INFECTION AND IMMUNITY, Dec. 2008, p. 5862–5872
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 12
Staphylococcus aureus Elicits Marked Alterations in the Airway
Proteome during Early Pneumonia?‡
Christy L. Ventura,1,6,10† Roger Higdon,2,3Laura Hohmann,4Daniel Martin,4,5Eugene Kolker,2,3,7
H. Denny Liggitt,8Shawn J. Skerrett,9and Craig E. Rubens1,6*
Division of Infectious Diseases, Center for Childhood Infections and Prematurity Research,1and Center for Developmental Therapeutics,2
Seattle Children’s Hospital Research Institute, BIATECH Institute,3InstituteforSystemsBiology,4FredHutchinsonCancerResearchCenter,5
and Departments of Pediatrics,6Medical Education and Biomedical Informatics,7Comparative Medicine,8and Medicine,9University of
Washington School of Medicine, Seattle, Washington, and Laboratory of Human Bacterial Pathogenesis,
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, 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 insight
into the mechanisms used by the host to protect itself from infection. An adult mouse model of Staphylococcus
aureus pneumonia was utilized to define the early events in the innate immune response and to assess the
changes in the airway proteome during the first 6 h of pneumonia. S. aureus actively replicated in the lungs of
mice 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 h
postinoculation. Bronchoalveolar lavage fluids from mice inoculated with phosphate-buffered saline (PBS) or
S. aureus were depleted of overabundant proteins and subjected to strong cation exchange fractionation
followed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the
airway. No significant changes in response to PBS inoculation or 30 min following S. aureus inoculation were
observed. 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 here
provide a comprehensive evaluation of the rapid and vigorous innate immune response mounted in the host
airway during the earliest stages of S. aureus pneumonia.
Staphylococcus aureus is a leading cause of hospital-acquired
and health care-associated pneumonia and may be increasing
in 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-associated
and hospital-acquired pneumonia (1, 9, 31). In addition, S.
aureus is a frequent cause of health care-associated pneumonia
occurring in residents of long-term-care facilities, individuals
recently discharged from acute-care hospitals, and patients
receiving 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-acquired
pneumonia underscores the importance of identifying host and
bacterial factors that facilitate the progression of staphylococ-
Mice have been used extensively to study pneumonia caused
by a variety of bacteria (2, 6, 26, 35, 36, 45, 55, 63, 64). Murine
models of airborne infection with S. aureus have been useful in
characterizing host responses during the first 4 to 8 h of lung
infection but do not mimic the natural route of infection and
result in self-limited disease, even in immunocompromised
animals (28, 53, 56). In these studies, proinflammatory cyto-
kines and chemokines were released and neutrophils (poly-
morphonuclear leukocytes [PMNs]) were rapidly recruited to
the site of infection; however, the mice were able to clear the
infection within 24 to 36 h (53). Bolus infection models in
which 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 and
infected i.n., leading to 100% morbidity and 30% mortality
following 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 the
airway. 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 surgical
trauma (13, 42). One goal of the present study was to develop
a staphylococcal pneumonia model in immunocompetent adult
mice by using a nasal inoculation and aspiration approach that
mimics 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:
† 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
?Published ahead of print on 13 October 2008.
immune response to S. aureus in the airway. Similar models
were developed simultaneously by other groups to study the
requirement for specific S. aureus virulence factors in pneumo-
nia (32, 60).
Shotgun proteomics has proven to be a very useful tool for
determining the global protein profile in a particular organ or
body fluid in the context of various disease states. A study by
Guo et al. utilized one-dimensional (1D) electrophoresis with
mass spectrometry (MS) and two-dimensional liquid chroma-
tography-MS (LC-MS) to define the airway proteome of a
healthy mouse (20). In addition, a proteomics approach has
been used to define the proteins present in the airway in
patients 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 the
effects of acute infection on the airway proteome and the ways
these effects change over time. We hypothesized that early host
responses to S. aureus infection of the lung, including changes
in the airway proteome, could be critical determinants of the
course and severity of pneumonia. To address this, we devel-
oped a mouse model of acute staphylococcal pneumonia and
utilized cell biological, immunological, and proteomics tech-
niques to examine the host response and changes in the airway
proteome during the first 6 h of S. aureus pneumonia. We
demonstrate that S. aureus elicits a vigorous airway inflamma-
tory response characterized by the rapid release and influx of
inflammatory 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 used
in these studies. RN6390 is a commonly used laboratory strain (49) that was
kindly provided by David Heinrichs (University of Western Ontario), and JP1 is
a human blood isolate obtained from the microbiology laboratory of the Veter-
ans Affairs Puget Sound Health Care System (53). S. aureus was grown in
Luria-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 then
diluted 1/100 into fresh LB broth (4:1 flask-to-medium ratio) and grown for an
additional 16 to 18 h with shaking (180 rpm). Stationary-phase bacteria were
harvested 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 by
optical density and confirmed by quantitative plate counting.
