diminished virulence of an alpha-toxin mutant of ...mouse (19) brain abscess models. however, the...

INFECTION AND IMMUNITY,0019-9567/01/$04.00�0 DOI: 10.1128/IAI.69.11.6902–6911.2001

Nov. 2001, p. 6902–6911 Vol. 69, No. 11

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Diminished Virulence of an Alpha-Toxin Mutant ofStaphylococcus aureus in Experimental Brain Abscesses

TAMMY KIELIAN,1* AMBROSE CHEUNG,2 AND WILLIAM F. HICKEY1

Departments of Pathology1 and Microbiology and Immunology,2 Dartmouth-Hitchcock Medical Center,Dartmouth Medical School, Lebanon, New Hampshire 03756

Received 23 March 2001/Returned for modification 8 June 2001/Accepted 23 July 2001

Staphylococcus aureus is one of the major etiologic agents of brain abscesses in humans, occasionally leadingto focal neurological deficits and even death. The objective of the present study was to identify key virulencedeterminants contributing to the pathogenesis of S. aureus in the brain using a murine brain abscess model.The importance of virulence factor production in disease development was demonstrated by the inability ofheat-inactivated S. aureus to induce proinflammatory cytokine or chemokine expression or brain abscessformation in vivo. To directly address the contribution of virulence determinants in brain abscess development,the abilities of S. aureus strains with mutations in the global regulatory loci sarA and agr were examined. AnS. aureus sarA agr double mutant exhibited reduced virulence in vivo, as demonstrated by attenuated proin-flammatory cytokine and chemokine expression and bacterial replication. Subsequent studies focused on theexpression of factors that are altered in the sarA agr double mutant. Evaluation of an alpha-toxin mutantrevealed a phenotype similar to that of the sarA agr mutant in vivo, as evidenced by lower bacterial burdens andattenuation of cytokine and chemokine expression in the brain. This suggested that alpha-toxin is a centralvirulence determinant in brain abscess development. Another virulence mechanism utilized by staphylococci isintracellular survival. Cells recovered from brain abscesses were shown to harbor S. aureus intracellularly,providing a means by which the organism may establish chronic infections in the brain. Together, these dataidentify alpha-toxin as a key virulence determinant for the survival of S. aureus in the brain.

Staphylococcus aureus is a potent and versatile pathogen ofhumans. The frequencies of both nosocomial and community-acquired staphylococcal infections have increased steadily overthe years (22). In addition, treatment of these infections hasbecome more challenging due to the emergence of multidrug-resistant strains (8, 29). S. aureus infection may be manifestedin a wide variety of forms, including focal abscesses, arthritis,endocarditis, and septicemia. Moreover, S. aureus has a diversearsenal of virulence factors that contribute to the pathogenesisof disease. These can be broadly subdivided into surface andextracellular secreted proteins. Surface proteins include bothstructural components of the bacterial cell wall, such as pep-tidoglycan and lipoteichoic acid, and surface proteins prefer-entially expressed during exponential growth, including proteinA, fibronectin-binding protein, and clumping factor. Secretedproteins are generally elaborated during the stationary phaseof bacterial growth and include such proteins as alpha-toxin,enterotoxin B, lipase, and V8 protease.

The differential regulation of surface and extracellular viru-lence factors during the growth of S. aureus is controlled by atleast three global regulatory systems, including sarA, agr, andsae (4, 10, 20). The sarA locus is involved in the expression ofexoproteins and cell wall proteins that are potential virulencedeterminants in experimental infections (4, 6, 13). The agrlocus up-regulates the production of extracellular proteinswhile repressing the synthesis of surface proteins (20, 24, 27,

28). The sae regulatory locus activates the production of sev-eral exoproteins, including alpha- and beta-toxin, coagulase,and protein A (10). As an alternative to dealing with antibiotic-resistant strains, the effective targeting and inactivation ofthese global regulatory loci could have a profound impact ondisease therapy. Therefore, an understanding of the host re-sponse to S. aureus global regulatory mutants in complex dis-ease models may reveal the importance of key virulence de-terminants in disease progression.

One virulence mechanism utilized by staphylococci is intra-cellular survival (21). The intracellular environment protectsstaphylococci from host defense mechanisms as well as thebactericidal effects of antibiotics. Intracellular survival of S.aureus has been demonstrated in both epithelial cells and neu-trophils (12, 16). Staphylococci also produce cytotoxins, such asalpha-toxin, which cause pore formation and induce proinflam-matory changes in mammalian cells (11, 30). Both the intra-cellular survival of S. aureus and the production of virulencefactors, such as alpha-toxin, most probably play an importantrole in the complex response to S. aureus in the host.

