Analysis of the Transcriptome of Group A Streptococcus in Mouse Soft Tissue Infection
2006; Elsevier BV; Volume: 169; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.060112
ISSN1525-2191
AutoresMorag Graham, Kimmo Virtaneva, Stephen F. Porcella, Donald J. Gardner, Rui Long, Diane M. Welty, William T. Barry, Claire A. Johnson, Larye D. Parkins, Fred A. Wright, James M. Musser,
Tópico(s)Neonatal and Maternal Infections
ResumoMolecular mechanisms mediating group A Streptococcus (GAS)-host interactions remain poorly understood but are crucial for diagnostic, therapeutic, and vaccine development. An optimized high-density microarray was used to analyze the transcriptome of GAS during experimental mouse soft tissue infection. The transcriptome of a wild-type serotype M1 GAS strain and an isogenic transcriptional regulator knockout mutant (covR) also were compared. Array datasets were verified by quantitative real-time reverse transcriptase-polymerase chain reaction and in situ immunohistochemistry. The results unambiguously demonstrate that coordinated expression of proven and putative GAS virulence factors is directed toward overwhelming innate host defenses leading to severe cellular damage. We also identified adaptive metabolic responses triggered by nutrient signals and hypoxic/acidic conditions in the host, likely facilitating pathogen persistence and proliferation in soft tissues. Key discoveries included that oxidative stress genes, virulence genes, genes related to amino acid and maltodextrin utilization, and several two-component transcriptional regulators were highly expressed in vivo. This study is the first global analysis of the GAS transcriptome during invasive infection. Coupled with parallel analysis of the covR mutant strain, novel insights have been made into the regulation of GAS virulence in vivo, resulting in new avenues for targeted therapeutic and vaccine research. Molecular mechanisms mediating group A Streptococcus (GAS)-host interactions remain poorly understood but are crucial for diagnostic, therapeutic, and vaccine development. An optimized high-density microarray was used to analyze the transcriptome of GAS during experimental mouse soft tissue infection. The transcriptome of a wild-type serotype M1 GAS strain and an isogenic transcriptional regulator knockout mutant (covR) also were compared. Array datasets were verified by quantitative real-time reverse transcriptase-polymerase chain reaction and in situ immunohistochemistry. The results unambiguously demonstrate that coordinated expression of proven and putative GAS virulence factors is directed toward overwhelming innate host defenses leading to severe cellular damage. We also identified adaptive metabolic responses triggered by nutrient signals and hypoxic/acidic conditions in the host, likely facilitating pathogen persistence and proliferation in soft tissues. Key discoveries included that oxidative stress genes, virulence genes, genes related to amino acid and maltodextrin utilization, and several two-component transcriptional regulators were highly expressed in vivo. This study is the first global analysis of the GAS transcriptome during invasive infection. Coupled with parallel analysis of the covR mutant strain, novel insights have been made into the regulation of GAS virulence in vivo, resulting in new avenues for targeted therapeutic and vaccine research. Skin and soft tissue infections are most commonly caused by bacteria and account for ∼7 to 10% of hospitalizations in North America.1DiNubile MJ Lipsky BA Complicated infections of skin and skin structures: when the infection is more than skin deep.J Antimicrob Chemother. 2004; 53: 37-50Google Scholar Substantial portions are caused by Streptococcus pyogenes (group A streptococci; GAS). A major human pathogen, GAS is associated with millions of skin and throat infections each year.2Musser JM Krause RM Krause RM The revival of group A streptococcal diseases, with a commentary on staphylococcal toxic shock syndrome. Emerging Infections. Academic Press, New York1998: 185-218Google Scholar Serious complications after infection, namely rheumatic fever, glomerulonephritis, and reactive arthritis, can follow even relatively mild GAS infections and cause significant morbidity worldwide.3Cunningham MW Pathogenesis of group A streptococcal infections.Clin Microbiol Rev. 2000; 13: 470-511Crossref PubMed Scopus (1607) Google Scholar, 4Bisno AL Brito MO Collins CM Molecular basis of group A streptococcal virulence.Lancet Infect Dis. 2003; 3: 