Animals. Specific-pathogen-free male and female C57BL/6 mice, aged 9 to 11
weeks, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals
were group housed in filtered, ventilated cages containing autoclaved bedding
and were permitted ad libitum access to sterile food and water. Cage changes and
animal handling occurred in a laminar flow hood. All experimental procedures
were approved by the Institutional Animal Care and Use Committee of the
University of Washington.
Mouse model of pneumonia and tissue harvest. Mice were anesthetized with
isoflurane, held vertically, and inoculated i.n. with S. aureus in 50 ?l endotoxin-
free PBS. Occasionally, lightly anesthetized mice flipped their heads during
inoculation, which may have affected bacterial deposition; these mice were re-
moved from the study. To determine the dose at which S. aureus would replicate
in the lungs, 12 mice each were inoculated with 3 ? 107, 1 ? 108, or 3 ? 108CFU
JP1 and monitored at least twice daily. At 30 min after inoculation (all doses), at
24 h, 48 h, and 96 h after inoculation (3 ? 107and 108CFU), or when mice
reached a moribund state (3 ? 108CFU), defined by hunched posture, piloerec-
tion, labored breathing, immobility, and loss of resistance to handling, mice were
euthanized by intraperitoneal injection of an overdose of pentobarbital. Both
lungs were harvested and homogenized for quantitative culture as described
previously (53). For analysis of the host response to S. aureus, 7 or 10 mice were
inoculated with a dose of 3 ? 108to 5 ? 108CFU JP1 or with endotoxin-free
PBS, as described above. At 30 min and 6 h postinoculation, the mice in each
group were euthanized, and bronchoalveolar lavage (BAL) was performed as
described previously (53, 54). Lungs were inflated in situ to approximately 15 cm
pressure with 4% paraformaldehyde and stored at 4°C in the same fixative.
BAL cultures and differential cell counts. An aliquot of BAL fluid from each
animal was removed for quantitative culture, cytokine analysis, and differential
counts; 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 1640
containing 10% heat-inactivated fetal calf serum (HyClone Laboratories, Logan,
UT), and cells were counted with a hemacytometer. Differential cell counts were
determined from cytocentrifuge specimens stained with Diff-Quik (Dade-Be-
hring, Dudigen, Switzerland).
Measurement of cytokines. Levels of immunoreactive tumor necrosis factor
alpha (TNF-?), interleukin-1? (IL-1?), macrophage inflammatory protein 2
(MIP-2), keratinocyte-derived chemokine (KC), monocyte chemotactic protein
1, 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 the
means. The Mann-Whitney test was performed to determine whether the median
times 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 test
with 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 infected
mice in a manner blinded to time after inoculation and inoculum.
Depletion of BAL fluid. Twenty mice were inoculated with PBS or S. aureus as
described above, and BAL was performed on 10 mice per treatment at 30 min
and 6 h postinoculation; the experiment was performed twice. Eukaryotic cells
were removed by centrifugation at 300 ? g, and BAL fluids were pooled by
treatment 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 room
temperature. The amount of total protein in each pooled BAL sample was
determined by a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Each
sample was concentrated and exchanged into Agilent buffer A (Agilent Tech-
nologies, Inc., Santa Clara, CA) containing 0.2% TX-100 by use of an Amicon
Ultra 5000 nominal molecular weight limit spin concentrator (Millipore, Bil-
lerica, MA). Two hundred micrograms of protein from each sample was depleted
of albumin, transferrin, and immunoglobulin (Ig) by use of a mouse multiple
affinity removal system (Agilent). The depletion was performed according to the
manufacturer’s recommendations except that Agilent buffer A was replaced with
Agilent buffer A containing 0.2% TX-100 to reduce protein aggregation. Each
depleted BAL sample was concentrated and exchanged into 50 mM ammonium
bicarbonate by use of an Amicon Ultra 5000 nominal molecular weight limit spin
concentrator (Millipore) and frozen at ?80°C.
