Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis

Abstract

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Keywords: RiPP biosynthesis; Staphylococcus aureus; bacterial pathogenesis; quorum sensing; synthetic biology.

Fig. 1.

SpsB does not directly participate in AIP biosynthesis. (A) Overview of the AIP biosynthetic pathway. AgrD (group-I sequence shown) is processed by AgrB to yield the biosynthetic intermediate, AgrD (1–32) thiolactone. This intermediate is converted into the mature AIP through the action of a second protease whose identity remains unclear. (B) SpsB cleavage of a validated fluorogenic substate, DABCYL-SceD-EDANS, in the presence and absence of a known SpsB inhibitor, M131. SpsB proteoliposomes were incubated with the substrate ± inhibitor and the cleavage reaction monitoring by fluorescence (510 nm) over time. Data presented as the mean ± SD (n = 3 replicates). (C) In vitro assay for AIP production. SpsB proteoliposomes were treated with indicated AgrD (1–32) thiolactone intermediates in the presence or absence of M131 inhibitor. Reaction mixtures were then subjected to a solid phase extraction (SPE) step, prior to which an internal standard (AIP derivative) was added, and analyzed by LC–MS. Shown are the extracted ion currents (EIC) traces for the expected AIP and the internal standard. A synthetic AIP treated with empty liposomes served as a positive control (top trace). (D) Cell-based assay for AIP production. Indicated S. aureus strains (group-I background) were grown for 8 h (the optimal timepoint for AIP-I production in the WT strain) at which point an internal standard was added and the media subjected to SPE and analyzed by LC–MS. Shown are the EIC traces for AIP-I and an internal standard. As a control, a synthetic AIP-I was added to media and subjected to the same purification protocol (black trace). (E) AIP-I produced by indicated S. aureus strains was quantified by LC–MS using a standard curve approach employing synthetic AIP-I. Data presented as the mean ± SD (n = 3–4 biological replicates). (F and G) Comparative LC–MS/MS analysis of AIP-I produced by WT, Δ cro/cI, and Δ cro/cI:Δ spsB variants of group-I S. aureus and a synthetic AIP-I standard. Color coding as in (D).

Fig. 2.

Genetic complementation approach to explore the role of mroQ in AIP biosynthesis. (A) Relative growth of WT and Δ mroQ strains containing inducible plasmids expressing mroQ, mroQ^mut, or an empty plasmid, generated in agr group-I, -II, and -III background strains over 48 h, monitored by OD₆₀₀. Data presented as the mean ± SD (n = 3 biological replicates). (B) Cell-based assay for AIP production. Indicated S. aureus strains were grown for 8 h (group I) and 16 h (group II, group III) at which point an internal standard was added and the media subjected to SPE and analyzed by LC–MS. Shown are the EIC traces for the AIPs and the internal standard. As a control, a synthetic AIP was added to media and subjected to the same purification protocol (black traces). (C) AIP levels produced by indicated S. aureus strains were quantified by LC–MS using a standard curve approach employing synthetic AIPs. Data presented as the mean ± SD (n = 3–8 biological replicates). (D and E) Comparative LC–MS/MS analysis of AIPs generated by WT and mutant-complemented strains. Color coding as in (C).

Fig. 3.

In vitro reconstitution of AIP biosynthesis and sensing. (A) MroQ proteoliposomes were combined with indicated AgrD (1–32) thiolactone intermediates. Reaction mixtures containing a spike-in internal standard were then subjected to a SPE step and analyzed by LC–MS. Shown are the EIC traces for the possible cleavage products and the internal standard. *Indicates correct AIP product from each specificity group. (B) AIP-I and -II levels produced by MroQ, MroQ^mut, or SpsB proteoliposomes were quantified by LC–MS using a standard curve approach employing synthetic AIPs. Data presented as the mean ± SD (n = 3 biological replicates). (C) Reconstituted AIP biosynthesis. AgrD-I or AgrD-II was incubated with cognate AgrB and MroQ proteoliposomes. Reaction mixtures containing a spike-in internal standard were then subjected to a SPE step and analyzed by LC–MS. Shown are the EIC traces for the expected AIP and the internal standard. A synthetic AIP treated with empty liposomes served as a positive control (bottom trace). (D) Overview of one-pot in vitro AIP biosynthesis and sensing assay. Input AgrD is processed by AgrB and MroQ proteoliposomes into mature AIP which then binds and activates nanodisc-reconstituted AgrC. (E) Autoradiography of AgrC-I autophosphorylation employing ATP-γ-³²P. All samples contained AgrD and AgrC in addition to the indicated proteins. Inhibitor refers to a known tight binding antagonist of AgrC-AIP interaction. Note, AgrC-I is known have basal autokinase activity (14). (F) Quantification of AgrC-I autokinase activity as determined by densitometry analysis of the autoradiographs. All samples contained AgrD and AgrC in addition to the indicated proteins and peptides. Maximal activation of AgrC-I was determined by addition of synthetic AIP-I to the mixtures. Data presented as the mean ± SD (n = 3 biological replicates).

Fig. 4.

The AgrD-I/II linker domain dictates MroQ specificity in AIP biosynthesis. (A) Overview of the in vitro MroQ cleavage assay and in vivo agrD mutant AIP production assays. AgrD-I/II thiolactone substrates, in which the linker-tail region of one specificity group is swapped for another, were coincubated with proteoliposome-reconstituted MroQ. In vivo studies utilized chimeric AgrD peptides containing the N-terminal helix domain, AIP macrocycle domain, and C-terminal domain from one agr specificity group, and a linker domain sequence from a different specificity group, expressed under P2 promoter control in an agr-null S. aureus background. Identity of cleaved or secreted chimeric AIPs was determined by LC–MS. (B) MroQ proteoliposomes were treated with AgrD-I-II-I (1–32) thiolactone. Reaction mixtures containing a spike-in internal standard were then subjected to a SPE step and analyzed by LC–MS. Shown are the EIC traces for the possible AIP cleavage products and the internal standard. Synthetic chimeric AIPs treated with empty liposomes served as positive controls (black traces). (C) EIC traces of chimeric AIP cleavage products and internal standard from LC–MS analysis of growth media from S. aureus cells expressing AgrD-I-II-I-I chimera. Synthetic chimeric AIP-II-I served as a positive control (black trace).