191-200Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar Some GAS infections rapidly progress to life-threatening invasive diseases such as septicemia, streptococcal toxic shock syndrome, and necrotizing fasciitis, requiring life-saving interventions in the form of aggressive fluid replacement, general supportive measures, and/or emergent surgical debridement.2Musser JM Krause RM Krause RM The revival of group A streptococcal diseases, with a commentary on staphylococcal toxic shock syndrome. Emerging Infections. Academic Press, New York1998: 185-218Google Scholar, 3Cunningham MW Pathogenesis of group A streptococcal infections.Clin Microbiol Rev. 2000; 13: 470-511Crossref PubMed Scopus (1607) Google Scholar, 4Bisno AL Brito MO Collins CM Molecular basis of group A streptococcal virulence.Lancet Infect Dis. 2003; 3: 191-200Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar Nearly 15,000 cases of invasive GAS disease and an estimated 1300 deaths occur annually in the United States.5O'Brien KL Beall B Barrett NL Cieslak PR Reingold A Farley MM Danila R Zell ER Facklam R Schwartz B Schuchat A Epidemiology of invasive group A Streptococcus disease in the United States, 1995–1999.Clin Infect Dis. 2002; 35: 268-276Crossref PubMed Scopus (307) Google Scholar Epidemiological data also suggest increased incidence and severity of GAS infections in recent years.2Musser JM Krause RM Krause RM The revival of group A streptococcal diseases, with a commentary on staphylococcal toxic shock syndrome. Emerging Infections. Academic Press, New York1998: 185-218Google Scholar, 6Stevens DL The flesh-eating bacterium: what's next?.J Infect Dis. 1999; 179: S366-S374Crossref PubMed Scopus (132) Google Scholar, 7Stevens D Streptococcal toxic shock syndrome associated with necrotizing fasciitis.Annu Rev Med. 2000; 51: 271-288Crossref PubMed Scopus (154) Google Scholar Because progression to systemic toxicity and shock can occur throughout a span of hours, early recognition and initiation of aggressive therapies are essential. Molecular mechanisms mediating GAS-host interactions remain poorly understood but are crucial for rapid diagnostic, therapeutic, and vaccine development.3Cunningham MW Pathogenesis of group A streptococcal infections.Clin Microbiol Rev. 2000; 13: 470-511Crossref PubMed Scopus (1607) Google Scholar, 4Bisno AL Brito MO Collins CM Molecular basis of group A streptococcal virulence.Lancet Infect Dis. 2003; 3: 191-200Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar As a consequence, concerted efforts have been made to identify regulatory mechanisms involved in coordinating the in vitro expression of GAS virulence determinants. Genes have been identified that regulate production of proven and putative virulence factors, and several of these regulators are known to respond to environmental changes in vitro or to specific phases of the bacterial growth cycle.3Cunningham MW Pathogenesis of group A streptococcal infections.Clin Microbiol Rev. 2000; 13: 470-511Crossref PubMed Scopus (1607) Google Scholar, 8Kreikemeyer B McIver KS Podbielski A Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions.Trends Microbiol. 2003; 11: 224-232Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar However, far less is known about gene expression in vivo during GAS infection of mammalian hosts.9Hynes W Virulence factors of the group A streptococci and genes that regulate their expression.Front Biosci. 2004; 9: 3399-3433Crossref PubMed Scopus (40) Google Scholar, 10Virtaneva K Porcella SF Graham MR Gardner DJ Bailey JR Parnell MJ Musser JM Longitudinal analysis of group Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.Proc Natl Acad Sci USA. 2005; 102: 9014-9019Crossref PubMed Scopus (157) Google Scholar, 11Loughman JA Caparon M Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a model for in vivo gene expression.J Bacteriol. 2006; 188: 399-408Crossref PubMed Scopus (71) Google Scholar Infection sites or the host circulation environment are complex, and their influence on bacterial gene regulation cannot be simulated in vitro.12Handfield M Progulske-Fox A Hillman J In vivo induced genes in human diseases.Periodontol 2000. 