Fractionation and LC–MS-MS analysis of depleted BAL samples. Depleted
BAL samples were fractionated by strong cation exchange (SCX) or 1D sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to LC-
tandem MS (LC–MS-MS). Prior to SCX fractionation, depleted BAL samples
were lyophilized and then dissolved in 0.5 ml 50 mM ammonium bicarbonate and
reduced 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 room
temperature in the dark. The alkylation reaction was quenched with 20 mM
dithiothreitol (Sigma) for 5 min at room temperature. Each sample was digested
with 1 ?g of sequencing-grade trypsin (Promega, Madison, WI) at pH 8 for 18 h
at 37°C. Digestion was confirmed using SDS-PAGE. Each digested sample was
fractionated using SCX (polysulfolethyl A; PolyLC, Inc., Columbia, MD). Each
sample was brought up to 1 ml of buffer A (5 mM KH2PO4, 25% acetonitrile, pH
2.7), and the pH was adjusted to 2 with 10% phosphoric acid. A 40-min gradient
was 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 seven
final fractions based upon UV absorbance signal. The seven fractions were
desalted using C18ultramicrospin columns (The Nest Group, Inc., Southbor-
One depleted BAL sample pooled from 10 mice that were mock infected for
30 min was fractionated by 1D SDS-PAGE rather than SCX due to the presence
of interfering and unidentifiable contaminants that made LC–MS-MS analysis
impossible. The sample was separated in a Novex NuPAGE 4 to 12% bis-Tris gel
(Invitrogen, Carlsbad, CA) and stained with Coomassie blue. The lane was
excised 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, 2008AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA 5863
pieces, washed three times with water followed by 50% acetonitrile, and then
dehydrated with pure acetonitrile. The Coomassie stain was removed with two
washes with 100 mM ammonium bicarbonate mixed 1:1 with acetonitrile. The gel
slices were reduced with 10 mM dithiothreitol at 50°C and alkylated with 55 mM
iodoacetamide for 45 min in the dark at room temperature. The pieces were
dried, rehydrated with 1 ?g sequencing-grade trypsin (Promega) in 50 mM
ammonium bicarbonate, and incubated for 18 h at 37°C. The digested peptides
were 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 and
an 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-size
Magic C18AQ material (Michrom Bioresources, Auburn, CA), as described
elsewhere (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 capillary
column packed with 100-Å-pore-size Magic C18AQ material (Michrom Biore-
sources), and then delivered into the mass spectrometer at a constant column tip
flow rate of 250 nl/min. Eluting peptides were analyzed by micro-LC-MS and
data-dependent micro-LC–MS-MS acquisition, selecting three precursor ions for
MS with a dynamic exclusion of 1 (21).
Proteomics data analysis. The MS-MS scans from each LC–MS-MS run were
converted from the .RAW file format to mzXML files by use of the program
ReAdW.exe (version 1.0; Institute for Systems Biology, Seattle, WA). The da-
tabase search program X!Tandem (12), included in the Computational Portal
and Analysis System (CPAS version 1.4) (48), was used for peptide identification
of the MS-MS spectra. The Comet scoring function (38) was used in place of the
default X!Tandem scoring function. The following parameters were used in the
database search: trypsin enzyme specificity, peptide mass tolerance of 2.5 Da,
fragment ion tolerance of 0.5 Da, monoisotopic molecular weight for both
peptide 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 searched
against 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!Tandem
output based upon a combination of the Comet score, delta (relative difference
to the second-best match), expectation value, percentage of matching ions,
charge state, peptide length, and delta mass (difference between the observed
and theoretical masses) by use of the logistic identification of peptide sequences
(LIPS) model (24). Experiment-specific peptide identification probabilities were
generated from the distribution of reshuffled peptide matches (25). A minimum
of 90% identification certainty was used to accept a peptide spectrum identifi-
cation. This resulted in an overall peptide false-positive identification rate of
0.8% (with a 95% confidence interval of 0.7 to 0.9%) based on reshuffled
database matches. Protein identification for each experimental condition was
based on four levels of certainty (very high, high, medium, and low). All proteins
identified by two or more unique peptides were classified with a “very high” level
of certainty. Single-hit proteins were classified in the remaining three certainty
levels based on the LIPS model by using peptide identification probability and
peptide length (25). The estimated false discovery rates for the four categories
are 0.1%, 1.5%, 24%, and 48% (with 95% confidence intervals of 0.0 to 0.4%, 0.3
to 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 reshuffled
sequences, resulting in the reshuffled database having more unique sequences.
