Publications by authors named "Michael S Chaussee"

32 Publications

The signaling peptide SpoV regulates streptolysin O and enhances survival in murine blood.

J Bacteriol 2021 Mar 15. Epub 2021 Mar 15.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota

(Group A , GAS) is a human pathogen that causes a wide range of diseases. For successful colonization within a variety of host niches, GAS must sense and respond to environmental changes. Intercellular communication mediated by peptides is one way GAS coordinates gene expression in response to diverse environmental stressors, which enhances bacterial survival and contributes to virulence. Using peptidomics we identified SpoV (treptococcal eptide cntrolling irulence) in culture supernatant fluids. SpoV is a secreted peptide encoded near the gene encoding the extracellular cholesterol-dependent cytolysin streptolysin O ( The addition of synthetic SpoV peptide derivatives, but not control peptides, increased transcript abundance in an M49 isolate but not in an M3 isolate. Deletion of decreased transcript abundance, extracellular SLO protein levels, and SLO-specific hemolytic activity. Complementation of the mutant increased transcript abundance. Lastly, a mutant was deficient in the ability to survive in murine blood compared to the parental strain. Moreover, pre-incubation of the mutant with synthetic SpoV peptide derivatives increased GAS survival. Our findings show that expression is regulated, in part, by the GAS-specific signaling peptide SpoV.GAS secretes signaling peptides that can alter gene expression and impact virulence. We used peptidomics to identify a signaling peptide designated SpoV. Further, we showed that SpoV altered the expression of the cholesterol-dependent cytolysin SLO. Peptide signaling plays an important regulatory role during disease progression among several bacterial pathogens, including GAS. The therapeutic potential of manipulating peptide-controlled regulatory networks is an attractive option for the development of novel therapeutic strategies that disrupt virulence gene expression.
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http://dx.doi.org/10.1128/JB.00586-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8117530PMC
March 2021

Recurrent Streptococcal Cellulitis: A Case Report and Review.

S D Med 2020 Jul;73(7):318-322

Division of Basic Biomedical Sciences, University of South Dakota Sanford School of Medicine.

Cellulitis is an infection of the dermis and subcutaneous layers of the skin. One challenge in treating the disease is that it is often difficult to identify the causative agent; although β-hemolytic Streptococci and Staphylococcus aureus are the most common causes. In addition, patients who recover from the disease are susceptible to recurrent infections. Here, we briefly review cellulitis and describe a patient's 24-year struggle with recurrent streptococcal cellulitis noting how the patient was negatively affected by changes in care.
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July 2020

Immunotherapy targeting the Streptococcus pyogenes M protein or streptolysin O to treat or prevent influenza A superinfection.

PLoS One 2020 23;15(6):e0235139. Epub 2020 Jun 23.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, SD, United States of America.

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235139PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310742PMC
September 2020

The Streptococcus pyogenes fibronectin/tenascin-binding protein PrtF.2 contributes to virulence in an influenza superinfection.

Sci Rep 2018 08 14;8(1):12126. Epub 2018 Aug 14.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota, USA.

Influenza A virus (IAV) and Streptococcus pyogenes (the group A Streptococcus; GAS) are important contributors to viral-bacterial superinfections, which result from incompletely defined mechanisms. We identified changes in gene expression following IAV infection of A549 cells. Changes included an increase in transcripts encoding proteins with fibronectin-type III (FnIII) domains, such as fibronectin (Fn), tenascin N (TNN), and tenascin C (TNC). We tested the idea that increased expression of TNC may affect the outcome of an IAV-GAS superinfection. To do so, we created a GAS strain that lacked the Fn-binding protein PrtF.2. We found that the wild-type GAS strain, but not the mutant, co-localized with TNC and bound to purified TNC. In addition, adherence of the wild-type strain to IAV-infected A549 cells was greater compared to the prtF.2 mutant. The wild-type strain was also more abundant in the lungs of mice 24 hours after superinfection compared to the mutant strain. Finally, all mice infected with IAV and the prtF.2 mutant strain survived superinfection compared to only 42% infected with IAV and the parental GAS strain, indicating that PrtF.2 contributes to virulence in a murine model of IAV-GAS superinfection.
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http://dx.doi.org/10.1038/s41598-018-29714-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092322PMC
August 2018

Contributions of Influenza Virus Hemagglutinin and Host Immune Responses Toward the Severity of Influenza Virus: Streptococcus pyogenes Superinfections.

Viral Immunol 2018 Jul/Aug;31(6):457-469. Epub 2018 Jun 5.

1 Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota , Vermillion, South Dakota.

