Using Quantitative Spectrometry to Understand the Influence of Genetics and Nutritional Perturbations On the Virulence Potential of Staphylococcus aureus
2017; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês
10.1074/mcp.o116.065581
ISSN1535-9484
AutoresJessica R. Chapman, Divya Balasubramanian, Kayan Tam, Manor Askenazi, Richard Copin, Bo Shopsin, Victor J. Torres, Beatrix Ueberheide,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoStaphylococcus aureus (Sa) is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of Sa as a human pathogen is contributed to its ability to adapt to different environments by changing expression, production, or secretion of virulence factors. Although Sa immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the Sa exoproteins (i.e. exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for in vitro studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties, highlighting the power of label-free quantitative mass spectrometry to distinguish Sa strains. Staphylococcus aureus (Sa) is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of Sa as a human pathogen is contributed to its ability to adapt to different environments by changing expression, production, or secretion of virulence factors. Although Sa immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the Sa exoproteins (i.e. exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for in vitro studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties, highlighting the power of label-free quantitative mass spectrometry to distinguish Sa strains. Staphylococcus aureus (Sa) 1The abbreviations used are: Sa, Staphylococcus aureus; agr, accessory gene regulator; Asn, asparagines; CA, community associated; CC, clonal complex; Cys, cysteine; FDR, false discovery rate; Gln, glutamine; HA, hospital associated; hPMNs, human polymorphonuclear leukocytes or neutrophils; LFQ, label-free quantitation as calculated by MaxQuant; Met, methionine; MLST, multilocus sequence typing; MRSA, Methicillin-resistant Staphylococcus aureus; MSSA, Methicillin-sensitive Staphylococcus aureus; NETs, neutrophil extracellular nets; PSM, peptide spectral match; pvl, Panton–Valentine leukocidin gene; Rot, repressor of toxins; RPMI, Roswell Park Memorial Institute medium-Cassamino acid; Sae, Sa exoprotein expression; SCCmec, staphylococcal cassette chromosome mec; ST, strain type; TSB, Tryptic Soy Broth; WT, wild type; TCS, Two-component system.1The abbreviations used are: Sa, Staphylococcus aureus; agr, accessory gene regulator; Asn, asparagines; CA, community associated; CC, clonal complex; Cys, cysteine; FDR, false discovery rate; Gln, glutamine; HA, hospital associated; hPMNs, human polymorphonuclear leukocytes or neutrophils; LFQ, label-free quantitation as calculated by MaxQuant; Met, methionine; MLST, multilocus sequence typing; MRSA, Methicillin-resistant Staphylococcus aureus; MSSA, Methicillin-sensitive Staphylococcus aureus; NETs, neutrophil extracellular nets; PSM, peptide spectral match; pvl, Panton–Valentine leukocidin gene; Rot, repressor of toxins; RPMI, Roswell Park Memorial Institute medium-Cassamino acid; Sae, Sa exoprotein expression; SCCmec, staphylococcal cassette chromosome mec; ST, strain type; TSB, Tryptic Soy Broth; WT, wild type; TCS, Two-component system. asymptomatic colonization of the nares, skin or gastrointestinal tract is detected in ∼30% of humans (1.Diekema D.J. 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The well-characterized, agr locus encodes for a "self-recognizing" two-component system (TCS) that detects increases in cell density via a secreted signaling peptide, resulting in the production of an effector regulatory RNA known as RNAIII (19.Novick R.P. Autoinduction and signal transduction in the regulation of staphylococcal virulence.Mol. Microbiol. 2003; 48: 1429-1449Crossref PubMed Scopus (1001) Google Scholar, 20.Novick R.P. Geisinger E. Quorum sensing in staphylococci.Annu. Rev. Genet. 2008; 42: 541-564Crossref PubMed Scopus (656) Google Scholar). RNAIII is a powerful activator of enzymes and cytotoxins that are thought to promote Sa survival in vivo (19.Novick R.P. Autoinduction and signal transduction in the regulation of staphylococcal virulence.Mol. Microbiol. 2003; 48: 1429-1449Crossref PubMed Scopus (1001) Google Scholar, 20.Novick R.P. Geisinger E. Quorum sensing in staphylococci.Annu. Rev. 