(160af) Development of a Luciferase Reporter Assay for Real-Time Monitoring of Streptococcus Pyogenes Cysteine Protease Speb Transcription

Background: Many bacterial pathogens secrete protein toxins as part of the disease process, often facilitating evasion of the human immune system. An improved understanding of the critical transcriptional regulators that influence toxin secretion can identify possible targets in an attempt to inhibit their expression. We are interested in understanding how certain transcriptional regulators and how some epigenetic processes, such as DNA and RNA methylation, influence the secretory protein expression of Streptococcal Pyrogenic Exotoxin (SpeB) within a major bacterial pathogen of humans, Streptococcus pyogenes. SpeB is a cysteine proteinase with a known role in virulence through the degradation of the extracellular matrix proteins, cytokines, chemokines, immunoglobulins, and serum complement components. In animal models, SpeB-deficient mutant strains of Streptococcus pyogenes have displayed a decrease in virulence. To investigate factors influencing speB transcription, we have developed a genetic reporter assay expressing firefly luciferase (luc) with real-time monitoring.

Methods: A speB::luc reporter was constructed through the genetic fusion of the firefly luciferase gene open reading frame to the speB gene promoter from a representative clinical isolate of S. pyogenes from a child with streptococcal pharyngitis (MEW123). The construct was assembled by PCR and cloned into an E. coli to S. pyogenes shuttle plasmid. The purified plasmid was transformed into S. pyogenes, and the luciferase gene replaced the native speB gene on the chromosome through an allelic exchange. When the speB promoter is activated, the mRNA for luciferase is transcribed and then translated into functional luciferase, which emits light in the presence of the substrate luciferin. Light emission can be detected on a microplate reader with luminescence detection over real-time under a variety of growth conditions. Strain MEW549 is a derivative of strain MEW123 expressing the speB-luciferase gene fusion. Additional strains of S. pyogenes with mutations of various transcription factors were also constructed with the speB::luc reporter plasmid to permit monitoring of the effect of those gene mutations on speB transcription. Environmental conditions known to influence speB gene transcription (e.g., carbohydrate concentration in media, pH, salt concentration) were used to validate our speB::luc reporter assay.

Results: In optimal growth conditions in minimal C-Media, maximal luciferase activity from strain MEW549 coincided with the late exponential-early stationary phases of growth; this expression pattern is consistent with known SpeB protein expression. Using previously known environmental factors that influence SpeB expression, we were able to validate that the speB::luc reporter assay functioned similarly to known SpeB expression patterns. Luciferase expression from the speB promoter was significantly stronger when recombinant strains were grown in media at a pH of 6 versus a pH of 7 or 8; low pH is known to stimulate maximal SpeB protein secretion. Also, we found the luciferase expression to significantly decrease when strains were grown in high sodium chloride and glucose concentrations, again consistent with known conditional influencers of SpeB expression. These environmental factors and their influence on luciferase expression support our use of the speB::luc reporter assay as a tool to investigate SpeB protease transcriptional expression. Ongoing experiments are examining the role of the Mga transcriptional regulator and DNA genomic methylation on speB expression.

Conclusion: Real-time monitoring of speB gene expression using a luc reporter assay will help future studies find novel regulators of SpeB expression and identify small-molecule inhibitors of S. pyogenes transcriptional activators. Small molecule inhibitors could potentially be used as adjuvant treatments, alongside standard antibiotics, to inhibit S. pyogenes pathogenesis and treat severe infections.