-129 Summerson colorimeter (A filter). Alternatively, overnight cultures of GAS (10 ml) were 130 adjusted to OD600 of 0.2 in saline, appropriate carbohydrate was added, and 50 µl aliquots were 131 transferred a 24-well microplate (Corning) containing CDM with various sugars and covered 132 with sealing tape (BioRad). Growth was followed for 12-24 hours at 37ºC using a FLUOstar 133 Omega microplate spectrophotometer (BMG), with measurements taken at 30 min intervals after 134 shaking (10 sec). Escherichia coli strain DH5α and C41 [DE3] were used as the host for plasmid 135 constructions (Table 1). All E. coli strains were grown in Luria-Bertani (LB) broth (EMD 136 Chemicals). 137

Antibiotics were used at the following concentrations: erythromycin at 500 µg/ml for E. 138 coli and 1.0 µg/ml for GAS, spectinomycin at 100 µg/ml for both E. coli and GAS, kanamycin at 139 50 µg/ml for E. coli and 300 µg/ml for GAS 140 141 DNA manipulations 142 Plasmid DNA was isolated from E. coli using the Wizard® Plus SV Miniprep system (Promega). 143 DNA fragments were gel purified from agarose using the Qiaquick Gel Extraction kit (Qiagen) 144 or the Wizard® SV Gel and PCR Clean-Up system (Promega). PCR for cloning and generating 145 probes was performed using Phusion (Finnzymes) according to manufacturers protocol. All 146 DNA sequencing was done by Genewiz, Inc. Genomic DNA was extracted from GAS using the 147 MasterPure Complete DNA and RNA Purification Kit for Gram-positive bacteria (Epicentre). 148 149 150

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Construction of a ∆ptsI mutant in GAS 151 The plasmid to generate the ∆ptsI allele was created as previously described (3). Briefly, the 152 primers ptsI-1a and ptsI-1b (Table 2) were used to amplify from MGAS5005 genomic DNA 153 (gDNA) an 819 bp upstream region containing 578 bp of ptsI, and a BglII restriction site. 154 Primers ptsI-2a and ptsI-2b (Table 2) were used to amplify an 812 bp region containing the 3′of 155 ptsI with BglII ends and a 10 bp overlap with the first fragment at the 5′end. These fragments 156 were combined as template DNA for PCR SOEing (32) with ptsI-3a and ptsI-3b (Table 2) to 157 generate the ptsI deletion. The resulting product was blunt end ligated into pBluescript II KS- to 158 create pBlueΔptsI (Table 1). The non-polar aad9 spectinomycin resistance cassette was 159 amplified from pSL60-1 using primers aad9L2-bglII and aad9R2-bglII (Table 2), digested with 160 BglII, and ligated into BglII-digested pBlueΔptsI (Table 1). The resulting XbaI/XhoI ΔptsI::aad9 161 fragment from pBlueΔptsI was ligated into XbaI/XhoI-digested pCRK to yield pKSM645, which 162 was used to construct the ∆ptsI mutant MGAS5005.∆ptsI (Table 1). Additional mutants were 163 constructed in 5448 (5448.∆ptsI and 5448AP (5448AP.∆ptsI) using the same protocol (Table 1). 164 GAS ∆ptsI mutants were screened for sensitivity to kanamycin and verified by PCR on genomic 165 DNA for specific junction regions. 166 167 Single-copy complementation of MGAS5005.∆ptsI 168 A thermosensitive integration plasmid for single-copy complementation was constructed using 169 primers ptsIcomplementL and ptsIcomplementR (Table 2) to amplify a 1919 bp fragment 170 containing a promoterless wild type copy of ptsI and BamHI restriction site on both ends. The 171 resulting PCR fragment was digested with BamHI, ligated into BamHI digested pCRK to yield 172 pKSM456 (Table 1), and confirmed by sequence analysis. pKSM456 was electroporated into the 173

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mutant MGAS5005ΔptsI at the permissive temperature (30°C) and integrants were isolated 174 following growth at the non-permissive temperature (37°C) to allow for complementation of the 175 ΔptsI allele in single copy. The resulting strain MGAS5005.∆ptsIc was verified by PCR and 176 sequence analysis. 177 178 Murine infections 179 An overnight culture (10 ml) was used to inoculate 75 ml of THY and incubated static at 37°C 180 until late logarithmic phase. Approximately 3 x 109 CFU/ml, as determined by microscope 181 counts and verified by plating for viable colonies, were used to infect 5 to 6-week-old female 182 CD-1 mice (Charles River Laboratories). The mice were anaesthetized with ketamine and 183 depilated for ~ 3 cm2 area of the haunch with Nair (Carter Products) and 100 μl of a cell 184 suspension in saline (3 x 108 CFU/mouse) was injected subcutaneously. Mice were monitored 185 twice daily for 7 days and were euthanized by CO2 asphyxiation upon signs of morbidity. Lesion 186 sizes (L x W) were measured at 36 h post infection with length (L) determined at the longest 187 point of the lesion and width (W) as the widest point. Lesion size data was analyzed using 188 GraphPad Prism (GraphPad Software) and tested for significance using an unpaired two-tailed t-189 test with 99% confidence. 190 191 RNA isolation 192 GAS from an overnight culture were inoculated 1:20 into 10 ml THY supplemented with 193 appropriate antibiotics and grown to the appropriate Klett unit (ca. 80-100). Cells were then 194 pelleted by centrifugation at 8,000 x g for 20 min at 4°C. Cells were resuspended in 1 ml of TE 195 buffer (10 mM Tris pH 7.4 and 1 mM EDTA) with 0.2% (v/v) Triton X-100 and boiled for 10 196

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min. The lysate was extracted three times using chloroform-isoamyl alcohol, EtOH precipitated 197 for 20 min at -80°C, pelleted at 13,000 x g for 15 min at 4°C, and the RNA pellet was 198 resuspended in DEPC treated H2O. RNA quality was assessed on a formaldehyde gel (18% (v/v) 199 formaldehyde, 1% (w/v) agarose, 72% (v/v) DEPC-treated H2O, and 10% (v/v) 10x MOPS 200 buffer (0.4 M 3-[N-Morpholino] propanesulfonic acid pH 7.0, 0.1 M sodium acetate, 0.01 M 201 EDTA). To quantify RNA, absorbance at 260/280 was determined using a spectrophotometer. 202 RNA (5 ug) was treated with DNase using the Turbo DNA Free kit (Ambion) per manufacturer’s 203 protocol. Treated RNA was assessed for quality as mentioned above and for gDNA 204 contamination by PCR analysis. 205 206 Real-time RT-PCR 207 25 ng of DNase-treated total RNA was added to SYBR Green Master mix (Applied Biosystems) 208 along with 6.5μl of each gene specific real-time primer from a stock of 20 nM (Table 2) using 209 the 1-step protocol. The real-time RT-PCR experiments were completed using a Light Cycler 210 480 (Roche) and levels presented represent ratios of wild type/experimental relative to the level 211 of gyrA transcript as the internal control. The real-time primers were designed using Primer 3: 212 WWW Primer tool (biotools.umassmed.edu/bioapps/primer3_www.cgi). 213 214 BIOLOG phenotype microarrays 215 Carbohydrate metabolic profiles were determined using the BIOLOG Omnilog system (Biolog 216 Inc.) per the manufacturers protocol. Tests were performed utilizing the PM1 and PM2 carbon 217 panels, each as standard 96-well microplates containing 95 different carbon sources and one 218 negative control. Each well contained a redox dye (tetrazolium violet) for colorimetric 219

