Quorum-Sensing Agr Mediates Bacterial Oxidation Response Via an Intramolecular Disulﬁde Redox Switch in the Response Regulator Agra

Quorum-sensing agr mediates bacterial oxidation response via an intramolecular disulﬁde redox switch in the response regulator AgrA

Fei Suna,1, Haihua Lianga,1, Xiangqian Kongb, Sherrie Xiea, Hoonsik Choc, Xin Denga, Quanjiang Jia, Haiyan Zhanga, Sophie Alvarezd, Leslie M. Hicksd, Taeok Baec, Cheng Luob, Hualiang Jiangb, and Chuan Hea,2

aDepartment of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; bState Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; cDepartment of Microbiology and Immunology, Indiana University School of Medicine-Northwest, Gary, IN 46408; and dDonald Danforth Plant Science Center, St. Louis, MO 63132

Edited by Richard P. Novick, New York University School of Medicine, New York, NY, and approved April 16, 2012 (received for review January 12, 2012) Oxidation sensing and quorum sensing significantly affect bacterial virulence factor δ-toxin (Hld) but also functions as a small regu- physiology and host–pathogen interactions. However, little attention latory RNA (sRNA) per se to modulate target gene expression; has been paid to the cross-talk between these two seemingly orthog- RNAII comprises a typical bacterial TCS consisting of the sensor onal signaling pathways. Here we show that the quorum-sensing agr kinase AgrC and the response regulator AgrA. In addition, it system has a built-in oxidation-sensing mechanism through an intra- encodes AgrD, the precursor of the quorum signal that can further molecular disulfide switch possessed by the DNA-binding domain of be processed and exported as a thiolactone-containing oligopep- the response regulator AgrA. Biochemical and mass spectrometric tide autoinducer (autoinducing peptide, AIP) by the cotranscribed analysis revealed that oxidation induces the intracellular disulfide AgrB. Upon binding to the extracellular sensory domain of AgrC, bond formation between Cys-199 and Cys-228, thus leading to disso- AIP activates the kinase activity of AgrC, subsequently leading to ciation of AgrA from DNA. Molecular dynamics (MD) simulations sug- phosphorylation of the response regulator AgrA (13, 14). Phos- gest that the disulfide bond formation generates a steric clash phorylated AgrA regulates transcription of genes encoding met- responsible for the abolished DNA binding of the oxidized AgrA. abolic factors and phenol-soluble modulin (PSM) peptides (15) Mutagenesis studies further established that Cys-199 is crucial for and, more importantly, triggers the expression of the agr operon oxidation sensing. The oxidation-sensing role of Cys-199 is further by binding to the promoter regions P2 (for RNAII) and P3 (for supported by the observation that the mutant Staphylococcus aureus RNAIII), thereby forming an autoinduction genetic circuit to strain expressing AgrAC199S is more susceptible to H2O2 owing to ensure a timely rearrangement of target gene expression at a repression of the antioxidant bsaA gene under oxidative stress. To- certain threshold level of population density. gether, our results show that oxidation sensing is a component of the In addition to this prominent agr-mediated quorum sensing in quorum-sensing agr signaling system, which serves as an intrinsic S. aureus, a number of studies have demonstrated that S. aureus checkpoint to ameliorate the oxidation burden caused by intense uses oxidation-sensing global transcriptional regulators, including metabolic activity and potential host immune response. MgrA, SarZ, and SarA, to control global gene expression via the redox active Cys residue (16–19). Additionally, there are two any pathogenic bacteria are dependent on their ability to S. aureus TCSs found to be capable of sensing oxidation. One is Mswiftly sense and respond to surrounding population den- the [4Fe-4S]-containing TCS NreABC (20). NreABC is a spe- sity and changing host microenvironments. Bacterial physiologi- cialized TCS that regulates a set of genes involved in anaerobic cal rearrangements can be controlled by quorum-sensing systems nitrate/nitrite uptake but with negligible effects on virulence gene in response to increasing population density (1–3). Meanwhile, in expression (20). We also identified a [2Fe-2S]-containing redox- – the context of host pathogen interactions, host immune systems responsive TCS, AirSR, that globally impacts gene expression such as macrophages and neutrophils generate a burst of oxidants under oxygen-limited conditions (21). So far specialized regula- − • (O2 ,HO,H2O2, HClO, NO, etc.) to kill invading pathogens. tory systems have been shown to execute the oxidation-sensing Oxidation sensing, on the other hand, is exploited by pathogenic process in S. aureus independently of quorum sensing. bacteria as a key signaling strategy to adapt and evade the hostile In this work we discover that the S. aureus quorum-sensing agr – immune system (4 7). Although both quorum sensing and oxida- system has integrated another level of the signaling pathway of tion sensing have been extensively studied in bacterial pathogenesis oxidation sensing into its predominant quorum-sensing mode to and related virulence regulation (3, 8), these two distinct signaling counter oxidative stress. We demonstrate that the DNA-binding processes are often regarded as independent events in gene regu- domain of the response regulator AgrA contains a redox-active lation, and little is known about the interplay between them. Cys-199, which forms an intramolecular disulfide bond with the Staphylococcus aureus, a major human pathogen that is the most spatially proximate Cys-228 under oxidative stress. The oxidized common source of nosocomial and community-acquired infec- AgrA dissociates from its cognate DNA, leading to down-regu- tions, causes a variety of diseases, ranging from minor skin infec- lation of the expression of RNAIII and up-regulation of that of tions to life-threatening blood infections (9). The success of this bacterium in pathogenesis is largely owing to the sophisticated regulatory network composed of several global transcriptional CHEMISTRY Author contributions: F.S., H.L., and C.H. designed research; F.S., H.L., X.K., S.X., H.C., X.D., regulators (e.g., SigB, Rot, MgrA, SarA, and SarA homologs) and Q.J., and H.Z. performed research; F.S., S.A., L.M.H., T.B., C.L., and H.J. contributed new 16 two-component systems (TCSs) (e.g., agr, srrAB, arlRS, vraRS, reagents/analytic tools; F.S. and H.L. analyzed data; and F.S., H.L., and C.H. wrote hssRS,andsaeRS) (10–12), which enable the bacterium to rapidly the paper. sense and adapt to changing environment. Central to this regula- The authors declare no conflict of interest. tory network is the quorum-sensing agr system, which controls the This article is a PNAS Direct Submission. expression of bacterial virulence in response to changes in cell 1F.S. and H.L. contributed equally to this work. density (10). Transcriptional expression of the agr system gen- 2To whom correspondence should be addressed. E-mail: [email protected].

