scholarly journals Genome-Wide Identification of Genes Required for Fitness of Group A Streptococcus in Human Blood

2013 ◽  
Vol 81 (3) ◽  
pp. 862-875 ◽  
Author(s):  
Yoann Le Breton ◽  
Pragnesh Mistry ◽  
Kayla M. Valdes ◽  
Jeffrey Quigley ◽  
Nikhil Kumar ◽  
...  
Keyword(s):  
Human Blood ◽  
De Novo ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Wide Spectrum ◽  
Sugar Metabolism ◽  
Nucleotide Synthesis ◽  
Rich Media ◽  
Group A ◽  
Virulence Regulator

ABSTRACTThe group A streptococcus (GAS) is a strict human pathogen responsible for a wide spectrum of diseases. Although GAS genome sequences are available, functional genomic analyses have been limited. We developed amariner-based transposon,osKaR, designed to perform Transposon-Site Hybridization (TraSH) in GAS and successfully tested its use in several invasive serotypes. A complexosKaRmutant library in M1T1 GAS strain 5448 was subjected to negative selection in human blood to identify genes important for GAS fitness in this clinically relevant environment. Mutants underrepresented after growth in blood (output pool) compared to growth in rich media (input pool) were identified using DNA microarray hybridization of transposon-specific tagsen masse. Using blood from three different donors, we identified 81 genes that met our criteria for reduced fitness in blood from at least two individuals. Genes known to play a role in survival of GAS in blood were found, including those encoding the virulence regulator Mga (mga), the peroxide response regulator PerR (perR), and the RofA-like regulator Ralp-3 (ralp3). We also identified genes previously reported for their contribution to sepsis in other pathogens, such asde novonucleotide synthesis (purD,purA,pyrB,carA,carB,guaB), sugar metabolism (scrB,fruA), zinc uptake (adcC), and transcriptional regulation (cpsY). To validate our findings, independent mutants with mutations in 10 different genes identified in our screen were confirmed to be defective for survival in blood bactericidal assays. Overall, this work represents the first use of TraSH in GAS to identify potential virulence genes.

scholarly journals Regulation of Polysaccharide Utilization Contributes to the Persistence of Group A Streptococcus in the Oropharynx

2007 ◽  
Vol 75 (6) ◽  
pp. 2981-2990 ◽  
Author(s):  
Samuel A. Shelburne ◽  
Nnaja Okorafor ◽  
Izabela Sitkiewicz ◽  
Paul Sumby ◽  
David Keith ◽  
...  
Keyword(s):  
Parental Strain ◽  
Group A Streptococcus ◽  
Transcriptional Regulator ◽  
Human Saliva ◽  
Transcript Levels ◽  
Expression Of Genes ◽  
Rich Media ◽  
Genes Encoding ◽  
Group A ◽  
Mutant Derivative

ABSTRACT Group A Streptococcus (GAS) genes that encode proteins putatively involved in polysaccharide utilization show growth phase-dependent expression in human saliva. We sought to determine whether the putative polysaccharide transcriptional regulator MalR influences the expression of such genes and whether MalR helps GAS infect the oropharynx. Analysis of 32 strains of 17 distinct M protein serotypes revealed that MalR is highly conserved across GAS strains. malR transcripts were detectable in patients with GAS pharyngitis, and the levels increased significantly during growth in human saliva compared to the levels during growth in glucose-containing or nutrient-rich media. To determine if MalR influenced the expression of polysaccharide utilization genes, we compared the transcript levels of eight genes encoding putative polysaccharide utilization proteins in the parental serotype M1 strain MGAS5005 and its ΔmalR isogenic mutant derivative. The transcript levels of all eight genes were significantly increased in the ΔmalR strain compared to the parental strain, especially during growth in human saliva. Following experimental infection, the ΔmalR strain persistently colonized the oropharynx in significantly fewer mice than the parental strain colonized, and the numbers of ΔmalR strain CFU recovered were significantly lower than the numbers of the parental strain CFU recovered. These data led us to conclude that MalR influences the expression of genes putatively involved in polysaccharide utilization and that MalR contributes to the persistence of GAS in the oropharynx.

scholarly journals Conversion of an M- group A streptococcus to M+ by transfer of a plasmid containing an M6 gene.