Influenza virus infections can be complicated by bacterial superinfections, which are medically relevant because of a complex interaction between the host, the virus, and the bacteria. Studies to date have implicated several influenza virus genes, varied host immune responses, and bacterial virulence factors, however, the host-pathogen interactions that predict survival versus lethal outcomes remain undefined. Previous work by our group showed that certain influenza viruses could yield a survival phenotype (A/swine/Texas/4199-2/98-H3N2, TX98), whereas others were associated with a lethal phenotype (A/Puerto Rico/8/34-H1N1, PR8). Based on this observation, we developed the hypothesis that individual influenza virus genes could contribute to a superinfection, and that the host response after influenza virus infection could influence superinfection severity. The present study analyzes individual influenza virus gene contributions to superinfection severity using reassortant viruses created using TX98 and PR8 viral genes. Host and pathogen interactions, relevant to survival and lethal phenotypes, were studied with a focus on pathogen clearance, host cellular infiltrates, and cytokine levels after infection. Specifically, we found that the hemagglutinin gene expressed by an influenza virus can contribute to the severity of a secondary bacterial infection, likely through modulation of host proinflammatory responses. Altogether, these results advance our understanding of molecular mechanisms underlying influenza virus-bacteria superinfections and identify viral and corresponding host factors that may contribute to morbidity and mortality.
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http://dx.doi.org/10.1089/vim.2017.0193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6043403PMC
October 2018

Binding host proteins to the M protein contributes to the mortality associated with influenza- superinfections.

Microbiology (Reading) 2017 Oct;163(10):1445-1456

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota, USA.

The mortality associated with influenza A virus (IAV) is often due to the development of secondary bacterial infections known as superinfections. The group A streptococcus (GAS) is a relatively uncommon cause of IAV superinfections, but the mortality of these infections is high. We used a murine model to determine whether the surface-localized GAS M protein contributes to the outcome of IAV-GAS superinfections. A comparison between wild-type GAS and an M protein mutant strain (emm3) showed that the M3 protein was essential to virulence. To determine whether the binding, or recruitment, of host proteins to the bacterial surface contributed to virulence, GAS was suspended with BALF collected from mice that had recovered from a sub-lethal infection with IAV. Following intranasal inoculation of naïve mice, the mortality associated with the wild-type strain, but not the emm3 mutant strain, was greater compared to mice inoculated with GAS suspended with either BALF from uninfected mice or PBS. Further analyses showed that both albumin and fibrinogen (Fg) were more abundant in the respiratory tract 8 days after IAV infection, that M3 bound both proteins to the bacterial surface, and that suspension of GAS with either protein increased GAS virulence in the absence of antecedent IAV infection. Overall, the results showed that M3 is essential to the virulence of GAS in an IAV superinfection and suggested that increased abundance of albumin and Fg in the respiratory tract following IAV infection enhanced host susceptibility to secondary GAS infection.
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http://dx.doi.org/10.1099/mic.0.000532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845574PMC
October 2017

The Association between Invasive Group A Streptococcal Diseases and Viral Respiratory Tract Infections.

Front Microbiol 2016 21;7:342. Epub 2016 Mar 21.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota Vermillion, SD, USA.

Viral infections of the upper respiratory tract are associated with a variety of invasive diseases caused by Streptococcus pyogenes, the group A streptococcus, including pneumonia, necrotizing fasciitis, toxic shock syndrome, and bacteremia. While these polymicrobial infections, or superinfections, are complex, progress has been made in understanding the molecular basis of disease. Areas of investigation have included the characterization of virus-induced changes in innate immunity, differences in bacterial adherence and internalization following viral infection, and the efficacy of vaccines in mitigating the morbidity and mortality of superinfections. Here, we briefly summarize viral-S. pyogenes superinfections with an emphasis on those affiliated with influenza viruses.
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http://dx.doi.org/10.3389/fmicb.2016.00342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4800185PMC
April 2016

Inactivation of the Rgg2 transcriptional regulator ablates the virulence of Streptococcus pyogenes.

PLoS One 2014 8;9(12):e114784. Epub 2014 Dec 8.

Department of Molecular Microbiology, Institute of Experimental Medicine, Saint-Petersburg, Russia; Saint-Petersburg State University, Saint-Petersburg, Russia.