In this study we have utilized a murine experimental brainabscess model using S. aureus, one of the major etiologicagents of brain abscesses in humans (23, 31). The course ofbrain abscess progression in the rodent model closely parallelswhat is observed in human disease in terms of histologicalappearance, infiltrating leukocytes, and chronicity (7, 19).Therefore, evaluating the role of bacterial virulence determi-nants and the host immune response to S. aureus in this modelsystem should approximate conditions encountered during hu-man disease. We have previously demonstrated that S. aureusinduces rapid and sustained expression of numerous proin-flammatory cytokines and chemokines in both the rat (18) and

* Corresponding author. Present address: University of Arkansas forMedical Sciences, Department of Anatomy and Neurobiology, B118Biomedical Research Center, Slot 510, 4301 W. Markham St., LittleRock, AR 72205. Phone: (501) 526-6348. Fax: (501) 686-6382. E-mail:[email protected].

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mouse (19) brain abscess models. However, the role of bacte-rial virulence factors in the expression of these mediators re-mains to be defined.

The present study was designed to examine the role of S.aureus virulence determinants in brain abscess development.The results demonstrate that staphylococcal strains that lackboth the sarA and agr global regulatory loci or alpha-toxinexhibit reduced virulence in vivo. Examination of proinflam-matory cytokine and chemokine expression revealed that al-though both sarA agr and alpha-toxin mutants are capable ofinducing mediator expression during the acute phase of infec-tion, this response is rapidly attenuated compared to the strongand sustained expression detected in response to isogenicstrains. Moreover, cells recovered from brain abscesses werefound to harbor S. aureus intracellularly, providing a mecha-nism by which this organism can establish chronicity and anti-biotic resistance, both features of human central nervous sys-tem (CNS) abscesses. These results reveal the importance ofS. aureus-derived virulence factors, in particular alpha-toxin, inbrain abscess development.

MATERIALS AND METHODS

Mice. Male AKR/J mice 6 to 8 weeks of age were obtained from JacksonLaboratories (Bar Harbor, Maine). The animal use protocol has been approvedby the Dartmouth College Institutional Animal Care and Use Committee and isin accord with National Institutes of Health guidelines for the use of rodents.

Bacterial strains. The bacterial strains used in this study are listed in Table 1.

All of the mutants utilized were derived from the S. aureus strain RN6390.Preparation of S. aureus-laden agarose beads. Live S. aureus cells were en-

capsulated in agarose beads prior to implantation in the brain as previouslydescribed (18, 19). The use of agarose beads prevents bacterial dissemination orrapid wound sterilization by the host. Briefly, bacterial strains were grown topostexponential phase at 37°C in brain heart infusion (BHI) medium (BectonDickinson, Sparks, Md.). A total of 109 bacteria were added to a solution of 1.4%low-melting-point agarose (type XII; Sigma, St. Louis, Mo.) at 40°C. The mixturewas then added to rapidly swirling heavy mineral oil (Sigma) prewarmed to 37°Cand quickly cooled to 0°C on crushed ice. Beads were washed four times in 1�Dulbecco’s phosphate-buffered saline (DPBS) (Mediatech Cellgro, Herndon,Va.) to remove mineral oil. Beads with diameters between 50 and 100 �m, asdetermined by phase-contrast microscopy, were used for implantation into thebrain. Heat-inactivated bacteria were prepared by incubating organisms for 1 hat 56°C prior to encapsulation. The bacterial viability or sterility of bead prep-arations was confirmed by overnight culture in BHI medium and quantitativeculture on blood agar plates (Becton Dickinson, Franklin Lakes, N.J.).

Induction of experimental brain abscesses. Mice were anesthetized with aver-tin (z-z-z-tribromoethanol) intraperitoneally, and a 1-cm longitudinal incisionwas made along the vertex of the skull extending from the ear to the eye,exposing the frontal sutures. A burr hole was drilled 1 mm anterior and 1 mmlateral to the frontal suture of the calvarium. A Hamilton syringe fitted withflexible tubing and a pulled, fine-tipped glass micropipette (diameter � 0.1 mm)was used to deliver beads into the brain parenchyma. A total of 3 �l of beads (105

CFU) was slowly infused 3 mm deep from the external surface of the calvariumto prevent reflux during injection. Using this approach, bacteria were reproduc-ibly deposited into the head of the caudate or adjacent frontal lobe white matter.To collect brain abscess tissues for analysis, lesion sites were demarcated by thestab wound created during injections. Brain tissues were sectioned 0.5 mm on allsides of the stab wound, and cortical material was removed in order to focus onchanges occurring in the white matter. Previous studies have established thatimplantation of agarose beads alone induces minimal proinflammatory cytokineor chemokine expression or cellular infiltration, indicating that neither the stabwound nor the deposition of foreign material (agarose beads) induces inflam-matory changes in the brain (18, 19). The mortality rate associated with brainabscess induction was minimal, with �95% of animals surviving the procedure.