1986 ◽  
Vol 164 (5) ◽  
pp. 1641-1651 ◽  
Author(s):  
J R Scott ◽  
P C Guenthner ◽  
L M Malone ◽  
V A Fischetti
Keyword(s):  
Structural Gene ◽  
Human Blood ◽  
Group A Streptococcus ◽  
M Protein ◽  
Gene Encoding ◽  
Hyperimmune Serum ◽  
Amino Terminal ◽  
Group A ◽  
Hyperimmune Rabbit ◽  
Streptococcal Strain

An M28-derived group A streptococcal strain deleted for the gene encoding M protein was converted to M+ by introduction of a plasmid carrying emm6, the structural gene for type 6 M protein from strain D471. The reconstituted M+ strain, JRS2, resists phagocytosis in human blood and is opsonized by anti-M6 hyperimmune serum, but not by anti-M28 serum. Immunofluorescent microscopy and ELISA demonstrate the presence of M protein on its surface. In addition, JRS2 removes opsonic antibodies from hyperimmune rabbit sera generated by immunization with purified ColiM6 protein and with a synthetic amino-terminal peptide derived from M6. Immunization of rabbits with JRS2 generates opsonic anti-M6 antibodies. These results indicate that the cloned emm6 gene contains the information necessary to convert a phagocytosis-sensitive streptococcus to phagocytosis resistance. Furthermore, it also contains the determinants for M type specificity and those required to elicit opsonic antibodies. It thus appears to determine all the traits associated with M protein.

scholarly journals Dextromethorphan Efficiently Increases Bactericidal Activity, Attenuates Inflammatory Responses, and Prevents Group A Streptococcal Sepsis

2011 ◽  
Vol 55 (3) ◽  
pp. 967-973 ◽  
Author(s):  
Ming-Han Li ◽  
Yueh-Hsia Luo ◽  
Chiou-Feng Lin ◽  
Yu-Tzu Chang ◽  
Shiou-Ling Lu ◽  
...  
Keyword(s):  
Bactericidal Activity ◽  
Inflammatory Responses ◽  
Group A Streptococcus ◽  
Wide Spectrum ◽  
Serum Levels ◽  
Streptococcal Toxic Shock Syndrome ◽  
Necrosis Factor Alpha ◽  
Bacterial Killing ◽  
Air Pouch ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases, ranging from mild throat and skin infections to severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), a dextrorotatory morphinan and a widely used antitussive drug, has recently been reported to possess anti-inflammatory properties. In this study, we investigated the potential protective effect of DM in GAS infection using an air pouch infection mouse model. Our results showed that DM treatment increased the survival rate of GAS-infected mice. Bacterial numbers in the air pouch were lower in mice treated with DM than in those infected with GAS alone. The bacterial elimination efficacy was associated with increased cell viability and bactericidal activity of air-pouch-infiltrating cells. Moreover, DM treatment prevented bacterial dissemination in the blood and reduced serum levels of the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-1β and the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and RANTES. In addition, GAS-induced mouse liver injury was reduced by DM treatment. Taken together, DM can increase bacterial killing and reduce inflammatory responses to prevent sepsis in GAS infection. The consideration of DM as an adjunct treatment in combination with antibiotics against bacterial infection warrants further study.

scholarly journals CovR Activation of the Dipeptide Permease Promoter (PdppA) in Group A Streptococcus

2006 ◽  
Vol 189 (4) ◽  
pp. 1407-1416 ◽  
Author(s):  
Asiya A. Gusa ◽  
Barbara J. Froehlich ◽  
Devak Desai ◽  
Virginia Stringer ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Activation Of Transcription ◽  
Genes Encoding ◽  
Group A ◽  
Transcription In Vitro ◽  
Dna Template ◽  
Regulated Promoters

ABSTRACT CovR, the two-component response regulator of Streptococcus pyogenes (group A streptococcus [GAS]) directly or indirectly represses about 15% of the genome, including genes encoding many virulence factors and itself. Transcriptome analyses also showed that some genes are activated by CovR. We asked whether the regulation by CovR of one of these genes, dppA, the first gene in an operon encoding a dipeptide permease, is direct or indirect. Direct regulation by CovR was suggested by the presence of five CovR consensus binding sequences (CBs) near the putative promoter. In this study, we identified the 5′ end of the dppA transcript synthesized in vivo and showed that the start of dppA transcription in vitro is the same. We found that CovR binds specifically to the dppA promoter region (PdppA) in vitro with an affinity similar to that at which it binds to other CovR-regulated promoters. Disruption of any of the five CBs by a substitution of GG for TT inhibited CovR binding to that site in vitro, and binding at two of the CBs appeared cooperative. In vivo, CovR activation of transcription was not affected by individual mutations of any of the four CBs that we could study. This suggests that the binding sites are redundant in vivo. In vitro, CovR did not activate transcription from PdppA in experiments using purified GAS RNA polymerase and either linear or supercoiled DNA template. Therefore, we propose that in vivo, CovR may interfere with the binding of a repressor of PdppA.

