INFECTION AND IMMUNITY, Feb. 2007, p. 1040–1044 Vol. 75, No. 20019-9567/07/$08.00�0 doi:10.1128/IAI.01313-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Surface Proteins and Exotoxins Are Required for the Pathogenesisof Staphylococcus aureus Pneumonia�

Juliane Bubeck Wardenburg,1,2 Ravi J. Patel,1 and Olaf Schneewind1*Departments of Microbiology1 and Pediatrics,2 University of Chicago, Chicago, Illinois 60637

Received 14 August 2006/Returned for modification 29 September 2006/Accepted 2 November 2006

A model of Staphylococcus aureus-induced pneumonia in adult, immunocompetent C57BL/6J mice is de-scribed. This model closely mimics the clinical and pathological features of pneumonia in human patients.Using this system, we defined a role for S. aureus strain Newman surface proteins and secreted exotoxins inpneumonia-related mortality.

Staphylococcus aureus is an important bacterial pathogencausing pneumonia in both adult and pediatric populations. Inrecent reports, workers have described the growing incidenceof severe S. aureus pneumonia in otherwise healthy individuals,often caused by multi-drug-resistant strains (8, 9). In addition,S. aureus remains one of the most common causes of ventila-tor-associated pneumonia, contributing to significant morbid-ity and mortality (18). At present, little is known about the S.aureus virulence factors that play a role in lower respiratorytract disease. The development of an adult, immunocompetentanimal model system recapitulating S. aureus pneumoniawould provide a useful tool for investigating such factors.

To date, small-animal models of S. aureus pneumonia haverelied on the use of surgical inoculation methods or infectionof immunocompromised animals (6, 17). While these modelshighlight the inflammatory response to intrapulmonary S. au-reus, detailed characterization of S. aureus-encoded virulencefactors has not been possible as the organisms are rapidlycleared from the lungs. A murine model of pulmonary infec-tion with agar-embedded S. aureus defined a role for coagulasein hematogenous infection (34), while a neonatal mouse modelof S. aureus pneumonia revealed the importance of the acces-sory gene regulator A (agrA), sarA, and staphylococcal proteinA (spa) in the development of disease (10, 13). Together, thedata suggest that multiple S. aureus virulence factors contrib-ute to the pathogenesis of pneumonia.

We sought to develop a transnasal murine model of S. au-reus pneumonia in adult, immunocompetent animals to permitinvestigation of virulence factors. To define infection parame-ters leading to evidence of pneumonia in 7-week-old C57BL/6Jmice (Jackson Laboratories), groups of 20 animals were inoc-ulated via the intranasal route with either phosphate-bufferedsaline (PBS) or one of three doses of S. aureus Newman, ahuman clinical isolate (7). Following 1:100 dilution of an over-night culture into fresh tryptic soy broth, staphylococci weregrown with shaking at 37°C to an optical density at 660 nm of0.5. Culture aliquots (50 ml) were sedimented by centrifuga-tion, and staphylococci were washed and suspended in 750 �l

PBS. Animals were anesthetized with ketamine and xylazine aspreviously described (21). After appropriate anesthesia wasdocumented, 30 �l of bacterial slurry was inoculated into theleft nare, and animals were held upright for 1 min postinocu-lation. All animals were given food and water ad libitum andobserved continually for 72 h. Immediately following inocula-tion, all animals displayed labored breathing marked by a highrespiratory rate and exaggerated chest wall excursion. Thisinitial physiologic change resolved within 6 h, and all live an-imals at this initial time were ambulatory and appeared to bewell. A small percentage of animals routinely succumbedwithin the first 6 h following inoculation, likely from the com-bined effects of aspiration and anesthesia. These animals werenot included in subsequent analyses. Inoculation with 4 � 108

CFU of S. aureus Newman resulted in a mortality rate ofapproximately 50% at 24 h, and an additional 20% of theanimals succumbed to infection within 48 h following inocula-tion (Fig. 1A). Importantly, all infected animals appeared to beill, having an increased respiratory rate, hunched posture, anddecreased mobility at 24 h. A smaller bacterial inoculum, 8 �107 CFU, resulted in no mortality, although the infected ani-mals appeared to be ill. The condition of this group of animalsimproved markedly by 48 h, and the animals resembled unin-fected animals. Similar results were obtained with an inoculumof 1.3 � 108 CFU of S. aureus Newman (data not shown).Inoculation with 8 � 108 CFU of S. aureus Newman resulted innearly 90% mortality by 24 h, which was significantly greaterthan the mortality observed for an inoculum of 4 � 108 CFUat the same time (P � 0.02); the surviving animals appeared tobe ill until 72 h postinfection.