erates two adjacent mRNAs corresponding to RNAII and RNAIII This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MICROBIOLOGY in opposite directions (Fig. 1A): RNAIII not only encodes a 1073/pnas.1200603109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1200603109 PNAS | June 5, 2012 | vol. 109 | no. 23 | 9095–9100 Downloaded by guest on October 1, 2021 Moreover, a detailed study revealed that oxidation of C-terminal methionine in the AIP by strong oxidants including HClO and ONOO, but not mild ones such as H2O2, represses the agr regulon (23). We confirmed that millimolar levels of H2O2 could down- regulate the transcription of the agr operon (Fig. S1A) in strain Newman independently of the redox-responsive SarA (24, 25), another global transcriptional regulator required for agr expression (26). Repression of RNAIII upon H2O2 treatment was observed in S. aureus strain Newman as well as in USA300 (Fig. S2A). Clearly, the model of AIP inactivation by oxidation, which is mainly responsible for extracellular factors that influence the agr regulation, accounts for the response of the agr system to certain types of reactive oxygen/nitrogen species (ROS/RNS), which prompted us to identify an alternative mechanism un- derlying the redox response of agr toward the most common H2O2 stress that S. aureus encounters. The solved crystal structure of the DNA-binding domain of the response regulator AgrA complexed with DNA (27) shows a unique topology with 10 β-strands arranged into three antipar- allel β-sheets and three small α-helices (Fig. 1B). Intriguingly, a close view of the crystal structure reveals two spatially proximate Cys residues, Cys-199 and Cys-228, residing in strands β6 and β10, respectively (Fig. 1B). The two sulfur atoms are approximately separated by 4.2 Å (Fig. 1B), an arrangement suggesting the presence of an intracellular disulfide switch similar to the OxyR type regulators (28, 29). However, the corresponding sulfur atoms in the reduced OxyR (Cys-199 and Cys-208) are ∼17 Å away from each other, far more separated than that of AgrA (30). The much shorter distance between Cys-199 and Cys-228 in AgrA makes the oxidation-induced formation of the disulfide bond highly conceivable. Given that the disulfide bond formation has been observed in many oxidation-sensing/defense proteins in addition to OxyR (31–34), we envisioned that this prospective disulfide switch might also play critical roles in redox regulation (Fig. 1A).