Cysteine proteinase SpeB from Streptococcus pyogenes – a potent modifier of immunologically important host and bacterial proteins

2011 ◽  
Vol 392 (12) ◽  
pp. 1077-1088 ◽  
Author(s):  
Daniel C. Nelson ◽  
Julia Garbe ◽  
Mattias Collin
Keyword(s):  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Wide Spectrum ◽  
Cysteine Proteinase ◽  
Complement Components ◽  
Bacterial Surface ◽  
Bacterial Proteins ◽  
Disease States ◽  
Group A ◽  
Important Host

AbstractGroup A streptococcus (Streptococcus pyogenes) is an exclusively human pathogen that causes a wide spectrum of diseases ranging from pharyngitis, to impetigo, to toxic shock, to necrotizing fasciitis. The diversity of these disease states necessitates thatS. pyogenespossess the ability to modulate both the innate and adaptive immune responses. SpeB, a cysteine proteinase, is the predominant secreted protein fromS. pyogenes. Because of its relatively indiscriminant specificity, this enzyme has been shown to degrade the extracellular matrix, cytokines, chemokines, complement components, immunoglobulins, and serum protease inhibitors, to name but a few of the known substrates. Additionally, SpeB regulates other streptococcal proteins by degrading them or releasing them from the bacterial surface. Despite the wealth of literature on putative SpeB functions, there remains much controversy about this enzyme because many of reported activities would produce contradictory physiological results. Here we review all known host and bacterial protein substrates for SpeB, their cleavage sites, and discuss the role of this enzyme in streptococcal pathogenesis based on the current literature.

scholarly journals RscA, a Member of the MDR1 Family of Transporters, Is Repressed by CovR and Required for Growth of Streptococcus pyogenes under Heat Stress

2006 ◽  
Vol 188 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Tracy L. Dalton ◽  
Julie T. Collins ◽  
Timothy C. Barnett ◽  
June R. Scott
Keyword(s):  
Streptococcus Pyogenes ◽  
Abc Transporters ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Transcriptional Analysis ◽  
Sensor Kinase ◽  
Two Component System ◽  
Transcription Profiles ◽  
Group A

ABSTRACT The ability of Streptococcus pyogenes (group A streptococcus [GAS]) to respond to changes in environmental conditions is essential for this gram-positive organism to successfully cause disease in its human host. The two-component system CovRS controls expression of about 15% of the GAS genome either directly or indirectly. In most operons studied, CovR acts as a repressor. We previously linked CovRS to the GAS stress response by showing that the sensor kinase CovS is required to inactivate the response regulator CovR so that GAS can grow under conditions of heat, acid, and salt stress. Here, we sought to identify CovR-repressed genes that are required for growth under stress. To do this, global transcription profiles were analyzed by microarrays following exposure to increased temperature (40°C) and decreased pH (pH 6.0). The CovR regulon in an M type 6 strain of GAS was also examined by global transcriptional analysis. We identified a gene, rscA (regulated by stress and Cov), whose transcription was confirmed to be repressed by CovR and activated by heat and acid. RscA is a member of the MDR1 family of ABC transporters, and we found that it is required for growth of GAS at 40°C but not at pH 6.0. Thus, for GAS to grow at 40°C, CovR repression must be alleviated so that rscA can be transcribed to allow the production of this potential exporter. Possible explanations for the thermoprotective role of RscA in this pathogen are discussed.

scholarly journals Distinct Structural Features of the Peroxide Response Regulator from Group A Streptococcus Drive DNA Binding

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89027 ◽  
Author(s):  
Chang Sheng-Huei Lin ◽  
Shi-Yu Chao ◽  
Michal Hammel ◽  
Jay C. Nix ◽  
Hsiao-Ling Tseng ◽  
...  
Keyword(s):  
Dna Binding ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Structural Features ◽  
Group A

scholarly journals A Response Regulator That Represses Transcription of Several Virulence Operons in the Group A Streptococcus

1999 ◽  
Vol 181 (12) ◽  
pp. 3649-3657 ◽  
Author(s):  
Michael J. Federle ◽  
Kevin S. McIver ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Regulatory System ◽  
Insertion Mutant ◽  
Multiple Gene ◽  
Streptolysin S ◽  
Streptolysin O ◽  
Group A ◽  
Complement C5a ◽  
Global Regulatory System

ABSTRACT A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209–219, 1998), caused the strain to produce mucoid colonies and to increase transcription ofhasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase),sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo(streptolysin O), mga (multiple gene regulator of GAS),emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system inEscherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for “control of virulence genes” in GAS. Transcription of thecovR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen.