Quantification of viable bacteria associated with brain abscesses in vivo. Toquantify the numbers of viable bacteria associated with brain abscesses in vivo,homogenates were prepared by disrupting brain abscess tissues (consisting ofboth solid tissue and purulent material) in 0.5 ml of DPBS supplemented with acomplete protease inhibitor cocktail tablet (Roche, Indianapolis, Ind.). Serial10-fold dilutions of homogenates were plated onto blood agar plates (BectonDickinson). Titers were calculated by enumerating colony growth and are ex-pressed as the mean log10 CFU per milliliter of homogenate.

FIG. 1. Brain abscesses are induced by live, but not heat-inactivated, S. aureus. Mice were implanted with live (A) or heat-inactivated (B)encapsulated organisms as described in Materials and Methods. Animals were euthanized 7 days later, and brain lesions were collected forhistological analysis. Brain tissues (5-�m sections) were stained using hematoxylin and eosin to reveal changes in tissue architecture. (A) Note theformation of a large, well-demarcated abscess in the animal which received live S. aureus. The arrows delineate the margin between surroundingbrain parenchyma and the abscess, which is denoted by an asterisk. (B) The arrow denotes a small inflammatory focus associated with the stabwound created during the injection of heat-inactivated organisms. Results presented in both panels are representative of three independentexperiments. Original magnification, �22.5.

TABLE 1. S. aureus strains used in this study

Strain designation Genotype Antibiotic resistance

ALC132 RN6390 isogenic strain NoneALC136 sarA mutant Ermr

ALC134 agr mutant Tetr

ALC135 sarA agr double mutant Ermr Tetr

ALC837 Alpha-toxin Ermr

ALC812 Lipase-negative Ermr Tetr

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Immunohistochemistry. To prepare tissues for immunohistochemistry, ani-mals were perfusion fixed with 4% paraformaldehyde in 0.1 M phosphate buffer,pH 7.4. The brain was removed, postfixed in paraformaldehyde for 30 min, andwashed in 0.2 M phosphate buffer, pH 7.4, overnight. Tissues were cryoprotectedin 30% sucrose for 24 h and then snap frozen at the optimal cutting temperaturefor immunohistochemistry.

Frozen sections of fixed tissues were processed for immunohistochemistryusing the avidin-peroxidase method as previously described (14). The followingantibodies were used for analysis: anti-GR-1 (neutrophil-specific); anti-CD11b,which reacts with the beta-integrin subunit expressed on neutrophils, monocytes/macrophages, and microglia; and the isotype control antibody rat immunoglob-ulin G2b (all from BD PharMingen). Sections were then incubated with abiotinylated secondary anti-rat immunoglobulin G antibody (Vector Laborato-ries) and developed using the substrate 3,3�-diaminobenzidene.

Isolation of cells from brain abscesses. Brain abscesses were collected fromanimals at days 5 and 7 following bacterial exposure to recover infiltrating andresident cells for gentamicin protection assays. Briefly, mice were perfused trans-cardially with DPBS to eliminate intravascular leukocytes. Tissue blocks contain-ing the brain abscesses were pooled, minced into fine pieces using forceps, andincubated with collagenase type II (final concentration, 1 mg/ml; Sigma) for 20min at 37°C. The resulting cell suspension was layered onto a discontinuousPercoll gradient (Amersham Pharmacia Biotech, Piscataway, N.J.) to separatemyelin debris from cells. Cells were washed twice with 1� DPBS, and viabilitywas determined by using trypan blue exclusion dye analysis.

Gentamicin protection assay. To determine whether cells recovered frombrain abscesses harbored viable S. aureus intracellularly, gentamicin protectionassays were performed. The S. aureus parental strain used in this study, RN6390,is sensitive to gentamicin and rifampin (T. Kielian, unpublished observations).Gentamicin kills extracellular S. aureus, but because its ability to permeate theeukaryotic cell membrane is limited, intracellular organisms are protected fromits bactericidal activity. Following the isolation of cells from brain abscesses, analiquot of cells was taken to determine the total bacterial titer (extra- plusintracellular organisms). In addition, cells were treated with gentamicin (100�g/ml) for 2 h at 37°C to kill extracellular organisms. After 2 h, cells were washed

twice to remove the gentamicin and serial dilutions of each treatment wereperformed to determine bacterial titers (log10 CFU). To ensure that intracellularbacteria were sensitive to an antibiotic which can penetrate mammalian cellmembranes, cells were incubated with rifampin (1 mg/ml), which effectivelyreduced the number of intracellular CFU.