Streptococcus pyogenes adapts to different niches encountered in the human host via the activity of numerous regulatory proteins including the Rgg family of transcriptional regulators. The S. pyogenes chromosome encodes four Rgg paralogues designated Rgg1 (RopB), Rgg2 (MutR), Rgg3, and Rgg4 (ComR). In order to understand the role of the Rgg2 protein in the regulation of metabolic and virulence-associated properties of S. pyogenes, the rgg2 gene was inactivated in the M1 serotype strain SF370. Inactivation of rgg2 increased the growth yield of S. pyogenes in THY broth, increased biofilm formation, and increased production of SIC, which is an important virulence factor that inhibits complement mediated lysis. To identify Rgg2-regulated genes, the transcriptomes of SF370 and the rgg2 mutant strains were compared in the middle-exponential and post-exponential phases of growth. Rgg2 was found to control the expression of dozens of genes primarily in the exponential phase of growth, including genes associated with virulence (sse, scpA, slo, nga, mf-3), DNA transformation, and nucleotide metabolism. Inactivation of rgg2 decreased the ability of S. pyogenes to adhere to epithelial cells. In addition, the mutant strain was more sensitive to killing when incubated with human blood and avirulent in a murine bacteremia model. Finally, inoculation of mice with the avirulent rgg2 mutant of S. pyogenes SF370 conferred complete protection to mice subsequently challenged with the wild-type strain. Restoration of an intact rgg2 gene in mutant strain restored the wild-type phenotypes. Overall, the results demonstrate that Rgg2 is an important regulatory protein in S. pyogenes involved in controlling genes associated with both metabolism and virulence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114784PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259489PMC
January 2016

Vaccination against the M protein of Streptococcus pyogenes prevents death after influenza virus: S. pyogenes super-infection.

Vaccine 2014 Sep 29;32(40):5241-9. Epub 2014 Jul 29.

Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD, United States. Electronic address:

Influenza virus infections are associated with a significant number of illnesses and deaths on an annual basis. Many of the deaths are due to complications from secondary bacterial invaders, including Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pyogenes. The β-hemolytic bacteria S. pyogenes colonizes both skin and respiratory surfaces, and frequently presents clinically as strep throat or impetigo. However, when these bacteria gain access to normally sterile sites, they can cause deadly diseases including sepsis, necrotizing fasciitis, and pneumonia. We previously developed a model of influenza virus:S. pyogenes super-infection, which we used to demonstrate that vaccination against influenza virus can limit deaths associated with a secondary bacterial infection, but this protection was not complete. In the current study, we evaluated the efficacy of a vaccine that targets the M protein of S. pyogenes to determine whether immunity toward the bacteria alone would allow the host to survive an influenza virus:S. pyogenes super-infection. Our data demonstrate that vaccination against the M protein induces IgG antibodies, in particular those of the IgG1 and IgG2a isotypes, and that these antibodies can interact with macrophages. Ultimately, this vaccine-induced immunity eliminated death within our influenza virus:S. pyogenes super-infection model, despite the fact that all M protein-vaccinated mice showed signs of illness following influenza virus inoculation. These findings identify immunity against bacteria as an important component of protection against influenza virus:bacteria super-infection.
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http://dx.doi.org/10.1016/j.vaccine.2014.06.093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146501PMC
September 2014

Transcriptional regulation of a bacteriophage encoded extracellular DNase (Spd-3) by Rgg in Streptococcus pyogenes.

PLoS One 2013 17;8(4):e61312. Epub 2013 Apr 17.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota, United States of America.

The Streptococcus pyogenes transcriptional regulator Rgg controls the expression of virulence-associated genes encoded both within the core genome and within horizontally transmissible DNA such as temperate bacteriophage. Previously, we showed that Rgg binds to the non-coding DNA upstream of the bacteriophage gene encoding an extracellular DNase Spd-3. In the current study, we further characterized Rgg-mediated regulation of spd-3 expression. Two spd-3 transcripts were identified by northern blotting. The 5' ends were 27 and 594 nucleotides upstream of the start codon as determined with primer extension analysis and 5' RACE (rapid amplification of c-DNA ends), respectively. Results obtained with gel shift assays showed that purified Rgg bound specifically to non-coding DNA containing the promoters of both transcripts. Transcriptional fusion analyses confirmed the presence of Rgg-repressible promoters within these DNA regions. In addition, repression was associated with direct DNA binding by Rgg as determined with chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (qPCR). The results show that the chromosomally encoded transcriptional regulator, Rgg, directly represses both bacteriophage promoters controlling the expression of Spd-3. The results provide new information regarding the regulation of prophage encoded virulence factors of S. pyogenes and highlight the complex evolutionary history of S. pyogenes and temperate bacteriophage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061312PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629212PMC
November 2013

CodY-mediated regulation of Streptococcus pyogenes exoproteins.

BMC Microbiol 2012 Jun 21;12:114. Epub 2012 Jun 21.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, SD, USA.

Background: The production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene.

Results: We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain.

Conclusions: Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.
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http://dx.doi.org/10.1186/1471-2180-12-114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438106PMC
June 2012

Growth phase-dependent modulation of Rgg binding specificity in Streptococcus pyogenes.

J Bacteriol 2012 Aug 25;194(15):3961-71. Epub 2012 May 25.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota, USA.

