•  
  •  
 

Abstract

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a human pathogen associated with a variety of diseases such as strep throat, scarlet fever, toxic shock syndrome, and necrotizing fasciitis. One of the virulence factors released by GAS during an invasive infection is a cytotoxic peptide, streptolysin S (SLS), which inhibits the immune response to necrotizing fasciitis. The streptolysin S associated gene A product, SagA, is modified to produce SLS. Thesag operon includes sagA and the genes required for enzyme-mediated post-translational modifications of SagA and the export of SLS. The sagA gene is contained within the pleiotropic effect locus (pel), which produces a small RNA (sRNA) that regulates the expression of other virulence factors. Potential mRNA interactions with the Pel sRNA have been mapped to the 5' and 3' untranslated regions (UTRs) of sagA. Our studies aim to identify and characterize RNA structural motifs in Pel/sagA that regulate the expression of sagA and other virulence factors. Several RNA constructs of Pel/sagA were designed to include regions predicted to contain secondary structure. The corresponding sequences were isolated by PCR from genomic DNA to create templates for in vitro transcription. After purification, the RNA constructs were analyzed by gel electrophoresis to verify size, and by RNase T1 digestion to assay for secondary structure. Three-dimensional models were generated using the FARFAR algorithm in Rosetta in order to identify regions of Pel/sagA that may be involved in regulatory interactions. Differential scanning fluorimetry provided evidence that the 5' and 3' UTRs of Pel/sagA contain stable structural regions. It is expected that the identification of structural motifs necessary for the regulation of gene expression will aid in the design of therapeutic strategies to inhibit the production of streptolysin S and other virulence factors.

Download

Share

COinS