RPA. Cytokine and chemokine mRNA expression in brain abscess tissues wereexamined by RNase protection assay (RPA) using the RiboQuant RPA kit (BDPharMingen). The multiprobe template sets used for analysis include mCK-2,mCK-3b, and mCK-5. All template sets contain probes for the housekeepinggenes L32 and glyceraldehyde-3-phosphate dehydrogenase, which serve as inter-nal controls for the assay. Probes were synthesized using [�-33P]UTP, resulting inan average specific activity of 4 � 106 cpm/�l. RPA was performed according tothe manufacturer’s instructions, using 10 �g of total RNA per sample. Productswere resolved on a 6% acrylamide gel, dried, and exposed to film (BioMax MR;Kodak, Rochester, N.Y.).

Statistics. Significant differences between experimental groups were deter-mined by using the Mann-Whitney rank sum test at the 95% confidence interval.

RESULTS

Brain abscesses are induced by live, but not heat-inactivated,S. aureus. To determine whether brain abscess formation re-quires ongoing bacterial replication and/or the production ofextracellular virulence factors, the ability of heat-inactivatedbacteria to induce abscesses was examined. An advantage ofusing heat-inactivated organisms as the inflammatory stimulusis that the contribution of an evolving or resolving infectiousprocess can be eliminated. In addition, virulence factors arenot produced by heat-inactivated organisms, allowing the di-rect assessment of the importance of these mediators in ab-scess pathogenesis. Heat inactivation of S. aureus was con-

FIG. 2. Live, but not heat-inactivated, S. aureus induces potent proinflammatory cytokine and chemokine expression in the brain. Mice wereimplanted with live or heat-inactivated (H-I) encapsulated organisms as described in Materials and Methods. Animals were euthanized at theindicated time points, and inoculation sites were collected for RNA extraction and analysis by RPA. The identity of each experimental mRNA isdenoted at the left. Results presented are representative of three independent experiments.

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firmed by the inability of organisms to grow in BHI medium oron blood agar plates (data not shown). As shown in Fig. 1A,animals receiving live S. aureus developed large brain abscessesassociated with a significant neutrophil and mononuclear cellinfiltrate. In contrast, there was no evidence of abscess forma-tion or any cellular infiltrates in those animals receiving heat-inactivated S. aureus (Fig. 1B). To determine whether theinability of heat-inactivated organisms to induce abscess for-mation was related to the number of bacteria inoculated intothe brain, animals were challenged with 1-log-greater numbersof heat-inactivated S. aureus. Even increasing the number ofheat-inactivated bacteria by 1 log was not sufficient to induceabscess formation (data not shown).

We have previously demonstrated that live S. aureus inducesthe rapid and sustained expression of proinflammatory cyto-kines and chemokines in the brain (18, 19). The inability ofheat-inactivated organisms to induce abscess formation sug-gested that their ability to initiate proinflammatory cytokineand chemokine production might be impaired. Indeed, bothproinflammatory cytokine and chemokine induction in thebrain was significantly attenuated in those animals receivingheat-inactivated compared to live organisms (Fig. 2). Increas-ing the number of heat-inactivated organisms inoculated intothe brain by 1 log was not sufficient to attain the levels ofproinflammatory cytokine and chemokine expression observedin response to live bacteria (data not shown). These resultssuggest that some factor(s) produced by viable organisms iscritical to the induction of proinflammatory cytokine and che-mokine expression and brain abscess development.

A sarA agr S. aureus global regulatory mutant exhibits re-duced virulence in an experimental brain abscess model. Thefindings obtained with heat-inactivated organisms suggestedthat S. aureus produces a virulence factor(s) which participatesin brain abscess formation. The sarA and agr global regulatoryloci are two major regulators of virulence factor expression inS. aureus. To determine what effect global regulatory loci playin brain abscess development, the ability of a sarA, agr, andsarA agr double mutant to induce disease was examined. Sinceeach global regulatory loci mutant displays a particular viru-lence factor phenotype, analysis of each mutant would allowidentification of a smaller subset of specific factors for furtherexamination. The replication of a sarA agr double mutant wasmarkedly attenuated compared to its isogenic control strainRN6390 at day 5 following bacterial exposure (Fig. 3). Inter-estingly, both sarA and agr single mutants replicated to thesame extent as RN6390 in the brain parenchyma, suggesting anadditive effect by the mutations in both regulatory loci. Todetermine whether the attenuated virulence of the sarA agrdouble mutant was related to an inability to replicate versusenhanced bacterial clearance, the kinetics of bacterial replica-tion was evaluated for this mutant. As shown in Fig. 4, thenumber of viable organisms recovered from the brains of an-imals inoculated with the sarA agr double mutant was reducedas early as 24 h following bacterial exposure compared to itsisogenic strain RN6390. However, the sarA agr double mutantwas capable of replication to a limited extent, as evidenced bythe small increase in bacterial titers observed at day 3 followingbacterial exposure (Fig. 4). By day 5, the number of viableorganisms in the brains of animals receiving the sarA agr dou-ble mutant was dramatically reduced compared to that in

brains of animals receiving the wild type, with 3-log-fewer CFUin the former.

