INFECTION AND IMMUNITY, Dec. 2008, p. 5862–5872 Vol. 76, No. 120019-9567/08/$08.00�0 doi:10.1128/IAI.00865-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Staphylococcus aureus Elicits Marked Alterations in the AirwayProteome during Early Pneumonia�‡

Christy L. Ventura,1,6,10† Roger Higdon,2,3 Laura Hohmann,4 Daniel Martin,4,5 Eugene Kolker,2,3,7

H. Denny Liggitt,8 Shawn J. Skerrett,9 and Craig E. Rubens1,6*Division of Infectious Diseases, Center for Childhood Infections and Prematurity Research,1 and Center for Developmental Therapeutics,2

Seattle Children’s Hospital Research Institute, BIATECH Institute,3 Institute for Systems Biology,4 Fred Hutchinson Cancer Research Center,5

and Departments of Pediatrics,6 Medical Education and Biomedical Informatics,7 Comparative Medicine,8 and Medicine,9 University ofWashington School of Medicine, Seattle, Washington, and Laboratory of Human Bacterial Pathogenesis,

Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes ofHealth, Hamilton, Montana10

Received 14 July 2008/Returned for modification 5 August 2008/Accepted 3 October 2008

Pneumonia caused by Staphylococcus aureus is a growing concern in the health care community. We hypoth-esized that characterization of the early innate immune response to bacteria in the lungs would provide insightinto the mechanisms used by the host to protect itself from infection. An adult mouse model of Staphylococcusaureus pneumonia was utilized to define the early events in the innate immune response and to assess thechanges in the airway proteome during the first 6 h of pneumonia. S. aureus actively replicated in the lungs ofmice inoculated intranasally under anesthesia to cause significant morbidity and mortality. By 6 h postinocu-lation, the release of proinflammatory cytokines caused effective recruitment of neutrophils to the airway.Neutrophil influx, loss of alveolar architecture, and consolidated pneumonia were observed histologically 6 hpostinoculation. Bronchoalveolar lavage fluids from mice inoculated with phosphate-buffered saline (PBS) orS. aureus were depleted of overabundant proteins and subjected to strong cation exchange fractionationfollowed by liquid chromatography and tandem mass spectrometry to identify the proteins present in theairway. No significant changes in response to PBS inoculation or 30 min following S. aureus inoculation wereobserved. However, a dramatic increase in extracellular proteins was observed 6 h postinoculation with S.aureus, with the increase dominated by inflammatory and coagulation proteins. The data presented hereprovide a comprehensive evaluation of the rapid and vigorous innate immune response mounted in the hostairway during the earliest stages of S. aureus pneumonia.

Staphylococcus aureus is a leading cause of hospital-acquiredand health care-associated pneumonia and may be increasingin importance as a cause of severe community-acquired pneu-monia. In the inpatient setting, it is the most common gram-positive bacterium implicated in cases of ventilator-associatedand hospital-acquired pneumonia (1, 9, 31). In addition, S.aureus is a frequent cause of health care-associated pneumoniaoccurring in residents of long-term-care facilities, individualsrecently discharged from acute-care hospitals, and patientsreceiving outpatient treatment at hospitals and dialysis centers(1, 27, 30). A steady increase in the isolation of methicillin-resistant strains of S. aureus from patients with hospital-ac-quired pneumonia and, more recently, community-acquiredpneumonia underscores the importance of identifying host andbacterial factors that facilitate the progression of staphylococ-cal pneumonia.

Mice have been used extensively to study pneumonia causedby a variety of bacteria (2, 6, 26, 35, 36, 45, 55, 63, 64). Murine

models of airborne infection with S. aureus have been useful incharacterizing host responses during the first 4 to 8 h of lunginfection but do not mimic the natural route of infection andresult in self-limited disease, even in immunocompromisedanimals (28, 53, 56). In these studies, proinflammatory cyto-kines and chemokines were released and neutrophils (poly-morphonuclear leukocytes [PMNs]) were rapidly recruited tothe site of infection; however, the mice were able to clear theinfection within 24 to 36 h (53). Bolus infection models inwhich mice are challenged by intratracheal or intranasal (i.n.)inoculation have been more successful in producing intrapul-monary bacterial replication and host mortality (13, 17, 23, 32,42, 60). Heyer et al. utilized an infant mouse model of staph-ylococcal pneumonia, which mimics disease in immunocom-promised individuals, in which the mice were anesthetized andinfected i.n., leading to 100% morbidity and 30% mortalityfollowing inoculation with virulent strains of S. aureus (23).They observed an increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) and an influx of PMNs in theairway. Earlier studies established a lethal S. aureus pneumo-nia model in adult mice; however, they infected the mice in-tratracheally, which introduces the additional factor of surgicaltrauma (13, 42). One goal of the present study was to developa staphylococcal pneumonia model in immunocompetent adultmice by using a nasal inoculation and aspiration approach thatmimics a common route of natural infection in order to pro-vide a system in which to define the earliest events in the host

* Corresponding author. Present address: Seattle Children’s Hospi-tal, Metropolitan Park West Bldg., 1100 Olive Way, M/S MPW10,Seattle, WA 98101. Phone: (206) 884-2777. Fax: (206) 884-7594. E-mail:craig.rubens@seattlechildrens.org.

† Present address: Department of Microbiology and Immunology,Uniformed Services University of the Health Sciences, Bethesda, MD.

‡ Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 13 October 2008.

5862

immune response to S. aureus in the airway. Similar modelswere developed simultaneously by other groups to study therequirement for specific S. aureus virulence factors in pneumo-nia (32, 60).

Shotgun proteomics has proven to be a very useful tool fordetermining the global protein profile in a particular organ orbody fluid in the context of various disease states. A study byGuo et al. utilized one-dimensional (1D) electrophoresis withmass spectrometry (MS) and two-dimensional liquid chroma-tography-MS (LC-MS) to define the airway proteome of ahealthy mouse (20). In addition, a proteomics approach hasbeen used to define the proteins present in the airway inpatients with a variety of conditions (3, 4, 7, 15, 16, 39, 41, 44,46, 50, 59, 61, 62, 65, 70). However, little is known about theeffects of acute infection on the airway proteome and the waysthese effects change over time. We hypothesized that early hostresponses to S. aureus infection of the lung, including changesin the airway proteome, could be critical determinants of thecourse and severity of pneumonia. To address this, we devel-oped a mouse model of acute staphylococcal pneumonia andutilized cell biological, immunological, and proteomics tech-niques to examine the host response and changes in the airwayproteome during the first 6 h of S. aureus pneumonia. Wedemonstrate that S. aureus elicits a vigorous airway inflamma-tory response characterized by the rapid release and influx ofinflammatory mediators during the first 6 h of pneumonia.Further, we show that this inflammatory response causes sig-nificant changes in the host airway proteome during the devel-opment of pneumonia.

MATERIALS AND METHODS

Bacteria and growth conditions. S. aureus strains RN6390 and JP1 were usedin these studies. RN6390 is a commonly used laboratory strain (49) that waskindly provided by David Heinrichs (University of Western Ontario), and JP1 isa human blood isolate obtained from the microbiology laboratory of the Veter-ans Affairs Puget Sound Health Care System (53). S. aureus was grown inLuria-Bertani (LB) broth at 37°C under aerobic conditions. For mouse infec-tions, bacteria were grown from a frozen stock for 6 to 10 h in LB broth and thendiluted 1/100 into fresh LB broth (4:1 flask-to-medium ratio) and grown for anadditional 16 to 18 h with shaking (180 rpm). Stationary-phase bacteria wereharvested by centrifugation at room temperature, washed twice with endotoxin-free phosphate-buffered saline (PBS) (Mediatech, Herndon, VA), and resus-pended in endotoxin-free PBS to the desired concentration as estimated byoptical density and confirmed by quantitative plate counting.

Animals. Specific-pathogen-free male and female C57BL/6 mice, aged 9 to 11weeks, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animalswere group housed in filtered, ventilated cages containing autoclaved beddingand were permitted ad libitum access to sterile food and water. Cage changes andanimal handling occurred in a laminar flow hood. All experimental procedureswere approved by the Institutional Animal Care and Use Committee of theUniversity of Washington.

Mouse model of pneumonia and tissue harvest. Mice were anesthetized withisoflurane, held vertically, and inoculated i.n. with S. aureus in 50 �l endotoxin-free PBS. Occasionally, lightly anesthetized mice flipped their heads duringinoculation, which may have affected bacterial deposition; these mice were re-moved from the study. To determine the dose at which S. aureus would replicatein the lungs, 12 mice each were inoculated with 3 � 107, 1 � 108, or 3 � 108 CFUJP1 and monitored at least twice daily. At 30 min after inoculation (all doses), at24 h, 48 h, and 96 h after inoculation (3 � 107 and 108 CFU), or when micereached a moribund state (3 � 108 CFU), defined by hunched posture, piloerec-tion, labored breathing, immobility, and loss of resistance to handling, mice wereeuthanized by intraperitoneal injection of an overdose of pentobarbital. Bothlungs were harvested and homogenized for quantitative culture as describedpreviously (53). For analysis of the host response to S. aureus, 7 or 10 mice wereinoculated with a dose of 3 � 108 to 5 � 108 CFU JP1 or with endotoxin-freePBS, as described above. At 30 min and 6 h postinoculation, the mice in each

group were euthanized, and bronchoalveolar lavage (BAL) was performed asdescribed previously (53, 54). Lungs were inflated in situ to approximately 15 cmpressure with 4% paraformaldehyde and stored at 4°C in the same fixative.