Oxidation Leads to Dissociation of AgrA from DNA. To substantiate that AgrA is responsive to oxidation, we cloned, expressed, and purified the DNA-binding domain of AgrA (Fig. S3) and examined its DNA-binding activity in the absence or presence of Fig. 1. S. aureus AgrA. (A) Model of gene regulation by the quorum-sensing H2O2 (unless mentioned otherwise, we use AgrA to refer to the agr. AIP, with its thiolactone structure, is encoded by agrD, processed, and truncated version of this protein containing residues 137–238 for exported by AgrB. AIP serves as the quorum signal to activate the TCS consisting of the sensor kinase AgrC and the response regulator AgrA. The in vitro assays throughout this article). We performed EMSA reported crystal structure of the DNA-binding domain of AgrA complexed with AgrA and the promoter region of the agr operon. As shown with DNA (27) revealed two spatially proximate Cys residues (Cys-199 and in Fig. 2A, treating the wild-type AgrA with H2O2 fully abolished Cys-228) with two sulfur atoms 4.2 Å away from each other (PDB entry: its DNA binding (lane 3), which was restored by the addition of 3BS1), suggesting a potential oxidation-sensing mechanism that impacts the excessive DTT (lane 4), indicating that oxidation modification of AgrA regulon, such as RNAIII, or the bsaA gene encoding the antioxidant, AgrA disrupts its DNA-binding activity. To determine which glutathione peroxidase. (B) Sequence and structure of the C-terminal DNA- of the two Cys residues contribute to the observed dissociation binding domain of AgrA (residues 136–238) with two Cys residues high- of AgrA from DNA, we sought to overexpress and obtain both lighted. Strands are colored red, and helices are colored cyan. mutant proteins AgrAC199S and AgrAC228S. We successfully expressed and purified recombinant AgrAC199S in Escherichia the glutathione peroxidase gene (bsaA), which is shown to be coli. However, the recombinant AgrAC228S expressed in E. coli essential for bacterial resistance to oxidative stress. We further was found to be insoluble as inclusion bodies (Fig. S3A), suggesting that Cys-228 might be structurally important for the show that abolishing the oxidation-sensing ability of AgrA through folding of the AgrA protein. AgrAC228A and AgrAC228F also mutation of Cys-199 to Ser significantly impairs staphylococcal form inclusion bodies when overexpressed in E. coli (Fig. S3B), resistance toward oxidative stress. This discovery showcases a further indicating that Cys-228 is critical for the stability of the quorum-sensing system that has an intrinsic oxidation-sensing protein. With the purified AgrAC199S in hand, we tested its mechanism to coordinate its gene regulation. Moreover, the in- DNA-binding activity and found it was not affected by 1 mM terplay between two seemingly independent signal transduction H O (Fig. 2A, lane 8), indicating that Cys-199 in AgrA is in- — — 2 2 pathways quorum sensing and oxidation sensing has broad volved in the response to oxidation. implications for understanding bacterial gene regulation. Disulfide Bond Formation Between Cys-199 and Cys-228 upon H O Results 2 2 Treatment. As aforementioned, EMSA revealed that H2O2 Potential Disulfide Redox Switch in AgrA. Previous studies have treatment impairs the DNA-binding activity of AgrA in a Cys- demonstrated that transcription of the quorum-sensing agr system 199 dependent manner. Moreover, the proximity (4.2 Å) be- is significantly repressed under the oxidative stress exerted by tween Cys-199 and Cys-228 in AgrA is highly indicative of the − oxidants such as Cu(II), H2O2, HClO, ONOO , etc. (22, 23). existence of an intracellular disulfide switch. To determine the

9096 | www.pnas.org/cgi/doi/10.1073/pnas.1200603109 Sun et al. Downloaded by guest on October 1, 2021 theoretical molecular mass, 2,279.0557 Da) was identified (Fig. 2B). MS/MS fragmentation of this 4+ charged peptide gave rise to a variety of cross-linked fragment peaks, including b5, y7, y8, B2, B3, B4, and B5, further confirming the presence of a disulfide bond between Cys-199 and Cys-228 (Fig. 2 C and D). We failed to detect any intermonomer “homo-crosslink” between Cys-199 and Cys’-199, or Cys-228 and Cys’-228, which rules out the possibility that the disulfide-containing peptide captured by MS resulted from random oxidation reactions. Overall, this mass spectrometry characterization strongly indicates the formation of a specific intramolecular disulfide bond between Cys-199 and Cys-228 under oxidative stress.

Molecular Dynamics (MD) Simulations of the Reduced and Oxidized AgrA. The aforementioned EMSA and MS analysis suggests that the attenuated DNA binding of AgrA is caused by the formation of an intramolecular disulﬁde bond. To unveil how this disulﬁde bond could impact DNA binding, we constructed an initial structural model of the oxidized AgrA based on the crystal structure of the reduced AgrA in the Protein Data Bank (PDB entry: 3BS1) as described in SI Experimental Procedures. To investigate its relative stability and obtain a stable conformation of oxidized AgrA, 200-ns MD simulations were performed on both forms of AgrA. The time evolutions of weighted rmsd for the backbone atoms of AgrA from their initial positions (t = 0) were monitored. As illustrated in Fig. 3, the reduced AgrA displayed very little conformational change after MD simulation, and steady rmsd for the backbone atoms in

Fig. 2. Dissociation of oxidized AgrA from DNA and mass spectrometric mapping of the disulﬁde bond in the oxidized AgrA. (A) EMSA with different forms of AgrA (1.0 μM). Both wild-type AgrA and AgrAC199S shifted

with DNA in the absence of H2O2 (lanes 2 and 7). The presence of 1 mM H2O2 led to dissociation of the wild type (lane 3) but not AgrAC199S (lane 8) from DNA, indicating that Cys-199 is responsible for oxidation sensing. The DNA

binding of H2O2-treated AgrA was restored by addition of 10 mM DTT (lane 4). Lanes 1 and 6 are controls with DNA alone. The intergenic region of RNAII and RNAIII was used as DNA probe. (B) ESI-Q-TOF mass spectrum (m/z 350– 750) of an unfractionated tryptic peptide mixture. Inset: The 4+ charged peak (m/z 570–573) corresponding to the disulﬁde-containing peptide of interest (theoretical molecular mass, 2,279.0557 Da). (C) MS/MS fragmentation of the 4+ charged peptide (m/z 570). (D) Graphical fragment map cor- relating the fragmentation ions to the peptide sequence. The disulﬁde- linked cysteines are circled.

