Abstract
Background
Streptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors have been found to contribute to persistent colonization of this niche, and many of these factors are important in mucosal immunity and vaccine development. In this work, we infected mice intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system—a peptide-based signaling system conserved in all sequenced isolates of S. pyogenes.
Methods
Three-week-old CD1 mice were intranasally infected with ~107 CFU of S. pyogenes strain MGAS315. Calcium alginate throat swabs were used to monitor nasopharyngeal colonization by the bacteria over time. Luciferase reporters used alongside an IVIS camera were able to show quorum sensing activity levels after inoculation into the mouse nose. Bacterial RNA was isolated from the throat of the mice and quantitative RT–PCR was performed on the samples to corroborate the luciferase reporter data. The nasal-associated lymphoid tissue (NALT) was excised and its supernatants were subjected to 32-plex murine cytokine and chemokine analysis (Millipore).
Results
Deletion of the QS system’s transcriptional activator (Δrgg2) dramatically diminished the percentage of colonized mice. Deletion of the transcriptional repressor (Δrgg3) increased the percentage of colonized mice compared with wild type. Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that activity of the QS system is responsive to conditions of the host nasopharynx. Mice inoculated with QS-dependent luciferase reporters were subjected to in vivo imaging and showed activation within 1 hour. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative RT–PCR subsequently confirmed QS upregulation within 1 hour of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the Δrgg2 bacteria suggests that their rapid elimination fails to elicit the previously characterized response to intranasal inoculation of GAS.
Conclusions
Deletion of the Rgg2 transcriptional activator of the Rgg 2/3 quorum sensing system eliminates colonization of the murine nasopharynx and changes the transcriptional profile of the bacteria in this niche. An existing small-molecule inhibitor of the Rgg2/3 system was unable to inhibit QS activation in vivo, likely due to the suboptimal achievable doses; however, results of our study indicate inhibition of QS may diminish the oropharyngeal colonization of S. pyogenes and argue for further development.
Non-Native Peptides Capable of Pan-Activating the agr Quorum Sensing System across Multiple Specificity Groups of Staphylococcus epidermidis
