Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia

Dual function of pneumolysin in the early

pathogenesis of murine pneumococcal

pneumonia.

J B Rubins, … , P W Andrew, E N Janoff

J Clin Invest.

1995;

95(1)

:142-150.

https://doi.org/10.1172/JCI117631

.

Streptococcus pneumoniae is one of the most common etiologic agents of

community-acquired pneumonia, particularly bacteremic pneumonia. Pneumolysin, a multifunctional

cytotoxin, is a putative virulence factor for S. pneumoniae; however, a direct role for

pneumolysin in the early pathogenesis of pneumococcal pneumonia has not been

confirmed in vivo. We compared the growth of a pneumolysin-deficient (PLY[-]) type 2 S.

pneumoniae strain with its isogenic wild-type strain (PLY[+]) after direct endotracheal

instillation of bacteria into murine lungs. Compared with PLY(-) bacteria, infection with

PLY(+) bacteria produced greater injury to the alveolar-capillary barrier, as assayed by

albumin concentrations in alveolar lavage, and substantially greater numbers of PLY(+)

bacteria were recovered in alveolar lavages and lung homogenates at 3 and 6 h after

infection. The presence of pneumolysin also contributed to the development of bacteremia,

which was detected at 3 h after intratracheal instillation of PLY(+) bacteria. The direct effects

of pneumolysin on lung injury and on the ability of pneumococci to evade local lung

defenses was confirmed by addition of purified recombinant pneumolysin to inocula of

PLY(-) pneumococci, which promoted growth of PLY(-) bacteria in the lung to levels

comparable to those seen with the PLY(+) strain. We further demonstrated the contributions

of both the cytolytic and the complement-activating properties of pneumolysin on enhanced

bacterial growth in murine lungs using genetically modified pneumolysin […]

Research Article

Dual Function

of

Pneumolysin

in

the Early Pathogenesis of Murine

Pneumococcal

Pneumonia

JeffreyB.Rubins,* Darlene Charboneau,* James C. Paton,11 Timothy J.Mitchell,IPeter W.Andrew,I

and Edward N. Janoff "

*Pulmonary DiseaseDivisionand*InfectiousDiseaseDivision, Department of Medicine, Veterans Affairs Medical Center, and 'Department of Medicine, University ofMinnesota School of Medicine,Minneapolis, Minnesota 55417; I'DepartmentofMicrobiology, Women's and Children's Hospital, Adelaide,Australia; andIDepartmentofMicrobiologyandImmunology, University of Leicester, Leicester, LEI-9HN, UnitedKingdom

Abstract

Streptococcuspneumoniaeis one of the most common etio-logic agents of

community-acquired

pneumonia, particu-larly bacteremic pneumonia. Pneumolysin, a multifunc-tional cytotoxin, is a putative virulence factor for S. pneu-moniae; however, a direct role for pneumolysin in the

early

pathogenesis of pneumococcal pneumonia has not been

con-firmedinvivo. We compared the growth of a

pneumolysin-deficient (PLY[-

_])

type 2 S. pneumoniae strain with its isogenic

wild-type

strain (PLY [+

])

after direct endotra-cheal instillation ofbacteria into murine lungs. Compared with PLY(-) bacteria,infectionwith PLY( +) bacteria pro-duced greater injury to the alveolar-capillary barrier, as assayed by albumin concentrations in alveolar lavage, and

substantiallygreaternumbers of PLY( +) bacteria were re-covered in alveolarlavagesand lung homogenates at 3 and 6 h afterinfection. The presence ofpneumolysin also con-tributed to thedevelopment ofbacteremia, which was de-tectedat3hafterintratracheal instillation of PLY( +) bac-teria.