scholarly journals CcpA-Mediated Repression of Streptolysin S Expression and Virulence in the Group A Streptococcus

2008 ◽  
Vol 76 (8) ◽  
pp. 3451-3463 ◽  
Author(s):  
Traci L. Kinkel ◽  
Kevin S. McIver
Keyword(s):  
Group A Streptococcus ◽  
Intraperitoneal Administration ◽  
Subcutaneous Administration ◽  
Sugar Metabolism ◽  
Virulence Gene ◽  
Systemic Infection ◽  
Lesion Size ◽  
Logarithmic Phase ◽  
Streptolysin S ◽  
Group A

ABSTRACT CcpA is the global mediator of carbon catabolite repression (CCR) in gram-positive bacteria, and growing evidence from several pathogens, including the group A streptococcus (GAS), suggests that CcpA plays an important role in virulence gene regulation. In this study, a deletion of ccpA in an invasive M1 GAS strain was used to test the contribution of CcpA to pathogenesis in mice. Surprisingly, the ΔccpA mutant exhibited a dramatic “hypervirulent” phenotype compared to the parental MGAS5005 strain, reflected as increased lethality in a model of systemic infection (intraperitoneal administration) and larger lesion size in a model of skin infection (subcutaneous administration). Expression of ccpA in trans from its native promoter was able to complement both phenotypes, suggesting that CcpA acts to repress virulence in GAS. To identify the CcpA-regulated gene(s) involved, a transcriptome analysis was performed on mid-logarithmic-phase cells grown in rich medium. CcpA was found to primarily repress 6% of the GAS genome (124 genes), including genes involved in sugar metabolism, transcriptional regulation, and virulence. Notably, the entire sag operon necessary for streptolysin S (SLS) production was under CcpA-mediated CCR, as was SLS hemolytic activity. Purified CcpA-His bound specifically to a cre within sagAp, demonstrating direct repression of the operon. Finally, SLS activity is required for the increased virulence of a ΔccpA mutant during systemic infection but did not affect virulence in a wild-type background. Thus, CcpA acts to repress SLS activity and virulence during systemic infection in mice, revealing an important link between carbon metabolism and GAS pathogenesis.

scholarly journals Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Anupama Ramalinga ◽  
Jessica L. Danger ◽  
Nishanth Makthal ◽  
Muthiah Kumaraswami ◽  
Paul Sumby
Keyword(s):  
Transcription Factor ◽  
Virulence Factor ◽  
Human Blood ◽  
Biochemical Characterization ◽  
Regulatory Mechanism ◽  
Group A Streptococcus ◽  
Regulatory Activity ◽  
Biochemical Basis ◽  
Content Type ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) (Streptococcus pyogenes) causes more than 700 million human infections each year. The significant morbidity and mortality rates associated with GAS infections are in part a consequence of the ability of this pathogen to coordinately regulate virulence factor expression during infection. RofA-like protein IV (RivR) is a member of the Mga-like family of transcriptional regulators, and previously we reported that RivR negatively regulates transcription of the hasA and grab virulence factor-encoding genes. Here, we determined that RivR inhibits the ability of GAS to survive and to replicate in human blood. To begin to assess the biochemical basis of RivR activity, we investigated its ability to form multimers, which is a characteristic of Mga-like proteins. We found that RivR forms both dimers and a higher-molecular-mass multimer, which we hypothesize is a tetramer. As cysteine residues are known to contribute to the ability of proteins to dimerize, we created a library of expression plasmids in which each of the four cysteines in RivR was converted to serine. While the C68S RivR protein was essentially unaffected in its ability to dimerize, the C32S and C377S proteins were attenuated, while the C470S protein completely lacked the ability to dimerize. Consistent with dimerization being required for regulatory activity, the C470S RivR protein was unable to repress hasA and grab gene expression in a rivR mutant. Thus, multimer formation is a prerequisite for RivR activity, which supports recent data obtained for other Mga-like family members, suggesting a common regulatory mechanism. IMPORTANCE The modulation of gene transcription is key to the ability of bacterial pathogens to infect hosts to cause disease. Here, we discovered that the group A Streptococcus transcription factor RivR negatively regulates the ability of this pathogen to survive in human blood, and we also began biochemical characterization of this protein. We determined that, in order for RivR to function, it must self-associate, forming both dimers (consisting of two RivR proteins) and higher-order complexes (consisting of more than two RivR proteins). This functional requirement for RivR is shared by other regulators in the same family of proteins, suggesting a common regulatory mechanism. Insight into how these transcription factors function may facilitate the development of novel therapeutic agents targeting their activity.

Sign in / Sign up

Export Citation Format

Share Document