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determination in response to metabolizing the carbon source. Strains were first cultured on TSA 220 5% blood agar plates for 24 h at 37°C. Cells were swabbed off the plate and resuspended in 221 Inoculating Fluid (BIOLOG) to adjust to an absorbance at 600 nm of 0.14. An aliquot of 100 μl 222 of the suspension was immediately dispensed into each well of the PM microplate with a 223 multichannel pipette. The plate was incubated at 37°C for 48 h and the data was analyzed using 224 the Omnilog software (BIOLOG). 225 226 Hemolysis assay 227 GAS were grown in THY supplemented with 10% heat-inactivated horse serum (Sigma). 228 Samples were taken every hour for a total of 8 to 14 hours and immediately frozen at −80°C, 229 bacterial cells were pelleted, and a 1:10 dilution was made of the supernatant in saline. 500 µl of 230 this dilution was added to an equal volume of 2.5% (v/v) difibrinated sheep red blood cells 231 (RBC, Sigma) and washed three times with sterile PBS, pH 7.5. This mixture was incubated at 232 37°C for 1 h and cleared by centrifugation at 3,000 x g. Supernatants were measured at 541 nm 233 by spectrophotometer (Molecular Dynamics) to determine release of hemoglobin by lysed RBCs. 234 Percent hemolysis was defined as follows: [(sample A − blank A)/(100% lysis A)] × 100. To 235 assay for streptolysin O-specific hemolytic activity, the Streptolysin S inhibitor trypan blue (13 236 μg/μl) was added to samples prior to incubation. 237 238

RESULTS 239 GAS EI mutant (ΔptsI) is defective for growth in several carbohydrates. To assess the role 240 of the PEP phosphotransferase system (PTS) in GAS, an EI mutant (ΔptsI) was constructed in 241

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the M1T1 strain MGAS5005 (Table 1), a representative strain of the most common GAS 242 serotype associated with severe invasive disease worldwide (29). The genetic composition of the 243 PTS in GAS is comparable to that found in many bacterial species (1). The ‘general’ PTS 244 proteins Hpr (encoded by ptsH) and EI (encoded by ptsI) are highly conserved among low G+C 245 Gram-positive bacteria, including multiple species of Streptococcus. The ptsHI 246 (M5005_Spy1121 and M5005_Spy1120, respectively) genes are typically found in an operon 247 (Fig. 1A). We replaced wild type ptsI with an in-frame deletion (ΔptsI) containing a non-polar 248 aad9 spectinomycin resistance cassette (33) in the MGAS5005 genome (Fig. 1A). The resulting 249 MGAS5005.ΔptsI mutant (Table 1) was verified by PCR (data not shown) and qRT-PCR (Fig. 250 4A). MGAS5005.ΔptsI exhibited a small colony phenotype compared to wild type MGAS5005 251 when grown on TSA agar supplemented with 5% sheep blood, but not on TSA or THY agar 252 alone (data not shown). Although MGAS5005.ΔptsI had a slightly increased lag phase 253 compared to wild type in rich THY medium, the growth kinetics were comparable (data not 254 shown). 255

Growth of the parental MGAS5005 and the MGAS5005.ΔptsI mutant was further 256 analyzed by phenotypic microarray (BIOLOG) using the carbon panels PM1 and PM2, which 257 contain a total of 190 different carbon sources. Each well was inoculated with either strain and 258 the plates were incubated at 37ºC in an Omnilog plate incubator for 48 h to allow generation of 259 independent growth curves (data not shown). Wild type MGAS5005 showed reasonable growth 260 in multiple carbon sources (Table 3). Of the 45 carbon sources able to support MGAS5005 261 growth, the ∆ptsI mutant showed poor or no growth in 19 (Table 3), including both PTS-262 transported (bold) and non-PTS-transported sugars. Carbohydrate-specific phenotypes observed 263 by phenotype microarray were confirmed by growth assays in chemically defined medium 264

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(CDM) supplemented with 1% w/v of certain carbohydrate sources as the sole carbon source. 265 MGAS5005.ΔptsI showed comparable growth to MGAS5005 (wild type) when grown in CDM 266 supplemented with 0.5% glucose (Fig. 1B). However, MGAS5005.ΔptsI was unable to grow 267 when the PTS-transported sugars sucrose and fructose (Fig. 1CD, Table 3) or lactose, galactose, 268 trehalose, and mannose were tested using CDM (data not shown, Table 3). These results 269 strongly support that MGAS5005.ΔptsI lacks a functional PTS. The inability of the ∆ptsI mutant 270 to grow on some non-PTS sugars (Table 3) indicates the importance of a functional PTS for 271 uptake and utilization of non-PTS sugars. 272

To confirm that the growth defect phenotype was specific to the ΔptsI allele, we 273 generated a complemented ΔptsI strain by introducing a wild type ptsI allele into the 274 chromosome of MGAS5005.ΔptsI (MGAS5005.ΔptsIc, Table 1). The MGAS5005.ΔptsIc 275 complemented strain showed a growth profile in all PTS specific sugars comparable to wild type 276 MGAS5005 (Fig. 1CD, data not shown). 277 ΔptsI mutant shows increased soft tissue invasion in a mouse model of GAS skin infection. 278 The role of EI (ptsI) and the PTS in GAS virulence was assessed using a murine model of 279 streptococcal soft tissue infection. MGAS5005 and MGAS5005.ΔptsI were grown to late log 280 phase and then injected subcutaneously (~3 x 108 CFU) into the haunches of 5-6 week old, 281 female CD1 mice. For 7 days post infection, the progression of disease was monitored for both 282 lesion size (at 38 h post infection) and survival. Mice infected with wild type MGAS5005 283 developed purulent abscessed lesions with minimal ulceration after 38 h (Fig. 2A). In contrast, 284 mice infected with a comparable dose of MGAS5005.ΔptsI developed lesions at 16 h equivalent 285 in size to wild type at 38 h, and these lesions became fully ulcerative by 24 h post infection (data 286 not shown). The visual difference in both the size and severity of lesions between the wild type 287

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and the ΔptsI mutant at 38 h post infection was quite striking (Fig. 2A). This coincided with a 288 significant increase in the mean lesion size for mice infected with MGAS5005.ΔptsI compared to 289 MGAS5005 measured at the same time post infection (Fig. 2B). Interestingly, a corresponding 290 increase in lethality over 7 days was not observed in mice infected with MGAS5005.ΔptsI, 291 suggesting there was no enhancement of dissemination from the skin or in systemic lethality 292 (Fig. 2C). Thus, the ΔptsI mutant exhibits a hyper virulent phenotype at the site of infection 293 compared to wild type; however, systemic progression and lethality is not altered. 294 ΔptsI mutant exhibits a comparable phenotype in multiple M1T1 GAS strains. To explore 295 the role of EI in different genetic backgrounds, we constructed independent ΔptsI mutants in two 296 additional M1T1 strains, 5448 and 5448AP (covS), with the same mutagenic plasmid used for 297 MGAS5005.ΔptsI (Table 1). Similar to MGAS5005.ΔptsI, a small colony phenotype on blood 298 agar plates was observed for 5448.ΔptsI and 5448AP.ΔptsI (data not shown). Furthermore, these 299 ΔptsI mutants exhibited growth defects when grown in CDM supplemented with PTS sugars 300 identical to that seen for MGAS5005.ΔptsI (Fig. S1, data not shown). Finally, similar growth 301 phenotypes were observed in at least two independently isolated MGAS5005.ΔptsI, 5448.ΔptsI 302 and 5448AP.ΔptsI mutants. Thus, biologically independent ΔptsI mutants exhibit comparable 303 phenotypes in multiple M1T1 backgrounds. 304