BAL cultures and differential cell counts. An aliquot of BAL fluid from eachanimal was removed for quantitative culture, cytokine analysis, and differentialcounts; the remaining BAL fluid was centrifuged at 300 � g, and the superna-tants were frozen at �80°C. The cell pellets were resuspended in RPMI 1640containing 10% heat-inactivated fetal calf serum (HyClone Laboratories, Logan,UT), and cells were counted with a hemacytometer. Differential cell counts weredetermined from cytocentrifuge specimens stained with Diff-Quik (Dade-Be-hring, Dudigen, Switzerland).

Measurement of cytokines. Levels of immunoreactive tumor necrosis factoralpha (TNF-�), interleukin-1� (IL-1�), macrophage inflammatory protein 2(MIP-2), keratinocyte-derived chemokine (KC), monocyte chemotactic protein1, IL-6, IL-10, IL-12p70, IL-17, and GM-CSF were measured with antibody-coated microbeads (R&D Systems, Minneapolis, MN) and a BioPlex analyzer(Bio-Rad, Hercules, CA).

Statistical analysis. Data are expressed as means � standard errors of themeans. The Mann-Whitney test was performed to determine whether the mediantimes to death for mice at each dose were statistically different. Statistical anal-ysis of cytokines and BAL fluid cells was performed using the Kruskal-Wallis testwith Dunn’s posttest. A P value of �0.05 was considered significant.

Histopathology. Paraformaldehyde-fixed lung tissue was embedded in paraffin,sectioned, and stained with hematoxylin and eosin (H&E). A veterinary pathol-ogist examined two to four sections from each lung of mock-infected and infectedmice in a manner blinded to time after inoculation and inoculum.

Depletion of BAL fluid. Twenty mice were inoculated with PBS or S. aureus asdescribed above, and BAL was performed on 10 mice per treatment at 30 minand 6 h postinoculation; the experiment was performed twice. Eukaryotic cellswere removed by centrifugation at 300 � g, and BAL fluids were pooled bytreatment and time point and frozen at �80°C. After the BAL fluid was thawed,Triton X-100 (TX-100) was added to 0.2%, each sample was vortexed for 15 s,and bacteria were removed by centrifugation at 10,000 � g for 10 min at roomtemperature. The amount of total protein in each pooled BAL sample wasdetermined by a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Eachsample was concentrated and exchanged into Agilent buffer A (Agilent Tech-nologies, Inc., Santa Clara, CA) containing 0.2% TX-100 by use of an AmiconUltra 5000 nominal molecular weight limit spin concentrator (Millipore, Bil-lerica, MA). Two hundred micrograms of protein from each sample was depletedof albumin, transferrin, and immunoglobulin (Ig) by use of a mouse multipleaffinity removal system (Agilent). The depletion was performed according to themanufacturer’s recommendations except that Agilent buffer A was replaced withAgilent buffer A containing 0.2% TX-100 to reduce protein aggregation. Eachdepleted BAL sample was concentrated and exchanged into 50 mM ammoniumbicarbonate by use of an Amicon Ultra 5000 nominal molecular weight limit spinconcentrator (Millipore) and frozen at �80°C.

Fractionation and LC–MS-MS analysis of depleted BAL samples. DepletedBAL samples were fractionated by strong cation exchange (SCX) or 1D sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to LC-tandem MS (LC–MS-MS). Prior to SCX fractionation, depleted BAL sampleswere lyophilized and then dissolved in 0.5 ml 50 mM ammonium bicarbonate andreduced in 5 mM Tris(2-carboxyethyl) phosphine (Sigma) for 30 min at 50°C.Cysteines were alkylated with 20 mM iodoacetamide (Sigma) for 60 min at roomtemperature in the dark. The alkylation reaction was quenched with 20 mMdithiothreitol (Sigma) for 5 min at room temperature. Each sample was digestedwith 1 �g of sequencing-grade trypsin (Promega, Madison, WI) at pH 8 for 18 hat 37°C. Digestion was confirmed using SDS-PAGE. Each digested sample wasfractionated using SCX (polysulfolethyl A; PolyLC, Inc., Columbia, MD). Eachsample was brought up to 1 ml of buffer A (5 mM KH2PO4, 25% acetonitrile, pH2.7), and the pH was adjusted to 2 with 10% phosphoric acid. A 40-min gradientwas run from 100% buffer A to 100% buffer B (5 mM KH2PO4, 25% acetonitrile,0.35 M KCl, pH 2.7), and absorbance was recorded at 214 nm and 284 nm.Fractions were collected every 2 min and were combined into a total of sevenfinal fractions based upon UV absorbance signal. The seven fractions weredesalted using C18 ultramicrospin columns (The Nest Group, Inc., Southbor-ough, MA).

One depleted BAL sample pooled from 10 mice that were mock infected for30 min was fractionated by 1D SDS-PAGE rather than SCX due to the presenceof interfering and unidentifiable contaminants that made LC–MS-MS analysisimpossible. The sample was separated in a Novex NuPAGE 4 to 12% bis-Tris gel(Invitrogen, Carlsbad, CA) and stained with Coomassie blue. The lane wasexcised into seven slices: 10 to 20 kDa, 20 to 40 kDa, 40 to 50 kDa, 50 to 60 kDa,60 to 85 kDa, 85 to 120 kDa, and 120 to 190 kDa. Each slice was cut into 1-mm3

VOL. 76, 2008 AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA 5863

pieces, washed three times with water followed by 50% acetonitrile, and thendehydrated with pure acetonitrile. The Coomassie stain was removed with twowashes with 100 mM ammonium bicarbonate mixed 1:1 with acetonitrile. The gelslices were reduced with 10 mM dithiothreitol at 50°C and alkylated with 55 mMiodoacetamide for 45 min in the dark at room temperature. The pieces weredried, rehydrated with 1 �g sequencing-grade trypsin (Promega) in 50 mMammonium bicarbonate, and incubated for 18 h at 37°C. The digested peptideswere extracted from the gel using 20 mM ammonium bicarbonate and acetoni-trile washes followed by 5% acetic acid and acetonitrile washes.

An LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, CA)was used with an in-house-fabricated micro-electrospray ionization source andan HP1100 nanoflow solvent delivery system (Agilent). Samples were automat-ically delivered by an Agilent microwell plate autosampler to a 100-�m-internal-diameter fused silica capillary precolumn packed with 2 cm of 200-Å-pore-sizeMagic C18AQ material (Michrom Bioresources, Auburn, CA), as describedelsewhere (68). The samples were washed with solvent A (0.1% formic acid, 5%acetonitrile) on the precolumn, eluted with a gradient of 10 to 35% solvent B(100% acetonitrile) over 30 min to a 75-�m by 10-cm fused silica capillarycolumn packed with 100-Å-pore-size Magic C18AQ material (Michrom Biore-sources), and then delivered into the mass spectrometer at a constant column tipflow rate of 250 nl/min. Eluting peptides were analyzed by micro-LC-MS anddata-dependent micro-LC–MS-MS acquisition, selecting three precursor ions forMS with a dynamic exclusion of 1 (21).

Proteomics data analysis. The MS-MS scans from each LC–MS-MS run wereconverted from the .RAW file format to mzXML files by use of the programReAdW.exe (version 1.0; Institute for Systems Biology, Seattle, WA). The da-tabase search program X!Tandem (12), included in the Computational Portaland Analysis System (CPAS version 1.4) (48), was used for peptide identificationof the MS-MS spectra. The Comet scoring function (38) was used in place of thedefault X!Tandem scoring function. The following parameters were used in thedatabase search: trypsin enzyme specificity, peptide mass tolerance of 2.5 Da,fragment ion tolerance of 0.5 Da, monoisotopic molecular weight for bothpeptide and fragment ion masses, b/y ion search, variable modification at M of�15.995, and static modification at C of �57.1. The database was searchedagainst a combined database consisting of the mouse International Protein Index(IPI) version 3.22, S. aureus COL (version NC_002951.1), and a list of contam-inants. In addition, randomly reshuffled versions of each database were ap-pended. This resulted in a database of 113,804 sequences being searched.