Thedirect effects ofpneumolysinonlung

injury

andon

theabilityofpneumococci toevadelocallungdefenseswas

confirmedbyaddition ofpurifiedrecombinantpneumolysin

toinoculaof PLY( -) pneumococci,whichpromotedgrowth

ofPLY(-)bacteria in thelungtolevelscomparabletothose seen withthePLY( +)strain. We further demonstrated the contributions of both thecytolyticand the complement-acti-vating properties of pneumolysin on enhanced bacterial

growthinmurine lungsusing genetically modified

pneumo-lysincongeners andgenetically complement-deficientmice.

Thus, pneumolysin facilitates intraalveolar replication

of pneumococci, penetration ofbacteria from alveoli into theinterstitium ofthelung,and dissemination of pneumo-cocciintothe bloodstream

during experimental pneumonia.

Moreover,both thecytotoxicand the

complement-activating

activities ofpneumolysin may contribute independently to

theacutepulmonaryinjuryand thehighratesofbacteremia which characterize pneumococcalpneumonia. (J. Clin.

In-Address correspondence to J. B. Rubins, M.D., Pulmonary

(11

IN),

Minneapolis VA Medical Center, One Veterans Drive, Minneapolis,

MN55417.

Receivedfor publication17May1994 and inrevisedform15

Sep-tember 1994.

TheJournal of ClinicalInvestigation,Inc.

Volume 95, January 1995, 142-150

vest.1995.95:142-150.) Key words: Streptococcus pneumon-iae *streptolysin -complement * mice * lung

Introduction

Pneumococcal pneumonia isassociated with high rates of inva-sive disease and the attendant early mortality which accompan-ies bacteremic infections (1-3). Pneumolysin, a major pneu-mococcal cytotoxin, is a putative virulence factor in Streptococ-cuspneumoniae infection. Pneumolysin is a potent cytotoxin thatinjures immune(4,5) and respiratory cells in vitro (6-8). Instillation ofpneumolysin into murine lungs reproduces many of the histological findings of pneumococcal pneumonia (9), perhaps through itsabilitytodirectlyactivate the classical

com-plement system (10) and cellular phospholipase A (Rubins, J.B.,manuscript submitted for publication), and stimulate cyto-kine release from monocytes ( 11). Moreover, genetically de-signed functionally pneumolysin-deficient (PLY[-])' mutant

pneumococcal strains have been demonstratedtobe less virulent thanisogenicwild-type(PLY [ +]) strains afterintraperitoneal

or intranasal injection in mice (12, 13). Also, immunization

with pneumolysin protects animals by delaying orpreventing death afterchallenge with virulentpneumococci (14, 15).

However,adirectrole ofpneumolysin in theearly pathogen-esis ofpneumococcal pneumonia hasnotbeencompletely

de-fined in vivo. It isnotknown whether theamountsof pneumo-lysin requiredtoproducecytolytic andpro-inflammatoryeffects

invitroareactually released by pneumococciduring infection

in vivo. Also, pneumolysin is knowntobereadilyinactivated by inhibitors which may be present in thepulmonary alveolus,

suchasoxidantsincluding hydrogenperoxide produced bythe pneumococcus itself(16),andbycholesterol (17)derived from surfactant, cellularmembranes, and serumlipoproteins (18).

We nowreport thatpneumolysinincreases bacterial

multi-plication in thelung and facilitates tissue invasion and bacter-emiaduring the initialphaseof murinepneumococcal pneumo-nia.Inaddition,weshow that thecytotoxicityandthe comple-ment-activating activities of

pneumolysin

appear to have

distinct roles in supporting bacterial replication and invasion

duringexperimental pneumonia.