To determine whether the reduced virulence of the sarA agrdouble mutant correlated with an attenuated host immuneresponse in the brain, proinflammatory cytokine and chemo-kine expression was evaluated in animals inoculated with ei-ther the sarA agr double mutant or RN6390. Initially, therewere no observable differences in the amount of proinflamma-

FIG. 3. Replication of a sarA agr S. aureus mutant is attenuated inthe brain. Animals were implanted with either sarA (n � 5), agr (n �7), or sarA agr (n � 7) mutants or the isogenic strain RN6390 (n � 7)as described in Materials and Methods. Animals were euthanized 5days following bacterial exposure, and the number of viable organismsin the brain was determined by quantitative culture. Titers are ex-pressed as the mean log10 CFU per milliliter of brain abscess homog-enate from three separate experiments. Significant differences are de-noted with asterisks (��, P � 0.001). Error bars, standard deviations.

FIG. 4. Comparison of growth kinetics between the sarA agr andRN6390 strains. Animals were implanted with either the sarA agrdouble mutant (hatched bars) or RN6390 isogenic strain (open bars)as described in Materials and Methods. Animals (n � 3 to 6 per group)were euthanized at the indicated time points, and the number of viableorganisms in the brain was determined by quantitative culture. Titersare expressed as the mean log10 CFU per milliliter of brain abscesshomogenate from two separate experiments. Significant differencesare denoted with asterisks (�, P � 0.05). Error bars, standard devia-tions.


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tory cytokine and chemokine expression elicited by eitherstrain (Fig. 5). However, within 48 h following bacterial expo-sure, proinflammatory cytokine and chemokine expression wasundetectable in the brains of animals inoculated with the sarAagr double mutant compared to the continued induction inresponse to its isogenic strain RN6390 (Fig. 5). The cytokineand chemokine response to RN6390 was still detected at days3 and 5 following bacterial exposure, whereas the response tothe sarA agr mutant remained negative (data not shown). Eventhough bacterial burdens and mediator expression were atten-uated in response to the sarA agr double mutant, these animalsstill developed rudimentary abscesses, albeit the lesions weredramatically smaller compared to those of animals receivingRN6390 (data not shown). This suggests that a virulence fac-tor(s), whose expression is reduced or absent in the sarA agrmutant, must play an important role in the pathogenic re-sponse to S. aureus in the brain.

Diminished virulence of an alpha-toxin mutant of S. aureusin experimental brain abscesses. The reduced virulence of thesarA agr double mutant in the brain suggested that any one ofa number of virulence factors could be involved in mediatingtissue damage in this model. An important factor, the expres-sion of which is greatly attenuated in the sarA agr doublemutant, is alpha-toxin. Previous studies have demonstratedthat alpha-toxin expression by the sarA agr double mutant issignificantly lower compared to that by either sarA or agr singlemutants (5). Alpha-toxin mediates its activity through formingpores in mammalian cell membranes, resulting in cell destruc-tion by osmotic lysis. Because of its importance in other diseasemodels (3, 15, 17), the role of alpha-toxin in brain abscessdevelopment was evaluated.

Replication of an S. aureus alpha-toxin mutant was markedlyattenuated in the brain compared to that of RN6390, with the

number of viable bacteria recovered approximately 3 to 4 logslower in the former (Fig. 6). To determine whether the re-duced virulence of the alpha-toxin mutant correlated with anattenuated host immune response in the brain, proinflamma-tory cytokine and chemokine expression was evaluated inanimals inoculated with either the alpha-toxin mutant or

FIG. 5. Proinflammatory cytokine and chemokine expression in the brain is attenuated in response to an S. aureus sarA agr double mutant. Micewere implanted with either the sarA agr mutant or RN6390 isogenic strain as described in Materials and Methods. Animals were euthanized at theindicated time points, and inoculation sites were collected for RNA extraction and analysis by RPA. The identity of each experimental mRNA isdenoted at the left. Results presented are representative of two independent experiments.