Therefore, the false-positive error rates need to be multiplied by the ratio of
unique peptide sequences in the target database to the number of unique se-
quences in the reshuffled database; otherwise, the number of false positives will
be overestimated. The ratio of unique peptide sequences in the target database
to the number of unique sequences in the reshuffled database was estimated to
be 60%, so the false discovery rate is estimated to be the number of reshuffled
peptides divided by the number of target peptides multiplied by the percentage
of unique sequences. Confidence intervals were generated by assuming Poisson
distributions for the numbers of reshuffled and false target peptide or protein
Table S1 in the supplemental material contains all of the proteins identified
from MS-MS spectra using X!Tandem. Because the same protein can have
multiple IPI entries, the information in Table S1 in the supplemental material
was condensed into Table S2 in the supplemental material. Condensation of the
protein list in Table S1 in the supplemental material was accomplished by
searching 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), and
the 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 Table
S2 in the supplemental material. The numbers of unique peptides and total
peptides identified for each protein were combined in Table S2 in the supple-
mental material, and the confidence of the protein identification was adjusted if
necessary. Only proteins that were identified with high or very high confidence
under 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 proteins
identified in the airway were eliminated due to low confidence of protein iden-
SDS-PAGE and Western immunoblotting. A 30-?l aliquot of each pooled
BAL sample (30 min and 6 h mock infected, 30 min and 6 h infected) was mixed
with 10 ?l 4? Laemmli buffer (33) and boiled for 5 min. The samples were
separated 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 h
prior 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 incubations
were carried out with 5% skim milk and 0.05% Tween 20 (Fisher Scientific,
Pittsburgh, PA) in PBS at room temperature. Detecting antibodies were
IRDye800-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) (Affinity
Bioreagents, 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 680
and donkey anti-goat Ig–Alexa Fluor 680 (Invitrogen). Fluorescence was de-
tected using an Odyssey infrared imaging system (LI-COR Biotechnology, Lin-
Gelatin zymography. A 30-?l aliquot of each BAL sample (30 min and 6 h
mock infected, 30 min and 6 h infected) was mixed with 10 ?l 4? Laemmli buffer
without reducing agent (33). Samples were separated by SDS-PAGE in 10% gels
containing 1% gelatin (Bio-Rad, Hercules, CA). Following electrophoresis, gels
were washed twice at room temperature with 2.5% TX-100 for 30 min each and
then 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% Coomassie
brilliant blue (Bio-Rad), destained briefly with 40% methanol and 10% acetic
acid, and imaged using the gel documentation system described above.
RESULTS AND DISCUSSION
S. aureus replicates in the lungs of mice. To explore the early
host-pathogen interactions that occur during the development
of acute S. aureus pneumonia, we sought to develop a mouse
model in which the bacteria actively replicate in the lungs to
cause pneumonia. Mice were infected i.n. under anesthesia
with 3 ? 107, 1 ? 108, or 3 ? 108CFU of JP1 to identify a dose
that would result in bacterial replication in the lungs. All of the
mice 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 ? 107CFU and 1 ? 108CFU
were ill for 24 to 36 h and then cleared the infection. In
contrast, a dose of 3 ? 108CFU caused mortality in 71% of the
mice, with a median time to death of 32.5 h (P ? 0.0008
compared to mice inoculated with 3 ? 107or 1 ? 108CFU).
Lungs were harvested from mice sacrificed 0.5 h, 24 h, 48 h,
and 96 h postinoculation (doses of 3 ? 107CFU and 1 ? 108
CFU) or from mice sacrificed 0.5 h postinoculation or follow-
ing death due to S. aureus infection (dose of 3 ? 108CFU).
Enumeration of S. aureus bacteria in the lungs at 30 min
postinoculation revealed deposition of 3 to 10% of the i.n.
5864 VENTURA ET AL.INFECT. IMMUN.
inoculum (Fig. 1). Bacterial replication was observed in the
lungs of mice infected with 3 ? 108CFU, reaching a mean
density of 1.54 ? 108? 1.44 ? 108CFU/lungs at death (com-
pared to a mean deposition of 2.91 ? 107? 1.29 ? 107
CFU/lungs) (Fig. 1). In contrast, bacteria were gradually
cleared from the lungs of mice inoculated with 3 ? 107or 1 ?
