H-063. Identification of Ligand Specificity Determinants in AgrC, the S. aureus Quorum Sensing Receptor

E. Geisinger1, E. A. George2, T. W. Muir2, R. P. Novick1;
1New York Univ. Sch. of Med., New York, NY, 2Rockefeller Univ., New York, NY.

Activation of the agr system, a major regulator of staphylococcal_virulence,_is initiated by the binding of a specific autoinducing peptide (AIP) to the extracellular domain of AgrC, a classical receptor-histidine protein_kinase._There are 4 known agr specificity groups in Staphylococcus aureus and we_have previously localized the determinant of AIP-receptor specificity to_the_C-terminal half of the AgrC sensor domain. We have now identified the_specific amino acid residues that determine ligand activation specificity for agr groups I and IV, the two most closely related. Comparison of the_AgrC-I and AgrC-IV sequences revealed a set of 5 divergent residues in_the_region of the receptor's second extracellular loop that could be_responsible. Accordingly, we exchanged these residues between AgrC-I and_AgrC-IV and tested the resulting constructs for activation by the respective AIPs, measuring activation kinetics with a transcriptional fusion of_blaZ to_the principal agr promoter, P3. Exchange of all 5 residues caused a_complete switch in receptor specificity. Replacement of two of the_AgrC-IV_residues by the corresponding residues in AgrC-I caused the receptor to_be_activated by AIP-I nearly as well as the wild type AgrC-I receptor._Replacement of two different AgrC-I residues by the corresponding_AgrC-IV_residues broadened receptor recognition specificity to include both_AIPs, a finding with important implications for the evolution of agr specificity._Various types of intermediate activity were observed with othe replacement_mutations. Preliminary characterization of the AgrC-I/AIP-I interaction_suggests that ligand specificity may be sterically determined.