Streptococcus pyogenes Rgg is a transcriptional regulator that interacts with the cofactor LacD.1 to control growth phase-dependent expression of genes, including speB, which encodes a secreted cysteine protease. LacD.1 is thought to interact with Rgg when glycolytic intermediates are abundant in a manner that prevents Rgg-mediated activation of speB expression via binding to the promoter region. When the intermediates diminish, LacD.1 dissociates from Rgg and binds to the speB promoter to activate expression. The purpose of this study was to determine if Rgg bound to chromatin during the exponential phase of growth and, if so, to identify the binding sites. Rgg bound to 62 chromosomal sites, as determined by chromatin immunoprecipitation coupled with DNA microarrays. Thirty-eight were within noncoding DNA, including sites upstream of the genes encoding the M protein (M49), serum opacity factor (SOF), fibronectin-binding protein (SfbX49), and a prophage-encoded superantigen, SpeH. Each of these sites contained a promoter that was regulated by Rgg, as determined with transcriptional fusion assays. Purified Rgg also bound to the promoter regions of emm49, sof, and sfbX49 in vitro. Results obtained with a lacD.1 mutant showed that both LacD.1 and Rgg were necessary for the repression of emm49, sof, sfbX49, and speH expression. Overall, the results indicated that the DNA binding specificity of Rgg is responsive to environmental changes in a LacD.1-dependent manner and that Rgg and LacD.1 directly control virulence gene expression in the exponential phase of growth.
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http://dx.doi.org/10.1128/JB.06709-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416553PMC
August 2012

Identification of Rgg binding sites in the Streptococcus pyogenes chromosome.

J Bacteriol 2011 Sep 15;193(18):4933-42. Epub 2011 Jul 15.

Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Lee Medical Building, 414 East Clark Street, Vermillion, SD 57069-2390, USA.

Streptococcus pyogenes Rgg is a regulatory protein that controls the transcription of 588 genes in strain NZ131 during the post-exponential phase of growth, including the virulence-associated genes encoding the extracellular SpeB protease, pullulanase A (PulA), and two extracellular nucleases (SdaB and Spd-3). Rgg binds to DNA proximally to the speB promoter (PspeB) to activate transcription; however, it is not known if Rgg binds to the promoters of other genes to influence expression, or if the perturbation of other global regulons accounts for the genome-wide changes in expression associated with the mutant. To address this issue, chromatin immunoprecipitation followed by DNA microarray analysis (ChIP-chip) was used to identify the DNA binding sites of Rgg. Rgg bound to 65 sites in the chromosome. Thirty-five were within noncoding DNA, and 43% of these were adjacent to genes previously identified as regulated by Rgg. Electrophoretic mobility shift assays were used to assess the binding of Rgg to a subset of sites bound in vivo, including the noncoding DNA upstream of speB, the genes encoding PulA, Spd-3, and a transcriptional regulator (SPY49_1113), and prophage-associated genes encoding a putative integrase (SPY49_0746) and a surface antigen (SPY49_0396). Rgg bound to all target DNAs in vitro, consistent with the in vivo results. Finally, analyses with a transcriptional reporter system showed that the DNA bound by Rgg contained an active promoter that was regulated by Rgg. Overall, the results indicate that Rgg binds specifically to multiple sites in the chromosome, including prophage DNA, to influence gene expression.
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http://dx.doi.org/10.1128/JB.00429-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165660PMC
September 2011

Inactivated and live, attenuated influenza vaccines protect mice against influenza: Streptococcus pyogenes super-infections.

Vaccine 2011 May 8;29(21):3773-81. Epub 2011 Apr 8.

Division of Basic Biomedical Sciences, University of South Dakota, Vermillion, SD, United States.

Mortality associated with influenza virus super-infections is frequently due to secondary bacterial complications. To date, super-infections with Streptococcus pyogenes have been studied less extensively than those associated with Streptococcus pneumoniae. This is significant because a vaccine for S. pyogenes is not clinically available, leaving vaccination against influenza virus as our only means for preventing these super-infections. In this study, we directly compared immunity induced by two types of influenza vaccine, either inactivated influenza virus (IIV) or live, attenuated influenza virus (LAIV), for the ability to prevent super-infections. Our data demonstrate that both IIV and LAIV vaccines induce similar levels of serum antibodies, and that LAIV alone induces IgA expression at mucosal surfaces. Upon super-infection, both vaccines have the ability to limit the induction of pro-inflammatory cytokines within the lung, including IFN-γ which has been shown to contribute to mortality in previous models of super-infection. Limiting expression of these pro-inflammatory cytokines within the lungs subsequently limits recruitment of macrophages and neutrophils to pulmonary surfaces, and ultimately protects both IIV- and LAIV-vaccinated mice from mortality. Despite their overall survival, both IIV- and LAIV-vaccinated mice demonstrated levels of bacteria within the lung tissue that are similar to those seen in unvaccinated mice. Thus, influenza virus:bacteria super-infections can be limited by vaccine-induced immunity against influenza virus, but the ability to prevent morbidity is not complete.
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http://dx.doi.org/10.1016/j.vaccine.2011.03.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084433PMC
May 2011

The Streptococcus pyogenes proteome: maps, virulence factors and vaccine candidates.