A composite peptide identification score was generated from the X!Tandemoutput based upon a combination of the Comet score, delta (relative differenceto the second-best match), expectation value, percentage of matching ions,charge state, peptide length, and delta mass (difference between the observedand theoretical masses) by use of the logistic identification of peptide sequences(LIPS) model (24). Experiment-specific peptide identification probabilities weregenerated from the distribution of reshuffled peptide matches (25). A minimumof 90% identification certainty was used to accept a peptide spectrum identifi-cation. This resulted in an overall peptide false-positive identification rate of0.8% (with a 95% confidence interval of 0.7 to 0.9%) based on reshuffleddatabase matches. Protein identification for each experimental condition wasbased on four levels of certainty (very high, high, medium, and low). All proteinsidentified by two or more unique peptides were classified with a “very high” levelof certainty. Single-hit proteins were classified in the remaining three certaintylevels based on the LIPS model by using peptide identification probability andpeptide length (25). The estimated false discovery rates for the four categoriesare 0.1%, 1.5%, 24%, and 48% (with 95% confidence intervals of 0.0 to 0.4%, 0.3to 2.9%, 13 to 36%, and 40 to 58%, respectively).

The IPI mouse database contains a large number of redundant peptide se-quences, and these redundant sequences generate different randomly reshuffledsequences, resulting in the reshuffled database having more unique sequences.Therefore, the false-positive error rates need to be multiplied by the ratio ofunique peptide sequences in the target database to the number of unique se-quences in the reshuffled database; otherwise, the number of false positives willbe overestimated. The ratio of unique peptide sequences in the target databaseto the number of unique sequences in the reshuffled database was estimated tobe 60%, so the false discovery rate is estimated to be the number of reshuffledpeptides divided by the number of target peptides multiplied by the percentageof unique sequences. Confidence intervals were generated by assuming Poissondistributions for the numbers of reshuffled and false target peptide or proteinidentifications.

Table S1 in the supplemental material contains all of the proteins identifiedfrom MS-MS spectra using X!Tandem. Because the same protein can havemultiple IPI entries, the information in Table S1 in the supplemental materialwas condensed into Table S2 in the supplemental material. Condensation of the

protein list in Table S1 in the supplemental material was accomplished bysearching the following online databases: the mouse IPI database (http://www.ebi.ac.uk/IPI/IPIhelp.html), the Swiss-Prot and TrEMBL databases (www.expasy.org), the Gene Ontology (GO) database (www.geneontology.org), andthe PubMed database (www.ncbi.nlm.nih.gov). When a single protein had mul-tiple IPI entries, all of the lines were combined into a single line entry in TableS2 in the supplemental material. The numbers of unique peptides and totalpeptides identified for each protein were combined in Table S2 in the supple-mental material, and the confidence of the protein identification was adjusted ifnecessary. Only proteins that were identified with high or very high confidenceunder at least one treatment condition (30 min or 6 h, mock infected or infected)were retained in Table S2 in the supplemental material. All keratin identifica-tions were eliminated as well, because they are likely a result of keratin contam-ination during sample processing. By use of these criteria, all S. aureus proteinsidentified in the airway were eliminated due to low confidence of protein iden-tification.

SDS-PAGE and Western immunoblotting. A 30-�l aliquot of each pooledBAL sample (30 min and 6 h mock infected, 30 min and 6 h infected) was mixedwith 10 �l 4� Laemmli buffer (33) and boiled for 5 min. The samples wereseparated by SDS-10% PAGE. Gels were stained for 16 h with Sypro ruby(Bio-Rad) and destained in methanol-acetic acid-water (10:7:83) for at least 1 hprior to visualization using a gel documentation system (Bio-Rad Laboratories,Inc., Hercules, CA). For Western blotting, proteins were transferred to nitro-cellulose membranes by use of a semidry transblotter (Bio-Rad). All incubationswere carried out with 5% skim milk and 0.05% Tween 20 (Fisher Scientific,Pittsburgh, PA) in PBS at room temperature. Detecting antibodies wereIRDye800-conjugated goat anti-mouse IgG (Rockland Immunochemicals, Inc.,Gilbertsville, PA), rabbit anti-human transferrin (Research Diagnostics, Inc.,Concord, MA), rabbit anti-mouse matrix metalloproteinase 9 (MMP-9) (AffinityBioreagents, Golden, CO), rabbit anti-mouse plasminogen (Molecular Innova-tions, Southfield, MI), and goat anti-mouse C3 (Bethyl Laboratories, Inc., Mont-gomery, TX). The secondary antibodies were goat anti-rabbit Ig–Alexa Fluor 680and donkey anti-goat Ig–Alexa Fluor 680 (Invitrogen). Fluorescence was de-tected using an Odyssey infrared imaging system (LI-COR Biotechnology, Lin-coln, NE).

Gelatin zymography. A 30-�l aliquot of each BAL sample (30 min and 6 hmock infected, 30 min and 6 h infected) was mixed with 10 �l 4� Laemmli bufferwithout reducing agent (33). Samples were separated by SDS-PAGE in 10% gelscontaining 1% gelatin (Bio-Rad, Hercules, CA). Following electrophoresis, gelswere washed twice at room temperature with 2.5% TX-100 for 30 min each andthen incubated for 16 to 18 h at 37°C in buffer composed of 50 mM Tris, pH 7.5,10 mM CaCl2, and 150 mM NaCl. Gels were stained with 0.5% Coomassiebrilliant blue (Bio-Rad), destained briefly with 40% methanol and 10% aceticacid, and imaged using the gel documentation system described above.

RESULTS AND DISCUSSION

S. aureus replicates in the lungs of mice. To explore the earlyhost-pathogen interactions that occur during the developmentof acute S. aureus pneumonia, we sought to develop a mousemodel in which the bacteria actively replicate in the lungs tocause pneumonia. Mice were infected i.n. under anesthesiawith 3 � 107, 1 � 108, or 3 � 108 CFU of JP1 to identify a dosethat would result in bacterial replication in the lungs. All of themice exhibited signs of illness, including hunched posture, pi-loerection, labored breathing, immobility, and loss of resis-tance to handling, by 6 h postinoculation at each dose tested.Mice inoculated with doses of 3 � 107 CFU and 1 � 108 CFUwere ill for 24 to 36 h and then cleared the infection. Incontrast, a dose of 3 � 108 CFU caused mortality in 71% of themice, with a median time to death of 32.5 h (P 0.0008compared to mice inoculated with 3 � 107 or 1 � 108 CFU).Lungs were harvested from mice sacrificed 0.5 h, 24 h, 48 h,and 96 h postinoculation (doses of 3 � 107 CFU and 1 � 108

CFU) or from mice sacrificed 0.5 h postinoculation or follow-ing death due to S. aureus infection (dose of 3 � 108 CFU).Enumeration of S. aureus bacteria in the lungs at 30 minpostinoculation revealed deposition of 3 to 10% of the i.n.

5864 VENTURA ET AL. INFECT. IMMUN.

inoculum (Fig. 1). Bacterial replication was observed in thelungs of mice infected with 3 � 108 CFU, reaching a meandensity of 1.54 � 108 � 1.44 � 108 CFU/lungs at death (com-pared to a mean deposition of 2.91 � 107 � 1.29 � 107

CFU/lungs) (Fig. 1). In contrast, bacteria were graduallycleared from the lungs of mice inoculated with 3 � 107 or 1 �108 CFU (Fig. 1). Similar levels of bacterial clearance, mor-bidity, and mortality were observed when mice were inoculatedwith the laboratory strain RN6390 at the same doses (data notshown), indicating that the results obtained with JP1 were notstrain specific. These data demonstrate that a dose of 3 � 108

CFU of JP1 or RN6390 was sufficient to cause pneumonia thatcould result in mortality, while a dose of 1 � 108 CFU or lowerresulted in symptoms of pneumonia that lasted for 24 to 36 h,followed by bacterial clearance and disease resolution. Thisdose-related mortality is similar to what has been reported withintratracheal inoculation of adult mice (13, 42) and, morerecently, with i.n. inoculation using exponentially growing S.aureus bacteria (32, 60). A dose of 3 � 108 CFU JP1 was usedfor all subsequent experiments.

Proinflammatory cytokines and chemokines recruit neutro-phils during early infection. The concentrations of airway cy-tokines and chemokines were determined as a measure of theinitial inflammatory response to inoculation with S. aureus orPBS. The levels of proinflammatory TNF-�, MIP-2, and KC inthe BAL fluid were elevated 30 min postinoculation with S.aureus compared to levels for the mock-infected controls. Inaddition, the levels of TNF-�, KC, MIP-2, IL-1�, IL-6, andGM-CSF in the airways of infected mice were significantlyhigher 6 h postinoculation than the levels in BAL fluid from30-min-infected and 6-h-mock-infected animals (Fig. 2A to F).In contrast, levels of anti-inflammatory IL-10, IL-12p70, IL-17,and gamma interferon were not increased significantly abovebackground in any of the groups during the first 6 h of infection(data not shown). These data show that a measurable proin-flammatory cytokine and chemokine response was initiated by30 min and increased significantly by 6 h postinoculation withS. aureus.