To assess the kinetics of bacterial growth and clearance inthe lung, animals were infected with 3 � 108 to 4 � 108 CFUof wild-type S. aureus Newman. At different times postinfec-tion, animals were killed by forced CO2 inhalation, in compli-ance with the University of Chicago Institute of Animal Careand Use Committee guidelines. The right lung of each animalwas excised using aseptic techniques and suspended in 1 ml ofPBS, and the tissue was homogenized. Serial dilution and plat-ing were performed to determine the staphylococcal burden inthe lung tissue. Immediately following infection, approximatelyone-third of the inoculum could be recovered from the lungs(Fig. 1B); this level of recovery was not significantly differentfrom that at 6 h postinfection. Interestingly, by 24 h, in most

* Corresponding author. Mailing address: Department of Microbi-ology, University of Chicago, 920 E. 58th St., Chicago, IL 60637.Phone: (773) 834-9060. Fax: (773) 834-8150. E-mail: oschnee@bsd.uchicago.edu.

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animals there were significant increases in the number ofstaphylococci in lung tissues (P � 0.05), indicating that S.aureus Newman proliferated following infection. The level ofrecovery of S. aureus decreased at 48 to 72 h, corresponding toclinical improvement in the animals.

To discern whether pulmonary infection with S. aureus inthis murine model was capable of causing pathological lesionsobserved in human patients, we examined the lungs of infectedanimals for gross pathological changes, as well as histopatho-logic evidence of infection. The lung tissue of infected animalswas red and had a firm texture (Fig. 2A). In contrast, the lungsof uninfected animals were light pink and spongy. Inspection ofthe dissected left lung from a representative infected animal

further revealed a heterogeneous red color, consistent withmarked congestion (Fig. 2B, right panel).

For histopathologic analysis, the left lung was dissected andplaced in 1% formalin. Formalin-fixed tissues were processed,stained with hematoxylin and eosin, and visualized by lightmicroscopy. Histopathologic examination revealed the conse-quences of S. aureus infection for lung parenchyma. As a con-trol, we observed normal alveolar architecture in uninfectedanimals, in which thin-walled air spaces were defined by asingle layer of pneumocytes (Fig. 3A). As early as 6 h followinginoculation with S. aureus, aggregates of dark purple-stainedimmune cells were observed in the lungs of infected animals(Fig. 3B). The overall lung architecture was preserved at thistime, and no bacteria were evident in tissues. In contrast, by24 h, significant alveolar destruction had occurred along withinfiltration of large numbers of immune cells (Fig. 3C). Inter-estingly, large foci of staphylococci were found in lung tissuesat this time, consistent with bacterial proliferation. Dense,eosinophilic staining consistent with proteinaceous edema wasobserved to fill the alveolar space in infected animals (Fig. 3D).By 48 h, the size of these bacterial foci was reduced or fociwere absent, and the reemergence of air-filled spaces was ev-ident (Fig. 3E). At 72 h, significant air space had been restored;however, the alveolar walls remained thickened (Fig. 3F). To-gether, these data established a murine model of S. aureuspneumonia that closely mimics the clinical and histopathologicfindings for human patients. It is likely that both the size of theinoculum and the mouse strain utilized contribute to the de-velopment of pneumonia in this animal model. This combina-tion was not examined in previous studies. The large inoculum

FIG. 1. Inoculum-based mortality and proliferation of S. aureusNewman in murine lung tissue. (A) C57BL/6J animals were inoculatedwith either PBS or various doses of live S. aureus Newman via theintranasal route. The levels of survival were recorded at 24, 48, and72 h postinfection. Animals that appeared to be moribund were killedand counted as dead animals at the appropriate time. The results wereanalyzed to determine statistical significance using Fisher’s exact test(P � 0.02) (indicated by asterisks). (B) Animals were inoculated with3 � 108 to 4 � 108 CFU of S. aureus Newman, and the bacterial CFUin both lungs (5 min) or the right lung (6, 24, 48, and 72 h) wereenumerated at different times postinfection. Data were analyzed todetermine significance using Student’s t test.

FIG. 2. Gross pathology of animals infected with S. aureus via theintranasal route. Representative infected animals were compared touninfected animals in order to obtain gross pathological findings forlungs in situ (A) or following dissection of the left lung (B).

