Date of Award
Doctor of Philosophy (PhD)
Quorum sensing (QS) plays a central role in virulence induction in the commensal pathogen, Staphylococcus aureus. This bacterium secretes an auto-inducer peptide (AIP), a small, cyclic peptide containing a thiolactone linkage as an indicator of its population density, and up-regulates virulence gene expression in response to high extracellular AIP levels. We have investigated two key biochemical events in S. aureus QS and revealed several underlying regulatory mechanisms. The first such event, formation of the high energy thiolactone in AIP, is unusual in that it occurs directly through proteolysis of the precursor peptide, AgrD, without free-energy input from ATP hydrolysis. We showed that this proteolysis is, in line with the thermodynamic prediction, unfavorable and strongly reversible in vitro. As a consequence, rapid degradation of the concomitantly released C-terminal fragment of AgrD is required to power efficient AIP production in vivo. This observation provides a novel connection between protein homeostasis and QS in S. aureus. The second study focused on the AIP-sensing receptor histidine kinase (HK), AgrC, whose auto-phosphorylation exhibits several remarkable properties in our reconstitution system based on nanometer-scale lipid-bilayer discs (nanodiscs). Activation of this receptor by its native activator, for instance, requires a membrane environment enriched of anionic lipids mimicking the electrostatic property of the S. aureus cell membrane. This strong dependence on lipid composition might explain why homologous QS systems exist only in low-GC Gram-positive bacteria, or Firmicutes, whose cell membranes are predominantly highly anionic. AgrC also binds to ATP at an exceptionally weak affinity, likely due to its distinct adenine-binding pocket conserved only in a small subfamily of HK receptors existing also exclusively in Firmicutes. The low affinity to the nucleotide cofactor likely enables AgrC to sense the energy condition of the bacterium and shut down the QS regardless of the population density when energy starvation drives down the cellular ATP level. Even more intriguing is the plasticity of AgrC auto-kinase activity when bound to different ligands. This behavior contrasts with the generally accepted two-state model of HKs. To understand the plasticity of AgrC, we systematically perturbed the conformation of the AgrC kinase domain using a fusion protein strategy. We demonstrated that the conformational state of a helical linker preceding the kinase domain exercises rheostat-like control over the kinase activity. Using full-length AgrC embedded in nanodiscs, we showed that binding of activator and inhibitor peptides results in twisting of the linker in different directions. These findings provide the first view on molecular motions triggered by ligand binding on a membrane bound receptor HK. The smooth input-response landscape of the AgrC kinase domain also sheds new light on the mechanism of HK evolution through domain shuffling.
Wang, Boyuan, "Reconstitution and Mechanistic Studies on the Staphylococcal agr Quorum Sensing Circuit" (2016). Student Theses and Dissertations. 306.