Molecular Mechanisms of Virulence Regulation in <i>Staphylococcus aureus</i>: A Journey into Reconstitutive Biochemistry

ConspectusMethodological development in the fields of genetics, chemical biology, and biochemistry over the last several decades has provided researchers with a diverse set of powerful tools to investigate biological processes. Leveraging these innovations in concert, scientists can now characterize biological pathways at a level of complexity ranging from systems biology down to molecular and atomic detail.Throughout this Account, we illustrate how discoveries made using these tools build on each other to develop a comprehensive understanding of biological pathways. Advancements in genetic sequencing facilitates association of genotypes and phenotypes, independent of biochemical mechanism. Through the biochemical reconstitution of the interactions between biological macromolecules─including the small molecules (ligands and metabolites) and proteins─that participate in these biological pathways, scientists can characterize the specific molecular features that link genotype and phenotype. This facilitates identification of targets within these pathways that can be manipulated to achieve a greater understanding of the biological process or to develop interventions to improve human health outcomes.Specifically, we describe how this toolbox was leveraged to discover and characterize the molecular biochemistry underlying control of pathogenicity in the Gram-positive bacterium <i>Staphylococcus aureus</i>. Concurrent with advancements in the investigative tools available to the scientific community, we and others reported on the genetic, molecular, and biochemical/biophysical components of this regulatory system. Virulence control in <i>S. aureus</i> is achieved through a chemical system of bacterial cell-to-cell communication indexed to local population density, referred to as quorum sensing (QS). We and our collaborators identified that this QS system is encoded in the <i>a</i>ccessory <i>g</i>ene <i>r</i>egulator (<i>agr</i>) operon and functions via the biosynthesis, secretion, and accumulation of a short peptide signaling molecule─the autoinducing peptide (AIP)─in the local environment correlated with the growth of <i>S. aureus</i> in the same biological niche. Above a threshold concentration, these AIPs bind and activate a cell-surface receptor to stimulate an intracellular response resulting in altered gene expression and bacterial group behaviors. We discovered that chemical modification of these AIPs often generates molecules that exhibit potent inhibition of <i>agr</i> QS, with demonstrated therapeutic potential to treat <i>S. aureus</i> infections. We went on to characterize the biochemical mechanism of signaling molecule biosynthesis and receptor activation in controlled systems through <i>in vitro</i> reconstitution of the constituent enzymes and substrates. Biochemical reconstitution enabled quantitative assessment of biophysical parameters. These efforts culminated in the comprehensive characterization and functional <i>in vitro</i> reconstitution of <i>agr</i> QS in a synthetic system in a minimal model at the interface of genotype, mechanism, and phenotype.