FIG. 6. The replication of an S. aureus alpha-toxin mutant is atten-uated in the brain. Animals were implanted with either the alpha-toxinmutant (hatched bars) or RN6390 isogenic strain (open bars) as de-scribed in Materials and Methods. Animals (n � 4 to 6 per group) wereeuthanized at the indicated time points, and the number of viableorganisms in the brain was determined by quantitative culture. Titersare expressed as the mean log10 CFU per milliliter of brain abscesshomogenate from two separate experiments. Significant differencesare denoted with asterisks (�, P � 0.05). Error bars, standard devia-tions.


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RN6390. Similar to the results obtained with the sarA agr doublemutant, the expression of proinflammatory cytokines and chemo-kines was attenuated in animals receiving the alpha-toxin mutantat 48 h following bacterial exposure (Fig. 7). However, the alpha-toxin mutant was capable of inducing mediator expression asdemonstrated by the production of numerous mediators as earlyas 24 h, but this response was short-lived. The cytokine and che-mokine response to RN6390 was still detected at days 3 and 5following bacterial exposure, whereas the response to the alpha-toxin mutant remained negative (data not shown).

Since both bacterial burdens and proinflammatory cytokineand chemokine responses were attenuated in animals receiving analpha-toxin mutant, the ability of these organisms to producebrain abscesses was investigated. We were able to detect onlysmall inflammatory foci in the brains of animals inoculated withthe alpha-toxin mutant, compared to the large, well-formed ab-scesses in those mice receiving the isogenic strain RN6390 (Fig.8). Immunohistochemical analysis revealed a paucity of neutro-phils in the brains of animals injected with the alpha-toxin mutant,whereas these cells were the predominant type infiltrating ab-scesses in response to RN6390 (Fig. 8). Together, these dataindicate that alpha-toxin is an important, and possibly pivotal,virulence determinant in CNS abscess formation.

Lipase is not a critical virulence determinant in brain ab-scess formation. In our experimental model, brain abscessesare induced in the white matter, which contains a large amountof lipid, namely, myelin. Therefore, we reasoned that bacteriallipase expression might be a key virulence determinant in-volved in the invasion and spread of bacteria throughout thebrain parenchyma. To examine this possibility, the virulence of

an S. aureus lipase mutant was evaluated. The growth kineticsof both the lipase mutant and RN6390 were identical (data notshown), suggesting that lipase does not contribute significantlyto the virulence of S. aureus in the brain.

S. aureus survives intracellularly within cells recovered frombrain abscesses. S. aureus has the ability to survive and repli-cate intracellularly within neutrophils and epithelial cells (12,16). Currently, it is not known whether bacteria persist withincells associated with brain abscesses. To determine whethercells isolated from abscesses harbor viable S. aureus, gentami-cin protection assays were performed on cells recovered fromabscesses 5 days following bacterial injection. This time pointwas selected for evaluation since it is when bacterial loads aremaximal or on the decline. Viable bacteria were still detectedin abscess-derived cells treated with gentamicin, despite thefact that the antibiotic effectively reduced the overall numberof bacteria by elimination of extracellular organisms (Fig. 9).Treatment of cells with rifampin further reduced the numberof viable S. aureus cells, demonstrating the sensitivity of bac-teria to an antibiotic capable of penetrating mammalian cells,and thus supporting the argument for their probable intracel-lular location. These findings indicate that S. aureus can surviveintracellularly within cells associated with brain abscesses invivo, providing a mechanism by which this organism can estab-lish chronic infections in the CNS.

DISCUSSION

Staphylococci produce a wide array of virulence determi-nants that play a role in the complex interactions between the

FIG. 7. Proinflammatory cytokine and chemokine expression in the brain is attenuated in response to an S. aureus alpha-toxin mutant. Micewere implanted with either alpha-toxin mutant or RN6390 encapsulated organisms as described in Materials and Methods. Animals wereeuthanized at the indicated time points, and inoculation sites were collected for RNA extraction and analysis by RPA. The identity of eachexperimental mRNA is denoted at the left. Results presented are representative of two independent experiments.


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organism and its host. While the in vivo function of thesevirulence factors is incompletely understood, it is probable thatthe identification of key factors required for disease progres-sion may lead to novel therapies in the treatment of staphylo-coccal infections. This study investigates the importance ofvirulence factors produced by S. aureus in experimental brainabscess development.

To establish whether ongoing bacterial replication and/orvirulence factor production was required for brain abscess in-duction, the response to heat-inactivated organisms was exam-ined. Heat-inactivated S. aureus itself was not sufficient toinduce proinflammatory cytokine or chemokine expression orabscess formation in the brain. These findings suggested thatthe active secretion of a virulence factor(s) was important fordisease induction. However, it was also conceivable that brainabscess formation was influenced by structural components ofthe bacterial cell wall which were limiting in these experiments.To ensure that the inability to induce cytokine or chemokineexpression was not merely a result of suboptimal concentra-

tions of cell wall products, we increased the amount of heat-inactivated organisms inoculated into the brain. We were un-able to induce significant mediator expression or abscessformation following the introduction of a 1-log-greater numberof heat-inactivated organisms, suggesting that virulence factorproduction is important for brain abscess formation. However,our results cannot discount a potential additive effect betweenvirulence determinants and an increasing mass of cell wallproducts produced by viable organisms. We are currently eval-uating the ability of purified peptidoglycan and lipoteichoicacid to induce pathology in the brain. Nonetheless, these datasuggest an important role for virulence factor expression in thehost response to S. aureus in the brain.