One of the major functions of proinflammatory cytokines

and chemokines is to recruit PMNs from the bloodstream tothe site of an infection. To determine the kinetics of the PMNinflux during early S. aureus pneumonia, total cell counts anddifferential counts of the BAL fluids from infected and mock-infected mice were performed. The total number of BAL fluidcells increased 10-fold by 6 h postinoculation with S. aureus(Fig. 2G) as a result of PMN influx (Fig. 2H). In contrast, thetotal number of BAL fluid cells from mock-infected mice didnot change significantly during the first 6 h, despite a modestPMN response after instillation of PBS into the lung. Thenumbers of mononuclear cells remained relatively constant(Fig. 2I), regardless of treatment or time point. These dataindicate that mice respond rapidly to S. aureus airway chal-lenge by releasing proinflammatory cytokines and chemokines,which act to recruit PMNs to the affected area.

S. aureus causes consolidated pneumonia. To assess the con-sequences of S. aureus infection of the airway histologically,lung specimens were stained with H&E and examined micro-scopically in a blind manner. H&E-stained lung sections takenfrom mice at 30 min postinoculation with either PBS or S.aureus were histologically similar except for the presence of afew, widely scattered intra-alveolar macrophages containing S.aureus in infected mice (Fig. 3, 30 min S. aureus). Otherwise,the lungs of the 30-min-mock-infected and 30-min-infectedmice were normal. In the 6-h-mock-infected animals, minimalneutrophilic inflammation was observed in widely scatteredlocations (Fig. 3, 6 h PBS). In contrast, lungs from 6-h-infectedmice had multiple, frequently confluent foci of inflammation,with various degrees of severity (Fig. 3, 6 h S. aureus). Theinflux of PMNs into small vessels and capillaries was pro-nounced (Fig. 3, 6 h S. aureus, �40 inset), leading to thethickening of alveolar walls and, in more severely affectedareas, the diffuse accumulation of PMNs within alveolarspaces. In some areas, consolidation of the air spaces withconcomitant loss of alveolar detail was observed. In the mostsevere foci, fibrin accumulation, thrombosis, and necrosis wereevident, with an increase in the number of free S. aureusbacteria. Our data demonstrate that S. aureus infection of theairway results in the rapid development of consolidating pneu-monia.

The composition of the airway proteome changes duringearly pneumonia. One of the hallmarks of an inflammatoryresponse is an increase in the amount of total protein presentin the infected area as a result of the local production andinflux of inflammatory mediators in response to cytokine andchemokine recruitment. To determine whether this occurredin our system, the total protein contents of BAL fluids frommock-infected and infected mice were measured using a BCAassay. As shown in Fig. 4A, the amounts of protein in BALsamples at 30 min, regardless of treatment, and in 6-h-mock-infected BAL samples were between 82 and 104 �g/ml. Incontrast, more than double that amount (213 �g/ml) waspresent in the 6-h-infected animals (P � 0.05). SDS-PAGEanalysis of BAL samples from mock-infected (30 min and 6 h)and infected (30 min and 6 h) mice indicated the presence of50-, 67-, and 78-kDa proteins in high abundance (Fig. 4B, lane1). Based upon molecular mass and immunoblot analyses,these proteins were predicted to be immunoglobulin (Fig. 4C),albumin, and transferrin (Fig. 4D), respectively. An affinityremoval system was used to deplete the BAL samples of these

FIG. 1. S. aureus replicates in the lungs of mice infected with 3 �108 CFU. Twelve mice at each dose were inoculated i.n. with 3 � 107

CFU (f), 1 � 108 CFU (Œ), or 3 � 108 CFU (�) S. aureus JP1.Three mice inoculated with 3 � 107 and 1 � 108 CFU were sacri-ficed at each of four time points (0.5, 24, 48, and 96 h postinocu-lation). Three mice inoculated with 3 � 108 CFU were sacrificed0.5 h postinoculation; the remaining mice succumbed to the infec-tion. Bacteria were enumerated from homogenized lungs. Eachsymbol represents three mice, except 3 � 108 CFU at 17 h (n 4),24 h (n 2), 41 h (n 2), and 46 h (n 1).

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proteins, which represent 75 to 85% of the total BAL protein(3), so as to increase the probability of identifying the less-abundant proteins during subsequent MS sampling. SDS-PAGE (Fig. 4B, lane 2) and Western blot (not shown) analysesof native and depleted samples demonstrated that depletionremoved all of the detectable immunoglobulin, albumin, andtransferrin.

A shotgun proteomics approach was utilized to determinehow the composition of the airway proteome changed duringthe course of early pneumonia. The depleted BAL sampleswere digested with trypsin, and the peptides were subjected toSCX fractionation and LC–MS-MS. The peptide sequencesgenerated by analysis of the MS-MS data were matched tomouse and S. aureus proteins using the combined databases ofthe mouse IPI and the S. aureus COL proteome. A total of1,096 mouse and 19 S. aureus proteins were identified in theairways of mice inoculated for 30 min or 6 h with PBS or S.aureus (see Table S1 in the supplemental material). A fewpeptides were identified as serum albumin, while no peptides

were assigned as transferrin or immunoglobulin, showing thatthe depletion of these three overabundant proteins was re-markably efficient. One potential concern with depletion is thatwe might remove a significant number of proteins that areassociated with albumin, transferrin, or immunoglobulin (ei-ther in the BAL fluid or attached to the cartridge resin). Be-cause our goal in this study was to identify potential targets forthe molecular characterization of specific host-pathogen inter-actions, we determined that the advantages of depleting theoverabundant proteins from all BAL samples prior to MS-MSanalysis outweighed the potential losses that may have oc-curred.

The list of proteins in Table S1 in the supplemental materialwas refined into Table S2 in the supplemental material bycombining multiple entries for a given protein into a single lineof the table, as described in Materials and Methods, so as toobtain a more biologically useful list of proteins. A total of 727unique host proteins were identified with high or very highconfidence in the airway under one or more of the treatment

FIG. 2. Proinflammatory cytokines and chemokines are released and recruit PMNs to the airway in response to S. aureus. Each symbolrepresents one mouse. The data are combined from three independent experiments with seven to eight mice per experiment. The bar for each dataset represents the median value for 21 or 22 mice per condition. Statistical comparisons were made using the Kruskal-Wallis test with Dunn’sposttest. SA, S. aureus; MN, mononuclear cells. �, P � 0.05; †, P � 0.01; #, P � 0.001.

5866 VENTURA ET AL. INFECT. IMMUN.

conditions (see Table S2 in the supplemental material). All ofthe identified S. aureus proteins were disregarded because theconfidence of the identifications for these proteins was belowthe cutoff for further consideration (identification using a sin-gle peptide with low to medium confidence, which resulted infalse discovery rates of 48 and 24%, respectively). Of the 727total proteins, 458 (63%) were identified using two or morepeptides, which increases the confidence that the protein towhich the peptide was assigned was correct (the false discoveryrate for identification using at least two peptides was 0.1%,compared with 1.5% for high-confidence identifications using asingle peptide).

Relative levels of abundance of airway cytoplasmic and ex-tracellular proteins are reversed as a result of S. aureus infec-tion. The identified proteins were assigned to the cellular-component and biological-process GO categories. Many of themouse proteins identified in this proteomics screen were notassigned by GO to cellular-component or biological-processcategories, so the assignments were generated manually duringthe refinement process described above. We chose to analyzethe percentages of proteins present in given GO categoriesbecause the raw numbers of identified proteins in those cate-gories were not meaningful, as a result of experimental vari-ability in the total numbers of proteins identified per condition.The relative percentages of proteins in different GO categorieswere more stable and, therefore, more meaningful to compare.There was no difference in the percentages of total proteinsassigned to any of the GO subcategories among both 30-minsamples (mock infected and infected) and the 6-h-mock-in-fected sample (analysis not shown). The similarity of the sam-ples from these treatment conditions suggests that any changesin the airway proteome immediately following inoculation withS. aureus and in the first 6 h following PBS inoculation are toosubtle to be detected using current methodologies. In partic-

ular, the cytokine response observed within 30 min postinocu-lation with S. aureus (Fig. 2A to F) was not detected on aproteomic level, most likely due to the low molecular weightand low relative abundance of cytokines and chemokines.Thus, the data from the 30-min-mock-infected, 30-min-in-fected, and 6-h-mock-infected samples were combined intoone “control” group to provide for a more rigorous analysis ofthe inflammatory response elicited by S. aureus 6 h followinginoculation (Fig. 5). We also observed that the airway pro-teome from uninfected mice was nearly identical to the pro-teome of samples obtained from mice subjected to any of thetreatment conditions in the control group (our unpublishedobservations). In the airway proteome of the control group,27% of the proteins were extracellular (Fig. 5A) and the re-maining 73% were localized to various compartments withinthe host cell, including the cell membrane (10%). In contrast,41% of the proteins identified in the 6-h-infected BAL fluidwere extracellular, while only 25% were cytoplasmic (com-pared to 38% in the control airway proteome). The increase inrelative abundance of extracellular proteins between the 6-h-infected and control samples (41% versus 27%) is indicative ofthe local production and release of proinflammatory proteins,as well as the influx of acute-phase reactants from the blood;both events occur rapidly during acute inflammation. The pres-ence of cytoplasmic and other intracellular proteins in theextracellular milieu of the airway likely results from lysis ofhost cells as a result of normal cell turnover, apoptosis,

X X

FIG. 3. Histopathology shows signs of consolidated pneumonia ininfected animals but not mock-infected animals. Representative low(�10)- and high (�40)-power histologic sections of lungs from miceinfected with S. aureus for 30 min and 6 h or mock infected (PBS) for6 h. The inset (6 h S. aureus, �40) shows a smaller vessel that isthrombosed. Bar 100 �m.