Methods

Bacterial strains andpreparationofinocula. APLY( -)mutanttype

2 S.pneumoniaestrain(PLN-A)waspreviouslyconstructedfrom strain

1.Abbreviations used in thispaper: e.t.,endotracheal; HU,hemolytic

units; LD50,50% lethaldose; PLY(+)and (-),pneumolysin-sufficient

D39byinsertion-duplicationmutagenesis asdescribed(12).The

mu-tant PLN-A strain produced a type 2 capsule as characterized by

Quellung reactionusingantiserum obtained from Statens Seruminstitut

(Copenhagen,Denmark),and thesizeofthecapsulewas

indistinguish-able from that of theparenteral D39 strainonblood agarplates.PLY( +)

wild-type and PLY(-)mutant strainswerestoredat -700Conglass

beads in trypticase soy broth supplemented with 10% glycerol, and

were passed through mice immediately before experiments to ensure

virulence. Mutant strainswereselected onerythromycinplatesas de-scribed(12). Bacteria were growntomid-logarithmic phaseunder mi-cro aerobic conditions in brain-heart infusion broth (DIFCO

Labora-tories,Detroit, MI),washed,andthen resuspendedin Dulbecco'sPBS

for inoculation. Inoculum sizewas confirmed by quantitative culture on blood agarplates (trypticase soy agarplates containing5% sheep

erythrocytes, GIBCO BRL,Gaithersburg, MD).

Animals.Specificpathogen-freewhitefemaleNationalInstitutesof Health Swiss outbred mice (20-30 g) were obtained from Harlan

Sprague-Dawley,Inc.(Indianapolis, IN).InbredB10.D2/oSnmice ge-netically deficient in the fifth component ofcomplement (C5-) and thecongenicwild-type BlO.D2/nSnmice (C5+)wereprocuredfrom Jackson Laboratories(Bar Harbor, ME) (19). Animals werehoused in a pathogen-free barrier facility fully accredited by the American Association for the Accreditation ofLaboratoryAnimal Care. Animal studies wereperformed in accordance withtheguidelines established in the NIH "Guide for the Care and Use of Laboratory Animals"

(Departmentof Health,Education andWelfarePublication No.[NIH] 85-23, Office ofScience and Health Reports, Division of Research

Resources,Bethesda,MD),andthe researchwasapprovedbythe Ani-malStudySubcommittee of theMinneapolis Veterans Affairs Medical Center(Minneapolis, MN).

Purification of recombinantpneumolysins. Recombinant pneumo-lysins congeners, modified to have either reduced cytolyticactivity(Trp 433 > Phe) or reduced ability to activate complement (Asp 385

>Asn), were produced by single-point site-directed mutagenesis as

described(20,21).Modifiedandwild-typerecombinantpneumolysins

were purified from lysates of Escherichia coli JM109 harboring the

pneumolysingene in theexpression vectorpKK233-2byhydrophobic chromatographyonaTSKphenyl-SPW highpressureliquid chromatog-raphy column(22). Purified recombinantpneumolysin migrated as a singleband withapproximatemolecularmassof 53 kDonsilver-stained SDS-polyacrylamide gels. Wild-typerecombinantpneumolysinhada specific activity of 3 x 105 hemolytic units (HU) per mg protein, assayedasdescribed (7).

Endotrachealinstillationofbacterial inocula. Afterinducing anes-thesia by i.p. injection of 50mg/kg sodium pentobarbital, mice were suspended vertically by supporting the lower incisor teeth on a wire

loop andretaining the upperincisorswith a rubber band as described (23). While using a small retractor to displace the tongue anteriorly, theoropharynx wastransilluminatedandunderdirectvisualization the trachea was cannulated with a blunt-tipped 22 gauge metal needle attached to amicroliter syringe. During inspiration, the bacterial suspen-sion inPBS (50,.d)followedby an equal volume of air was injected, and the animals were maintained upright at a

450

angle until fully recovered from anesthesia (- 30 min). This technique reproducibly delivered greater than 99% of the inoculum to the lungs, as determined by quantitative culture of lungsimmediatelyafter infection, and equally distributedthe inocula to both lower lobes, as determined by endotra-cheal (e.t.) instillation of India ink.

Determination of 50% lethal dose after i.p. and e.t. infection.