108CFU (Fig. 1). Similar levels of bacterial clearance, mor-
bidity, and mortality were observed when mice were inoculated
with the laboratory strain RN6390 at the same doses (data not
shown), indicating that the results obtained with JP1 were not
strain specific. These data demonstrate that a dose of 3 ? 108
CFU of JP1 or RN6390 was sufficient to cause pneumonia that
could result in mortality, while a dose of 1 ? 108CFU or lower
resulted in symptoms of pneumonia that lasted for 24 to 36 h,
followed by bacterial clearance and disease resolution. This
dose-related mortality is similar to what has been reported with
intratracheal inoculation of adult mice (13, 42) and, more
recently, with i.n. inoculation using exponentially growing S.
aureus bacteria (32, 60). A dose of 3 ? 108CFU JP1 was used
for 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 the
initial inflammatory response to inoculation with S. aureus or
PBS. The levels of proinflammatory TNF-?, MIP-2, and KC in
the BAL fluid were elevated 30 min postinoculation with S.
aureus compared to levels for the mock-infected controls. In
addition, the levels of TNF-?, KC, MIP-2, IL-1?, IL-6, and
GM-CSF in the airways of infected mice were significantly
higher 6 h postinoculation than the levels in BAL fluid from
30-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 above
background 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 by
30 min and increased significantly by 6 h postinoculation with
One of the major functions of proinflammatory cytokines
and chemokines is to recruit PMNs from the bloodstream to
the site of an infection. To determine the kinetics of the PMN
influx during early S. aureus pneumonia, total cell counts and
differential counts of the BAL fluids from infected and mock-
infected mice were performed. The total number of BAL fluid
cells increased 10-fold by 6 h postinoculation with S. aureus
(Fig. 2G) as a result of PMN influx (Fig. 2H). In contrast, the
total number of BAL fluid cells from mock-infected mice did
not change significantly during the first 6 h, despite a modest
PMN response after instillation of PBS into the lung. The
numbers of mononuclear cells remained relatively constant
(Fig. 2I), regardless of treatment or time point. These data
indicate 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 taken
from mice at 30 min postinoculation with either PBS or S.
aureus were histologically similar except for the presence of a
few, 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-infected
mice were normal. In the 6-h-mock-infected animals, minimal
neutrophilic inflammation was observed in widely scattered
locations (Fig. 3, 6 h PBS). In contrast, lungs from 6-h-infected
mice had multiple, frequently confluent foci of inflammation,
with various degrees of severity (Fig. 3, 6 h S. aureus). The
influx of PMNs into small vessels and capillaries was pro-
nounced (Fig. 3, 6 h S. aureus, ?40 inset), leading to the
thickening of alveolar walls and, in more severely affected
areas, the diffuse accumulation of PMNs within alveolar
spaces. In some areas, consolidation of the air spaces with
concomitant loss of alveolar detail was observed. In the most
severe foci, fibrin accumulation, thrombosis, and necrosis were
evident, with an increase in the number of free S. aureus
bacteria. Our data demonstrate that S. aureus infection of the
airway results in the rapid development of consolidating pneu-
The composition of the airway proteome changes during
early pneumonia. One of the hallmarks of an inflammatory
response is an increase in the amount of total protein present
in the infected area as a result of the local production and
influx of inflammatory mediators in response to cytokine and
chemokine recruitment. To determine whether this occurred
in our system, the total protein contents of BAL fluids from
mock-infected and infected mice were measured using a BCA
assay. As shown in Fig. 4A, the amounts of protein in BAL
samples at 30 min, regardless of treatment, and in 6-h-mock-
infected BAL samples were between 82 and 104 ?g/ml. In
contrast, more than double that amount (213 ?g/ml) was
present in the 6-h-infected animals (P ? 0.05). SDS-PAGE
analysis of BAL samples from mock-infected (30 min and 6 h)
and infected (30 min and 6 h) mice indicated the presence of
50-, 67-, and 78-kDa proteins in high abundance (Fig. 4B, lane
1). Based upon molecular mass and immunoblot analyses,
these proteins were predicted to be immunoglobulin (Fig. 4C),
albumin, and transferrin (Fig. 4D), respectively. An affinity
removal system was used to deplete the BAL samples of these
FIG. 1. S. aureus replicates in the lungs of mice infected with 3 ?
108CFU. Twelve mice at each dose were inoculated i.n. with 3 ? 107
CFU (f), 1 ? 108CFU (Œ), or 3 ? 108CFU (?) S. aureus JP1.