Future Microbiol 2010 Oct;5(10):1539-51

Department of Molecular Microbiology, Institute of Experimental Medicine. acad. Pavlov str., 12, Saint-Petersburg, 197376, Russia.

Streptococcus pyogenes is an important cause of human morbidity and mortality worldwide. A wealth of genomic information related to this pathogen has facilitated exploration of the proteome, particularly in response to environmental conditions thought to mimic various aspects of pathogenesis. Proteomic approaches are also used to identify immunoreactive proteins for vaccine development and to identify proteins that may induce autoimmunity. These studies have revealed new mechanisms involved in regulating the S. pyogenes proteome, which has opened up new avenues in the study of S. pyogenes pathogenesis. This article describes the methods used, and progress being made towards characterizing the S. pyogenes proteome, including studies seeking to identify potential vaccine candidates.
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http://dx.doi.org/10.2217/fmb.10.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092638PMC
October 2010

Community-based intervention to manage an outbreak of MRSA skin infections in a county jail.

J Correct Health Care 2010 Jul 12;16(3):205-15. Epub 2010 May 12.

Sioux Falls Family Medicine Program, Center for Family Medicine, Sioux Falls, South Dakota, USA.

This article describes a community-based intervention to manage an outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) skin infections in a midwestern county jail. A systematic investigation conducted by a family medicine residency program identified 64 total cases and 19 MRSA cases between January 1 and December 31, 2007. Factors contributing to MRSA transmission included inadequate surveillance, lack of antibacterial soap, and a defective laundry process. All 19 isolates were CA-MRSA and all seven tested by pulsed-field gel electrophoresis (PFGE) were USA300. Four of the seven isolates showed variation of their PFGE patterns. A primary care approach using community-based resources effectively reduced the number of cases in this heterogeneous outbreak of CA-MRSA, with the last MRSA being isolated in October 2007.
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http://dx.doi.org/10.1177/1078345810366679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390025PMC
July 2010

Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.

PLoS Pathog 2009 Dec 24;5(12):e1000708. Epub 2009 Dec 24.

Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.

Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC) causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome) for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia) utilize a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3), a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well as an AP-1-dependent reporter. We identified a region of NleH1 (N40-K45) that is at least partially responsible for the inhibitory activity of NleH1 toward RPS3. Deleting nleH1 from E. coli O157:H7 produced a hypervirulent phenotype in a gnotobiotic piglet model of Shiga toxin-producing E. coli infection. We suggest that NleH may disrupt host innate immune responses by binding to a cofactor of host transcriptional complexes.
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http://dx.doi.org/10.1371/journal.ppat.1000708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2791202PMC
December 2009

A naturally occurring Rgg variant in serotype M3 Streptococcus pyogenes does not activate speB expression due to altered specificity of DNA binding.

Infect Immun 2009 Dec 14;77(12):5411-7. Epub 2009 Sep 14.

Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota 57069-2390, USA.

The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg(103P)); in contrast, all other strains encode a serine at this position (Rgg(103S)). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg(103S), Rgg(103P) does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg(103P) retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.
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http://dx.doi.org/10.1128/IAI.00373-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786450PMC
December 2009

Inter- and intraserotypic variation in the Streptococcus pyogenes Rgg regulon.

FEMS Microbiol Lett 2008 Jul;284(1):43-51

Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Vermillion, SD 57069-2390, USA.

Human isolates of Streptococcus pyogenes, a Gram-positive bacterium, are characterized by significant genetic and phenotypic variation. The rgg locus, also known as ropB, is a global transcriptional regulator of genes associated with metabolism, stress responses, and virulence in S. pyogenes strain NZ131 (serotype M49). To assess the breadth of the Rgg regulon, the rgg gene was inactivated in three additional strains representing serotypes M1 (strains SF370 and MGAS5005) and M49 (strain CS101). Changes in gene expression were identified in the postexponential phase of growth using Affymetrix NimbleExpress Arrays. The results identified an Rgg core-regulon consisting of speB and adjacent hypothetical protein gene, spy2040, and a variable and strain-specific subregulon, ranging in size from a single gene (spy1793) in strain MGAS5005 to 43 genes in strain SF370. Thus, both interserotypic and intraserotypic variation is characteristic of the Rgg regulon in S. pyogenes.
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http://dx.doi.org/10.1111/j.1574-6968.2008.01171.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2561273PMC
July 2008

Proteomic analysis of proteins secreted by Streptococcus pyogenes.