Groups of 4-6 mice were infected with increasing concentrations of PLY(+) and PLY(-) bacteria by either i.p. or e.t. injection. Survival

wasrecorded at 8-h intervals for 96 h, and the 50% lethal dose

_(LDm)

wascalculated by the method of Reed and Muench (24).

Recoveryofbacteriafromblood and lung. Mice were sacrificed by cervical dislocationatselected times after infection. After opening the

thorax, 100

_MI

ofbloodfor culture was obtained by cardiac puncture. The trachea was then surgically exposed and cannulated, and the lungs

were lavaged with 1 ml of PBS. Approximately 75% of the lavage fluidwascollectedby aspirationafter several seconds. Thissingle-bolus technique reproducibly recovered >95% of the instilledinoculum, as

assessedby quantitativecultureimmediately afterinstillation, and had the advantage ofdecreasing transudation of serum proteins into the

lavage,which mightoccurduring repetitive lavage. After removal of analiquotfor quantitativeculture, thelavage wascleared of cells by centrifugationandfrozenat-20'C.

Afterlavage,thelungsweredissected frommajorvessels and

bron-chi, rinsed,andhomogenized in 2 ml of sterile PBS in aglass tissue

grinder. The numberof viable bacteria in samples ofblood, alveolar

lavage, andlung homogenate wasdeterminedby quantitative culture of serial dilutions on blood agar plates. Bacteria were identified as

pneumococci by colony morphologyofa-hemolytic organismsandby Optochin sensitivity,andPLY(-) bacteria were identifiedby growth

on erythromycin plates and by lack ofhemolysin activity. The total number of bacteria in each murinelungwascalculatedasthesumof the total numberof bacteria in alveolar lavage plus lung homogenate samples.

Determinationof albumin concentration in lavage. The concentra-tion ofalbumin inlavage supernatants wasmeasured as an index of alveolar-capillary barrierdisruption. Lavageproteins

_(40-,ul

aliquots)

and albumin standards were separated by 10% SDS-polyacrylamide gel electrophoresis. After Coomassie staining, gels wereanalyzed by video densitometry (model 620;Bio-RadLaboratories, Hercules, CA),

and the concentration of albumin determined by comparison to stan-dards.

Assay of endotoxin activity. Endotoxin activities in recombinant

pneumolysin preparations were quantified using aLimulus amoebocyte

lysate test(Pyrotell; AssociationofCapeCod, WoodsHole, MA).

Statistics. Geometric means and standarderrors ofmeans(SE) of

bacterial colony forming units (CFU) were calculated on

log-trans-formed data, and then data was converted by exponentialtransformation

toCFU.Where indicated, statistical significance was calculated by com-parison of geometric means by unpaired two-tailed t-test. Each datum point in figures and tables represents the mean±SE of 3-4 animals,

and results of eachexperiment were confirmed by at least one repeat

experiment.

Results

Relative virulence ofPLY(+) wild-type and PLY(-) mutant strainsby systemic and mucosal infection. After systemic infec-tion by i.p. infecinfec-tion, thetype2 S. pneumoniae PLY( +) strain

was highly virulent, with an

_LDm

of25 CFU. In contrast, the

PLY(-) mutant strain was substantially less virulent, with an

LDm

i.p. doseof500CFU. Furthermore, death was appreciably delayed in thegroups infected with the PLY(-) mutant com-paredwith the PLY(+) strain (median survival times 72 and

20 h, respectively).