We also tested the 5448.ΔptsI and 5448AP.ΔptsI mutants and their respective parental strains 305 in the murine model of streptococcal soft tissue infection (Fig. 3). As seen with 306 MGAS5005.∆ptsI, mice infected by either ΔptsI mutant exhibited a more severe (Fig. 3A) and 307 significantly larger (Fig. 3B) ulcerative lesion at 38 h post infection compared to infection with 308 its parental strain. In contrast to MGAS5005.∆ptsI and 5448-AP.∆ptsI (covS mutants), only 309 mice infected with 5448.ΔptsI (intact covS) showed a significant increase in systemic lethality 310

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compared to wild type (Fig. 3C). These data suggest that EI is important in controlling virulence 311 at the site of localized skin infections, as well as potentially serving a CovS-dependent role in 312 limiting dissemination to sterile sites of the body. 313

314 ∆ptsI mutant increases secretion of SpeB in a strain-specific manner. The cysteine protease 315 SpeB is a well-characterized secreted virulence factor that has been shown to be a key 316 contributor in various models of GAS skin infection, including ulcerative lesion formation (34-317 36). To investigate whether the M1T1 ΔptsI mutants resulted in altered speB transcript levels, 318 qRT-PCR was performed on mRNA isolated from wild type and the corresponding isogenic 319 ΔptsI mutant from all three M1T1 strains at transition phase of growth. No significant differences 320 (greater than 2-fold) in speB transcript levels were observed for any of the three ΔptsI mutants 321 compared to their respective parental strain (data not shown), suggesting that transcription of 322 speB is not altered in any of the mutants. 323

Increased secretion of active SpeB by GAS has been shown to negatively effect the 324 formation of biofilms (36-38) and can be used as an indirect reporter for SpeB activity. A 325 biofilm formation assay (supplemental Methods) was performed on each M1T1 wild type and its 326 respective ∆ptsI mutant. Cells were grown in a 6-well plate and allowed to grow for 24 h at 327 37°C and biofilm formation was quantified by crystal violet staining. MGAS5005.∆ptsI and 328 5448AP.∆ptsI, both covS mutant backgrounds, showed significantly less biofilm than their wild 329 type parental strains (Fig. S2A-C, left). Importantly, the reduction seen in these ∆ptsI mutants 330 could be reversed with the addition of the cysteine protease inhibitor E64 (Fig. S2AC, left). In 331 contrast, 5448.∆ptsI showed no significant difference compared to the wild type 5448. It should 332 be noted that 5448 is reduced in its ability to form biofilm compared to MGAS5005 and 333

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5448AP, and an MGAS5005.∆speB control exhibited no significance difference in biofilm 334 formation when compared to 5448 (Fig S2A). 335

Secretion of SpeB into the supernatant of all three ΔptsI mutants and their wild type 336 strains was assayed by western blot using a polyclonal SpeB antibody. No differences were 337 observed in the amount of cell-bound SpeB of ΔptsI mutants compared with their corresponding 338 wild type strain (data not shown). However, dramatically increased levels of secreted SpeB (both 339 40-kDa zymogen and 28-kDa mature forms) were detected in the culture supernatant of 340 MGAS5005.∆ptsI and 5448AP.∆ptsI compared to their isogenic covS parental strains (Fig. S2A-341 C, right). Interestingly, 5448.∆ptsI had decreased levels of secreted SpeB compared to wild type 342 5448, which correlates with the biofilm forming ability. Although this strongly suggests that EI 343 (PTS) may reduce SpeB secretion in a CovS-dependent manner (MGAS5005∆ptsI and 344 5448AP∆ptsI), this does not provide a mechanism for how increased lesion severity occurs in 345 5448.∆ptsI. 346 347 ΔptsI mutants exhibit expression of SLS during log phase growth. Streptolysin S (SLS) is a 348 secreted virulence factor that contributes to the formation of lesions in soft tissue models of 349 mouse infection and GAS virulence in general (34, 39, 40). Biosynthesis and secretion of SLS in 350 GAS requires the 9-gene sag operon, with sagA encoding the toxin precursor and the first 351 position in the locus (41). To determine whether the ΔptsI mutation affects sag operon 352 expression, qRT-PCR was performed on the isolated mRNA of MGAS5005, MGAS5005.ΔptsI, 353 and the complemented MGAS5005.ΔptsIc to quantify sagA transcript levels at late log phase 354 cells in THY. Changes in transcript levels greater than two-fold were considered significant. A 355 modest yet significant increase in sagA transcript levels was observed in the ΔptsI mutants 356

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compared to their corresponding wild type strains (Fig. 4A-C) at this time point. 357 SLS-specific hemolytic activity was assayed using 2.5% defibrinated sheep RBCs 358

incubated with culture supernatants from the wild type MGAS5005 and mutant 359 MGAS5005.ΔptsI GAS taken at 1 h intervals across growth. Addition of the SLS inhibitor, 360 trypan blue, blocked all RBC lysis in each experiment, indicating that the observed hemolytic 361 activity was due to SLS (data not shown). SLS hemolytic activity in the MGAS5005.ΔptsI 362 mutant supernatants showed a dramatic increase early in logarithmic phase and remained 363 elevated during stationary phase (Fig 4D). In comparison, SLS hemolytic activity from the 364 supernatants of wild type and the complemented ∆ptsI showed little activity during logarithmic 365 phase and increased to maximum levels at the transition to stationary phase (Fig. 4D), as 366 previously observed for SLS (19). Similarly, an early onset of SLS hemolytic activity was 367 observed for the in 5448.∆ptsI and 5448AP.∆ptsI mutants in comparison to their respective wild 368 type strains (Fig. 4EF). These data suggest that the early onset of SLS activity during exponential 369 phase growth in all three M1T1 GAS strains lacking a functional PTS could lead to the increased 370 severity of localized lesions observed in mice (Fig. 2 and 3). 371

372 SLS is required for the lesion severity observed in M1T1 ∆ptsI mutants. The role of SLS in 373 the hypervirulence of lesions of mice infected with ∆ptsI mutants was investigated by creating a 374 ∆ptsI sagB double mutant in MGAS5005. Inactivation of sagB has been shown to block SLS 375 production in M1T1 GAS (19, 41). As expected, a ∆ptsI sagB double mutant resulted in the 376 absence of hemolytic activity both on blood agar plates (Fig. 5A) and also in RBC hemolysis 377 assays (data not shown) comparable to an established MGAS5005.sagB mutant (Fig. 5) (19). To 378 investigate the role of SLS in vivo, MGAS5005, MGAS5005.∆ptsI, MGAS5005.∆sagB, and the 379

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MGAS5005.∆ptsIsagB double mutant were tested in the murine model of streptococcal soft 380 tissue infection (Fig. 5BC). The non-hemolytic ∆sagB mutant (19) showed lesion sizes 381 comparable to the wild type MGAS5005 (Fig. 5C), indicating that SLS does not normally 382 contribute significantly to ulcer formation in this M1T1 background. Despite the absence of a 383 functional PTS, the ΔptsI sagB double mutant lacking expression of SLS produced purulent 384 lesions of the size and severity comparable to the wild type and ∆sagB strains, and not the ∆ptsI 385 mutant (Fig. 5BC). These results strongly suggest that altered temporal expression of SLS during 386 growth is the primary factor responsible for the increased lesion size and severity observed upon 387 subcutaneous infection of mice with the ∆ptsI mutant. 388 389

DISCUSSION 390 Although the connections between carbon metabolism and pathogenesis in GAS have been 391 recognized for many years, the contribution of the PTS pathway to GAS virulence has yet to be 392 investigated. In this study, we generated PTS mutants in multiple strains of GAS by deleting ptsI 393 (EI) and characterized their growth profiles to identify the PTS-dependent carbon sources that 394 support the growth of M1T1 GAS in vitro. Importantly, a PTS mutant was still able to colonize 395 and elicit disease in a murine model of disseminating soft tissue infection. However, the PTS 396 mutant resulted in a significant increase in localized lesion severity and size due to an increase in 397 sag operon expression and an altered temporal expression of Streptolysin S (SLS). Thus, a 398 functional PTS appears to limit the pathogenesis of GAS during invasive skin infection. 399 400 PTS pathway and carbon utilization in GAS 401