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required to cause pneumonia in these animals speaks to theremarkable ability of the murine immune system to eliminatethis pathogen from the lung, raising the possibility that anextension of this model system to other strains of immunocom-petent mice may enhance our understanding of pulmonaryimmunity against S. aureus.

To define S. aureus virulence factors critical for infection ofthe lower respiratory tract, mortality following pulmonary in-fection of mice with wild-type S. aureus Newman or isogenicmutants of this strain was assessed. S. aureus Newman strainscarrying a deletion in srtA and srtB have been described pre-viously (24, 27). agrA, spa, hla, and icaA mutants harboringbursa aurealis insertions were transduced into wild-type S.aureus Newman using isolates of the Phoenix transposon li-brary (2). All mutant strains were cultured in tryptic soy brothsupplemented with erythromycin (10 �g/ml). When mice were

inoculated with wild-type strain Newman, slightly more than70% of the infected animals succumbed over a 72-h period(Fig. 4A). Sortase A mutants (srtA) of S. aureus strain Newmanare unable to anchor surface proteins with LPXTG sortingsignals to the staphylococcal cell wall envelope; srtA mutationseffectively disrupt the surface display of 17 polypeptides (Spa,FnBPA, FnBPB, ClfA, ClfB, SdrC, SdrD, SdrE, IsdA [SasE],IsdB [SasJ], IsdH [SasI], SasA, SasB, SasC, SasD, SasF, andSasH) involved in staphylococcal adherence to host tissues orimmune evasive strategies (24, 25, 27). Compared to the mor-tality of animals challenged with the same dose of wild-typestaphylococci, there was a significant reduction in the mortalityof animals infected with sortase A mutants (P � 0.001). Pro-tein A, a surface protein with five immunoglobulin-bindingmodules, captures antibodies via their Fc portion (5, 14). S.aureus Newman insertion mutants with mutations in spa, with

FIG. 3. Histopathologic findings following intranasal inoculation of S. aureus. Lung tissue harvested from animals infected with S. aureusNewman was prepared and visualized by hematoxylin and eosin staining. Representative histology for an uninfected control having normal lungparenchyma is shown along with a series of images obtained for animals examined at the times indicated. Aggregates of purple-stained immunecells were seen as early as 6 h postinfection (arrowhead), and dense accumulation of bacteria was evident in tissues at 24 h postinfection(arrowhead).

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defects in protein A synthesis and in staphylococcal binding toimmunoglobulin, also displayed a significant defect for S. au-reus-induced mortality. These data corroborate previous ob-servations concerning the requirement of protein A for thepathogenesis of staphylococcal pneumonia in newborn mice(10). Sortase B (SrtB) anchors IsdC, a heme-binding protein,to the cell wall envelope, and mutants with a deletion in srtBhave defects in staphylococcal heme iron scavenging (22, 26).Deletion of srtB in S. aureus strain Newman resulted in only asmall reduction in mortality, suggesting that heme iron scav-enging may not be essential for the pathogenesis of staphylo-coccal pneumonia. The exopolysaccharide poly-N-acetylglu-cosamine (PNAG) is synthesized by icaABC products (12, 29).PNAG conjugates may function as a vaccine as immunizationof mice with this compound can protect the animals againstinvasive staphylococcal disease (20, 29). Furthermore, icaABCmutations cause a reduction in virulence in a mouse model ofabscess formation in kidney tissues (19). However, icaA mu-tants displayed no defect in virulence, suggesting that thePNAG exopolysaccharide is not required for the pathogenesisof staphylococcal pneumonia in mice.