FIG. 4. Depletion of BAL fluid removes overabundant proteins.(A) Total protein in pooled BAL samples from mock-infected (whitebars) and infected (gray bars) mice was measured by BCA assay.Results are from three pooled BAL samples. �, P � 0.05. (B) SDS-PAGE gel stained with Sypro ruby, showing native BAL fluid (lane 1)and depleted BAL fluid (lane 2) from 6-h-infected mice. Equivalentvolumes of each sample were separated in the gel. (C and D) Westernblots of BAL samples from 30-min-mock-infected (lane 1), 30-min-infected (lane 2), 6-h-mock-infected (lane 3), and 6-h-infected (lane 4)mice, probed with antibodies against mouse immunoglobulin (C) andhuman transferrin (D). Equivalent volumes of each native BAL samplewere separated in the gel prior to immunoblotting. Molecular massesin kilodaltons are shown on the right side of each panel.

VOL. 76, 2008 AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA 5867

necrosis, and/or S. aureus-mediated cytolysis (in the case ofthe 6-h-infected sample).

Inflammatory and coagulation proteins dominate the earlyresponse to S. aureus in the airway. Assignment of the identi-fied proteins into the biological-process GO category revealedan increase in the percentages of inflammatory and coagula-tion proteins in the airways of 6-h-infected mice compared tothose for mice in the control group (5% and 14%, respectively,at 6 h compared with 2% and 6% in control airways) (Fig. 5),which correlates with the influx of inflammatory cells and me-diators triggered by the early cytokine response (Fig. 2). Theinflammatory proteins identified in the airway are shown inTable 1. Antimicrobial peptides and peptidoglycan recognitionproteins have direct antibacterial properties on the S. aureusmembrane and cell wall peptidoglycan, respectively, that canultimately result in bacterial cell lysis. Multimers of cathelici-din antimicrobial peptide (CRAMP) and myeloid bactenecinbind to bacterial membranes and create pores (47). Peptidogly-can recognition proteins exhibit N-acetyl-muramoyl-L-alanine-amidase activity that cleaves the stem peptide of the pepti-doglycan, eventually resulting in bacterial cell lysis (51).Mannose binding lectin A (MBL-A) and MBL-C are lectinsthat have been shown to opsonize S. aureus to promote phago-cytosis (40). Nearly every component of the complement cas-cade was identified in the airway. Thus, the components re-quired for the activation of complement via the classical(immunoglobulins), alternative (C3, factors B and D, and pro-perdin), and lectin (MBL-A and MBL-C) pathways werepresent in the airway in response to S. aureus infection. WhileS. aureus is not susceptible to lysis by the membrane attackcomplex (11), opsonization by any of these proteins facilitatesphagocytic uptake of the bacteria by macrophages and PMNs.Complement-mediated opsonization of bacteria may also beaugmented by serum amyloid protein P (69), which was also

differentially present in the BAL fluids of infected versusmock-infected mice. In a related study, we found that S. aureuswas associated with or internalized by 65% of alveolar macro-phages within 30 min of infection and by 25% of PMNs after6 h in the airway; in addition, we found that C3, Ig, and MBL-Cwere associated with the surface of the bacteria (58). Previousstudies by other groups demonstrated that S. aureus wasphagocytosed by alveolar macrophages within 30 min followingaerosol inoculation as well (18, 28, 34).

The primary function of MMP-8 and -9 is to facilitate thedegradation of extracellular matrix components formed by thehost in response to injury; however, both have also been im-plicated in acute inflammation. Both MMPs are stored in PMNgranules and are released during an acute inflammatory re-sponse. MMP-8, also known as neutrophil collagenase, pro-motes balanced PMN recruitment during acute inflammationand resolution of PMN influx during chronic inflammation(57). The proteolytic activity of MMP-9, also known as gela-tinase B, cleaves the proforms of the early proinflammatorycytokines IL-8, TNF-�, transforming growth factor �, andIL-1� to their active forms (8, 43). In addition, MMP-9 isknown to form complexes with neutrophil gelatinase-associ-ated lipocalin to prevent the autodegradation of MMP-9 (66).Thus, MMPs play an active role in establishing and maintain-ing an appropriate inflammatory response.

The coagulation proteins identified in the airways of miceinoculated with PBS or S. aureus are shown in Table 2. Inter-estingly, in addition to their role in recruiting neutrophils andinflammatory mediators to the site of an infection, the proin-flammatory cytokines TNF-�, IL-1, and IL-6 have been shownto activate coagulation pathways and attenuate fibrinolytic ac-tivity (52), which are hallmarks of alveolar inflammation (10).IL-6 activates bronchoalveolar coagulation via the tissue factor(extrinsic) pathway (37). The increase in IL-6 that we observed

B .

7%

25%

2%

41%

12%

6%4%

2% 1%actin cytoskeleton

cytoplas m

endoplas mic re ticulum

extrace llular space

me mbrane

mitochondrion

nucleus

ribo some

unknown

A.

6%

38%

2%27%

10%

8%4%3%

2%

D. 8%6%

5%1%2%

2%

14%

17%

10%

4%

9%

6%

12% 1%

3%

C . 8%6%

2%1%2%1%

6%

22%

3%9%

11%

9%

7%

10% 3%

inflamma tion

cytoskeleton

ce ll adhe sion

extrace llular ma trix

s tress response

de velopm ent

coagulation

me ta bo lismnucleic acid inte ractionsproteo lys is

protein synthe s is

regu lation

signa lling

transport

unknown

FIG. 5. A total of 727 proteins were identified in the airways of control and/or 6-h-infected mice (see Table S2 in the supplemental materialfor a complete list of the proteins). Shown are cellular-component GO categories for proteins identified in the airways of control (A) and6-h-infected (B) mice and biological-process GO categories for proteins identified in the airways of control (C) and 6-h-infected (D) mice. A totalof 658 proteins were identified in the control samples, and a total of 396 proteins were identified in the 6-h-infected samples.

5868 VENTURA ET AL. INFECT. IMMUN.

during early S. aureus airway infection (Fig. 2E) correspondswith an increase in the abundance of proteins involved incoagulation (Fig. 5D; Table 2) and the appearance of fibrindeposits in the airway, as evidenced histologically (Fig. 3, 6 h S.aureus). We also identified several of the proteins necessary forfibrin accumulation via the contact factor (intrinsic) pathway,including plasma kallikrein and coagulation factors V and X.Heparin cofactor 2 and antithrombin are downstream proteinsin both pathways that are involved in the activation of fibrin-ogen to fibrin. Plasminogen and alpha-2 antiplasmin are fi-brinolytic proteins that serve to balance the formation anddissolution of fibrin clots. S. aureus secretes staphylokinase, aprotein that activates plasminogen to plasmin; this process canbe augmented by CRAMP (5), which was also identified in theBAL fluid from 6-h-infected animals. Further studies to inves-tigate the involvement of staphylokinase, CRAMP, and plas-minogen activation in acute staphylococcal pneumonia are on-going in our laboratory. In addition to their roles in initiatingcoagulation, proteases of the coagulation system are active ininducing a proinflammatory response (37). Taken together,these proteomics data show an increase in proteins involved ininflammation and coagulation processes during the first 6 h ofS. aureus airway infection.