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The PTS carbohydrate transport system is widespread in bacteria, where it shares the same basic 402 composition in all genera; two “general” cytoplasmic components, EI and HPr, and sugar-403 specific EII transporters located in the cytoplasmic membrane (10). Typically, the genes 404 encoding Hpr (ptsH) and EI (ptsI) are organized as a tightly regulated ptsHI operon (42) and this 405 appears to be the case for GAS as well. Several attempts to construct either a non-polar deletion 406 or a polar insertion mutation of ptsH were unsuccessful (data not shown), suggesting that Hpr 407 may be critical for GAS fitness under the conditions used here. This observation has been further 408 supported by Tn-Seq essentiality studies ongoing in our lab (Le Breton and McIver, unpublished 409 results). Our results also correlate to work done in closely related streptococci, such as S. 410 pneumonia (43) and S. mutans (23), as well as Staphylococcus aureus (44, 45) and Mycoplasma 411 species (46, 47), where ptsH (Hpr) has also been reported to also be essential for growth. In 412 contrast to ptsH, we were able to generate a non-polar ptsI (EI) deletion mutant in 3 different 413 strains of M1T1 GAS and in an M4 GAS strain (3), indicating that the PTS itself is not required 414 and that Hpr appears to possess a separate essential function in GAS. Despite being able to make 415 ptsI null mutations in GAS, attempts to clone ptsI on a multi-copy plasmid for complementation 416 proved to be difficult and we were only successful when the construct lacked a functional 417 promoter. This suggests that overexpression of ptsI can be deleterious in E. coli and that the 418 ptsHI operon is likely under tight regulation in GAS. 419 By screening with the BIOLOG phenotypic microarray platform, we established that 45 420 of 190 carbon sources tested were able to support the growth (defined as 2-fold over 421 background) of the M1T1 GAS invasive throat strain MGAS5005 (Table 3). In contrast, 422 Kreikemeyer et. al. identified only 21 carbon sources with BIOLOG that supported the growth 423 (defined as >= 10% of growth on glucose) of the M49 GAS "generalist" strain 591, a strain that 424

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is able to colonize both the throat and skin (48). Out of these 21 carbon sources, all but 5 (Tween 425 40, Tween 80, pectin, mannan, and gelatin) were in common with our studies in M1T1 (Table 3). 426 An older study using CDM found that an M3 strain of GAS did not grow on lactose and glycerol, 427 although both were found to support growth using BIOLOG (25). The different metrics used to 428 determine functional growth between the two studies could explain some of the disparity in 429 carbon sources. However, it could also indicate that growth requirements differ between strains 430 of GAS, likely due to their varied tissue sites of infection. S. pneumoniae, which is found 431 primarily in the nasopharynx, can grow on 29 different carbon sources in BIOLOG; 16 of which 432 overlapped with our M1T1 GAS findings (Table 3). Thus, many of the carbon sources are 433 similar between streptococci; however, they appear to possess niche- and species-specific 434 requirements. 435 The M1T1 MGAS5005 GAS genome possesses 14 putative EII loci (49) predicted for the 436 transport of specific PTS sugars, although the exact substrates transported by these systems has 437 not been experimentally determined. The isogenic ΔptsI M1T1 MGAS5005 mutant was still 438 able to utilize 26 of the 45 carbon sources required for wild type growth (Table 3), indicating that 439 GAS does not need a functional PTS to utilize these carbon sources. However, the ΔptsI mutant 440 did have a growth defect in 19 carbon sources (Table 3), including 8 predicted PTS-specific 441 carbohydrates (galactose, trehalose, fructose, mannose, lactose, sucrose, salicin, and maltotriose). 442 Growth defects in maltose and glucose were not found in our study likely due to the presence of 443 non-PTS uptake systems. Transport of maltose in GAS has been shown to occur through both the 444 MalE-dependent ABC transport system as well as the MalT-specific PTS pathway (49, 50). In L. 445 lactis, a non-PTS glucose uptake system (GlcU) has been reported; however, genes encoding a 446 homologous system have not yet been identified in the genomes of S. pyogenes or S. pneumoniae 447

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(51). Interestingly, there is a putative glucokinase gene present in the MGAS5005 genome that 448 could act to phosphorylate glucose uptake via a non-PTS transport system. The identity of the 449 non-PTS glucose transporter in GAS remains to be determined. In addition to the six carbon 450 compounds, ΔptsI blocked the growth in α-glucosides (trehalose, sucrose, maltotriose), β-451 galactosides (lactose, lactulose), β-glucosides (β-methyl-D-glucoside, salicin), cyclodextrins, 452 lactic acid and the three carbon compound glycerol (Table 3). The growth defect in non-PTS 453 glycerol can be explained by the fact that glycerol kinase of Firmicutes is phosphorylated by 454 PEP, EI and HPr, and that this modification is necessary for the activation of the enzyme (52). 455 The results suggest that the PTS plays a broader role in regulating PTS-dependent and non-PTS 456 dependent transport, as well as the subsequent utilization of these substrates in central carbon 457 metabolism. 458 459 PTS limits lesion severity during invasive skin infection in mice. 460 Despite the inability to grow on 19 PTS and non-PTS sugars (Table 3), all three independent 461 M1T1 ∆ptsI mutants were able to colonize mice following subcutaneous inoculation and elicit 462 both localized and systemic disease to at least the levels of their parental wild type strains. This 463 provides strong evidence that GAS does not require a functional PTS or these specific sugars to 464 infect at this tissue site and likely utilizes a carbon source that is not altered in the ∆ptsI mutant 465 (Table 3). In fact, lesion formation was significantly more rapid with increased necrosis and size 466 in mice infected with mutant strains (Fig. 2, 3, and 5). Furthermore, mice infected by ptsI 467 mutants exhibited highly ulcerative and spreading lesions that resulted in severe hemorrhaging 468 and tissue damage. This is in sharp contrast to the phenotype of a ∆ptsI mutant in Staphylococcus 469 aureus, where virulence was attenuated compared to wild type in an intraperitoneal (i.p.) model 470

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of systemic infection in BALB/c mice (53). The difference may merely reflect the different site 471 of infections. Regardless, a functional PTS acts to limit localized tissue damage in GAS during 472 invasive skin infection and this appears to be linked to the metabolism of sugars that require PTS 473 transport (Table 3). 474 Dissemination of GAS from the subcutaneous site of infection to the bloodstream and 475 organs leading to lethality was not significantly altered in MGAS5005.∆ptsI and 5448AP.∆ptsI 476 compared to their parental strains (Fig. 2C and 3C). Both of these M1T1 strains possess a 477 mutation in the histidine kinase gene covS that is associated with a molecular switch to a highly 478 invasive phenotype for GAS. In contrast, deletion of ptsI in the M1T1 strain 5448 harboring a 479 wild type covS gene exhibited a significant increase in systemic lethality due to dissemination 480 (Fig. 3C) that correlated with the increase in lesion severity observed in all of the mutants. Thus, 481 there might be a Cov-dependent influence on the ability of a functional PTS to limit systemic 482 spread as well as localized lesion formation, although other genetic differences unlinked to cov 483 could be involved. At this point, we do not know which Cov-regulated factor(s) may be 484 involved in this dissemination phenotype. The cysteine protease SpeB is Cov-regulated and has 485 been directly associated with the formation of severe lesions in skin models of GAS infection 486 (34-36). We observed a PTS-dependent regulation on secretion of SpeB, but it was not common 487 to all three M1T1 backgrounds regardless of Cov status (Fig. S2). 488

