Faculty Opinions recommendation of Genome-wide identification of genes required for fitness of group A Streptococcus in human blood.

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.

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Toward a Genome-Wide Systems Biology Analysis of Host-Pathogen Interactions in Group A Streptococcus

American Journal Of Pathology ◽

10.1016/s0002-9440(10)61232-1 ◽

2005 ◽

Vol 167 (6) ◽

pp. 1461-1472 ◽

Cited By ~ 73

Author(s):

James M. Musser ◽

Frank R. DeLeo

Keyword(s):

Systems Biology ◽

Group A Streptococcus ◽

Host Pathogen Interactions ◽

Genome Wide ◽

A Genome ◽

Host Pathogen ◽

Group A

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

Infection and Immunity ◽

10.1128/iai.00837-12 ◽

2013 ◽

Vol 81 (3) ◽

pp. 862-875 ◽

Cited By ~ 69

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.

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

Journal of Bacteriology ◽

10.1128/jb.00452-16 ◽

2016 ◽

Vol 199 (1) ◽

Cited By ~ 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.

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Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0337370100 ◽

2003 ◽

Vol 100 (4) ◽

pp. 1996-2001 ◽

Cited By ~ 117

Author(s):

J. M. Voyich ◽

D. E. Sturdevant ◽

K. R. Braughton ◽

S. D. Kobayashi ◽

B. Lei ◽

...

Keyword(s):

Polymorphonuclear Leukocytes ◽

Group A Streptococcus ◽

Protective Response ◽

Genome Wide ◽

Group A ◽

Human Polymorphonuclear Leukocytes

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Gene fitness landscape of group A streptococcus during necrotizing myositis

10.1101/432526 ◽

2018 ◽

Author(s):

Luchang Zhu ◽

Randall J. Olsen ◽

Stephen B. Beres ◽

Jesus M. Eraso ◽

Matthew Ojeda Saavedra ◽

...

Keyword(s):

Fitness Landscape ◽

Group A Streptococcus ◽

Insertion Site ◽

Pcr Analysis ◽

Genome Wide ◽

A Genome ◽

Necrotizing Myositis ◽

Genes Encoding ◽

Isogenic Mutant Strain ◽

Group A

ABSTRACTNecrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified novel GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon directed insertion-site sequencing (TraDIS) we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins, and exporters for toxins, quorum sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding five transporters confirmed that each isogenic mutant strain was significantly impaired in causing necrotizing myositis in NHPs. qRT-PCR analysis showed that these five genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface-exposed, suggesting that our findings have translational research implications.

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