To delineate virulence factors which potentially participatein abscess pathogenesis, the growth of S. aureus global regula-tory mutants was examined in the brain. Interestingly, an S.aureus sarA agr double mutant was markedly less virulent invivo, whereas both single mutants behaved similarly to theparental strain RN6390 in terms of bacterial replication and

FIG. 8. An S. aureus alpha-toxin mutant fails to induce abscess formation in the brain. Mice were implanted with either the isogenic strainRN6390 (A and C) or an alpha-toxin mutant (B and D) as described in Materials and Methods. Animals were euthanized at day 7 followingbacterial exposure to evaluate brain abscess formation and cellular infiltrates. Serial sections of brain lesions were stained with the neutrophil-specific antibody GR-1 (A and B) or anti-CD11b (C and D), which reacts with neutrophils, monocytes/macrophages, and resident microglia. Notethe outer edge of an abscess in the animal receiving RN6390 (dense staining area in the corners of panels A and C), whereas a well-defined abscesswas not detected in response to the alpha-toxin mutant. Results presented are representative of two independent experiments. Bars, 20 �m.


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immune activation in the brain. This finding cannot be ex-plained by impaired replication of the sarA agr double mutant,since previous studies have established that its growth rate isidentical to that of its isogenic strain RN6390 (5). Additionally,this mutant replicated successfully during the first three daysfollowing implantation into the brain. This suggested that vir-ulence factors, the expression of which are dramatically re-duced in the sarA agr double mutant, are pivotal for brainabscess induction.

The findings obtained with the sarA agr mutant led us toexamine the potential role of two virulence factors, alpha-toxinand lipase, in brain abscess development. Similar to the find-ings obtained with the sarA agr double mutant, the replicationof an alpha-toxin mutant was significantly attenuated com-pared to that of its isogenic strain RN6390. Importantly, thevirulence of a lipase mutant was equivalent to that of its iso-genic strain RN6390, indicating that the results obtained withthe alpha-toxin mutant were specific. In addition to its im-paired replication and enhanced clearance, the alpha-toxinmutant did not induce well-defined abscesses in the brain;rather, minimal inflammation and few infiltrating cells wereobserved. This finding may be explained by the following. Therapid replication of wild-type S. aureus (RN6390) induces pro-longed cytokine and chemokine expression and direct damageto the brain parenchyma by bacterial products, leading to ab-scess formation. RN6390 produces alpha-toxin, which formssmall transmembrane pores spanning the plasma membrane ofmammalian cells, leading to osmotic lysis. Secretion of alpha-toxin is an effective way to eliminate infiltrating neutrophilsand other leukocytes, cells which play a pivotal role in contain-ing bacterial burdens. The lack of toxin expression in the al-pha-toxin mutant now allows more leukocytes to survive in thebrain, rapidly reducing bacterial loads. The quick and effective

containment of the alpha-toxin mutant in the brain preventsthese immune responses and bacteria from persisting, whichmost likely explains the absence of well-defined abscesses. Thestriking reduction in virulence associated with the alpha-toxinmutant also indicates that alpha-toxin is the major virulencedeterminant in the brain and its activity cannot be substitutedby the gamma- and delta-toxins which are still produced by thismutant. The finding that animals inoculated with the sarA agrdouble mutant develop microabscesses can also be explainedon the basis of alpha-toxin expression. Although alpha-toxinproduction is significantly decreased in the sarA agr mutant,some protein is still detected due to induction by other regu-latory systems (5). The small amount of alpha-toxin producedby the sarA agr double mutant may be sufficient to transientlycompromise the host response, which eventually contains theinfection without inducing much damage to the brain paren-chyma, resulting in microscopic abscesses. However, it is likelythat an additional factor(s) participates in S. aureus infection inthe brain since the alpha-toxin mutant was not completelyavirulent. Our findings demonstrating the importance of alpha-toxin in the brain are in agreement with others using variousmodel systems (3, 15, 17, 26).