2005; 38: 123-134Crossref PubMed Scopus (13) Google Scholar Hence, further understanding of GAS host/pathogen molecular interactions requires analysis of in vivo conditions that better approximate the natural history of infections. Streptococcal soft tissue infections, such as cellulitis involving the subdermal layers and superficial fascia, often commence with nonspecific symptoms such as erythema, heat, soft tissue edema, generalized pain, and sometimes serosanguinous bullae.6Stevens DL The flesh-eating bacterium: what's next?.J Infect Dis. 1999; 179: S366-S374Crossref PubMed Scopus (132) Google Scholar, 7Stevens D Streptococcal toxic shock syndrome associated with necrotizing fasciitis.Annu Rev Med. 2000; 51: 271-288Crossref PubMed Scopus (154) Google Scholar Progression to diffuse necrosis of subcutaneous and deep fascia (necrotizing fasciitis) often occurs without overt skin injury13Headley AJ Necrotizing soft tissue infections: a primary care review.Am Fam Physician. 2003; 68: 323-328PubMed Google Scholar and leads to development of vasculitis, intravascular thrombosis, and tissue gangrene. Histopathology of debrided soft tissue reveals acute inflammation of subcutaneous tissue with bacterial aggregates and multifocal necrosis. Mice subcutaneously injected with GAS also develop inflamed bullae that later ulcerate; diffuse inflammatory infiltrates with high bacterial counts, and diffuse tissue necrosis including thrombosed vessels.3Cunningham MW Pathogenesis of group A streptococcal infections.Clin Microbiol Rev. 2000; 13: 470-511Crossref PubMed Scopus (1607) Google Scholar In a previous study,14Graham MR Smoot LM Migliaccio CAL Virtaneva K Sturdevant DE Porcella SF Federle MJ Adams GJ Scott JR Musser JM Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.Proc Natl Acad Sci USA. 2002; 99: 13855-13860Crossref PubMed Scopus (274) Google Scholar we measured a subset (n = 17) of GAS transcripts expressed in vivo 2 days after inoculation with a wild-type (WT) serotype M1 GAS strain and a ΔcovR isogenic deletion mutant of a key GAS two-component regulatory system (TCS) referred to as CovR-CovS (Cov, control of virulence). In this present study we have expanded our investigation by using a custom high-density array for whole GAS transcriptome analysis in the murine soft tissue infection model. The data presented here provide substantial new information about GAS survival strategies in soft tissue. The most abundant GAS transcripts detected were found to encode extracellular proteins that are key virulence determinants. Other expressed GAS genes are potential targets for therapeutic intervention and warrant further study. Serotype M1 GAS strains commonly cause pharyngitis and invasive infections.2Musser JM Krause RM Krause RM The revival of group A streptococcal diseases, with a commentary on staphylococcal toxic shock syndrome. Emerging Infections. Academic Press, New York1998: 185-218Google Scholar Strain MGAS5005 (no. BAA-947; American Type Culture Collection, Rockville, MD), a WT clinical strain (serotype M1), and its isogenic ΔcovR derivative strain (JRS950) have been described.14Graham MR Smoot LM Migliaccio CAL Virtaneva K Sturdevant DE Porcella SF Federle MJ Adams GJ Scott JR Musser JM Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.Proc Natl Acad Sci USA. 2002; 99: 13855-13860Crossref PubMed Scopus (274) Google Scholar, 15Hoe NP Nakashima K Lukomski S Grigsby D Liu M Kordari P Dou SJ Pan X Vuopio-Varkila J Salmelinna S McGeer A Low DE Schwartz B Schuchat A Naidich S De Lorenzo D Fu YX Musser JM Rapid selection of complement-inhibiting protein variants in group A Streptococcus epidemic waves.Nat Med. 1999; 5: 924-929Crossref PubMed Scopus (66) Google Scholar, 16Sumby P Porcella SF Madrigal AG Barbian KD Virtaneva K Ricklefs SM Sturdevant DE Graham MR Vuopio-Varkila J Hoe NP Musser JM Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events.J Infect Dis. 2005; 192: 771-782Crossref PubMed Scopus (250) Google Scholar Bacteria were cultured statically on Trypticase soy agar containing 5% sheep blood agar (Becton-Dickinson, Cockeysville, MD) or in Todd-Hewitt broth (Becton-Dickinson) containing 0.2% (w/v) yeast extract (THY; Difco Laboratories, Detroit, MI), at 37°C in an atmosphere containing 5% CO2. For generating inoculum, cells were harvested from THY at late-exponential phase (OD600 ∼0.75) to limit infectivity differences across strains associated with up-regulated capsule biosynthesis in strain JRS950, which is maximal in the early-to-mid exponential growth phases. This model of GAS soft tissue infection has been used extensively to study bacterial-host interactions.14Graham MR Smoot LM Migliaccio CAL Virtaneva K Sturdevant DE Porcella SF Federle MJ Adams GJ Scott JR Musser JM Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.Proc Natl Acad Sci USA. 2002; 99: 13855-13860Crossref PubMed Scopus (274) Google Scholar, 17Sumby P Barbian KD Gardner DJ Whitney AR Welty DM Long RD Bailey JR Parnell MJ Hoe NP Adams GG DeLeo FR Musser JM Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response.Proc Natl Acad Sci USA. 2005; 102: 1679-1684Crossref PubMed Scopus (230) Google Scholar, 18Lukomski S Hoe NP Abdi I Rurangirwa J Kordari P Liu M Dou SJ Adams GG Musser JM Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization.Infect Immun. 