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When Sa adheres and colonizes various tissues, it rapidly grows, leading to activation of the Agr system. Activated Agr leads to suppression of Rot, and thus enhances enzyme and toxin production, while repressing production of immune modulators. Mutations in master regulators are often associated with clinical infections and can be reflective of the severity of infections. The ability to predict severity of infection of an emerging strain could potentially inform clinical prognostication, management and infection control. Current methods for cataloging Sa strains rely on utilizing genomic sequences to determine clonality, such as multilocus sequence typing (MLST) and spa typing. Strains are also screened for the presence of important genetic biomarkers, such as the staphylococcal cassette chromosome mec gene (SSCmec) and the Panton-Valentine Leukocidin gene (pvl) (34.Stefani S. Chung D.R. Lindsay J.A. Friedrich A.W. Kearns A.M. Westh H. Mackenzie F.M. Meticillin-resistant Staphylococcus aureus (MRSA): global epidemiology and harmonisation of typing methods.Int. J. Antimicrob. Agents. 2012; 39: 273-282Crossref PubMed Scopus (395) Google Scholar). MLST uses seven housekeeping genes to group the different Sa strains into sequence types (ST). STs sharing 5 of the 7 identical alleles are grouped as clonal complexes (CCs). Thus, MLST can provide information regarding the lineage of different Sa isolates in population and epidemiological studies (35.Enright M.C. Day N.P. Davies C.E. Peacock S.J. Spratt B.G. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus.J. Clin. Microbiol. 2000; 38: 1008-1015Crossref PubMed Google Scholar). Spa typing cataloges the variable tandem number of repeat polymorphisms in the 3′ coding region of Spa (36.Koreen L. Ramaswamy S.V. Graviss E.A. Naidich S. Musser J.M. 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The presence of pvl is often associated with CA-MRSA, such as strains from the USA300 lineage (3.David M.Z. Daum R.S. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic.Clin. Microbiol. Rev. 2010; 23: 616-687Crossref PubMed Scopus (1398) Google Scholar). Although genotypic methods have been reliable and reproducible in grouping the different Sa strains (34.Stefani S. Chung D.R. Lindsay J.A. Friedrich A.W. Kearns A.M. Westh H. Mackenzie F.M. Meticillin-resistant Staphylococcus aureus (MRSA): global epidemiology and harmonisation of typing methods.Int. J. Antimicrob. Agents. 2012; 39: 273-282Crossref PubMed Scopus (395) Google Scholar), they provide limited information on the production of virulence factors; thus the virulence potential of these strains cannot be accurately predicted based on typing alone. Since the controlled production of virulence factors is vital for the success of Sa as a human pathogen, we sought to test the utility of label-free quantitative proteomics for characterization of Sa exoproteomes under a variety of different conditions. We quantitatively compared the exoprotein profiles of Sa USA300 1) master regulator mutants, Δagr, Δrot, and Δsae grown to stationary phase in either minimal (RPMI) or rich (TSB) media; 2) wild type (WT) USA300 grown to either exponential, early stationary, or late-stationary phase; and 3) 13 different reference Sa strains belonging to four different clonal complexes. Altogether, our study provides a rich data set cataloging the exoproteome of the highly prevalent CA-MRSA in the United States, USA300, and other important Sa isolates. Sa strains were grown at 37 °C on Tryptic Soy Agar (TSA), then in Tryptic Soy Broth (TSB) or Roswell Park Memorial Institute media (RPMI, Invitrogen, Waltham, MA) supplemented with 1% Cassamino Acid. Liquid cultures were grown in 5 ml growth media in 15 ml tubes at a 45° angle or in 150 μl growth media in 96-well plates with shaking at 180 rpm overnight prior to subculture. The construction and validation of the isogenic agr::tet (26.Benson M.A. Lilo S. Nygaard T. Voyich J.M. Torres V.J. Rot and SaeRS cooperate to activate expression of the staphylococcal superantigen-like exoproteins.J. Bacteriol. 2012; 194: 4355-4365Crossref PubMed Scopus (50) Google Scholar, 39.Mootz J.M. Benson M.A. Heim C.E. Crosby H.A. Kavanaugh J.S. Dunman P.M. Kielian T. Torres V.J. Horswill A.R. Rot is a key regulator of Staphylococcus aureus biofilm formation.Mol. Microbiol. 2015; 96: 388-404Crossref PubMed Scopus (53) Google Scholar), rot::erm (26.Benson M.A. Lilo S. Nygaard T. Voyich J.M. Torres V.J. Rot and SaeRS cooperate to activate expression of the staphylococcal superantigen-like exoproteins.J. Bacteriol. 2012; 194: 4355-4365Crossref PubMed Scopus (50) Google Scholar), and sae::spec (29.Nygaard T.K. Pallister K.B. Ruzevich P. Griffith S. Vuong C. Voyich J.M. SaeR binds a consensus sequence within virulence gene promoters to advance USA300 pathogenesis.J. Infect. Dis. 2010; 201: 241-254Crossref PubMed Scopus (116) Google Scholar) have been described. For growth curves, Sa strain LAC was subcultured at 1:100 in 100 μl TSB or RPMI from 6 independent colonies grown in 150 μl TSB in a 96-well plate with shaking overnight at 180 rpm. Optical densities at 600 nm were read at the beginning of the subculture and at the indicated time points using a PerkinElmer Envision 2103 Multi-label reader (PerkinElmer, Waltham, MA). Leukopaks were obtained from de-identified donors from the New York Blood Center where written consents were obtained from all participants and human polymorphonuclear neutrophils (hPMNs) were purified as described previously (40.Reyes-Robles T. Lubkin A. Alonzo 3rd, F. Lacy D.B. Torres V.J. Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis.EMBO Rep. 2016; 17: 428-440Crossref PubMed Scopus (31) Google Scholar). hPMNs were resuspended in RPMI 1640 (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (FBS). Cytotoxicity assays were performed as described previously (21.Benson M.A. Ohneck E.A. Ryan C. Alonzo 3rd, F. Smith H. Narechania A. Kolokotronis S.O. Satola S.W. Uhlemann A.C. Sebra R. Deikus G. Shopsin B. Planet P.J. Torres V.J. Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element.Mol. Microbiol. 2014; 93: 664-681Crossref PubMed Scopus (60) Google Scholar, 41.Dumont A.L. Nygaard T.K. Watkins R.L. Smith A. Kozhaya L. Kreiswirth B.N. Shopsin B. Unutmaz D. Voyich J.M. Torres V.J. Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis.Mol. Microbiol. 2011; 79: 814-825Crossref PubMed Scopus (139) Google Scholar). Briefly, culture supernatants were collected from Sa strains sub cultured at 1:100 from overnight cultures in 5 ml TSB or 5 ml RPMI in 15 ml conical tubes at the indicated time points. The culture supernatants were serially diluted and added to 2 × 105 hPMN/well for a final volume of 100 μl/well. hPMNs were intoxicated with the culture supernatant from the indicated Sa strain for 1 h at 37 °C and 5% CO2. hPMN viability was determined using CellTiter 96 Aqueous One Solution (Promega, Madison, WI). Briefly, 10 μl/well of CellTiter was added and incubated at 37 °C and 5% CO2 for 2 h. Cell viability was measured by absorbance at 492 nm using a PerkinElmer Envision 2103 Multilabel reader (PerkinElmer). Sa cultures were grown in 5 ml TSB or RPMI in 15 ml conical tubes for 3, 5, or 8 h in a 1:100 subculture from overnight cultures grown in TSB. Isogenic mutants were grown in TSB for 5 h. All established reference strains were grown in TSB for 5 h. At the indicated time point post-subculture, cultures were normalized to the same optical density by adding respective media to dilute cultures with higher cell density. Culture supernatants were collected by centrifugation at 4000 rpm for 10 min to remove bacteria, followed by filtration through a 0.22 μm filter to remove cell debris. The proteins in the culture supernatants were precipitated in 10% (v/v) trichloroacetic acid (TCA) at 4 °C overnight. The precipitated proteins were sedimented by centrifugation and pellets were washed with ethanol. The protein pellets were centrifuged again, the remaining ethanol was removed and the pellets were allowed to air dry. Precipitated exoproteins were resuspended in 8 m urea for 30 min at RT, then diluted 1:1 with 2× SDS sample buffer and boiled for 10 min. exoproteins were separated in a 12% SDS-PAGE gel and protein visualized using either Coomassie, silver staining, or Instant Blue. The gels were imaged using the Gel Doc XR System (Bio-Rad, Hercules, CA). Reconstituted exoprotein isolates were reduced with 0.02 m dithiothreitol and alkylated with 0.05 m iodoacetamide. The exoproteins were in-gel digested as described in (42.Balasubramanian D. Ohneck E.A. Chapman J. Weiss A. Kim M.K. Reyes-Robles T. Zhong J. Shaw L.N. Lun D.S. Ueberheide B. Shopsin B. Torres V.J. Staphylococcus aureus coordinates leukocidin expression and pathogenesis by sensing metabolic fluxes via RpiRc.MBio. 2016; 7Crossref PubMed Scopus (39) Google Scholar) and the resulting peptide mixture desalted as previously described (see supplemental information for details) (43.Cotto-Rios X.M. Bekes M. Chapman J. Ueberheide B. Huang T.T. Deubiquitinases as a signaling target of oxidative stress.Cell Rep. 2012; 2: 1475-1484Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Aliquots of the peptide mixtures were loaded onto a Acclaim PepMap 100 precolumn (75 μm × 2 cm, C18, 3 μm, 100 Å) in-line with an EASY-Spray, PepMap column (75 μm × 50 cm, C18, 2 μm, 100 Å) with a 5 μm emitter using the autosampler of an EASY-nLC 1000 (Thermo Scientific, Waltham, MA). The samples were gradient eluted directly into an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) and analyzed in a data dependent manner using a top speed method. Complete details of the LC-MS acquisition can be found in the supplemental materials. The MaxQuant software suite (version 1.5.2.8) was used for peptide and protein identifications and label-free quantitation (44.Cox J. Hein M.Y. Luber C
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