In previous work, researchers reported that tracheal instil-lation of S. aureus strain 8325-4 into the lungs of anesthetizedSprague-Dawley rats causes damage to alveolar epithelia anderythrocytes in a manner requiring hla, which encodes staph-ylococcal alpha-toxin, the secreted hemolysin expressed by vir-tually all S. aureus strains (16, 28). After binding to receptorsites on cell surfaces, alpha-toxin forms a heptameric assemblyand funnel-shaped pore that perforates host cell membranes(3, 35). S. aureus mutants lacking hla have reduced virulence ininvasive disease models as larger numbers of staphylococci arerequired to kill mice following either intraperitoneal or intra-mammary infection (4, 33). These observations prompted us toexamine the virulence of S. aureus Newman hla mutants inmurine pneumonia. Interestingly, animals infected with the hlamutant strain appeared to be moderately ill within 24 h post-inoculation; however, only a small number of these animalssuccumbed to the infection (Fig. 4B). The death of these ani-mals was delayed, occurring more than 48 h postinoculation.Expression of many staphylococcal genes is regulated by agr,the accessory gene regulatory locus. This locus provides bothquorum sensing and regulatory control of virulence (31).Briefly, AgrA and AgrC, a response regulator and a sensorykinase, perceive the environmental abundance of autoinducerpeptide to activate expression of an array of genes, includinghla and other exotoxin genes, at the threshold level (15). Theautoinducer peptide, synthesized from an AgrD proinducer, isprocessed and secreted by AgrB (23). Mutations in agrA areknown to abrogate quorum sensing (32). S. aureus Newmanvariants carrying a bursa aurealis insertion in agrA are avirulentin the murine pneumonia model, as none of the experimentalanimals succumbed to infection (Fig. 4). These findings can beexplained by the regulatory defect of agrA mutations, whichabrogate expression of many virulence genes, including genesencoding �-hemolysin, �-hemolysin, �-hemolysin, and �-hemo-lysin, as well as leukocidins (31). S. aureus Newman cannotexpress �-hemolysin, as this strain has a phage insertion in thehlb gene (1). However, three secreted �-hemolysins (HlgA,HlgB, and HlgC) assemble into heterooligomeric toxins with astructure and function similar to the structure and functionreported for �-hemolysin (11). Thus, the observed virulencedefect of agrA mutants in the murine pneumonia model islikely due to the aggregate loss of all secreted hemolysins andtoxins (31).

The inability of agrA and hla mutant strains to contribute tolethality in experimental animals raises the interesting possi-bility that S. aureus exotoxins may play a pivotal role in lungparenchymal injury. It is readily appreciated that insults to thealveolar epithelium contribute to impaired gas exchange. Fur-thermore, there are detrimental systemic effects of pulmonaryinflammation, as patients with acute lung injury are susceptibleto multiple-organ dysfunction and increased mortality. Thesesystemic effects are likely mediated by the combined effects ofinflammatory cytokines, such as interleukin-1 and interleu-kin-8, along with the products of arachadonic acid metabolism,including thromboxane A2 and prostaglandins. Our observa-tion that agrA and �-hemolysin mutants do not induce mortal-ity may provide insight into the specific mechanism by which S.aureus-induced lung injury contributes to the significant mor-bidity and mortality associated with severe S. aureus pneumo-nia. Together with the observation that protein A is required

FIG. 4. S. aureus mutants lacking protein A (spa) or all surfaceproteins (srtA) or exoproteins (agrA and hla) are defective in the abilityto cause pneumonia-related mortality. Animals infected with 3 � 108

to 4 � 108 CFU of wild-type S. aureus Newman or isogenic mutantstrains were scored for acute lethal disease, which demonstrated thatthere was a significant reduction in mortality for animals infected withboth the srtA and spa strains (A). Analysis of mutants with bursaaurealis insertions in agrA and hla (alpha-toxin) likewise demonstratedthat there was a marked reduction in the ability to cause acute lethaldisease (B). Statistical significance was evaluated by Fisher’s exact test(one asterisk, P � 0.001; two asterisks, P 0.002).

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for inflammatory responses in the lung following S. aureusinfection, our data suggest that one of the principal functionsof S. aureus virulence factors may be to cause lung parenchy-mal insult, facilitating bacterial survival and evasion of hostdefenses.

Multiple recent studies have highlighted the association ofthe Panton-Valentine leukocidin (PVL) with S. aureus strainsisolated from patients with severe necrotizing pneumonia (8,30). Like alpha-toxin and other hemolysins, PVL is a pore-forming toxin whose expression is regulated by agr. The preciserole of PVL in pulmonary infection has not been elucidatedyet. Considering the data presented here, it is plausible tospeculate that S. aureus alpha-toxin and PVL may both havethe ability to induce pulmonary inflammation, resulting in sys-temic manifestations of disease and concomitant mortality.The murine model system described here should allow morerigorous assessment of the role of these cytotoxins and otherstaphylococcal virulence factors in the pathogenesis of pulmo-nary infection.

We thank the Department of Pathology at The University of Chi-cago for preparation of histology samples.

J.B.W. is an NICHD Fellow of the Pediatric Scientist DevelopmentProgram (NICHD grant K12-HD00850). Work on the role of surfaceproteins and sortases in the pathogenesis of S. aureus infections wassupported by United States Public Health Service grants AI38897 andAI52474 from the National Institute of Allergy and Infectious DiseasesDivision of Microbiology and Infectious Diseases to O.S.

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Editor: V. J. DiRita

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