2000; 68: 535-542Crossref PubMed Scopus (109) Google Scholar Our experimental protocol was approved by the Institutional Animal Care and Use Committee, National Institute of Allergy and Infectious Diseases. Outbred, immunocompetent, hairless male Crl:SKH1-hrBR mice (5 weeks old; 20 to 25 g) (Charles River Breeding Laboratories, Bar Harbor, ME), maintained on standard mouse food and water ad libitum, were randomly assigned to one of two treatment groups (WT or ΔcovR mutant GAS strain, n = 27 each; four mice per cage). Immediately before inoculation, the animals were weighed and anesthetized with isoflurane (Aerrane; Ohmeda Caribe, Guayama, Puerto Rico) inhalation. The animals were inoculated subcutaneously in the dorsal side with 0.1 ml of pyrogen-free phosphate-buffered saline (PBS) (diluent) or ∼3 × 107 colony-forming units of MGAS5005 (WT) or JRS950 (ΔcovR). The actual colony-forming units of viable bacteria inoculated were verified by growth on blood agar. To blind the investigator, cage numbers were reassigned after inoculation, and the blind was broken after data analysis. Methods for clinical measurements and analysis of gross pathologies have been reported previously for this soft tissue model of infection,14Graham MR Smoot LM Migliaccio CAL Virtaneva K Sturdevant DE Porcella SF Federle MJ Adams GJ Scott JR Musser JM Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.Proc Natl Acad Sci USA. 2002; 99: 13855-13860Crossref PubMed Scopus (274) Google Scholar, 19Lukomski S Montgomery CA Rurangirwa J Geske RS Barrish JP Adams GJ Musser JM Extracellular cysteine protease produced by Streptococcus pyogenes participates in the pathogenesis of invasive skin infection and dissemination in mice.Infect Immun. 1999; 67: 1779-1788Crossref PubMed Google Scholar and data are found in Supplementary Tables 1 and 2 (see http://ajp.amjpathol.org). Length (L) and width (W) values were used to calculate abscess volume [V = 4/3π(L/2)2 × (W/2)] and area [A = π(L/2) × (W/2)], using equations for a spherical ellipsoid as described.14Graham MR Smoot LM Migliaccio CAL Virtaneva K Sturdevant DE Porcella SF Federle MJ Adams GJ Scott JR Musser JM Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.Proc Natl Acad Sci USA. 2002; 99: 13855-13860Crossref PubMed Scopus (274) Google Scholar At 53 hours after inoculation, mice were euthanized and weighed, the infection site was swabbed to confirm GAS infection, and tissue was obtained from each animal via a biopsy that included dermis and underlying soft tissue lesions. Tissues were wrapped in aluminum foil, snap-frozen in liquid nitrogen, and stored at −80°C until RNA was isolated. Three additional control mice injected with sterile saline failed to show symptoms of clinical infection and did not grow GAS bacterial colonies on plating. A one-factor experimental design with two treatment (strain) levels was used. Samples were randomized at the start of procedures, and care was taken to ensure that batches of sample preparation, array hybridizations, and posthybridization washes were not confounded with treatment. Additional experimental design details are outlined in the Supplementary Materials and Methods (see http://ajp.amjpathol.org). Frozen tissue extracts were divided into three aliquots from which RNA was purified. Tissue extracts were pulverized with a series of sharp blows delivered with a 3-pound drill hammer (Razor-Back; Union Tools Inc., Columbus, OH). Cell lysis and RNA isolation were conducted with a FastRNA kit (MP Biomedicals, Irvine, CA) as described.20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar The concentrate was fragmented with a Qiashredder (Qiagen, Inc., Valencia, CA) and the isolated total RNA (containing both bacterial and host RNA) was purified in 96-well format using a plate centrifugation system (RNeasy 96; Qiagen), with on-column DNase I treatment and posttreatment with DNAFree (Ambion, Inc., Austin, TX) as described.10Virtaneva K Porcella SF Graham MR Gardner DJ Bailey JR Parnell MJ Musser JM Longitudinal analysis of group Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.Proc Natl Acad Sci USA. 2005; 102: 