The greater virulence of the PLY(+) wild-type compared with the PLY(-) mutant pneumococcal strain with systemic infection paralleled that with mucosal infection by e.t. instilla-tion.However,miceshowedamarked resistance to pneumococ-cal infection by mucosal compared to systemic infection. The

LD50 fore.t. infection with PLY(-) bacteria was again one log higher than that for PLY(+) bacteria (108 CFU versus 107

CFU), but each was five logs greater than the corresponding

i.p. doses. In summary, thePLY(-) mutant type 2 S. pneumo-niae strain was less virulent than the isogenic wild-type strain afterboth routes of infection, consistent with previous observa-tions (12). Moreover, the mouse lung appeared to be capable

of clearing extraordinary numbers of virulent bacteria, sug-gesting that itserves as amajor lineofdefense against

Table I. Alveolar-Capillary Barrier Injury after Infection with PLY(+) and PLY(-) Bacteria

Albuminconcentration (mg/ml) Time afterinfection (h)

Inoculum 0 3 6 24

PBS .06±.01 .09±.03 .18±.07 .22±.05

Pneumolysin (5 HU) .07±.02 .32±.141 .42±.12§ .34±.16

PLY(+) strain* .07±.01 .34±.11 .72±.14'11 .37±.12

PLY(-) straint .12±.07 .18±.04 .22±.01

+5 HU pneumolysin .52±0.8§11 .46±.34 .56±.39

+50 HUpneumolysin 1.12±.26§** 1.35±.321** 1.22±.341**

*Inocula= 5 x 106CFU in 0.05 ml PBS. Data representmean±SEof three animals.

tlnocula

= _{1.35 x} 107CFU in 0.05 ml PBS.Mean±SE,

n= _3. §P< .02compared

with

PBS; 11P < .02 compared wit PLY(-) strain; 1P < .05 compared with PBS; **_{P < .05 compared}

with

PLY(-)strain.

Comparison of recovery of PLY(+) and PLY(-) strains from lung and blood. We next investigated whether pneumo-lysin might disrupt the protective function of the lung in vivo. Purified pneumolysin injures alveolar epithelial cells in vitro and the alveolar-capillary barrier in isolated perfused murine lungs (7). We have speculated that pneumolysin injury to the alveolarepitheliummayproduce alveolar flooding with serous exudate, which may provide necessary nutrients and promote rapid multiplication ofpneumococci within the alveoli. To test this hypothesis in vivo, mice were inoculated e.t. with either the wild-type PLY(+) type 2 strain (5 x 106 CFU) or the PLY(-) mutant strain (1.35 X

10'

CFU). At selected times after infection, mice were sacrificed and samples ofalveolar lavage, lung homogenates, andblood were obtained for culture and biochemical studies. Basedonprevious histologicalstudies (25), times were selected to approximate the period before

significant neutrophilinflux(congestion,3h), theacutephase

ofneutrophil influx (red hepatization, 6 h), and theperiodof established neutrophil response (grayhepatization, 24h).

To determine whetherpneumolysin released from bacteria

wasactively cytotoxic invivo,weassayedalbumin

concentra-tions in the alveolarlavage samples as amarker of

alveolar-capillary injury.Infection withPLY(+) bacteriawasassociated witha3.7-fold increase inlavagealbuminat3 handafourfold increase at6 hcomparedwith control micereceivingPBS e.t.

(Table I). In contrast, no significantincrease in albumin was

detected inlavage from mice infected with thePLY(-) strain

ascomparedtoPBS-treated mice.

Therefore,

PLY( +) bacteria,

butnotPLY( -)bacteria, appearedtoinduceadegreeof

muco-salinjurysufficienttodisrupt thealveolar-capillarybarrier in murmnelungsin vivo.

Corresponding to the greater alveolar injury produced by

thePLY( +)bacteria, significantlygreater numbers of bacteria

wererecovered from murinelungsinfected withPLY( +) pneu-mococci at3 and 6 h afterinfection, compared with those in-fected with PLY(-) mutants

(Fig.

1). Recovery of

PLY(+)

bacteria fromlungswas morethanthreefoldhigherat3hand 30-foldhigherat6 hcomparedtoPLY(-)bacteria.However, despite the reported cytotoxicity of pneumolysin to immune

leukocytes (4, 5), the rate ofnet clearance of bothPLY(+)

andPLY(-)bacteria from 6to24 h afterinfectionwas very similar (14.5 and 13% perhour,respectively).