Three mice inoculated with 3 ? 107and 1 ? 108CFU were sacri-
ficed at each of four time points (0.5, 24, 48, and 96 h postinocu-
lation). Three mice inoculated with 3 ? 108CFU were sacrificed
0.5 h postinoculation; the remaining mice succumbed to the infec-
tion. Bacteria were enumerated from homogenized lungs. Each
symbol represents three mice, except 3 ? 108CFU at 17 h (n ? 4),
24 h (n ? 2), 41 h (n ? 2), and 46 h (n ? 1).
VOL. 76, 2008 AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA 5865
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) analyses
of native and depleted samples demonstrated that depletion
removed all of the detectable immunoglobulin, albumin, and
A shotgun proteomics approach was utilized to determine
how the composition of the airway proteome changed during
the course of early pneumonia. The depleted BAL samples
were digested with trypsin, and the peptides were subjected to
SCX fractionation and LC–MS-MS. The peptide sequences
generated by analysis of the MS-MS data were matched to
mouse and S. aureus proteins using the combined databases of
the mouse IPI and the S. aureus COL proteome. A total of
1,096 mouse and 19 S. aureus proteins were identified in the
airways of mice inoculated for 30 min or 6 h with PBS or S.
aureus (see Table S1 in the supplemental material). A few
peptides were identified as serum albumin, while no peptides
were assigned as transferrin or immunoglobulin, showing that
the depletion of these three overabundant proteins was re-
markably efficient. One potential concern with depletion is that
we might remove a significant number of proteins that are
associated 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 for
the molecular characterization of specific host-pathogen inter-
actions, we determined that the advantages of depleting the
overabundant proteins from all BAL samples prior to MS-MS
analysis outweighed the potential losses that may have oc-
The list of proteins in Table S1 in the supplemental material
was refined into Table S2 in the supplemental material by
combining multiple entries for a given protein into a single line
of the table, as described in Materials and Methods, so as to
obtain a more biologically useful list of proteins. A total of 727
unique host proteins were identified with high or very high
confidence 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 symbol
represents one mouse. The data are combined from three independent experiments with seven to eight mice per experiment. The bar for each data
set represents the median value for 21 or 22 mice per condition. Statistical comparisons were made using the Kruskal-Wallis test with Dunn’s
posttest. SA, S. aureus; MN, mononuclear cells. ?, P ? 0.05; †, P ? 0.01; #, P ? 0.001.
5866VENTURA ET AL.INFECT. IMMUN.
conditions (see Table S2 in the supplemental material). All of
the identified S. aureus proteins were disregarded because the
confidence of the identifications for these proteins was below
the cutoff for further consideration (identification using a sin-
gle peptide with low to medium confidence, which resulted in
false discovery rates of 48 and 24%, respectively). Of the 727
total proteins, 458 (63%) were identified using two or more
peptides, which increases the confidence that the protein to
which the peptide was assigned was correct (the false discovery
rate for identification using at least two peptides was 0.1%,
compared with 1.5% for high-confidence identifications using a
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 the
mouse proteins identified in this proteomics screen were not
assigned by GO to cellular-component or biological-process
categories, so the assignments were generated manually during
the refinement process described above. We chose to analyze
the percentages of proteins present in given GO categories
because 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 categories
were more stable and, therefore, more meaningful to compare.
There was no difference in the percentages of total proteins
assigned to any of the GO subcategories among both 30-min
samples (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 changes
in the airway proteome immediately following inoculation with
S. aureus and in the first 6 h following PBS inoculation are too
subtle 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 a
proteomic level, most likely due to the low molecular weight
and 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 into
one “control” group to provide for a more rigorous analysis of
the inflammatory response elicited by S. aureus 6 h following
inoculation (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 the
treatment conditions in the control group (our unpublished
observations). 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 within
the host cell, including the cell membrane (10%). In contrast,
41% of the proteins identified in the 6-h-infected BAL fluid
were extracellular, while only 25% were cytoplasmic (com-
pared to 38% in the control airway proteome). The increase in
relative abundance of extracellular proteins between the 6-h-
infected and control samples (41% versus 27%) is indicative of
the 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 the
extracellular milieu of the airway likely results from lysis of
host cells as a result of normal cell turnover, apoptosis,
FIG. 3. Histopathology shows signs of consolidated pneumonia in
infected animals but not mock-infected animals. Representative low
(?10)- and high (?40)-power histologic sections of lungs from mice
infected with S. aureus for 30 min and 6 h or mock infected (PBS) for
6 h. The inset (6 h S. aureus, ?40) shows a smaller vessel that is
thrombosed. Bar ? 100 ?m.