genuine oxidation modification occurring to AgrA after H2O2 CHEMISTRY treatment, we performed mass spectrometric mapping analysis. fi fi μ Fig. 3. Structural comparison of the reduced and oxidized AgrA during MD Speci cally, puri ed AgrA (20 M) was oxidized by 1 mM H2O2 simulations. (A) Snapshot structure of the reduced AgrA at t =0ns(Left)and at room temperature for 1 h, followed by excessive iodoaceta- t = 200 ns (Center), along with the optimal backbone alignment of the two mide treatment to block all unreacted cysteine residues. After structures (Right). (B) Snapshot structure of the oxidized AgrA at t =0ns trypsin digestion, the resulting tryptic peptide mixture was ana- (Left) and t = 200 ns (Center) along with the optimal backbone alignment of lyzed by electrospray ionization (ESI)–quadrupole (Q)-TOF the two structures (Right). For clarity, the residues involved in clash with + DNA are colored in yellow. (C) Structural alignment of the reduced and mass spectrometry. In the resulting spectrum a prominent 4 oxidized AgrA in the presence of DNA at t = 200 ns. (D) Close view of Cys-199 fi charged peak (m/z 570) corresponding to the disul de-contain- and Cys-228 in the reduced and oxidized AgrA. (E) Close view of the steric MICROBIOLOGY ing peptide of interest (cross-link between Cys-199 and Cys-228; clash between α2 and DNA backbone.