489 PTS represses sag expression and early SLS production 490 As mentioned above, the cytolysin SLS has been shown to contribute to the severity of skin 491 lesions during GAS infection in mice (34, 39, 40). A modest up regulation of sagA at late log 492 phase and increased SLS activity in the supernatants of the M1T1 ∆ptsI mutants early in growth 493

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correlates nicely with the rapid onset of ulcerative lesions observed in the mice infected with the 494 mutants. Importantly, inactivating sagB (an SLS-defective mutant) in the MGAS5005.∆ptsI 495 background reversed this phenotype and resulted in localized lesions comparable to the wild type 496 parental strain alone (Fig 5). These data strongly suggest that early expression of SLS during 497 growth is likely the primary factor responsible for the increased lesion size and severity observed 498 upon subcutaneous infection of mice with the MGAS5005.∆ptsI and other M1T1 strains. Despite 499 the absence of SLS hemolytic activity in the ∆ptsI sagB double mutant, lesion formation 500 comparable to wild type was still observed (Fig. 5). These results suggest that other virulence 501 factors besides SLS are contributing to the lesion development seen in the parental M1T1 502 MGAS5005. 503 A similar hypervirulent lesion phenotype, albeit not as significant, was previously 504 observed by our group when a ∆ccpA mutant in MGAS5005 was tested in the murine skin 505 infection model. Importantly, this phenotype was attributed to CcpA-dependent overproduction 506 of sagA and SLS (19). Other studies have actually found attenuation in virulence upon infection 507 with a ∆ccpA mutant in M1T1 MGAS5005 and other GAS strains (18, 21). However, all studies 508 observed an up regulation in sag operon transcription leading to increased SLS production in the 509 mutants in vitro. Kietzman et al. showed that CcpA regulation of the sag operon in the M14 510 GAS strain HSC5 was indirect and that the repression did not appear to occur in infected skin 511 tissue (18). The mechanism of indirect regulation of sag by CcpA has not been determined. 512 Overall, this indicated that absence of CCR in GAS modulates the regulation of SLS, but this 513 appears to vary between strains. Since the CCR pathway remains intact in our ∆ptsI mutants 514 (Hpr Kinase, Hpr, CcpA), our findings may reflect a novel pathway for influencing sag 515 expression and SLS production based on carbohydrate availability. However, we cannot rule out 516

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that both CcpA and the PTS pathways are impinging indirectly on the same regulatory pathway 517 and leading to repression of SLS production. 518 In addition to CcpA, the expression of sagA is under the transcriptional control of GAS 519 global regulators such as CovRS, Mga, RofA, FasBCA and Nra (54). Our study suggests the 520 effect of ptsI on SLS activity is CovS-independent; yet, there is published evidence that CovR 521 regulates the sag operon by direct binding to the PsagA promoter (55). Thus, although we think 522 that CovS is not involved, we cannot rule out a role for CovR. Other known regulators of the 523 sag operon include the newly described PTS regulatory domain (PRD)-containing virulence 524 regulators (PCVR) such as Mga and RofA (3). Thus, it is possible that signaling through PTS 525 might influence the expression of global regulatory networks to influence the expression of SLS 526 and other virulence factors in GAS. 527 In conclusion, we have used PTS-defective strains of M1T1 GAS to identify the PTS and 528 non-PTS carbon sources that allow these invasive isolates to grow in vitro. The ΔptsI mutants 529 exhibited more severe and larger ulcerative lesions at the site of infection in a subcutaneous 530 model of mouse infection. This phenotype was linked to up regulation of sagA and early onset 531 of Streptolysin S (SLS) activity during exponential phase growth in the mutant. Infection of 532 mice with a ∆ptsI sagB double mutant returned lesions to wild type levels, implicating SLS in 533 the observed phenotype. Therefore, a functional PTS is not required for subcutaneous skin 534 infection in mice; however, it does limit early expression of SLS and thus the overall severity of 535 lesions in vivo. 536

537 538

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ACKNOWLEDGEMENTS 539 We thank Yoann Le Breton, Surya Sundar, and Kayla Valdes for critical review of this 540 manuscript. This work was supported by a grant from the NIH National Institute of Allergy and 541 Infectious Diseases (AI047928) to K.S.M. 542 543 544

FIGURE LEGENDS 545 Figure 1: Construction of ∆ptsI mutant of MGAS5005. (A) Schematic of ptsHI genomic 546 region from GAS MGAS5005 with putative Ppts promoter (arrow) driving transcription of ptsH 547 (Hpr) and ptsI (EI). The ∆ptsI region (black bar) was replaced with a non-polar aad9 cassette 548 (spectinomycin-resistance. The region amplified for qRT-PCR analysis is shown (dashed line). 549 Growth curve of wild type MGAS5005 (squares), ∆ptsI mutant (grey circles) and complemented 550 ∆ptsI (triangles) in CDM supplemented with (B) 0.5% (w/v) glucose (C) 1% (w/v) sucrose, or 551 (D) 1% (w/v) fructose is shown. Data is representative of three independent experiments. 552 Figure 2: Effect of M1T1 MGAS5005.∆ptsI mutant in mouse model of GAS infection. Mice 553 were inoculated s.c. with ~3 x 108 CFU and monitored over 7 days (A) Representative images of 554 mice infected with wild type MGAS5005 (left) or the ∆ptsI mutant (right) at 38 hours post 555 infection. (B) Lesion sizes measured in mice infected with MGAS5005 (black) and ∆ptsI mutant 556 (red) at 38 hours post infection. One of two independent experiments is shown (total n = 40). 557 Each point represents a single animal and bars indicate the statistical mean. Significance was 558 determined by unpaired two-tailed t test of animals shown. (C) Survival plot of mice infected 559 with MGAS5005 or its ∆ptsI mutant over the course of 7 days. Significance was determined by 560

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Kaplan-Meier survival analysis and log rank test of animals shown. (*** p≤.001; NS, not 561 significant). 562 Figure 3: ∆ptsI mutants of additional M1T1 GAS strains. (A) Representative images of mice 563 infected s.c. with 5448 (CovS+) and its ∆ptsI mutant (left) or 5448AP (CovS-) and its ∆ptsI 564 mutant (right) at 38 h post infection. CFU used in infection are indicated. (B) Lesion sizes of 565 same strains measured at 38 h post infection from a representative experiment (total n = 60). 566 Each point represents a single animal, with bars indicating statistical mean. Significance was 567 determined by unpaired two-tailed t test. (C) Survival plot of mice infected with either wild type 568 5448 (left) and 5448AP (right) in black and their corresponding ∆ptsI mutants in red. 569 Significance was determined by Kaplan-Meier survival analysis and log rank test. (** p≤0.01, 570 *** p≤.001, and NS, not significant). 571 Figure 4: Influence of ∆ptsI on Streptolysin S (SLS) Production. Transcript levels of sagA 572 were measured by qRT-PCR at late-log phase in THY for wild type, and the complemented 573 MGAS5005.ΔptsIc and isogenic ptsI mutants of (A) MGAS5005, (B) 5448, and (C) 5448AP. 574 Two fold differences in expression for mutant compared to wild type (dashed line) were 575 considered significant. SLS hemolytic activity was measured in culture supernatants for wild 576 type (squares) and isogenic ptsI mutants (grey circles) of (D) MGAS5005, (E) 5448, and (F) 577 5448AP and the complemented ΔptsI (triangles). Strains were grown in THY supplemented with 578 10% heat-inactivated horse serum and supernatants isolated throughout growth for analysis of 579 SLS activity. Data are presented as percent hemolysis (solid lines) for three biological replicates. 580 Representative growth is shown as dashed lines. 581