FIG. 4. Depletion of BAL fluid removes overabundant proteins.
(A) Total protein in pooled BAL samples from mock-infected (white
bars) 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. Equivalent
volumes of each sample were separated in the gel. (C and D) Western
blots 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) and
human transferrin (D). Equivalent volumes of each native BAL sample
were separated in the gel prior to immunoblotting. Molecular masses
in kilodaltons are shown on the right side of each panel.
VOL. 76, 2008AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA5867
necrosis, and/or S. aureus-mediated cytolysis (in the case of
the 6-h-infected sample).
Inflammatory and coagulation proteins dominate the early
response to S. aureus in the airway. Assignment of the identi-
fied proteins into the biological-process GO category revealed
an increase in the percentages of inflammatory and coagula-
tion proteins in the airways of 6-h-infected mice compared to
those 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). The
inflammatory proteins identified in the airway are shown in
Table 1. Antimicrobial peptides and peptidoglycan recognition
proteins have direct antibacterial properties on the S. aureus
membrane and cell wall peptidoglycan, respectively, that can
ultimately result in bacterial cell lysis. Multimers of cathelici-
din antimicrobial peptide (CRAMP) and myeloid bactenecin
bind 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 lectins
that 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 were
present in the airway in response to S. aureus infection. While
S. aureus is not susceptible to lysis by the membrane attack
complex (11), opsonization by any of these proteins facilitates
phagocytic uptake of the bacteria by macrophages and PMNs.
Complement-mediated opsonization of bacteria may also be
augmented by serum amyloid protein P (69), which was also
differentially present in the BAL fluids of infected versus
mock-infected mice. In a related study, we found that S. aureus
was associated with or internalized by 65% of alveolar macro-
phages within 30 min of infection and by 25% of PMNs after
6 h in the airway; in addition, we found that C3, Ig, and MBL-C
were associated with the surface of the bacteria (58). Previous
studies by other groups demonstrated that S. aureus was
phagocytosed by alveolar macrophages within 30 min following
aerosol inoculation as well (18, 28, 34).
The primary function of MMP-8 and -9 is to facilitate the
degradation of extracellular matrix components formed by the
host in response to injury; however, both have also been im-
plicated in acute inflammation. Both MMPs are stored in PMN
granules and are released during an acute inflammatory re-
sponse. MMP-8, also known as neutrophil collagenase, pro-
motes balanced PMN recruitment during acute inflammation
and 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 proinflammatory
cytokines IL-8, TNF-?, transforming growth factor ?, and
IL-1? to their active forms (8, 43). In addition, MMP-9 is
known 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 mice
inoculated with PBS or S. aureus are shown in Table 2. Inter-
estingly, in addition to their role in recruiting neutrophils and
inflammatory mediators to the site of an infection, the proin-
flammatory cytokines TNF-?, IL-1, and IL-6 have been shown
to 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
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 material
for a complete list of the proteins). Shown are cellular-component GO categories for proteins identified in the airways of control (A) and
6-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 total
of 658 proteins were identified in the control samples, and a total of 396 proteins were identified in the 6-h-infected samples.
5868VENTURA ET AL.INFECT. IMMUN.
during early S. aureus airway infection (Fig. 2E) corresponds
with an increase in the abundance of proteins involved in
coagulation (Fig. 5D; Table 2) and the appearance of fibrin
deposits in the airway, as evidenced histologically (Fig. 3, 6 h S.
aureus). We also identified several of the proteins necessary for
fibrin accumulation via the contact factor (intrinsic) pathway,
including plasma kallikrein and coagulation factors V and X.