Methods Mol Biol 2008 ;431:15-24

Division of Basic Biomedical Sciences, The Stanford School of Medicine of the University of South Dakota, Vermillion, SD, USA.

Streptococcus pyogenes secretes various proteins to the extracellular environment. During infection, these proteins interact with human macromolecules and contribute to pathogenesis. We describe a proteomic approach routinely used in our laboratory to characterize culture supernatant proteins using small-format two-dimensional gel electrophoresis. Proteins are collected after overnight growth of the bacteria in broth media. Compounds that inhibit isoelectric focusing, such as salts, are removed by enzymatic treatment and precipitation with trichloroacetic acid and acetone. Following resuspension in denaturing solution, the proteins are separated by isoelectric focusing using a 7-cm immobilized strip with a pH gradient of 4-7. Subsequently, proteins are further separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with SYPRO Ruby. The small-gel format requires less time, reagents, and smaller culture volumes compared with large-format approaches, while still resolving and detecting a large proportion of the exoprotein fraction.
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http://dx.doi.org/10.1007/978-1-60327-032-8_2DOI Listing
July 2008

Growth phase-associated changes in the transcriptome and proteome of Streptococcus pyogenes.

Arch Microbiol 2008 Jan 31;189(1):27-41. Epub 2007 Jul 31.

Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Lee Medical Building, 414 East Clark Street, Vermillion, SD 57069-2390, USA.

Streptococcus pyogenes is responsible for approximately 500,000 deaths each year worldwide. Many of the associated virulence factors are expressed in a growth phase-dependent manner. To identify growth phase-associated changes in expression on a genomescale, the exponential and stationary phase transcriptomes and proteomes of S. pyogenes strain NZ131 (serotype M49) were compared by using Affymetrix NimbleExpress gene chips and two-dimensional gel electrophoresis. At the transcript level, the expression of 689 genes, representing approximately 40% of the chromosome, differed by twofold or more between the two growth phases. The majority of transcripts that were more abundant in the early-stationary phase encoded proteins involved in energy conversion, transport, and metabolism. At the protein level, an average of 527 and 403 protein spots were detected in the exponential and stationary phases of growth, respectively. Tandem mass spectrometry was used to identify 172 protein spots, 128 of which were growth phase regulated. Enzymes involved in glycolysis and pyruvate metabolism and several stress-responsive proteins were more abundant in the stationary phase of growth. Overall, the results identified growth phase-regulated genes in strain NZ131 and revealed significant post-transcriptional complexity associated with pathogen adaptation to the stationary phase of growth.
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http://dx.doi.org/10.1007/s00203-007-0290-1DOI Listing
January 2008

The Rgg regulator of Streptococcus pyogenes influences utilization of nonglucose carbohydrates, prophage induction, and expression of the NAD-glycohydrolase virulence operon.

J Bacteriol 2006 Oct;188(20):7230-41

Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Lee Medical Building, 414 East Clark Street, Vermillion, SD 57069-2390, USA.

The expression of many virulence-associated genes in Streptococcus pyogenes is controlled in a growth phase-dependent manner. Unlike the model organisms Escherichia coli and Bacillus subtilis, such regulation is apparently not dependent upon alternative sigma factors but appears to rely on complex interactions among several transcriptional regulators, including Rgg. The purpose of this study was to identify changes in gene expression associated with inactivation of the rgg gene in S. pyogenes strain NZ131 (serotype M49). To this end, the transcriptomes of wild-type and rgg mutant strains were analyzed during both the exponential and postexponential phases of growth using Affymetrix NimbleExpress gene chips. Genomewide differences in transcript levels were identified in both phases of growth. Inactivation of rgg disrupted coordinate expression of genes associated with the metabolism of nonglucose carbon sources, such as fructose, mannose, and sucrose. The changes were associated with an inability of the mutant strain to grow using these compounds as the primary carbon source. Bacteriophage transcript levels were also altered in the mutant strain and were associated with decreased induction of at least one prophage. Finally, transcripts encoding virulence factors involved in cytolysin-mediated translocation of NAD-glycohydrolase, including the immunity factor IFS and the cytolysin (streptolysin O [SLO]), were more abundant in the mutant strain, which correlated with the amount of NADase and SLO activities in culture supernatant fluids. The results provide further evidence that Rgg contributes to growth phase-dependent gene regulation in strain NZ131.
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http://dx.doi.org/10.1128/JB.00877-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1636216PMC
October 2006

Inactivation of the group A Streptococcus regulator srv results in chromosome wide reduction of transcript levels, and changes in extracellular levels of Sic and SpeB.