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Figure 5: Role of Streptolysin S in ∆ptsI increased lesion formation. (A) Zones of hemolysis 582 for WT MGAS5005, ∆ptsI single mutant, ∆sagB single mutant, and ∆sagB ∆ptsI double mutant 583 strains on 5% sheep blood agar plates after growth at 37°C. (B) Representative images of mice 584 infected s.c. with WT MGAS5005, MGAS5005.∆ptsI mutant, ∆sagB single mutant and ∆ptsI 585 sagB double mutant strains. CFU used in infection are indicated. (C) Lesion size of same 586 experiment measured at 38 hr post s.c. infection of MGAS5005 (black), ∆ptsI mutant (red), 587 ∆sagB mutant (blue), ∆ptsI sagB double mutant (green). Data represent two independent 588 experiments and significance was determined using the unpaired two-tailed t-test (*** p≤ .001; 589 NS, not significant). 590 591

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Table 1. Bacterial strains and plasmids 592 Name Description Reference

Strains

E. coli strains

DH5α hsdR17 recA1 gyrA endA1 relA1 (56)

C41[DE3] F– ompT gal dcm hsdSB(rB- mB

-)(DE3) (57)

S. pyogenes strains

MGAS5005 M1T1 (29)

5448 M1T1 (58)

5448AP M1T1 (58)

MGAS5005.∆ptsI ΔptsI mutant in MGAS5005 This study

MGAS5005.∆ptsIc ΔptsI complemented with pKSM456 insertion This study

5448.∆ptsI ΔptsI mutant in 5448 This study

5448AP.∆ptsI ΔptsI mutant in 5448AP This study

Plasmids

pCRK Temp. sensitive conditional vector, KmR (59)

pBluescript II KS- ColE1 ori Ampr lacZa Stratagene

pSL60-1 Vector containing non-polar aad9 gene (33)

pKSM645

pBlue.∆ptsI

∆ptsI mutagenic plasmid; non-polar aad9

∆ptsI in pBluescriptII KS-; bla (Apr)

(3)

(3)

pKSM456 promoterless ptsI integration plasmid; KmR This study

593 594

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Table 2. Primers used in this study 595

a Lowercase letters indicate sequences non complementary to the target DNA; Underline indicates restriction sites. 596 Italics reflect overlap region for SOEing. 597 598

Target PCR

Primers Sequence (5’-3’) Reference

ptsI ptsI-1a

ptsI-1b

ptsI-2a

ptsI-2b

ptsI-3a

ptsI-3b

ptsl-internal sense

ptsl-internal antisense

ptsl-external sense

ptsl-external antisense

5005.645jxn-sense

5005.645jxn-antisense

ptsIcomplementL

ptsIcomplementR

agatctGCTGAGTGACTTGTACGA

GGTATTCATGCGCGTCCA

gcgGCCTTGTGTTGGTGGTTTAAGAGCAAC

GTCACTCAGCagatctCGTGCGCTTACAGAATGT

tctagaGCGGCCTTGTGTTGGTGGTTT

ctcgagGGTATTCATGCGCGTCCA

CAAATTGGTCTGCAAGC

CAGATACTGCTCAACTTAACA

GCTAGCAAAAAAGAGCTGGTTTA

CTCTTGACTACAAAGGTAAAGCAGTAAA

GCGGGTTATTTTTTAAATGTTTCCGAAG

GGGCCATCTGCAAAATACAAAGCAT

cccggatccCATCACTCTTGACTACAA

cccggatccTTAATCTTCAGAAACGTA

This Study

This Study

This Study

This Study

This Study

This Study

This Study

This Study

This Study

This Study

This Study

This Study

This Study

ptsI M1 RT L ptsI M1 RT R CGGAAACCAAGGAATGGATTGGCAAACCTGTTGTGGTT This study

aad9

aad9L2-bglII

aad9R2-bglII

gcgcagatctGGGTGACTAAATAGTGAGGAG

gcgcagatctGGCATGTGATTTTCC

(19)

gyrA gyrA M1 RT L gyrA M1 RT R

CGACTTGTCTGAACGCCAAAGTATCACGTTCCAAACCAGTCAAAC

(60)

sagA

sagA M1 RT L sagA M1 RT R

GCTACTAGTGTAGCTGAAACAACTCAAAGCAACAAGTAGTACAGCAGCAA

(19)

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Table 3. Growth of MGAS5005.∆ptsI in various carbon sources by BIOLOG 599 Carbon Source MGAS5005 MGAS5005.∆ptsI

D-galactose + - D-trehalose + - D-fructose + - D-mannose + - α-D-lactose + - sucrose + - maltotriose + -

salicin + - β-methyl-D-glucoside + - L-lactic acid +/- - D-glucose-6-phosphate +/- - thymidine + - lactulose +/- -

D-fructose-6-phosphate + - N-acetyl-β-D-mannosamine + - α-cyclodextrin + - β-cyclodextrin +/- -

5-keto-D-gluconic acid + - glycerol + - L-arabinose + + N-actetyl-D-glucosamine + + D-xylose + + D-ribose + + α-D-glucose + + maltose + + uridine + + adenosine + + inosine + + L-lyxose + + pyruvic acid + + dextrin + + glycogen + + β-D-allose + +

D-arabinose + + 2-deoxy-D-ribose + + D-fucose + +

3-O-β-D-galactopyranosyl-D arabinose + +

3-methyl-glucose + + palatinose + +

L-sorbose + + D-tagatose + + turanose + +

D-glucosamine + + oxalomalic acid + +

dihydroxyacetone + + Bold indicates PTS sugars; + represents growth > 2x over background; +/- shows growth 2x over background. 600 601

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REFERENCES 602 1. Deutscher, J., C. Francke, and P. W. Postma. 2006. How phosphotransferase system-603

related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol. 604 Mol. Biol. Rev. 70:939-1031. 605

2. Gorke, B., and J. Stulke. 2008. Carbon catabolite repression in bacteria: many ways to 606 make the most out of nutrients. Nat. Rev. Microbiol. 6:613-624. 607

3. Hondorp, E. R., S. C. Hou, L. L. Hause, K. Gera, C. E. Lee, and K. S. McIver. 2013. 608 PTS phosphorylation of Mga modulates regulon expression and virulence in the group A 609 streptococcus. Mol. Microbiol. 88:1176-1193. 610

4. Cho, K. H., and M. G. Caparon. 2005. Patterns of virulence gene expression differ 611 between biofilm and tissue communities of Streptococcus pyogenes. Mol. Microbiol. 612 57:1545-1556. 613

5. Munoz-Elias, E. J., and J. D. McKinney. 2005. Mycobacterium tuberculosis isocitrate 614 lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11:638-615 644. 616

6. Rollenhagen, C., and D. Bumann. 2006. Salmonella enterica highly expressed genes are 617 disease specific. Infect. Immun. 74:1649-1660. 618

7. Iyer, R., and A. Camilli. 2007. Sucrose metabolism contributes to in vivo fitness of 619 Streptococcus pneumoniae. Mol. Microbiol. 66:1-13. 620

8. Son, M. S., W. J. Matthews, Jr., Y. Kang, D. T. Nguyen, and T. T. Hoang. 2007. In 621 vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the 622 lungs of cystic fibrosis patients. Infect. Immun. 75:5313-5324. 623

9. Lengeler, J. W., and K. Jahreis. 2009. Bacterial PEP-dependent carbohydrate: 624 phosphotransferase systems couple sensing and global control mechanisms. Contrib 625 Microbiol 16:65-87. 626

10. Postma, P. W., J. W. Lengeler, and G. R. Jacobson. 1993. 627 Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol 628 Rev 57:543-594. 629