The increased recovery of viable PLY( +) bacteria from murine lungs during early infection reflected the markedly greater numbers ofPLY( +) bacteria recovered from both alve-olar lavages andlunghomogenatesat3 and6 h afterinfection, compared with the PLY(-) strain (Fig. 2). Therewasa striking dissociation between the numbers of organisms in the alveolar and tissuesamplesat6 h after infection with PLY( +) bacteria (Fig. 2 A), suggesting increased penetration of pneumococci frompulmonary alveoli into interstitium. Incontrast, numbers of PLY (-)pneumococciin alveolarlavagedidnotincreaseat

any timepoint,nordid they show anysignificantinvasion into lung tissue (Fig. 2 B). Rather, numbers of PLY(-) bacteria continuedtodecrease inboth compartments after 3 h.

5)

51)

0 0

Q)

0

0

8

7

6

0

0 6 12 18 24

Time, h

Figure1.Recoveryofpneumococcifrom murinelungs.Totalnumber

of bacteria in murinelungswerecalculated fromquantitativeculture of

alveolarlavagesandlung homogenatesobtained at the indicated times

aftere.t.instillationofapneumolysin-deficientmutanttype2 strain(o)

ortheisogenic wild-typestrain(e).Eachdatumpoint representsthe

geometricmean±SE of the total CFUperlungforthreeanimals.

Com-parisonofgeometricmeanCFUforPLY(+) andPLY(-)strainsby unpairedt-test; *,P< .05;**,P< .01.

*PLY(+)

o PLY(-)

*2\

0

4

lavage

_ homogenate '0

a)

a)

0 C)

a)

S.)

0

to

0

3

2

1

0 6 12 18 24

Time, h

Figure 3. Recovery ofpneumococcifrom blood afterendotracheal instil-lation. Bloodsampleswere collected forquantitativecultureatthe indi-cated times after e.t. instillation ofPLY(-) (o)andPLY(+) (-)

strains. Each datumpointrepresents thegeometricmean±SE for three animals. Datumpointsbelow the axis break represent0bacteria. Data isrepresentativeoftwoexperiments. Statisticalcomparisonsareas de-scribed forFig. 1.

8

la

a) a)

0 C)

a)

No

0

-4

0

7

6

5

B.

0 3 6 24

Time, h

Figure2. Recoveryof pneumococci fromalveolarlavagesand lung

homogenates. Samples of alveolar lavage (u) and lung homogenates (*)werequantitatively culturedattheindicated intervals after infection

with(A) PLY(+) and (B) PLY(-) strains. Datum points and statistical comparisonsare asdescribed for Fig. 1.

Consistentwith theincreased invasion ofPLY( +) bacteria from alveoli into lung tissue, PLY(+) bacteria also produced

earlier andhigher grade bacteremia thandidPLY(-) bacteria

(Fig. 3). PLY( +)bacteriaappearedinbloodat3 and 6h,and

numbers dramatically increased at 24 h. PLY(+) bacteremia increased to 106_107 CFU/ml at48 h, and the animalsbegan

todieduringthesubsequent 24 h. Incontrast,PLY(-)bacteria

were not detected in blood before 24 h, when more modest

numbers of bacteria were recovered. Also, the number of

PLY(-)bacteria in the bloodat48and 72 hwashighlyvariable

betweenanimals. Aminority ofanimals who appeared overtly

illwerebacteremicwith 106 CFU/mlat72h,andsomeofthese

died during thenext48 h. However, the majority of the mice

infected with PLY(-) appeared well and had no detectable

bacteremia at72 h.

To ascertain whether the enhanced growth of PLY(+) pneumococci in infected murine lungs reflectedanintrinsic

de-fect in the growth ofthePLY(-) mutants, we compared the

in vitrogrowthof thetwostrains inseveral substrates. In

con-trast to growth in vivo, the PLY(-) mutant straingrowth in vitro when incubated in brain-heart infusion broth, in

Todd-Hewittmedia, and in homogenates ofmurine lung (doubling

timesof32, 37,and20 min,respectively),wasequalorgreater than that of thePLY(+) strain (doublingtimesof32, 37, and 22 min, respectively).

