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Proteome-Wide Quantification and Characterization of Oxidation View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell Host & Microbe Resource Proteome-wide Quantification and Characterization of Oxidation-Sensitive Cysteines in Pathogenic Bacteria Xin Deng,1 Eranthie Weerapana,2,5,6 Olesya Ulanovskaya,5,6 Fei Sun,1 Haihua Liang,1 Quanjiang Ji,1 Yan Ye,3 Ye Fu,1 Lu Zhou,1 Jiaxin Li,3 Haiyan Zhang,1 Chu Wang,5,6 Sophie Alvarez,4 Leslie M. Hicks,4 Lefu Lan,7 Min Wu,3 Benjamin F. Cravatt,5,6,* and Chuan He1,* 1Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA 2Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA 3Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, ND 58203, USA 4Donald Danforth Plant Science Center, St. Louis, MO 63132, USA 5The Skaggs Institute for Chemical Biology 6Department of Chemical Physiology The Scripps Research Institute, La Jolla, CA 92121, USA 7Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong Zhangjiang Hi-Tech Park, Shanghai 201203, China *Correspondence: [email protected] (B.F.C.), [email protected] (C.H.) http://dx.doi.org/10.1016/j.chom.2013.02.004 SUMMARY immune system produce high concentrations of reactive oxygen species (ROS), such as superoxide hydrogen peroxide (H2O2) Thiol-group oxidation of active and allosteric cyste- and hydroxyl radicals that bacterial pathogens must overcome ines is a widespread regulatory posttranslational for sustained virulence. These bacteria utilize multiple com- protein modification. Pathogenic bacteria, including ponents to defend against the oxidative stress produced by Pseudomonas aeruginosa and Staphylococcus host innate immunity pathways, including ROS-deactivating aureus, use regulatory cysteine oxidation to respond enzymes such as catalase, superoxide dismutase, thioredoxin, to and overcome reactive oxygen species (ROS) and glutaredoxin. Oxidation of the thiol group at active and allosteric cysteines encountered in the host environment. To obtain in proteins has emerged as a widespread and important a proteome-wide view of oxidation-sensitive cyste- posttranslational modification. To date, this modification has ines in these two pathogens, we employed a com- been found in more than two hundred proteins, including tran- petitive activity-based protein profiling approach to scription factors, signaling proteins, metabolic enzymes, and globally quantify hydrogen peroxide (H2O2) reactivity other cellular components in a wide range of organisms (Lindahl with cysteines across bacterial proteomes. We iden- et al., 2011). Pathogenic bacteria are known to mount a global tified 200 proteins containing H2O2-sensitive cyste- response to oxidative stress that is mediated by transcriptional ines, including metabolic enzymes, transcription regulators, including OxyR, MgrA, OhrR, and SarA, which factors, and uncharacterized proteins. Additional undergo oxidation of cysteine (Chen et al., 2006; Fuangthong biochemical and genetic studies identified an oxida- and Helmann, 2002; Fujimoto et al., 2009). In P. aeruginosa, tion-responsive cysteine in the master quorum- OxyR is important for oxidative-stress defense and is required for full virulence in rodent and insect models of infection (Lau sensing regulator LasR and redox-regulated activi- et al., 2005). P. aeruginosa also produces a group of multicolored ties for acetaldehyde dehydrogenase ExaC, arginine redox-active antibiotics, phenazines, which have been identified deiminase ArcA, and glyceraldehyde 3-phosphate as virulence factors (Mahajan-Miklos et al., 1999). Similarly, dehydrogenase. Taken together, our data indicate S. aureus possesses a number of thiol-based transcriptional that pathogenic bacteria exhibit a complex, multi- regulators that sense ROS stress and coordinate responses layered response to ROS that includes the rapid (Chen et al., 2006; Fujimoto et al., 2009). Our previous work adaption of metabolic pathways to oxidative-stress showed that these human pathogens utilize a thiol-based oxida- challenge. tion-sensing mechanism to sense the host immune response and regulate a global change of their properties (Chen et al., 2006, 2008). INTRODUCTION The modification of cysteines by cellular ROS can generate a number of chemical products, including reversible sulfenic Pseudomonas aeruginosa and Staphylococcus aureus are acid (SOH) modification and more stable sulfinic (SO2H) and ubiquitous, opportunistic human pathogens that cause life- sulfonic (SO3H) modifications, only a subset of which can be threatening human diseases (Govan and Harris, 1986; Lowy, detected with good sensitivity in proteomes (Leonard and 1998). In response to infection, phagocytic cells in the human Carroll, 2011). As a consequence of this