Heparin cofactor 2 and antithrombin are downstream proteins
in 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 and
dissolution of fibrin clots. S. aureus secretes staphylokinase, a
protein that activates plasminogen to plasmin; this process can
be augmented by CRAMP (5), which was also identified in the
BAL 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 initiating
coagulation, proteases of the coagulation system are active in
inducing a proinflammatory response (37). Taken together,
these proteomics data show an increase in proteins involved in
inflammation and coagulation processes during the first 6 h of
S. aureus airway infection.
Alternative approaches confirm changes in airway proteome
composition. Western blot analysis was performed to confirm
the presence and relative levels of abundance of complement
component C3, plasminogen, and MMP-9 in the different BAL
samples. As seen in Fig. 6A, the amount of C3, which was
detectable primarily as C3b and iC3b (based upon molecular
mass), in both 6-h samples was greater than that in the 30-min
samples. In addition, lower-molecular-mass degradation prod-
TABLE 1. Inflammatory proteins present in the airways of infected
and/or mock-infected animals
Level of protein
Advanced glycosylation end product-specific receptor
Cathelicidin antimicrobial peptide
Chitinase 3-like 1
Chitinase 3-like 3
Complement C8 alpha
Complement C8 beta
Complement C8 gamma
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
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
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
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
aBy use of the LIPS model (25), proteins were identified with very high confidence
(??) or high confidence (?) or not identified (?). The total number of inflammatory
total number for 6-h-infected mice was 54 (13.6% of the total proteins identified). The
changes in the proteome as is the percentage of total proteins identified in that category
because there were differences in the total numbers of proteins identified as a result of
differences in MS sampling.
TABLE 2. Coagulation proteins present in the airways of infected
and/or mock-infected animals
Level of protein identificationa
Coagulation factor V
Coagulation factor X
Coagulation factor XIII
Heparin cofactor 2
Hyaluronan-binding protein 2
Vitamin K-dependent protein S
Vitamin K-dependent protein Z
aBy use of the LIPS model (25), proteins were identified with very high
confidence (??) or high confidence (?) or not identified (?). The total number
of coagulation proteins identified in control mice was 15 (2.3% of the total
proteins identified), and the total number for 6-h-infected mice was 18 (4.5% of
the total proteins identified). The absolute number of proteins identified in a
given category is not as useful an indicator of changes in the proteome as is the
percentage of total proteins identified in that category because there were dif-
ferences in the numbers of total proteins identified as a result of differences in
VOL. 76, 2008AIRWAY PROTEOME CHANGES IN S. AUREUS PNEUMONIA5869
ucts of C3b were present in the 6-h-infected sample, indicating
that cleavage of C3b occurred in the airways of these animals.
Plasminogen and MMP-9, both of which have been shown to
increase during active infection (5, 10, 14, 19, 22, 67), were also
more abundant in the 6-h-infected samples than in the other
samples (Fig. 6B and C). Western blot analyses of these pro-
teins were performed on the BAL fluid of mice inoculated with
lower doses of S. aureus (3 ? 107and 1 ? 108CFU), with
similar results (data not shown). Further evidence for the pres-
ence of MMP-9 in the 6-h-infected sample was obtained using
gelatin zymography, which is more sensitive than Western blot-
ting (Fig. 6D). The zymogram showed that the predominant
form of MMP-9 in BAL fluid was the proform (92 kDa), which
is not uncommon, as the activated form (86 kDa) is typically
found tightly associated with extracellular matrix components
that are not removed during routine lavage. Several MMP-9
complexes were also observed using zymography; MMP-9 is
known to form homomultimers as well as heterodimers with
neutrophil gelatinase-associated lipocalin (29), which was
identified in our proteomics screen (see Table S2 in the sup-
plemental material). These data confirm the proteomics data
showing that C3 and plasminogen were present in the airways
of mock-infected mice and were increased in BAL fluid from
infected mice and that MMP-9 was present only in the airways
of mice infected for 6 h. Further, they demonstrate the utility
of a shotgun proteomics approach for characterizing changes
in the proteome of a biological fluid during the course of an
Conclusions. In this report, we describe marked alterations
in 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 events
in S. aureus pneumonia. The data presented here provide a
critical 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 within
the airway during the first 6 h following staphylococcal chal-
lenge. We have demonstrated for the first time that S. aureus
elicits a rapid and vigorous inflammatory response within the
first 6 h of infection. In fact, as early as 30 min after bacterial
inoculation, 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 of
which may play key roles in the pathogenesis of acute bacterial
pneumonia. These studies provide the foundation for future
analyses investigating specific host-pathogen interactions that
occur during the early stages of S. aureus pneumonia.
We thank Jeannette Crisostomo, Destry Taylor, and Michele Timko
for their expert technical assistance, Jimmy Eng for assistance with
proteomics data analysis, William Parks for advice about MMP-9 anal-
ysis, and Amanda Jones for insightful comments and critical review of
This work was supported by HL073996 from the National Institutes
of Health (C.E.R.).
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