S. aureus induces potent proinflammatory cytokine and che-mokine expression in the brain (18, 19). Since both sarA agrand alpha-toxin mutants exhibited a significant reduction intissue damage in the brain, we were interested in determiningwhether this correlated with an attenuation in the host immuneresponse. Both sarA agr and alpha-toxin mutants were initiallycapable of inducing proinflammatory cytokine and chemokineexpression. However, this response was transient in that me-diator production was undetectable 48 h following bacterialexposure, which correlated with a decrease in the numbers ofviable organisms in the brain. This suggests that during the

FIG. 9. Abscess-associated cells harbor viable S. aureus intracellularly. Mice were implanted with RN6390-containing agarose beads andeuthanized at day 5 following bacterial exposure. Cells were recovered from brain abscesses as described in Materials and Methods, and thepresence of intracellular organisms was demonstrated using gentamicin protection assays. An aliquot of cells was taken prior to antibiotic treatmentto demonstrate the total number of abscess-associated bacteria. Rifampin was included as a control to verify the susceptibility of intracellularorganisms to an antibiotic which can penetrate the mammalian cell membrane. Results are expressed as the mean log10 CFU per milliliter of brainabscess cells and are representative of five independent experiments.


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initial stage of infection, sufficient organisms are present withinthe brain parenchyma to trigger activation of host immuneresponses. However, as the replication of these mutants israpidly held in check, the immune response begins to diminish.Alternatively, there may be less of a quorum-sensing functionto activate hemolysin production as the number of organismsbegins to decline. This would, in effect, minimize damage tothe surrounding normal brain parenchyma resulting from anoveractive immune response which is thought to contribute toabscess severity in response to fully virulent strains of S. aureus.

Work by others has demonstrated a critical role(s) for theagr (1, 9) and sarA (25) loci in regulating S. aureus virulence invivo. However, our results differ from these studies in that weobserved a reduction in virulence only for an S. aureus strain inwhich both regulatory loci were inactivated. Our findings are inagreement with Cheung et al. who demonstrated diminishedvirulence of an S. aureus sarA agr double mutant in a rabbitmodel of endocarditis (5). In addition, Booth et al. also dem-onstrated that inactivation of both the sarA and agr loci led tonear-complete attenuation of virulence in experimental en-dophthalmitis (2). Our data in experimental brain abscessessuggest that the residual virulence factor expression detectedin either the sarA or agr single mutants is sufficient to allowthese organisms to replicate and induce tissue pathology sim-ilar to wild-type strains. Only the inactivation of both regula-tory loci effectively reduces virulence factor expression, effec-tively compromising bacterial replication and minimizingtissue damage in the brain.

Recently, S. aureus has been demonstrated to survive intra-cellularly within neutrophils and epithelial cells (12, 16).Therefore, we were interested in identifying whether viableorganisms were associated with abscess-derived cells. This vir-ulence mechanism could explain the phenomenon of daughterabscess formation in the brain which occurs in a small percent-age of affected individuals. As abscesses evolve, the fibroticwall surrounding the lesion may become weakened, allowingcontents to permeate neighboring tissue and establish a newnidus of infection. This seeding of small microabscesses resem-bles “beads on a string” and is life-threatening if intraventric-ular rupture occurs, emptying purulent material into the cere-brospinal fluid. The survival of bacteria within the initialabscess may be a prerequisite for daughter abscess formation.The question remains whether bacteria survive extracellularlyor persist within cells associated with brain abscesses. Indeed,we found that cells recovered from brain abscesses harboredviable organisms. However, the identity of these cells is cur-rently not known. One possibility is that S. aureus is containedwithin neutrophils infiltrating the brain parenchyma. Previousstudies have established that neutrophils constitute the major-ity of cells infiltrating acute brain abscesses (19). However, thehalf-life of an activated neutrophil is relatively short, whichdoes not fit the profile of a cell that would persist, allowing adaughter abscess to become established. Another possibility isthat S. aureus may survive within resident microglia, the resi-dent macrophage population in the brain. We are currentlyinvestigating the cellular localization of S. aureus in the brainusing a strain that constitutively expresses green fluorescentprotein. These studies should allow identification of cells thatare capable of supporting bacterial survival in the brain.

In summary, these studies have revealed the central impor-

tance of alpha-toxin in brain abscess development. It has beenwell documented that many immune responses in the CNS aredistinct from those observed in peripheral tissues. However, asshown here, the pivotal role of alpha-toxin in the CNS has alsobeen observed in other models of S. aureus infection in theperiphery. It will be interesting to determine whether othervirulence factors such as V8 protease and staphylococcal en-terotoxin B contribute to CNS disease as they do in the pe-riphery, or if this is where the similarities between these diver-gent compartments end.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes ofHealth (NS40730) and the Hitchcock Foundation (both to T.K.) andNIH grant NS-27321 (to W.F.H.).

We thank John Hutchins for image analysis and excellent technicalassistance.

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