Sun et al. PNAS | June 5, 2012 | vol. 109 | no. 23 | 9097 Downloaded by guest on October 1, 2021 the reduced AgrA was 1.68 Å (Fig. 3A), indicating that the con- gene in the complementation strain in which agrBDC was ex- formation of the reduced AgrA is relatively stable. However, cluded). However, introduction of pCL55-agrAC199S into ΔagrA a dramatic conformational change was observed in the oxidized fully restored the hemolytic activity comparable to that of wild- AgrA during MD simulation, and the corresponding rmsd for the type Newman (Fig. 4), showing that AgrAC199S is a hyperactive backbone atoms in the oxidized AgrA after 200-ns MD simulation transcriptional regulator compared with the wild-type AgrA, was up to 4.25 Å (Fig. 3B), suggesting that the disulfide bond most likely because of the resistance of AgrAC199S toward ox- formation between Cys-199 and Cys-228 introduces a significant idation. Although immuno-detection of the whole-cell extracts structural disturbance to AgrA. A close examination of structures showed that AgrAC228S can be detected in ΔagrA (Fig. S5), after MD simulation revealed that the elongated β-β-β sandwich introduction of pCL55-agrAC228S into ΔagrA completely failed featured in the DNA-binding domain of AgrA (27) is more open to restore hemolysis (Fig. 4), indicating that AgrAC228S, al- in the oxidized form than in the reduced form owing to the for- though expressed, is inactive inside S. aureus, which is in line with mation of the disulfide bond (Fig. 3C,rmsd=4.18Å).Particu- the fact that substitution of Cys-228 with Ser destabilizes and larly, the β10 strand where Cys-228 resides is displaced unfolds the protein. Taken together, we suspect that Cys-199 is approximately by 4.5 Å as a result of the cross-link with β6(Fig. the primary redox-active site that initiates the disulfide bond 3D). Moreover, the two-turn α2 helix in the oxidized AgrA after formation, whereas Cys-228 is structurally essential for the MD simulation is shifted allosterically and exerts an apparent folding of AgrA. steric clash with the DNA backbone (Fig. 3E). These results ex- plain the dissociation of the oxidized AgrA from DNA. AgrA Modulates Target Gene Expression in Response to Oxidative Stress. As mentioned above, millimolar levels of H2O2 repress Cys-199 and Cys-228 Play Different Roles in AgrA Function. Although the transcription of RNAIII in strain Newman in an AgrA-de- Cys-199 and Cys-228 form the disulfide bond in the oxidized pendent manner (Fig. S1A). However, expression of RNAIII in AgrA, the insolubility of AgrAC228S expressed in E. coli (Fig. the ΔagrA mutant complemented with pCL55-agrAC199S was S3) implies that Cys-228 is structurally essential for the folding of hardly affected by H2O2 (Fig. S6A), indicating that mutation of AgrA. To investigate the functional difference between Cys-199 Cys-199 to Ser renders the agr regulon unresponsive to oxidation, and Cys-228, we constructed the initial models of AgrAC199S consistent with the EMSA result that the binding of AgrAC199S and AgrAC228S as described in SI Experimental Procedures. to the promoter region of RNAIII is inert to oxidation (Fig. 2A). Subsequently, 200-ns MD simulations were performed on wild- A previous microarray study of the agr system showed that AgrA type AgrA, AgrAC199S, and AgrAC228S. As illustrated in Fig. down-regulates transcription of S. aureus glutathione peroxidase S4, after MD simulation, both wild-type AgrA and AgrAC199S (bsaA, SAV1306) (38), a putative enzyme responsible for de- exhibited minimal conformational changes, as reflected by their fense against oxidative stress. Our transcriptional analysis con- small rmds values (1.68 Å for wild-type and 1.70 Å for C199S) firmed this observation. As shown in Figs. S1B and S6, the ΔagrA (Fig. S4 A and B). By contrast, after MD simulation the β10 mutant displayed the increased expression of bsaA compared strand, where Cys-228 locates, and the adjacent α4 helix com- with wild-type Newman in the absence of H2O2. Transcription of pletely unfolded in AgrAC228S (Fig. S4C). The large rmsd value bsaA in wild-type Newman (Fig. S1B) and the complementation (as high as 5.75 Å) of AgrAC228S before and after MD simu- strain ΔagrA/pCL55-agrA (Fig. S6 A and B) can be triggered by lation reflects this drastic conformational change (Fig. S4C). The H2O2 treatment. However, in ΔagrA/pCL55-agrAC199S, tran- in silico result coincides with the experimental observation that scription of bsaA was unaffected by the presence of H2O2 (Fig. AgrAC228S forms inclusion bodies upon expression. Thus, the S6 A and B), supporting the redox-sensing role of Cys-199 in MD simulation results confirm that Cys-228 is structurally im- regulating bsaA expression. In addition, EMSAs established that portant for the proper folding of AgrA. AgrA is capable of binding to the promoter region of bsaA and The agr system is essential for expression of a number of that the binding is significantly weakened by H2O2 treatment, S. aureus virulence factors, including α-hemolysin, β-hemolysin, indicating that AgrA might control the expression of bsaA via δ-hemolysin, and PSMs (15, 35–37). Consistent with previous a direct DNA-binding mechanism (Fig. S7). results, we observed that the ΔagrA in-frame deletion mutant The redox-sensing mechanism of AgrA impacts not only the strain displayed almost no hemolytic activity compared with the bsaA gene but also the AgrA-activated psmα operon. As shown wild-type Newman strain, as indicated by zones of clearance on in Fig. S6C, a treatment of H2O2 repressed the expression of 5% (vol/vol) sheep blood agar (Fig. 4). Introduction of the in- psmα in wild-type Newman but had negligible effects on the ex- tegration single-copy pCL55-agrA into the ΔagrA mutant only pression of psmα in ΔagrA/pCL55-agrAC199S. Collectively, these partially restored hemolysis (Fig. 4), which might be attributed to results provide strong evidence that oxidative stress modulates the slightly altered expression level of AgrA due to changed transcription of RNAIII, bsaA,andpsmα through AgrA, of which genetic context (note that the intergenic region of RNAII and Cys-199 is required for its oxidation sensing. These results are RNAIII is directly fused with the ORF corresponding to the agrA also consistent with the observation that AgrAC199S triggers expression of hemolysins (α-hemolysin and PSMs) responsible for sheep erythrocyte lysis (Fig. 4).

AgrA Mediates S. aureus Defense Against Oxidative Stress via Cys- 199. S. aureus has developed an intricate regulatory system to counter oxidative stress. Our growth assays in the absence and presence of H2O2 revealed that the ΔbsaA mutant is extremely susceptible to H2O2, indicating that S. aureus glutathione peroxidase (BsaA) is essential for its survival under oxidative stress Fig. 4. Effects of Cys-199 and Cys-228 in AgrA on hemolysis. The strains (Fig. 5). Given that repression of the bsaA gene by AgrA can be tested were spotted on 5% sheep blood agar plate. Zones of clearance in- eradicated by H2O2 in a Cys-199 dependent manner (Fig. S6), we dicate hemolysis. WT/pCL55, wild-type Newman carrying an empty chro- envisioned that AgrA, with the redox-sensing capability, might mosome-integrated vector pCL55; ΔagrA/pCL55, agrA in-frame deletion mutant carrying an empty pCL55; ΔagrA/pCL55, agrA deletion mutant car- protect S. aureus against oxidative stress via derepressing anti- rying pCL55-agrA; ΔagrA/pCL55-agrAC199S, agrA deletion mutant carrying oxidant genes such as bsaA (Fig. 6). To test this hypothesis, we pCL55-agrAC199S; ΔagrA/pCL55-agrAC228S, agrA deletion mutant carrying compared the growth of various S. aureus strains in the absence pCL55-agrAC228S. and presence of H2O2 over 24 h. In the absence of H2O2, all

