title: Transcription of the Streptococcus pyogenes hyaluronic acid capsule biosynthesis operon is regulated by previously unknown upstream elements.
abstract: The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA.
authors.forename: Marina
authors.initials: M
authors.lastname: Falaleeva
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA.
authors.forename: Oliwia W
authors.initials: OW
authors.lastname: Zurek
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Watkins
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Reed
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA.
authors.forename: Hadeel
authors.initials: H
authors.lastname: Ali
authors.affiliation: Department of Molecular Microbiology & Immunology, University of Nevada, Reno, Nevada, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Sumby
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA.
authors.forename: Jovanka M
authors.initials: JM
authors.lastname: Voyich
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA nkorotkova@uky.edu.
authors.forename: Natalia
authors.initials: N
authors.lastname: Korotkova
mesh_terms: D000818:Animals; D016667:Bacterial Capsules / Q000378:metabolism*; D001770:Blood Bactericidal Activity; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D014644:Genetic Variation; D006801:Humans; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D057131:Immune Evasion; D008807:Mice, Inbred BALB C; D009876:Operon; D060888:Real-Time Polymerase Chain Reaction; D050436:Regulatory Elements, Transcriptional; D017384:Sequence Deletion; D013290:Streptococcal Infections / Q000276:immunology / Q000382:microbiology / Q000473:pathology; D013297:Streptococcus pyogenes / Q000235:genetics*; D014158:Transcription, Genetic; D014774:Virulence
chemical_list: D006820:Hyaluronic Acid