Alternative approaches confirm changes in airway proteomecomposition. Western blot analysis was performed to confirmthe presence and relative levels of abundance of complementcomponent C3, plasminogen, and MMP-9 in the different BALsamples. As seen in Fig. 6A, the amount of C3, which wasdetectable primarily as C3b and iC3b (based upon molecularmass), in both 6-h samples was greater than that in the 30-minsamples. In addition, lower-molecular-mass degradation prod-

TABLE 1. Inflammatory proteins present in the airways of infectedand/or mock-infected animals

Protein

Level of proteinidentificationa

Controlmice

6-h-infectedmice

Advanced glycosylation end product-specific receptor �� ��Alpha-1-acid glycoprotein �� ��C4b-binding protein � ��Cathelicidin antimicrobial peptide � ��CD44 antigen � �Chitinase 3-like 1 �� ��Chitinase 3-like 3 �� ��Complement C1r � ��Complement C1s � ��

Complement C3 �� ��Complement C4b �� ��Complement C5 �� ��Complement C5a �� ��Complement C6 �� ��Complement C7 �� ��Complement C8 alpha �� ��Complement C8 beta �� ��Complement C8 gamma �� ��

Complement C9 �� ��Complement factor B �� ��Complement factor D �� �Complement factor H �� ��Complement factor H-related protein � �Complement factor H-related protein � �Complement factor H-related protein C � �Complement factor I �� ��Complement factor P/properdin �� ��

Gamma interferon-inducible protein 30 �� �Leucine-rich alpha-2 glycoprotein �� ��Lipopolysaccharide-binding protein � �Long palate, lung, and nasal epithelium carcinoma-

associated protein 1�� ��

Long palate, lung, and nasal epithelium carcinoma-associated protein 3

� �

Macrophage migration inhibitory factor �� �Macrophage stimulatory protein � ��Mannose-binding lectin A � �Mannose-binding lectin C � ��

Matrix metalloproteinase 9 � ��Major histocompatibility complex �� ��Myeloid bactenecin �� ��Neutrophil collagenase/matrix metalloproteinase 8 � ��Neutrophil gelatinase-associated lipocalin �� ��Odorant binding protein 1F �� ��Odorant binding protein 1A �� ��Palate, lung, and nasal epithelium clone protein �� �Parotid secretory protein �� ��

Peptidoglycan recognition protein 1 � ��Peptidoglycan recognition protein 2 � ��Polymeric-immunoglobulin receptor �� ��Secreted phosphoprotein 1 �� ��Secretoglobin family 3A member 1 �� �Serum amyloid A-1 protein � ��Serum amyloid A-2 protein � �Serum amyloid A-4 protein � �Serum amyloid P component � ��

Small inducible cytokine B15 �� �Small inducible cytokine subfamily E, member 1 � �Whey acidic protein four-disulfide core domain

protein 12� �

a By use of the LIPS model (25), proteins were identified with very high confidence(��) or high confidence (�) or not identified (�). The total number of inflammatoryproteins identified in control mice was 41 (6.2% of the total proteins identified), and thetotal number for 6-h-infected mice was 54 (13.6% of the total proteins identified). Theabsolute number of proteins identified in a given category is not as useful an indicator ofchanges in the proteome as is the percentage of total proteins identified in that categorybecause there were differences in the total numbers of proteins identified as a result ofdifferences in MS sampling.

TABLE 2. Coagulation proteins present in the airways of infectedand/or mock-infected animals

Protein

Level of protein identificationa

Controlmice

6-h-infectedmice

Alpha-2 antiplasmin �� ��Antithrombin III �� ��Coagulation factor V � ��Coagulation factor X � ��Coagulation factor XIII � ��Factor XII �� ��Fibrinogen, alpha �� ��Fibrinogen, beta �� ��Fibrinogen, gamma �� ��

Heparin cofactor 2 �� ��High-molecular-weight

kininogen II�� ��

Hyaluronan-binding protein 2 � ��Kininogen 1 �� ��Plasma kallikrein �� ��Plasminogen �� ��Prothrombin �� ��Vitamin K-dependent protein S �� �Vitamin K-dependent protein Z � ��

a By use of the LIPS model (25), proteins were identified with very highconfidence (��) or high confidence (�) or not identified (�). The total numberof coagulation proteins identified in control mice was 15 (2.3% of the totalproteins identified), and the total number for 6-h-infected mice was 18 (4.5% ofthe total proteins identified). The absolute number of proteins identified in agiven category is not as useful an indicator of changes in the proteome as is thepercentage of total proteins identified in that category because there were dif-ferences in the numbers of total proteins identified as a result of differences inMS sampling.

VOL. 76, 2008 AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA 5869

ucts of C3b were present in the 6-h-infected sample, indicatingthat cleavage of C3b occurred in the airways of these animals.Plasminogen and MMP-9, both of which have been shown toincrease during active infection (5, 10, 14, 19, 22, 67), were alsomore abundant in the 6-h-infected samples than in the othersamples (Fig. 6B and C). Western blot analyses of these pro-teins were performed on the BAL fluid of mice inoculated withlower doses of S. aureus (3 � 107 and 1 � 108 CFU), withsimilar results (data not shown). Further evidence for the pres-ence of MMP-9 in the 6-h-infected sample was obtained usinggelatin zymography, which is more sensitive than Western blot-ting (Fig. 6D). The zymogram showed that the predominantform of MMP-9 in BAL fluid was the proform (92 kDa), whichis not uncommon, as the activated form (86 kDa) is typicallyfound tightly associated with extracellular matrix componentsthat are not removed during routine lavage. Several MMP-9complexes were also observed using zymography; MMP-9 isknown to form homomultimers as well as heterodimers withneutrophil gelatinase-associated lipocalin (29), which wasidentified in our proteomics screen (see Table S2 in the sup-plemental material). These data confirm the proteomics datashowing that C3 and plasminogen were present in the airwaysof mock-infected mice and were increased in BAL fluid frominfected mice and that MMP-9 was present only in the airwaysof mice infected for 6 h. Further, they demonstrate the utilityof a shotgun proteomics approach for characterizing changesin the proteome of a biological fluid during the course of aninfection.

Conclusions. In this report, we describe marked alterationsin the airway proteome that accompany the inflammatory re-sponse during the first 6 h of murine staphylococcal pneumo-nia. We have combined immunology and cell biology tech-niques with a proteomics approach to define the initial eventsin S. aureus pneumonia. The data presented here provide acritical first step toward understanding the complex interac-tions between S. aureus and the airway at the onset of pneu-monia. The use of shotgun proteomics provided us with an

unparalleled opportunity to define the protein changes withinthe airway during the first 6 h following staphylococcal chal-lenge. We have demonstrated for the first time that S. aureuselicits a rapid and vigorous inflammatory response within thefirst 6 h of infection. In fact, as early as 30 min after bacterialinoculation, we observed the release of proinflammatory cyto-kines and chemokines, which recruited PMNs and antimicro-bial mediators to the airway. Within 6 h postinoculation with S.aureus, the airway proteome was altered dramatically to in-clude an increase in antimicrobial peptides, opsonins, proin-flammatory mediators, and coagulation proteins, many ofwhich may play key roles in the pathogenesis of acute bacterialpneumonia. These studies provide the foundation for futureanalyses investigating specific host-pathogen interactions thatoccur during the early stages of S. aureus pneumonia.

ACKNOWLEDGMENTS

We thank Jeannette Crisostomo, Destry Taylor, and Michele Timkofor their expert technical assistance, Jimmy Eng for assistance withproteomics data analysis, William Parks for advice about MMP-9 anal-ysis, and Amanda Jones for insightful comments and critical review ofthe manuscript.

This work was supported by HL073996 from the National Institutesof Health (C.E.R.).

REFERENCES

1. American Thoracic Society. 2005. Guidelines for the management of adultswith hospital-acquired, ventilator-associated, and healthcare-associatedpneumonia. Am. J. Respir. Crit. Care Med. 171:388–416.

2. Balachandran, P., A. Brooks-Walter, A. Virolainen-Julkunen, S. K. Holling-shead, and D. E. Briles. 2002. Role of pneumococcal surface protein C innasopharyngeal carriage and pneumonia and its ability to elicit protectionagainst carriage of Streptococcus pneumoniae. Infect. Immun. 70:2526–2534.

3. Bell, D. Y., J. A. Haseman, A. Spock, G. McLennan, and G. E. Hook. 1981.Plasma proteins of the bronchoalveolar surface of the lungs of smokers andnonsmokers. Am. Rev. Respir. Dis. 124:72–79.

4. Bowler, R. P., B. Duda, E. D. Chan, J. J. Enghild, L. B. Ware, M. A. Matthay,and M. W. Duncan. 2004. Proteomic analysis of pulmonary edema fluid andplasma in patients with acute lung injury. Am. J. Physiol. Lung Cell. Mol.Physiol. 286:L1095–L1104.

5. Braff, M. H., A. L. Jones, S. J. Skerrett, and C. E. Rubens. 2007. Staphylo-coccus aureus exploits cathelicidin antimicrobial peptides produced duringearly pneumonia to promote staphylokinase-dependent fibrinolysis. J. Infect.Dis. 195:1365–1372.

6. Brown, J. S., T. Hussell, S. M. Gilliland, D. W. Holden, J. C. Paton, M. R.Ehrenstein, M. J. Walport, and M. Botto. 2002. The classical pathway is thedominant complement pathway required for innate immunity to Streptococ-cus pneumoniae infection in mice. Proc. Natl. Acad. Sci. USA 99:16969–16974.