9098 | www.pnas.org/cgi/doi/10.1073/pnas.1200603109 Sun et al. Downloaded by guest on October 1, 2021 Fig. 5. Mutation of Cys-199 to Ser renders S. aureus more susceptible to H2O2. Strains of wild-type Newman, ΔbsaA (SAV1306), ΔagrA/pCL55, ΔagrA/pCL55- agrA, and ΔagrA/pCL55-agrAC199S were inoculated and grown in the presence of (A) 0 mM, (B)6mMH2O2,or(C)10mMH2O2. The effects of H2O2 on the growth pattern of these strains were examined at 37 °C for 24 h. All measurements were done in sextuplicate. Mean and SD are presented. ΔbsaA, the bursa

transposon insertion mutant of S. aureus glutathione peroxidase gene (SAV1306) which is vulnerable to H2O2, served as a positive control.

strains (wild-type Newman, ΔagrA/pCL55, ΔagrA/pCL55-agrA, disulfide switch responsive to oxidative stress, which reveals the and ΔagrA/pCL55-agrAC199S) exhibited similar growth patterns presence of an additional layer of the redox signaling in this par- (Fig. 5A). In the presence of H2O2 (6 mM or 10 mM), although no adigmatic quorum-sensing system. The cross-talk between oxida- significant growth defect was observed in wild-type Newman, tion sensing and quorum sensing within a single regulatory system ΔagrA/pCL55, or ΔagrA/pCL55-agrA, the growth of ΔagrA/pCL55- fully demonstrates the complexity of bacterial signal transduction. agrAC199S was significantly inhibited, especially during the first It is conceivable that other quorum-sensing systems, such as N-acyl 10 h (Fig. 5C), confirming the redox switch role of Cys-199 in the homoserine lactones-based systems in Gram-negative bacteria, wild-type AgrA. With this defense mechanism, both wild-type might also have subordinate built-in signaling pathways to com- Newman and the complementation strain ΔagrA/pCL55-agrA pensate or modulate the cell density-dependent regulatory net- were able to survive a high dose of H2O2 (10 mM) as well as ΔagrA/ work. This study provides an example of additional oxidation sig- pCL55, the strain that constantly maintains a high expression level nals that affect the cell density-dependent quorum-sensing system. of bsaA because of the absence of AgrA (Fig. S1B). In ΔagrA/ The S. aurues agr system, like most quorum-sensing systems, pCL55-agrAC199S, mutation of Cys-199 to Ser abolishes the oxi- constitutes an autoinduction genetic circuit, resulting in a rapid dation-sensing ability of AgrA, which leads to repression of the burst of activity once a certain threshold of cell density has been bsaA gene under oxidative stress (Fig. 6 and Fig. S6) and an in- reached (10). Previous studies have demonstrated that the acti- creased susceptibility of ΔagrA/pCL55-agrAC199S to H2O2 (Fig. vation of this system is inevitably accompanied by an intense 5). It is noteworthy that the growth inhibition of ΔagrA/pCL55- metabolic burden, which could be detrimental and presumably agrAC199S by H2O2 is temporary and that the bacterium can responsible for frequent spontaneous agr mutations in the labo- eventually overcome this growth delay after 10 h (Fig. 5C), indi- ratory (10, 39). One severe consequence of the intense metabolic cating the presence of other unknown factors contributing to this activity is the generation of a heavy oxidation burden concomitant redox regulation in S. aureus. Taken together, our results dem- with increased concentrations of ROS/RNS (40). This metabolic onstrate that the oxidation sensing of AgrA is essential for S. au- burden and the concomitant oxidative stress necessitate a delicate reus defense against oxidative stress and that mutation of Cys-199 sensing system to enable the bacterium to avoid the detrimental to Ser renders the bacterium more susceptible to H2O2. effects caused by agr activation. Indeed, S. aureus, as a successful human pathogen, has developed a rapid adaptive response to a Discussion sublethal dose of oxidants (4, 6), thereby reorienting gene ex- The S. aureus agr system is one of the most thoroughly studied pression for antioxidation. Our discovery suggests that the agr quorum-sensing systems in Gram-positive bacteria, which uses system serves to trigger bacterial response (through activation of AIP-based signals to coordinate bacterial behaviors with the local bsaA gene in particular) to oxidative stress through an intracel- cell density (13, 14). Here we show that the DNA-binding domain lular disulfide switch contained in the response regulator AgrA. of the response regulator AgrA possesses an intrinsic intracellular In fact, the two Cys residues, Cys-199 and Cys-228, are only 4.2 Å CHEMISTRY

Fig. 6. Model of oxidation sensing for AgrA. For wild-type AgrA (Upper), Cys-199 plays a redox-sensing role, which can be converted to a sulfenic acid upon

H2O2 treatment and subsequently reacts with its spatially proximate residue Cys-228 to yield an intramolecular disulﬁde bond. Oxidation of AgrA leads to its

dissociation from DNA, thereby resulting in derepression of the bsaA gene and resistance to H2O2. For AgrAC199S (Lower), mutation of Cys-199 to Ser abolishes MICROBIOLOGY the oxidation-sensing ability of AgrA, causing repression of bsaA gene under oxidative stress, which renders the bacterium more susceptible to H2O2.