Takentogether,thesestudiessuggestedthatthepresenceof

endogenous production of pneumolysin was associated with

enhancement of specific pathogenic effects ofS. pneumoniae

infection, includingincreasedintraalveolar replication of bacte-ria,penetrationoforganismsfrom alveoli into the interstitium

of thelung, and dissemination ofpneumococci into the

blood-stream.

AdditionofrecombinantpneumolysintoinoculaofPLY(-)

bacteria. To confirm that differences inpneumolysin production

accounted for the observed differences in specific pathogenic

effects of thePLY(+)straincomparedwith thePLY( -) strain,

A.

8

**

**

7 '0

Q)

a)

0

C.)

a)

C)

r-4 0

no

0

*

T

6

6 24

a

a)

a)

V

0

C) a) 5-.

0 0

7

6k

5

8

a)

a)

~-0

C)

0 0

0 6 12 18 24

Time, h

Figure 4. Effect of added pneumolysinonrecoveryof PLY( -) bacteria

frommurine lungs. Total numbers of bacteria inmurinelungswere

determined from quantitative culture ofalveolar lavage and lung

homog-enatesobtainedattheindicated intervals aftere.t.instillation of inocula ofPLY(-)bacteria, either alone (0)orsupplementedwith purified recombinant pneumolysin (+rPLY)atconcentrations of5HU/ml(.)

or50HU/ml(*). Datum pointsare asdescribed for Fig. 1. Comparison

ofgeometricmeanCFU for PLY(-)rPLY byunpairedt-test. *,P

< .05, **,P< .01.

inocula of PLY(-) pneumococci (107 CFU) were

supple-mented with either 5 or 50 HU/ml of purified recombinant

pneumolysin. In preliminary studies, these concentrations of pneumolysin approximated the cytolytic activity released in vitro by 107 and 108 CFU/ml, respectively, of the PLY(+)

strain. Addition of recombinantpneumolysintoPLY(-)

pneu-mococciproducedadose-dependentincrease in alveolarlavage

albumin concentrations(Table I), confirming injurytothe

alve-olar-capillarybarrier. Recombinant pneumolysinalso induced

adose-dependentincrease inrecoveryofPLY( -)bacteria from

murinelungs earlyinthecourseofinfection,similartoPLY( +)

bacteria (Fig.4). Addition of50 HU/mlpneumolysin

substan-tially increased total numbers ofPLY(-) bacteria recovered from lungs at 3 and 6h, approximating the effects observed withPLY( +)bacteria.

To control for the effects of endotoxin contamination of recombinant pneumolysin, we added of 15 pg/ml endotoxin, equivalenttothe contaminant concentrationsmeasuredby

Lim-uluslysateassayin thepneumolysin preparations, toPLY(-)

bacteria. Thesesuspensionsneither caused albumin influx into alveolar lavage, norincreased numbers of bacteria in lavage, homogenateorblood(datanotshown). Thus,endotoxin

impu-rities didnot accountfor thepathogeniceffects ofpneumolysin

in this model.

Roleof cytolytic activity inpneumolysin-facilitated bacte-rialgrowth. In additiontoitspotentcytolyticactivity,

pneumo-lysin is also capable of activating the classical complement

system (10). Molecular studies have defined distinct peptide

sequencesfor thecytolyticandcomplement-stimulating active sites (20, 21). Furthermore, the functional independence of these two activities has been demonstrated by thepersistence

7

6

5

4

0

'V

1

oPLY(-) * + rPLY V + heat-rPLY

0

0 6 12 18 24

Time, h

Figure5. Effect ofheat-inactivated pneumolysinon recoveryof

PLY( -) bacteria from murine lungs. Total numbers of bacteria

recov-ered from murinelungsweredeterminedattheindicated times aftere.t.