FEMS Immunol Med Microbiol 2006 Nov 25;48(2):283-92. Epub 2006 Sep 25.

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

Group A Streptococcus is characterized by the ability to cause a diverse number of human infections including pharyngitis, necrotizing fasciitis, toxic shock syndrome, and acute rheumatic fever, yet the regulation of streptococcal genes involved in disease processes and survival in the host is not completely understood. Genome scale analysis has revealed a complex regulatory network including 13 two-component regulatory systems and more than 100 additional putative regulators, the majority of which remain uncharacterized. Among these is the streptococcal regulator of virulence, Srv, the first Group A Streptococcus member of the Crp/Fnr family of transcriptional regulators. Previous work demonstrated that the loss of srv resulted in a significant decrease in Group A Streptococcus virulence. To begin to define the gene products influenced by Srv, we combined microarray and two-dimensional gel electrophoresis analysis. Loss of srv results in a chromosome wide reduction of gene transcription and changes in the production of the extracellular virulence factors Sic (streptococcal inhibitor of complement) and SpeB (cysteine proteinase). Sic levels are reduced in the srv mutant, whereas the extracellular concentration and activity of SpeB is increased. These data link Srv to the increasingly complex GAS regulatory network.
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http://dx.doi.org/10.1111/j.1574-695X.2006.00150.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726989PMC
November 2006

Proteomic analysis of a penicillin-tolerant rgg mutant strain of Streptococcus pyogenes.

J Antimicrob Chemother 2006 Oct 5;58(4):752-9. Epub 2006 Aug 5.

Division of Basic Biomedical Sciences, The Stanford School of Medicine of the University of South Dakota 414 East Clark Street, Vermillion, SD 57069, USA.

Objectives: To determine whether the transcriptional regulator Rgg contributes to penicillin-induced killing of Streptococcus pyogenes by altering a regulatory response to penicillin.

Methods: Penicillin-induced killing of a wild-type and isogenic rgg mutant strain was assessed in broth and solid media and in the presence of cerulenin, which inhibits fatty acid biosynthesis (FAB). Proteins from wild-type and rgg mutant cultures, either exposed to penicillin or not, were characterized by two-dimensional gel electrophoresis. Proteins of interest were identified with tandem mass spectrometry.

Results: The MIC of penicillin was 0.012 mg/L for both the wild-type strain NZ131 and an isogenic rgg mutant strain. The wild-type strain lost 1.9 log(10) cfu/mL ( approximately 80-fold) after 24 h of exposure to 0.024 mg/L penicillin compared with controls; however, the mutant strain lost 0.3 log(10) cfu/mL ( approximately 2-fold) compared with controls. Changes in the proteome of wild-type and mutant cultures were assessed 1 and 4 h after exposure to penicillin. One hour exposure was associated with increased abundance (P < 0.05) of 12 proteins associated with FAB, the pentose phosphate pathway, glycolysis and stress responses in the wild-type strain. The abundance of 8 of 12 of these proteins was greater in samples obtained from the mutant strain, even prior to penicillin exposure. After 4 h of exposure, the abundance of 16 proteins was altered in one or both strains; however, a clear functional relationship was not evident. The addition of cerulenin slightly enhanced penicillin-induced killing of wild-type strain, which supported the proteomic results.

Conclusions: The results suggest that penicillin-independent changes in the cytoplasmic proteome of an rgg mutant strain of NZ131 confer tolerance to penicillin-mediated killing.
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http://dx.doi.org/10.1093/jac/dkl319DOI Listing
October 2006

Persistence of Streptococcus pyogenes in stationary-phase cultures.

J Bacteriol 2005 May;187(10):3319-28

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10(3) CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.
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http://dx.doi.org/10.1128/JB.187.10.3319-3328.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1111994PMC
May 2005

Rgg regulates growth phase-dependent expression of proteins associated with secondary metabolism and stress in Streptococcus pyogenes.

J Bacteriol 2004 Nov;186(21):7091-9

Division of Basic Biomedical Sciences, University of South Dakota College of Medicine, Lee Medical Building, 414 East Clark Street, Vermillion, SD 57069-2390, USA.