11. Kruger, S., S. Gertz, and M. Hecker. 1996. Transcriptional analysis of bglPH 630 expression in Bacillus subtilis: evidence for two distinct pathways mediating carbon 631 catabolite repression. J. Bacteriol. 178:2637-2644. 632

12. McAllister, L. J., A. D. Ogunniyi, U. H. Stroeher, and J. C. Paton. 2012. Contribution 633 of a genomic accessory region encoding a putative cellobiose phosphotransferase system 634 to virulence of Streptococcus pneumoniae. PLoS One 7:e32385. 635

13. Antunes, A., I. Martin-Verstraete, and B. Dupuy. 2011. CcpA-mediated repression of 636 Clostridium difficile toxin gene expression. Mol. Microbiol. 79:882-899. 637

14. Stoll, R., S. Mertins, B. Joseph, S. Muller-Altrock, and W. Goebel. 2008. Modulation 638 of PrfA activity in Listeria monocytogenes upon growth in different culture media. 639 Microbiology 154:3856-3876. 640

15. Tsvetanova, B., A. C. Wilson, C. Bongiorni, C. Chiang, J. A. Hoch, and M. Perego. 641 2007. Opposing effects of histidine phosphorylation regulate the AtxA virulence 642 transcription factor in Bacillus anthracis. Mol. Microbiol. 63:644-655. 643

16. Fujita, Y. 2009. Carbon catabolite control of the metabolic network in Bacillus subtilis. 644 Biosci Biotechnol Biochem 73:245-259. 645

on Novem

ber 28, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

Page 32: The PEP Phosphotransferase System (PTS) in the Group A ... · yz P~Hpr-Ser interacts with the catabolite control protein CcpA, ... sur Summerson colorimeter (A filter). Alternatively,

32

17. Iyer, R., N. S. Baliga, and A. Camilli. 2005. Catabolite control protein A (CcpA) 646 contributes to virulence and regulation of sugar metabolism in Streptococcus 647 pneumoniae. J. Bacteriol. 187:8340-8349. 648

18. Kietzman, C. C., and M. G. Caparon. 2010. CcpA and LacD.1 affect temporal 649 regulation of Streptococcus pyogenes virulence genes. Infect. Immun. 78:241-252. 650

19. Kinkel, T. L., and K. S. McIver. 2008. CcpA-mediated repression of streptolysin S 651 expression and virulence in the group A streptococcus. Infect. Immun. 76:3451-3463. 652

20. Seidl, K., M. Stucki, M. Ruegg, C. Goerke, C. Wolz, L. Harris, B. Berger-Bachi, and 653 M. Bischoff. 2006. Staphylococcus aureus CcpA affects virulence determinant 654 production and antibiotic resistance. Antimicrob. Agents Chemother. 50:1183-1194. 655

21. Shelburne, S. A., 3rd, D. Keith, N. Horstmann, P. Sumby, M. T. Davenport, E. A. 656 Graviss, R. G. Brennan, and J. M. Musser. 2008. A direct link between carbohydrate 657 utilization and virulence in the major human pathogen group A Streptococcus. Proc Natl 658 Acad Sci U S A 105:1698-1703. 659

22. Varga, J., V. L. Stirewalt, and S. B. Melville. 2004. The CcpA protein is necessary for 660 efficient sporulation and enterotoxin gene (cpe) regulation in Clostridium perfringens. J. 661 Bacteriol. 186:5221-5229. 662

23. Zeng, L., and R. A. Burne. 2010. Seryl-phosphorylated HPr regulates CcpA-663 independent carbon catabolite repression in conjunction with PTS permeases in 664 Streptococcus mutans. Mol. Microbiol. 75:1145-1158. 665

24. Carapetis, J. R., A. C. Steer, E. K. Mulholland, and M. Weber. 2005. The global 666 burden of group A streptococcal diseases. Lancet Infect. Dis. 5:685-694. 667

25. Venditti, V., and G. M. Clore. 2012. Conformational selection and substrate binding 668 regulate the monomer/dimer equilibrium of the C-terminal domain of Escherichia coli 669 enzyme I. J. Biol. Chem. 287:26989-26998. 670

26. Pine, L., and M. W. Reeves. 1978. Regulation of the synthesis of M protein by sugars, 671 Todd Hewitt broth, and horse serum, in growing cells of Streptococcus pyogenes. 672 Microbios 21:185-212. 673

27. Musser, J. M., and S. A. Shelburne, 3rd. 2009. A decade of molecular pathogenomic 674 analysis of group A Streptococcus. J. Clin. Invest. 119:2455-2463. 675

28. Loughman, J. A., and M. G. Caparon. 2006. A novel adaptation of aldolase regulates 676 virulence in Streptococcus pyogenes. EMBO J. 25:5414-5422. 677

29. Sumby, P., S. F. Porcella, A. G. Madrigal, K. D. Barbian, K. Virtaneva, S. M. 678 Ricklefs, D. E. Sturdevant, M. R. Graham, J. Vuopio-Varkila, N. P. Hoe, and J. M. 679 Musser. 2005. Evolutionary origin and emergence of a highly successful clone of 680 serotype M1 group a Streptococcus involved multiple horizontal gene transfer events. J. 681 Infect. Dis. 192:771-782. 682

30. Podbielski, A., M. Woischnik, B. Kreikemeyer, K. Bettenbrock, and B. A. Buttaro. 683 1999. Cysteine protease SpeB expression in group A streptococci is influenced by the 684 nutritional environment but SpeB does not contribute to obtaining essential nutrients. 685 Med. Microbiol. Immunol. 188:99-109. 686

31. van de Rijn, I., and R. E. Kessler. 1980. Growth characteristics of group A streptococci 687 in a new chemically defined medium. Infect. Immun. 27:444-448. 688

32. Horton, R. M. 1995. PCR-mediated recombination and mutagenesis. SOEing together 689 tailor-made genes. Mol. Biotechnol. 3:93-99. 690

on Novem

ber 28, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

Page 33: The PEP Phosphotransferase System (PTS) in the Group A ... · yz P~Hpr-Ser interacts with the catabolite control protein CcpA, ... sur Summerson colorimeter (A filter). Alternatively,

33

33. Deutscher, J., and M. H. Saier, Jr. 1983. ATP-dependent protein kinase-catalyzed 691 phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the 692 phosphotransferase system in Streptococcus pyogenes. Proc Natl Acad Sci U S A 693 80:6790-6794. 694

34. Engleberg, N. C., A. Heath, K. Vardaman, and V. J. DiRita. 2004. Contribution of 695 CsrR-regulated virulence factors to the progress and outcome of murine skin infections 696 by Streptococcus pyogenes. Infect. Immun. 72:623-628. 697

35. Lukomski, S., C. A. Montgomery, J. Rurangirwa, R. S. Geske, J. P. Barrish, G. J. 698 Adams, and J. M. Musser. 1999. Extracellular cysteine protease produced by 699 Streptococcus pyogenes participates in the pathogenesis of invasive skin infection and 700 dissemination in mice. Infect. Immun. 67:1779-1788. 701

36. Connolly, K. L., A. L. Roberts, R. C. Holder, and S. D. Reid. 2011. Dispersal of 702 Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease 703 severity in a murine model. PLoS One 6:e18984. 704

37. Doern, C. D., A. L. Roberts, W. Hong, J. Nelson, S. Lukomski, W. E. Swords, and S. 705 D. Reid. 2009. Biofilm formation by group A Streptococcus: a role for the streptococcal 706 regulator of virulence (Srv) and streptococcal cysteine protease (SpeB). Microbiology 707 155:46-52. 708

38. Roberts, A. L., R. C. Holder, and S. D. Reid. 2010. Allelic replacement of the 709 streptococcal cysteine protease SpeB in a ∆srv mutant background restores biofilm 710 formation. BMC Res Notes 3:281. 711