Sun et al. PNAS | June 5, 2012 | vol. 109 | no. 23 | 9099 Downloaded by guest on October 1, 2021 away from each other in the reduced AgrA, which are poised to provides a unique perspective for understanding the bacterial cell form a disulfide bond upon oxidation. This additional built-in density control and gene regulation during oxidative stress and oxidation-sensing ability of the primary quorum-sensing agr, host–pathogen interactions in general. which seems to be conserved in many Gram-positive bacteria (Fig. S8), enables the bacterium to timely adjust gene expression Experimental Procedures with the metabolic activity and oxidation burden. Bacterial Strains, Plasmids, and Culture Conditions. The bacterial strains and Cys-199 and Cys-228 are the only two Cys residues conserved in plasmids used in this study are listed in Table S1. Unless otherwise mentioned, the DNA-binding domain of AgrA. AgrA has three additional cys- S. aureus strain Newman was used in the study. S. aureus cells were grown in teines, Cys-6, Cys-55, and Cys-123 (Fig. S9A). Although no structure either tryptic soy broth or brain–heart infusion broth. E. coli cultures were of this domain is available, we built a virtual structural model (resi- grown in LB medium. Whenever required, antibiotics were added to the due range: 1–127) using SWISS-MODEL (http://swissmodel.expasy. culture medium (for E. coli,100μg/mL ampicillin; for S. aureus,10μg/mL org/). As shown in Fig. S9B, this model suggests that these three nalidixic acid or 5 μg/mL chloramphenicol). Cys residues are all located far apart. They are also less likely to form disulfide bonds with Cys-199 and Cys-228, which are buried Other Procedures. Detailed procedures are available in SI Experimental in the elongated β-β-β sandwich in the DNA-binding domain. It Procedures. would be interesting to see whether these other Cys residues play any regulatory role in the agr system in the future. ACKNOWLEDGMENTS. We thank Drs. O. Schneewind and D. Missiakas at the S. aureus University of Chicago for providing transposon mutants, and S. F. Reichard In summary, our study shows that has integrated an for editing the manuscript. This work was financially supported by National additional oxidation-sensing mechanism into the primary quo- Institutes of Health National Institute of Allergy and Infectious Diseases rum-sensing agr system through an intrinsic intracellular disulfide Grants AI074658 and P50GM081892 (to C.H.), a Burroughs Wellcome Fund switch in the response regulator AgrA. The oxidation-sensing Investigator in the Pathogenesis of Infectious Disease Award (to C.H.), Shanghai Committee of Science and Technology Grant 10410703900 (to ability of AgrA is shown to be critical for bacterial defense against C.L.), and 863 Program Grant SS2012AA021103 (to H.J.). F.S. is a Scholar of oxidative stress. This rare demonstration of the cross-talk be- the Chicago Biomedical Consortium with support from The Searle Funds at tween oxidation sensing and quorum sensing in human pathogen The Chicago Community Trust.

