agr receptor mutants reveal distinct modes of inhibition by staphylococcal autoinducing peptides

Abstract

Through the agr quorum-sensing system, staphylococci secrete unique autoinducing peptides (AIPs) and detect their concentration via the AgrC transmembrane receptor, coordinating local bacterial population density with global changes in gene expression. Unique AIP and AgrC variants exist within and between species, and although autologous interactions lead to agr activation, heterologous interactions usually lead to cross-inhibition, resulting in natural quorum-sensing interference. To gain insight into the mechanisms responsible for these phenomena at the level of the receptor, we used random mutagenesis to isolate variants of Staphylococcus aureus AgrC-I with constitutive activity. Constitutive mutations in the sensor domain of the receptor were localized to the last transmembrane helix, whereas those in the histidine kinase domain were mostly clustered to a region near the phosphorylation site histidine. Analysis of these mutants with a range of noncognate AIPs revealed that inhibition is manifested by inverse agonism in certain heterologous pairings and by neutral antagonism in others. In addition, we isolated and characterized an AgrC sensor domain mutant with dramatically broadened activation specificity and reduced sensitivity to inhibition, identifying a single amino acid as a critical determinant of ligand-mediated inhibition. These results suggest that certain noncognate AIPs stabilize an inhibitory receptor conformation that may be a critical feature of the ligand-receptor interaction not initially appreciated in previous analyses of agr inhibition.

Fig. 1.

AgrC-I topology, domain prediction, and mutagenized residues. AgrC-I is a 430-aa protein with a polytopic TM sensor domain (7, 28) and a cytoplasmic HK domain, which is further divided into the DHp and CA subdomains. Two cytoplasmic regions with high α-helical propensity predicted to form the DHp domain are boxed. The active site histidine (H239) is shown in gray. Residues analyzed in this study in which mutation (listed at right) resulted in constitutive activity are shown in black. Residue I171, in which mutation resulted in broadened specificity, is shown in spherical relief. (Inset) Helical wheel analysis of a region within the first predicted DHp α-helix.

Fig. 2.

AgrC-I constitutively active mutants. β-Lactamase reporter cells expressing WT AgrC-I, constitutive AgrC-I sensor domain mutants (A), or constitutive AgrC-I HK domain mutants (B) were incubated without or with the indicated AIP at 1 μM. Data are presented as BLU ± SEM.

Fig. 3.

Inverse agonism by staphylococcal AIPs. (A–C and F) β-Lactamase dose–response assays of AgrC-I-L205R. Reporter cells expressing AgrC-I-L205R were incubated with increasing concentrations of the indicated AIP alone (A and F) or in the presence of a constant dose (100 nM) of AIP-II (B and C). Data are presented as percent maximal activation ± SEM. (D and E) Assay of AgrC-I-L205R (D) or AgrC-II-L202R (E) in the absence or presence of the indicated AIP (at 1 μM) or the indicated non-aureus culture supernatant. cap, S. caprae; epi, S. epidermidis; sim, S. simulans. Data are presented as BLU ± SEM.

Fig. 4.

Probing charged residues in the DHp domain. Reporter cells expressing AgrC-I with mutation to R238 (A) or a DHp residue (B and C) were incubated without or with the indicated AIP at 1 μM. Data are presented as BLU ± SEM.

Fig. 5.

Tests of the altered specificity variant AgrC-I-I171K with various AIPs. (A) β-Lactamase reporter cells expressing AgrC-I-I171K were incubated with increasing doses of the indicated AIP. (B) Cells expressing AgrC-I WT or I171K were assayed without or with AIP-I (1 μM) or the indicated supernatant. (C–E) Inhibition assays. AgrC-I WT or I171K was assayed with increasing concentrations of AIP-II (C) or S. epidermidis AIP (D) in the presence of a constant dose (50 nM) of AIP-I. (E) The indicated supernatant was added in the presence of AIP-I at 25 nM. Data are presented as percent maximal activation ± SEM.