The transcriptional regulatory protein Rgg coordinates amino acid catabolism and virulence factor expression in Streptococcus pyogenes. We used a proteomic approach to compare cytoplasmic proteins isolated from S. pyogenes wild-type strain NZ131 (serotype M49) to proteins isolated from an rgg mutant strain during the exponential and stationary phases of growth. Proteins were separated by two-dimensional gel electrophoresis, and 125 protein spots of interest were identified by tandem mass spectrometry. Comparative analysis of proteins isolated from the isogenic strains revealed that growth phase-associated regulation of enzymes involved in the metabolism of arginine (ArcABC), histidine (HutI), and serine (SdhA) was abrogated in the rgg mutant strain, which synthesized the proteins in the exponential phase of growth. In contrast, the enzymes were detected only among wild-type proteins isolated from organisms in the stationary phase of growth. The differences in protein composition were correlated with previously described metabolic changes. In addition, proteins associated with thermal and oxidative stress responses, including ClpE and ClpL, were present in samples isolated from the rgg mutant strain but not in samples isolated from the wild-type strain. The rgg mutant strain was more tolerant to elevated temperature and puromycin than the wild-type strain; however, the mutant was less tolerant to paraquat. We concluded that Rgg is a global regulatory factor that contributes to growth phase-dependent synthesis of proteins associated with secondary metabolism and oxidative and thermal stress responses.
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http://dx.doi.org/10.1128/JB.186.21.7091-7099.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC523193PMC
November 2004

Rgg coordinates virulence factor synthesis and metabolism in Streptococcus pyogenes.

J Bacteriol 2003 Oct;185(20):6016-24

Division of Basic Biomedical Science, University of South Dakota College of Medicine, Vermillion, South Dakota 57069, USA.

Streptococcus pyogenes is a human-specific pathogen that relies on its host for metabolic substrates. Rgg-like proteins constitute a family of transcriptional regulators present in several gram-positive bacteria. In S. pyogenes, Rgg influences the expression of several virulence-associated proteins localized to the cell wall and extracellular environment. Secreted enzymes may degrade host macromolecules, thereby liberating metabolic substrates. To determine if Rgg regulation of exoprotein expression is associated with altered metabolism, the catabolic activities of S. pyogenes strain NZ131 (serotype M49) and an isogenic rgg mutant strain were analyzed during growth with complex and defined media. As expected, the wild-type strain preferentially used glucose and produced lactic acid during the exponential phase of growth. In contrast, the rgg mutant fermented arginine in the exponential phase of growth, even in the presence of glucose. Arginine degradation was associated with a neutral culture pH and excretion of NH(3) and ornithine. Arginine, serine, and asparagine were depleted from mutant cultures during growth. The addition of arginine and serine to culture media increased the growth yield and NH(3) production of mutant but not wild-type cultures. Addition of asparagine had no effect on the growth yield of either strain. Altered metabolism of arginine and serine in the mutant was associated with increased transcript levels of genes encoding arginine deiminase and a putative serine dehydratase. Thus, Rgg coordinates virulence factor synthesis and catabolic activity and may be important in the pathogen's adaptation to changes in the availability of metabolic substrates.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC225023PMC
http://dx.doi.org/10.1128/JB.185.20.6016-6024.2003DOI Listing
October 2003

Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival.

Infect Immun 2002 Nov;70(11):6373-82

Laboratory of Human Bacterial Pathogenesis. Rocky Mountain Microscopy Branch. Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840, USA.

Staphylococcus aureus preferentially catabolizes glucose, generating pyruvate, which is subsequently oxidized to acetate under aerobic growth conditions. Catabolite repression of the tricarboxylic acid (TCA) cycle results in the accumulation of acetate. TCA cycle derepression coincides with exit from the exponential growth phase, the onset of acetate catabolism, and the maximal expression of secreted virulence factors. These data suggest that carbon and energy for post-exponential-phase growth and virulence factor production are derived from the catabolism of acetate mediated by the TCA cycle. To test this hypothesis, the aconitase gene was genetically inactivated in a human isolate of S. aureus, and the effects on physiology, morphology, virulence factor production, virulence for mice, and stationary-phase survival were examined. TCA cycle inactivation prevented the post-exponential growth phase catabolism of acetate, resulting in premature entry into the stationary phase. This phenotype was accompanied by a significant reduction in the production of several virulence factors and alteration in host-pathogen interaction. Unexpectedly, aconitase inactivation enhanced stationary-phase survival relative to the wild-type strain. Aconitase is an iron-sulfur cluster-containing enzyme that is highly susceptible to oxidative inactivation. We speculate that reversible loss of the iron-sulfur cluster in wild-type organisms is a survival strategy used to circumvent oxidative stress induced during host-pathogen interactions. Taken together, these data demonstrate the importance of the TCA cycle in the life cycle of this medically important pathogen.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC130419PMC
http://dx.doi.org/10.1128/IAI.70.11.6373-6382.2002DOI Listing
November 2002

Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks.

Proc Natl Acad Sci U S A 2002 Apr 26;99(7):4668-73. Epub 2002 Mar 26.

Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59840, USA.

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.
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http://dx.doi.org/10.1073/pnas.062526099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC123705PMC
April 2002
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