1. Camilli A, Bassler BL (2006) Bacterial small-molecule signaling pathways. Science 311: 22. Baker J, et al. (2010) Copper stress induces a global stress response in Staphylococcus 1113–1116. aureus and represses sae and agr expression and biofilm formation. Appl Environ 2. Fuqua C, Greenberg EP (2002) Listening in on bacteria: Acyl-homoserine lactone Microbiol 76:150–160. signalling. Nat Rev Mol Cell Biol 3:685–695. 23. Rothfork JM, et al. (2004) Inactivation of a bacterial virulence pheromone by 3. Novick RP, Geisinger E (2008) Quorum sensing in staphylococci. Annu Rev Genet 42: phagocyte-derived oxidants: new role for the NADPH oxidase in host defense. Proc 541–564. Natl Acad Sci USA 101:13867–13872. 4. Chen PR, Brugarolas P, He C (2011) Redox signaling in human pathogens. Antioxid 24. Fujimoto DF, et al. (2009) Staphylococcus aureus SarA is a regulatory protein re- Redox Signal 14:1107–1118. sponsive to redox and pH that can support bacteriophage lambda integrase-medi- 5. Marshall HE, Merchant K, Stamler JS (2000) Nitrosation and oxidation in the regula- ated excision/recombination. Mol Microbiol 74:1445–1458. – tion of gene expression. FASEB J 14:1889 1900. 25. Ballal A, Manna AC (2009) Regulation of superoxide dismutase (sod) genes by SarA in 6. Mongkolsuk S, Helmann JD (2002) Regulation of inducible peroxide stress responses. Staphylococcus aureus. J Bacteriol 191:3301–3310. – Mol Microbiol 45:9 15. 26. Cheung AL, Projan SJ (1994) Cloning and sequencing of sarA of Staphylococcus au- 7. Liu Z, et al. (2011) Vibrio cholerae anaerobic induction of virulence gene expression is reus, a gene required for the expression of agr. J Bacteriol 176:4168–4172. controlled by thiol-based switches of virulence regulator AphB. Proc Natl Acad Sci 27. Sidote DJ, Barbieri CM, Wu T, Stock AM (2008) Structure of the Staphylococcus aureus – USA 108:810 815. AgrA LytTR domain bound to DNA reveals a beta fold with an unusual mode of 8. Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annu Rev Microbiol 55: binding. Structure 16:727–735. 165–199. 28. Zheng M, Aslund F, Storz G (1998) Activation of the OxyR transcription factor by 9. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339:520–532. reversible disulfide bond formation. Science 279:1718–1721. 10. Novick RP (2003) Autoinduction and signal transduction in the regulation of staph- 29. Helmann JD (2002) OxyR: A molecular code for redox sensing? Sci STKE 2002:pe46. ylococcal virulence. Mol Microbiol 48:1429–1449. 30. Choi H, et al. (2001) Structural basis of the redox switch in the OxyR transcription 11. Stauff DL, Skaar EP (2009) The heme sensor system of Staphylococcus aureus. Contrib factor. Cell 105:103–113. Microbiol 16:120–135. 31. Chen H, et al. (2008) The Pseudomonas aeruginosa multidrug efflux regulator MexR 12. Cheung AL, Nishina KA, Trotonda MP, Tamber S (2008) The SarA protein family of uses an oxidation-sensing mechanism. Proc Natl Acad Sci USA 105:13586–13591. Staphylococcus aureus. Int J Biochem Cell Biol 40:355–361. 32. Hutchison KA, Matic G, Meshinchi S, Bresnick EH, Pratt WB (1991) Redox manipulation 13. Geisinger E, Muir TW, Novick RP (2009) agr receptor mutants reveal distinct modes of of DNA binding activity and BuGR epitope reactivity of the glucocorticoid receptor. inhibition by staphylococcal autoinducing peptides. Proc Natl Acad Sci USA 106: J Biol Chem 266:10505–10509. 1216–1221. 33. Antelmann H, Helmann JD (2011) Thiol-based redox switches and gene regulation. 14. Mayville P, et al. (1999) Structure-activity analysis of synthetic autoinducing thio- Antioxid Redox Signal 14:1049–1063. lactone peptides from Staphylococcus aureus responsible for virulence. Proc Natl 34. Klomsiri C, Karplus PA, Poole LB (2011) Cysteine-based redox switches in enzymes. Acad Sci USA 96:1218–1223. – 15. Queck SY, et al. (2008) RNAIII-independent target gene control by the agr quorum- Antioxid Redox Signal 14:1065 1077. sensing system: Insight into the evolution of virulence regulation in Staphylococcus 35. McNamara PJ, Iandolo JJ (1998) Genetic instability of the global regulator agr ex- aureus. Mol Cell 32:150–158. plains the phenotype of the xpr mutation in Staphylococcus aureus KSI9051. J Bac- – 16. Chen PR, et al. (2006) An oxidation-sensing mechanism is used by the global regulator teriol 180:2609 2615. MgrA in Staphylococcus aureus. Nat Chem Biol 2:591–595. 36. Villaruz AE, et al. (2009) A point mutation in the agr locus rather than expression of 17. Chen PR, et al. (2009) A new oxidative sensing and regulation pathway mediated by the Panton-Valentine leukocidin caused previously reported phenotypes in Staphy- the MgrA homologue SarZ in Staphylococcus aureus. Mol Microbiol 71:198–211. lococcus aureus pneumonia and gene regulation. J Infect Dis 200:724–734. 18. Ballal A, Manna AC (2010) Control of thioredoxin reductase gene (trxB) transcription 37. Recsei P, et al. (1986) Regulation of exoprotein gene expression in Staphylococcus by SarA in Staphylococcus aureus. J Bacteriol 192:336–345. aureus by agr. Mol Gen Genet 202:58–61. 19. Luong TT, Dunman PM, Murphy E, Projan SJ, Lee CY (2006) Transcription profiling of 38. Dunman PM, et al. (2001) Transcription profiling-based identification of Staphylo- the mgrA regulon in Staphylococcus aureus. J Bacteriol 188:1899–1910. coccus aureus genes regulated by the agr and/or sarA loci. J Bacteriol 183:7341–7353. 20. Schlag S, et al. (2008) Characterization of the oxygen-responsive NreABC regulon of 39. Somerville GA, et al. (2002) In vitro serial passage of Staphylococcus aureus: Changes Staphylococcus aureus. J Bacteriol 190:7847–7858. in physiology, virulence factor production, and agr nucleotide sequence. J Bacteriol 21. Sun F, et al. (2011) AirSR, a [2Fe-2S] cluster-containing two-component system, me- 184:1430–1437. diates global oxygen sensing and redox signaling in Staphylococcus aureus. JAm 40. Somerville GA, Proctor RA (2009) At the crossroads of bacterial metabolism and vir- Chem Soc. ulence factor synthesis in Staphylococci. Microbiol Mol Biol Rev 73:233–248.

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