7. Candiano, G., M. Bruschi, N. Pedemonte, E. Caci, S. Liberatori, L. Bini, C.Pellegrini, M. Vigano, B. J. O’Connor, T. H. Lee, L. J. Galietta, and O.Zegarra-Moran. 2005. Gelsolin secretion in interleukin-4-treated bronchialepithelia and in asthmatic airways. Am. J. Respir. Crit. Care Med. 172:1090–1096.

8. Chakrabarti, S., and K. D. Patel. 2005. Matrix metalloproteinase-2 (MMP-2)and MMP-9 in pulmonary pathology. Exp. Lung Res. 31:599–621.

9. Chastre, J., and J. Y. Fagon. 2002. Ventilator-associated pneumonia. Am. J.Respir. Crit. Care Med. 165:867–903.

10. Choi, G., M. J. Schultz, J. W. van Till, P. Bresser, J. S. van der Zee, M. A.Boermeester, M. Levi, and T. van der Poll. 2004. Disturbed alveolar fibrinturnover during pneumonia is restricted to the site of infection. Eur. Respir.J. 24:786–789.

11. Cooper, N. R. 1991. Complement evasion strategies of microorganisms. Im-munol. Today 12:327–331.

12. Craig, R., and R. C. Beavis. 2004. TANDEM: matching proteins with tandemmass spectra. Bioinformatics 20:1466–1467.

13. DeMaria, T. F., and F. A. Kapral. 1978. Pulmonary infection of mice withStaphylococcus aureus. Infect. Immun. 21:114–123.

14. D’Haese, A., A. Wuyts, C. Dillen, B. Dubois, A. Billiau, H. Heremans, J. VanDamme, B. Arnold, and G. Opdenakker. 2000. In vivo neutrophil recruit-ment by granulocyte chemotactic protein-2 is assisted by gelatinaseB/MMP-9 in the mouse. J. Interferon Cytokine Res. 20:667–674.

15. Fajardo, I., L. Svensson, A. Bucht, and G. Pejler. 2004. Increased levels ofhypoxia-sensitive proteins in allergic airway inflammation. Am. J. Respir.Crit. Care Med. 170:477–484.

A .

75

37

50

1 2 3 4

C.

10075

1 2 3 4

B.

75

100

1 2 3 4

D.

Pro MMP-9MMP-9

MMP-9 comp lexes

FIG. 6. (A to C) Western blotting confirms the presence and rela-tive levels of abundance of C3 (A), plasminogen (B), and MMP-9 (C).Equal volumes of BAL samples from 30-min-mock-infected (lane 1),30-min-infected (lane 2), 6-h-mock-infected (lane 3), and 6-h-infected(lane 4) mice were separated by SDS-PAGE and subjected to Westernimmunoblotting. Molecular mass markers in kilodaltons are shown tothe right of each blot. (D) Gelatin zymography of BAL fluid from6-h-infected mice demonstrates the presence of activated MMP-9 (86kDa), proform (Pro) MMP-9 (92 kDa), and MMP-9 complexes (�100kDa).

5870 VENTURA ET AL. INFECT. IMMUN.

16. Fehniger, T. E., J. G. Sato-Folatre, J. Malmstrom, M. Berglund, C. Lind-berg, C. Brange, H. Lindberg, and G. Marko-Varga. 2004. Exploring thecontext of the lung proteome within the airway mucosa following allergenchallenge. J. Proteome Res. 3:307–320.

17. Gomez, M. I., A. Lee, B. Reddy, A. Muir, G. Soong, A. Pitt, A. Cheung, andA. Prince. 2004. Staphylococcus aureus protein A induces airway epithelialinflammatory responses by activating TNFR1. Nat. Med. 10:842–848.

18. Green, G. M., and E. H. Kass. 1964. The role of the alveolar macrophage inthe clearance of bacteria from the lung. J. Exp. Med. 119:167–176.

19. Gronlund, U., C. Hallen Sandgren, and K. Persson Waller. 2005. Haptoglobinand serum amyloid A in milk from dairy cows with chronic sub-clinicalmastitis. Vet. Res. 36:191–198.

20. Guo, Y., S. F. Ma, D. Grigoryev, J. Van Eyk, and J. G. Garcia. 2005. 1-DEMS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome.Proteomics 5:4608–4624.

21. Gygi, S. P., Y. Rochon, B. R. Franza, and R. Aebersold. 1999. Correlationbetween protein and mRNA abundance in yeast. Mol. Cell. Biol. 19:1720–1730.

22. Hartog, C. M., J. A. Wermelt, C. O. Sommerfeld, W. Eichler, K. Dalhoff, andJ. Braun. 2003. Pulmonary matrix metalloproteinase excess in hospital-ac-quired pneumonia. Am. J. Respir. Crit. Care Med. 167:593–598.

23. Heyer, G., S. Saba, R. Adamo, W. Rush, G. Soong, A. Cheung, and A. Prince.2002. Staphylococcus aureus agr and sarA functions are required for invasiveinfections but not inflammatory responses in the lung. Infect. Immun. 70:127–133.

24. Higdon, R., J. M. Hogan, G. Van Belle, and E. Kolker. 2005. Randomizedsequence databases for tandem mass spectrometry peptide and protein iden-tification. Omics 9:364–379.

25. Higdon, R., and E. Kolker. 2007. A predictive model for identifying proteinsby a single peptide match. Bioinformatics 23:277–280.

26. Jones, M. R., L. J. Quinton, B. T. Simms, M. M. Lupa, M. S. Kogan, and J. P.Mizgerd. 2006. Roles of interleukin-6 in activation of STAT proteins andrecruitment of neutrophils during Escherichia coli pneumonia. J. Infect. Dis.193:360–369.

27. Kaye, M. G., M. J. Fox, J. G. Bartlett, S. S. Braman, and J. Glassroth. 1990.The clinical spectrum of Staphylococcus aureus pulmonary infection. Chest97:788–792.

28. Kim, M., E. Goldstein, J. P. Lewis, W. Lippert, and D. Warshauer. 1976.Murine pulmonary alveolar macrophages: rates of bacterial ingestion, inac-tivation, and destruction. J. Infect. Dis. 133:310–320.

29. Kjeldsen, L., A. H. Johnsen, H. Sengelov, and N. Borregaard. 1993. Isolationand primary structure of NGAL, a novel protein associated with humanneutrophil gelatinase. J. Biol. Chem. 268:10425–10432.

30. Kollef, M. H., and S. T. Micek. 2005. Staphylococcus aureus pneumonia: a“superbug” infection in community and hospital settings. Chest 128:1093–1097.

31. Kollef, M. H., A. Shorr, Y. P. Tabak, V. Gupta, L. Z. Liu, and R. S. Johannes.2005. Epidemiology and outcomes of health-care-associated pneumonia:results from a large US database of culture-positive pneumonia. Chest 128:3854–3862.

32. Labandeira-Rey, M., F. Couzon, S. Boisset, E. L. Brown, M. Bes, Y. Benito,E. M. Barbu, V. Vazquez, M. Hook, J. Etienne, F. Vandenesch, and M. G.Bowden. 2007. Staphylococcus aureus Panton-Valentine leukocidin causesnecrotizing pneumonia. Science 315:1130–1133.

33. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

34. LaForce, F. M., W. J. Kelly, and G. L. Huber. 1973. Inactivation of staphy-lococci by alveolar macrophages with preliminary observations on the im-portance of alveolar lining material. Am. Rev. Respir. Dis. 108:784–790.

35. Lathem, W. W., S. D. Crosby, V. L. Miller, and W. E. Goldman. 2005.Progression of primary pneumonic plague: a mouse model of infection,pathology, and bacterial transcriptional activity. Proc. Natl. Acad. Sci. USA102:17786–17791.

36. Lawlor, M. S., J. Hsu, P. D. Rick, and V. L. Miller. 2005. Identification ofKlebsiella pneumoniae virulence determinants using an intranasal infectionmodel. Mol. Microbiol. 58:1054–1073.

37. Levi, M., M. J. Schultz, A. W. Rijneveld, and T. van der Poll. 2003. Bron-choalveolar coagulation and fibrinolysis in endotoxemia and pneumonia.Crit. Care Med. 31:S238–S242.

38. MacLean, B., J. K. Eng, R. C. Beavis, and M. McIntosh. 2006. Generalframework for developing and evaluating database scoring algorithms usingthe TANDEM search engine. Bioinformatics 22:2830–2832.

39. McMorran, B. J., S. A. Ouvry Patat, J. B. Carlin, K. Grimwood, A. Jones,D. S. Armstrong, J. C. Galati, P. J. Cooper, C. A. Byrnes, P. W. Francis, C. F.Robertson, D. A. Hume, C. H. Borchers, C. E. Wainwright, and B. J. Wain-wright. 2007. Novel neutrophil-derived proteins in bronchoalveolar lavagefluid indicate an exaggerated inflammatory response in pediatric cystic fi-brosis patients. Clin. Chem. 53:1782–1791.