9014-9019Crossref PubMed Scopus (157) Google Scholar, 20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar RNA integrity was assessed with a 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA)10Virtaneva K Porcella SF Graham MR Gardner DJ Bailey JR Parnell MJ Musser JM Longitudinal analysis of group Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.Proc Natl Acad Sci USA. 2005; 102: 9014-9019Crossref PubMed Scopus (157) Google Scholar, 20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar and by measurement of the A260/A280 ratios. Absence of contaminating bacterial genomic DNA was confirmed by real-time polymerase chain reaction (PCR). Two RNA aliquots were pooled to perform the microarrays; the remaining extract was used for real-time RT-PCR validation. Fifty-four microarray targets were prepared primarily as described.10Virtaneva K Porcella SF Graham MR Gardner DJ Bailey JR Parnell MJ Musser JM Longitudinal analysis of group Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.Proc Natl Acad Sci USA. 2005; 102: 9014-9019Crossref PubMed Scopus (157) Google Scholar, 20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Briefly, each RNA sample was divided into two to three aliquots of 10-μg total RNA (each) to which 0.8 μg of bacteriophage MS2 carrier RNA (Roche Applied Biosciences, Indianapolis, IN) was added. Control spike transcripts (130 pmol/L) were added to each RNA aliquot, and 5 μg of random primers (Invitrogen, Carlsbad, CA) were annealed (10 minutes at 70°C, 10 minutes at 25°C). cDNA synthesis reactions and postsynthesis RNA digestion were performed as described.10Virtaneva K Porcella SF Graham MR Gardner DJ Bailey JR Parnell MJ Musser JM Longitudinal analysis of group Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.Proc Natl Acad Sci USA. 2005; 102: 9014-9019Crossref PubMed Scopus (157) Google Scholar, 20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar In brief, the resultant cDNA was purified using Qiaquick 96 kit (Qiagen) according to the manufacturer's recommendation. For cDNA fragmentation, 3 μg of cDNA and 0.75 U of DNase I (Roche Biosciences) were used (10 minutes at 37°C, 10 minutes at 98°C). The desired cDNA size range of 50 to 200 bases was verified by separating 200 ng of cDNA on a RNA 6000 Nano LabChip (Agilent) using the 2100 BioAnalyzer (Agilent) with no added dye in the loading buffer. The fragmented cDNA was then end-labeled with biotin-ddUTP as per the Enzo BioArray terminal labeling kit (60 minutes at 37°C; Enzo Diagnostics, New York, NY) and pooled with other end-labeled cDNAs of the same sample. Samples were hybridized to 54 Rocky Mountain Laboratory (RML) Affymetrix custom GAS arrays, as described.10Virtaneva K Porcella SF Graham MR Gardner DJ Bailey JR Parnell MJ Musser JM Longitudinal analysis of group Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.Proc Natl Acad Sci USA. 2005; 102: 9014-9019Crossref PubMed Scopus (157) Google Scholar, 20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar The Affymetrix Inc. (Santa Clara, CA) custom high-density anti-sense oligonucleotide GeneChip array (designated RMLChip herein) has been described.10Virtaneva K Porcella SF Graham MR Gardner DJ Bailey JR Parnell MJ Musser JM Longitudinal analysis of group Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.Proc Natl Acad Sci USA. 2005; 102: 9014-9019Crossref PubMed Scopus (157) Google Scholar, 16Sumby P Porcella SF Madrigal AG Barbian KD Virtaneva K Ricklefs SM Sturdevant DE Graham MR Vuopio-Varkila J Hoe NP Musser JM Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events.J Infect Dis. 2005; 192: 771-782Crossref PubMed Scopus (250) Google Scholar It contains redundant probe sets representing more than 90% coverage of predicted coding regions (1869 ORFs) encoded by the MGAS5005 genome.16Sumby P Porcella SF Madrigal AG Barbian KD Virtaneva K Ricklefs SM Sturdevant DE Graham MR Vuopio-Varkila J Hoe NP Musser JM Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events.J Infect Dis. 2005; 192: 771-782Crossref PubMed Scopus (250) Google Scholar Each probe set is used to detect the presence of a single transcript, and several GAS transcripts (genes) are represented by more than one probe set. For in-depth RMLChip details, refer to the Supplementary Materials and Methods (see http://ajp.amjpathol.org). Target hybridizations, washing, staining, and scanning were performed by the National Institute of Allergy and Infectious Diseases Affymetrix core facility (Science Applications International Corp., Frederick, MD) using a GeneChip