complexity, our 358 Cell Host & Microbe 13, 358–370, March 13, 2013 ª2013 Elsevier Inc. Cell Host & Microbe Redox-Sensitive Cysteines in Pathogenic Bacteria understanding of the full spectrum of oxidation-sensitive cyste- 10 mM H2O2. After 20 min, cells were collected and bacterial ines in bacterial proteomes is incomplete and would greatly protein extracts were prepared by sonication. The extracts benefit from new technologies that can quantify cysteine were treated for 1 hr with the IA probe for alkylating reactive reactivity in native biological systems (Klomsiri et al., 2010; cysteines in the bacterial proteomes. An isotopically labeled, Leichert et al., 2008; Seo and Carroll, 2009). Recently, a method protease-cleavable azido-biotin tag (Weerapana et al., 2010) termed isotopic tandem orthogonal proteolysis-activity-based was then attached to the alkyne using click chemistry (Rostovt- protein profiling (isoTOP-ABPP) (Weerapana et al., 2010) was sev et al., 2002; Tornøe et al., 2002). Isotopically heavy (13C and 15 12 14 introduced for the identification and quantification of reactive N) and light ( C and N) tags were conjugated to the ÀH2O2 cysteines in native proteomes. Here, we have adapted this and +H2O2 samples, respectively. Equal amounts of the corre- chemoproteomic technology to quantitatively profile oxidation- sponding À and + H2O2 samples were mixed and subjected sensitive cysteines in the proteomes of P. aeruginosa and to successive protease digestion steps with trypsin and S. aureus. We identified approximately 200 oxidation-sensitive tobacco-etch virus (TEV) protease, affording isotopically tagged, cysteines across diverse classes of proteins and determined cysteine-containing peptides for mass spectrometry (MS) through targeted genetic and biochemical studies that several analysis. of these proteins perform important redox-active functions in The light/heavy (+H2O2/ÀH2O2) ratio calculated for each IA- bacteria, such as oxidation-responsive transcriptional regulation labeled cysteine in the bacterial proteomes provided a measure and oxidation-induced switching of metabolic pathways by of its relative reactivity in + versus À H2O2 samples. Oxidation modification of active-site and/or allosteric-cysteine residues of a cysteine by H2O2 should reduce its reactivity with the IA in enzymes. probe, resulting in a light/heavy peptide ratio of <1 (Figure 1A). The lower the measured ratio, the more reactive the cysteine RESULTS AND DISCUSSION was toward H2O2 in this experiment. The identity of each cysteine-containing peptide and its parent protein was then Quantifying Oxidation-Sensitive Cysteines in Bacterial determined by tandem MS analysis and database searches Proteomes using the SEQUEST search algorithm. Overall, 307 IA-probe- The original isoTOP-ABPP approach was used to quantify the reactive cysteines were identified in bacterial proteomes, 82 intrinsic reactivity of cysteine residues in proteomes with a click- and 113 of which were oxidation sensitive (defined as having able iodoacetamide (IA)-alkyne probe (Weerapana et al., 2010). a light/heavy ratio <0.67, representing a 1.5-fold change that is Here, we adapted this technology to quantify oxidation-sensitive commonly used as a threshold value for indicating significant cysteines in the proteomes of P. aeruginosa MPAO1, a Gram- difference) in H2O2-treated P. aeruginosa (Table S1) and negative bacterium, and S. aureus Newman, a Gram-positive S. aureus (Table S2), respectively (Chan et al., 2005; Leichert bacterium, by measuring the ability of H2O2 exposure to et al., 2008). We noticed that the ratios of the oxidation-sensitive ‘‘compete’’ away cysteine reactivity with the IA probe. We cysteines in P. aeruginosa were generally lower than those in reasoned that one advantage of this type of competitive profiling S. aureus. This difference was more apparent when applying approach is that it would be sensitive to multiple forms of a more stringent threshold for oxidation sensitivity (light/heavy cysteine oxidation (i.e., sensitive to any mode of modification ratio <0.33), which was satisfied by 62 cysteines (75.6% of all that impaired the nucleophilicity of the IA-probe-reactive oxidation-sensitive cysteines) in P. aeruginosa, but only 6 cyste- cysteines). ines (5.3%) in S. aureus (Table 1). We define these cysteines as We treated both pathogens with 10 mM H2O2, a concentration highly oxidation-sensitive cysteines. commonly used by other studies because both pathogens can Previously, 18 proteins were identified as possessing oxida- tolerate millimolar levels of H2O2 well (Chang et al., 2006; tion-sensitive thiols after H2O2 treatment in S. aureus through Salunkhe et al., 2005). We measured
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