40. Neth, O., D. L. Jack, M. Johnson, N. J. Klein, and M. W. Turner. 2002.Enhancement of complement activation and opsonophagocytosis by com-plexes of mannose-binding lectin with mannose-binding lectin-associated

serine protease after binding to Staphylococcus aureus. J. Immunol. 169:4430–4436.

41. Neumann, M., C. von Bredow, F. Ratjen, and M. Griese. 2002. Bronchoal-veolar lavage protein patterns in children with malignancies, immunosup-pression, fever and pulmonary infiltrates. Proteomics 2:683–689.

42. Onofrio, J. M., G. B. Toews, M. F. Lipscomb, and A. K. Pierce. 1983.Granulocyte-alveolar-macrophage interaction in the pulmonary clearance ofStaphylococcus aureus. Am. Rev. Respir. Dis. 127:335–341.

43. Parks, W. C., C. L. Wilson, and Y. S. Lopez-Boado. 2004. Matrix metallo-proteinases as modulators of inflammation and innate immunity. Nat. Rev.Immunol. 4:617–629.

44. Plymoth, A., Z. Yang, C. G. Lofdahl, A. Ekberg-Jansson, M. Dahlback, T. E.Fehniger, G. Marko-Varga, and W. S. Hancock. 2006. Rapid proteomeanalysis of bronchoalveolar lavage samples of lifelong smokers and never-smokers by micro-scale liquid chromatography and mass spectrometry. Clin.Chem. 52:671–679.

45. Polissi, A., A. Pontiggia, G. Feger, M. Altieri, H. Mottl, L. Ferrari, and D.Simon. 1998. Large-scale identification of virulence genes from Streptococcuspneumoniae. Infect. Immun. 66:5620–5629.

46. Rahman, S. M., Y. Shyr, P. B. Yildiz, A. L. Gonzalez, H. Li, X. Zhang, P.Chaurand, K. Yanagisawa, B. S. Slovis, R. F. Miller, M. Ninan, Y. E. Miller,W. A. Franklin, R. M. Caprioli, D. P. Carbone, and P. P. Massion. 2005.Proteomic patterns of preinvasive bronchial lesions. Am. J. Respir. Crit.Care Med. 172:1556–1562.

47. Ramanathan, B., E. G. Davis, C. R. Ross, and F. Blecha. 2002. Cathelicidins:microbicidal activity, mechanisms of action, and roles in innate immunity.Microbes Infect. 4:361–372.

48. Rauch, A., M. Bellew, J. Eng, M. Fitzgibbon, T. Holzman, P. Hussey, M. Igra,B. Maclean, C. W. Lin, A. Detter, R. Fang, V. Faca, P. Gafken, H. Zhang, J.Whiteaker, D. States, S. Hanash, A. Paulovich, and M. W. McIntosh. 2006.Computational Proteomics Analysis System (CPAS): an extensible, open-source analytic system for evaluating and publishing proteomic data and highthroughput biological experiments. J. Proteome Res. 5:112–121.

49. Recsei, P., B. Kreiswirth, M. O’Reilly, P. Schlievert, A. Gruss, and R. P.Novick. 1986. Regulation of exoprotein gene expression in Staphylococcusaureus by agr. Mol. Gen. Genet. 202:58–61.

50. Roh, G. S., Y. Shin, S. W. Seo, B. R. Yoon, S. Yeo, S. J. Park, J. W. Cho, andK. Kwack. 2004. Proteome analysis of differential protein expression inallergen-induced asthmatic mice lung after dexamethasone treatment. Pro-teomics 4:3318–3327.

51. Royet, J., and R. Dziarski. 2007. Peptidoglycan recognition proteins: pleio-tropic sensors and effectors of antimicrobial defences. Nat. Rev. Microbiol.5:264–277.

52. Schultz, M. J., J. J. Haitsma, H. Zhang, and A. S. Slutsky. 2006. Pulmonarycoagulopathy as a new target in therapeutic studies of acute lung injury orpneumonia—a review. Crit. Care Med. 34:871–877.

53. Skerrett, S. J., H. D. Liggitt, A. M. Hajjar, and C. B. Wilson. 2004. Cuttingedge: myeloid differentiation factor 88 is essential for pulmonary host de-fense against Pseudomonas aeruginosa but not Staphylococcus aureus. J. Im-munol. 172:3377–3381.

54. Skerrett, S. J., T. R. Martin, E. Y. Chi, J. J. Peschon, K. M. Mohler, andC. B. Wilson. 1999. Role of the type 1 TNF receptor in lung inflammationafter inhalation of endotoxin or Pseudomonas aeruginosa. Am. J. Physiol.276:L715–L727.

55. Tang, H., M. Kays, and A. Prince. 1995. Role of Pseudomonas aeruginosa piliin acute pulmonary infection. Infect. Immun. 63:1278–1285.

56. Toews, G. B., G. N. Gross, and A. K. Pierce. 1979. The relationship ofinoculum size to lung bacterial clearance and phagocytic cell response inmice. Am. Rev. Respir. Dis. 120:559–566.

57. Van Lint, P., and C. Libert. 2006. Matrix metalloproteinase-8: cleavage canbe decisive. Cytokine Growth Factor Rev. 17:217–223.

58. Ventura, C. L., R. Higdon, E. Kolker, S. J. Skerrett, and C. E. Rubens. 2008.Host airway proteins interact with Staphylococcus aureus during early pneu-monia. Infect. Immun. 76:888–898.

59. von Bredow, C., P. Birrer, and M. Griese. 2001. Surfactant protein A andother bronchoalveolar lavage fluid proteins are altered in cystic fibrosis. Eur.Respir. J. 17:716–722.

60. Wardenburg, J. B., R. J. Patel, and O. Schneewind. 2007. Surface proteinsand exotoxins are required for the pathogenesis of Staphylococcus aureuspneumonia. Infect. Immun. 75:1040–1044.

61. Wattiez, R., I. Noel-Georis, C. Cruyt, F. Broeckaert, A. Bernard, and P.Falmagne. 2003. Susceptibility to oxidative stress: proteomic analysis ofbronchoalveolar lavage from ozone-sensitive and ozone-resistant strains ofmice. Proteomics 3:658–665.

62. Wong, W. S., H. Zhu, and W. Liao. 2007. Cysteinyl leukotriene receptorantagonist MK-571 alters bronchoalveolar lavage fluid proteome in a mouseasthma model. Eur. J. Pharmacol. 575:134–141.

63. Wu, H., A. Kuzmenko, S. Wan, L. Schaffer, A. Weiss, J. H. Fisher, K. S. Kim,and F. X. McCormack. 2003. Surfactant proteins A and D inhibit the growthof Gram-negative bacteria by increasing membrane permeability. J. Clin.Investig. 111:1589–1602.

64. Wu, H., Z. Song, M. Hentzer, J. B. Andersen, A. Heydorn, K. Mathee, C.

VOL. 76, 2008 AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA 5871

Moser, L. Eberl, S. Molin, N. Hoiby, and M. Givskov. 2000. Detection ofN-acylhomoserine lactones in lung tissues of mice infected with Pseudomo-nas aeruginosa. Microbiology 146:2481–2493.

65. Wu, J., M. Kobayashi, E. A. Sousa, W. Liu, J. Cai, S. J. Goldman, A. J.Dorner, S. J. Projan, M. S. Kavuru, Y. Qiu, and M. J. Thomassen. 2005.Differential proteomic analysis of bronchoalveolar lavage fluid in asthmaticsfollowing segmental antigen challenge. Mol. Cell. Proteomics 4:1251–1264.

66. Yan, L., N. Borregaard, L. Kjeldsen, and M. A. Moses. 2001. The high molecularweight urinary matrix metalloproteinase (MMP) activity is a complex of gela-tinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Mod-ulation of MMP-9 activity by NGAL. J. Biol. Chem. 276:37258–37265.

67. Yang, S.-F., S.-C. Chu, I.-C. Chiang, W.-F. Kuo, H.-L. Chiou, F.-P. Chou,W.-H. Kuo, and Y.-S. Hsieh. 2005. Excessive matrix metalloproteinase-9 in

the plasma of community-acquired pneumonia. Clin. Chim. Acta 352:209–215.

68. Yi, E. C., H. Lee, R. Aebersold, and D. R. Goodlett. 2003. A microcapillarytrap cartridge-microcapillary high-performance liquid chromatography elec-trospray ionization emitter device capable of peptide tandem mass spectrom-etry at the attomole level on an ion trap mass spectrometer with automatedroutine operation. Rapid Commun. Mass Spectrom. 17:2093–2098.

69. Yuste, J., M. Botto, S. E. Bottoms, and J. S. Brown. 2007. Serum amyloid Paids complement-mediated immunity to Streptococcus pneumoniae. PLoSPathog. 3:1208–1219.

70. Zhao, J., H. Zhu, C. H. Wong, K. Y. Leung, and W. S. Wong. 2005. Increasedlungkine and chitinase levels in allergic airway inflammation: a proteomicsapproach. Proteomics 5:2799–2807.

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