hybridization oven and the Pseudomonas aeruginosa hybridization protocol (Affymetrix), as described.20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar The hybridization solution volume used was 200 μl because the RMLChip is a standard size array. Each array was scanned at 570 nm at 3-μm resolution with a GeneArray scanner. Scanned DAT-image files were analyzed with Affymetrix Microarray Suite (MAS) 5.0 software. The raw CEL files have been submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo). Downstream genome analysis was accomplished using MicrobesOnline (available at http://www.microbesonline.org)21Alm EJ Huang KH Price MN Koche RP Keller K Dubchak IL Arkin AP The MicrobesOnline web site for comparative genomics.Genome Res. 2005; 15: 1015-1022Crossref PubMed Scopus (162) Google Scholar and in-house bioinformatics analysis. Model-based expression estimates for each gene were obtained using the PM-MM difference model22Li C Wong WH Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection.Proc Natl Acad Sci USA. 2001; 98: 31-36Crossref PubMed Scopus (2641) Google Scholar of dCHIP software as described.20Graham MR Virtaneva K Porcella SF Barry WT Gowen BB Johnson CR Wright FA Musser JM Group A Streptococcus transcriptome dynamics during growth in human blood reveals bacterial adaptive and survival strategies.Am J Pathol. 2005; 166: 455-465Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar The gene expression estimates were normalized across samples by quadratic scaling to an artificial array with the median expression for each gene.23Yoon H Liyanarachchi S Wright FA Davuluri R Lockman JC de la Chapelle A Pellegata NS Gene expression profiling of isogenic cells with different TP53 gene dosage reveals numerous genes that are affected by TP53 dosage and identifies CSPG2 as a direct target of p53.Proc Natl Acad Sci USA. 2002; 99: 15632-15637Crossref PubMed Scopus (69) Google Scholar Two-dimensional scatterplots of expression estimates were generated for all pairs of samples within the same treatment group to examine uniformity across samples. Hierarchical clustering and principal components analyses also were performed (Supplementary Figure 1, see http://ajp.amjpath.org). Five arrays with poorer quality RNA and low within-strain correlation were removed as outliers. Downstream data analysis was performed with MGAS5005 genome-specific probe sets using Partek Pro (Partek Inc., St. Louis, MO). To evaluate expression rankings, absolute square root-transformed expression estimates were integer-ranked, starting from 1 for the most abundant GAS transcript and increasing correspondingly with decreasing transcript detection. To investigate expression correlations, standard Pearson correlation coefficients were determined for select genes versus all other genes using Partek Pro. To investigate strain effects, expression estimates were analyzed by analysis of variance with treatment (WT versus ΔcovR strain) as a fixed effect. Technical variables included in the analysis included batches for sample preparation, sample hybridization, and posthybridization washes (Supplementary Figure 2, see http://ajp.amjpath.org). Final results were subjected to multiple testing correction using Q ≤ 0.05 false discovery rate cutoff values.24Storey JD Tibshirani R Statistical significance for genomewide studies.Proc Natl Acad Sci USA. 2003; 100: 9440-9445Crossref PubMed Scopus (6243) Google Scholar, 25Yekutieli D Benjamini Y Resampling based FDR controlling multiple hypotheses testing.J Stat Plan Infer. 1999; 82: 171-196Crossref Google Scholar Using rigorous permutation-based statistics, we also performed significance analysis of function and expression (termed SAFE)26Barry WT Nobel AB Wright FA Significance analysis of functional categories in gene expression studies: a structured permutation approach.Bioinformatics. 2005; 21: 1943-1949Crossref PubMed Scopus (231) Google Scholar to assess the significance of multiple gene categories in GAS in vivo transcriptional responses across strains. A detailed description of the procedures used is provided in the Supplementary Materials and Methods (see http://ajp.amjpathol.org). Real-time reverse transcriptase (RT)-PCR assays were conducted to validate a subset of the microarray data. Eight oligonucleotide primer pairs and 6FAM-labeled probe sets (specific for cfa, dppA, emm1, sceD, sclA, sic, slo, and speA2) were used to perform target amplification and detection from cDNA templates in 20-μl mul
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