39. Fontaine, M. C., J. J. Lee, and M. A. Kehoe. 2003. Combined contributions of 712 streptolysin O and streptolysin S to virulence of serotype M5 Streptococcus pyogenes 713 strain Manfredo. Infect. Immun. 71:3857-3865. 714

40. Heath, A., V. J. DiRita, N. L. Barg, and N. C. Engleberg. 1999. A two-component 715 regulatory system, CsrR-CsrS, represses expression of three Streptococcus pyogenes 716 virulence factors, hyaluronic acid capsule, streptolysin S, and pyrogenic exotoxin B. 717 Infect. Immun. 67:5298-5305. 718

41. Nizet, V., B. Beall, D. J. Bast, V. Datta, L. Kilburn, D. E. Low, and J. C. De 719 Azavedo. 2000. Genetic locus for streptolysin S production by group A streptococcus. 720 Infect. Immun. 68:4245-4254. 721

42. Vadeboncoeur, C., M. Frenette, and L. A. Lortie. 2000. Regulation of the pts operon 722 in low G+C Gram-positive bacteria. J. Mol. Microbiol. Biotechnol. 2:483-490. 723

43. Song, J. H., K. S. Ko, J. Y. Lee, J. Y. Baek, W. S. Oh, H. S. Yoon, J. Y. Jeong, and J. 724 Chun. 2005. Identification of essential genes in Streptococcus pneumoniae by allelic 725 replacement mutagenesis. Mol Cells 19:365-374. 726

44. Forsyth, R. A., R. J. Haselbeck, K. L. Ohlsen, R. T. Yamamoto, H. Xu, J. D. 727 Trawick, D. Wall, L. Wang, V. Brown-Driver, J. M. Froelich, K. G. C, P. King, M. 728 McCarthy, C. Malone, B. Misiner, D. Robbins, Z. Tan, Z. Y. Zhu Zy, G. Carr, D. A. 729 Mosca, C. Zamudio, J. G. Foulkes, and J. W. Zyskind. 2002. A genome-wide strategy 730 for the identification of essential genes in Staphylococcus aureus. Mol. Microbiol. 731 43:1387-1400. 732

45. Chaudhuri, R. R., A. G. Allen, P. J. Owen, G. Shalom, K. Stone, M. Harrison, T. A. 733 Burgis, M. Lockyer, J. Garcia-Lara, S. J. Foster, S. J. Pleasance, S. E. Peters, D. J. 734 Maskell, and I. G. Charles. 2009. Comprehensive identification of essential 735

on Novem

ber 28, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

Page 34: The PEP Phosphotransferase System (PTS) in the Group A ... · yz P~Hpr-Ser interacts with the catabolite control protein CcpA, ... sur Summerson colorimeter (A filter). Alternatively,

34

Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation 736 (TMDH). BMC Genomics 10:291. 737

46. Glass, J. I., N. Assad-Garcia, N. Alperovich, S. Yooseph, M. R. Lewis, M. Maruf, C. 738 A. Hutchison, 3rd, H. O. Smith, and J. C. Venter. 2006. Essential genes of a minimal 739 bacterium. Proc Natl Acad Sci U S A 103:425-430. 740

47. French, C. T., P. Lao, A. E. Loraine, B. T. Matthews, H. Yu, and K. Dybvig. 2008. 741 Large-scale transposon mutagenesis of Mycoplasma pulmonis. Mol. Microbiol. 69:67-76. 742

48. Siemens, N., T. Fiedler, J. Normann, J. Klein, R. Munch, N. Patenge, and B. 743 Kreikemeyer. 2012. Effects of the ERES pathogenicity region regulator Ralp3 on 744 Streptococcus pyogenes serotype M49 virulence factor expression. J. Bacteriol. 745 194:3618-3626. 746

49. Shelburne, S. A., 3rd, D. B. Keith, M. T. Davenport, N. Horstmann, R. G. Brennan, 747 and J. M. Musser. 2008. Molecular characterization of group A Streptococcus 748 maltodextrin catabolism and its role in pharyngitis. Mol. Microbiol. 69:436-452. 749

50. Venditti, V., R. Ghirlando, and G. M. Clore. 2013. Structural Basis for Enzyme I 750 Inhibition by alpha-Ketoglutarate. ACS Chem Biol. 751

51. Bidossi, A., L. Mulas, F. Decorosi, L. Colomba, S. Ricci, G. Pozzi, J. Deutscher, C. 752 Viti, and M. R. Oggioni. 2012. A functional genomics approach to establish the 753 complement of carbohydrate transporters in Streptococcus pneumoniae. PLoS One 754 7:e33320. 755

52. Darbon, E., P. Servant, S. Poncet, and J. Deutscher. 2002. Antitermination by GlpP, 756 catabolite repression via CcpA and inducer exclusion triggered by P-GlpK 757 dephosphorylation control Bacillus subtilis glpFK expression. Mol. Microbiol. 43:1039-758 1052. 759

53. Kok, M., G. Bron, B. Erni, and S. Mukhija. 2003. Effect of enzyme I of the bacterial 760 phosphoenolpyruvate : sugar phosphotransferase system (PTS) on virulence in a murine 761 model. Microbiology 149:2645-2652. 762

54. Molloy, E. M., P. D. Cotter, C. Hill, D. A. Mitchell, and R. P. Ross. 2011. Streptolysin 763 S-like virulence factors: the continuing sagA. Nat. Rev. Microbiol. 9:670-681. 764

55. Gao, J., A. A. Gusa, J. R. Scott, and G. Churchward. 2005. Binding of the global 765 response regulator protein CovR to the sag promoter of Streptococcus pyogenes reveals a 766 new mode of CovR-DNA interaction. J. Biol. Chem. 280:38948-38956. 767

56. Saier, M. H., R. N. Hvorup, and R. D. Barabote. 2005. Evolution of the bacterial 768 phosphotransferase system: from carriers and enzymes to group translocators. Biochem. 769 Soc. Trans. 33:220-224. 770

57. Hvorup, R., A. B. Chang, and M. H. Saier, Jr. 2003. Bioinformatic analyses of the 771 bacterial L-ascorbate phosphotransferase system permease family. J. Mol. Microbiol. 772 Biotechnol. 6:191-205. 773

58. Aziz, R. K., M. J. Pabst, A. Jeng, R. Kansal, D. E. Low, V. Nizet, and M. Kotb. 2004. 774 Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent 775 proteolytic degradation of multiple virulence factors by SpeB. Mol. Microbiol. 51:123-776 134. 777

59. Le Breton, Y., P. Mistry, K. M. Valdes, J. Quigley, N. Kumar, H. Tettelin, and K. S. 778 McIver. 2013. Genome-wide identification of genes required for fitness of group A 779 Streptococcus in human blood. Infect. Immun. 81:862-875. 780

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60. Ribardo, D. A., and K. S. McIver. 2006. Defining the Mga regulon: Comparative 781 transcriptome analysis reveals both direct and indirect regulation by Mga in the group A 782 streptococcus. Mol. Microbiol. 62:491-508. 783 784 785

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1% sucrose 1% fructose

ptsI ptsH Ppts

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0.5% glucose

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0.5% glucose

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0 24 48 72 96 120 144 1680

25

50

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Time (Hours)

A.

B. C.

MGAS5005 ptsI

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0

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25

50

75

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Time (Hours)

**

0 25 50 75 100 125 150 175 2000

25

50

75

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Time (Hours)

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*** ***

A.

B.

C.

2.4 x108 2.6 x108 3.4 x108 3.2 x108

5448 ptsI 5448AP ptsI

5448 5448AP

0

250

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5005 ΔptsI ΔptsI sagBsagB

2.7 x 108 2.8 x 108 2.8 x 108

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