instillation of inoculaofPLY( -) bacteria, either alone (o)or

supple-mented withuntreated recombinant pneumolysin (rPLY, 10 HU/ml; *)

orheat-inactivated pneumolysin (heat-rPLY, A). Datum points and

sta-tisticalcomparisonsare asdescribed forFig. 4.

ofcomplementactivation after inhibition ofpneumolysin

cyto-toxicity (10).To examine the contributions of thesetwo activi-ties onpneumolysin-facilitated PLY(-) bacterial growth, we

first inactivated cytolytic activity by incubating recombinant

pneumolysin at560C for 30minbeforeadditiontoinocula of

PLY(-) bacteria. Hemolytic activity was reducedby > 99%

after heat inactivation(6). Incomparisonwithuntreated

pneu-molysin (10 HU/ml), heat-inactivated pneumolysin had less effectonrecoveryofPLY(-)bacteria fromlungsat3 h(Fig. 5). However,addition of heat-inactivatedpneumolysin signifi-cantlyincreased numbers ofPLY(-)bacteriaat6 h,identical tothe effect of untreatedpneumolysin.This increasedrecovery

ofPLY(-) bacteria at 6 h reflected a significant persistence

ofbacterial inoculum in alveolar lavage and increased tissue invasion(datanotshown).

Thus,the addition ofexogenouspurifiedrecombinant

pneu-molysintoPLY(-)bacteriareproducedmanyof the sequential

changes in bacterial growth and invasion seen with

pneumo-lysin-producing organisms. However,inthis in vivomodel,the

activity of pneumolysin was related only in part to its

heat-labilecytolytic activity, suggesting thatanadditional property

ofpneumolysin mightbe involved in these effects.

Role of complement activation in pneumolysin-facilitated

bacterialgrowth.Because thecomplement-activatingactivityof

pneumolysin canpersist after heat-inactivation ofcytotoxicity (Mitchell, T. J., unpublished data), we hypothesized that the

persistenteffects ofheat-inactivated pneumolysinon bacterial

growth mightbe related to its ability toactivate complement.

Totest thishypothesis,weadded modified recombinant

pneu-molysins to e.t. inocula of PLY( -) bacteria in our murine model. We usedtwomodifiedpneumolysinswithsingleamino

acid substitutions generated by site-directedmutagenesis; one oPLY(-)

* + rPLY (5 HU)

with fullcytolytic activity but reducedability to activate

com-plement (Asp 385 >Asn,complement deficient), and the other with reduced cytolytic activity (103 HU/mg,

represent-ing 0.33% of wild-type toxin activity) but normal

comple-ment-activating activity (Trp 433 > Phe, cytolytic deficient) (20, 21).

Aftere.t.infection,recovery of PLY(-) bacteria from

mu-rine lungs at 3 h was appreciably increased after addition of 1

_jig/ml

(equivalent to 15 HU of unmodified toxin) of the

complement-deficient

pneumolysin possessingfullcytolytic

ac-tivity (Fig.6A),whereas addition of the "cytolytic-deficient" pneumolysin producedno increase(Fig. 6 B). Conversely, re-covery ofPLY(-) bacteria at6 hwasnotincreasedafter addi-tionof thecomplement-deficient pneumolysin,butwas

appreci-ablyincreasedafter addition ofcytolytic-deficient pneumolysin havingnormalcomplement-stimulating activity. Similarto the effects of heat-inactivated pneumolysin, the increased number ofPLY( -)bacteriaat6 hreflectedanappreciable persistence

of bacterial inoculum, predominantly in alveolar lavage

sam-ples, in mice receiving pneumolysin with intact

complement-activating activity (datanot shown).

In summary, studies with recombinant pneumolysins

sug-gested that pneumolysin's cytolytic activity accelerated bacte-rialgrowthand invasion intolung tissue during the initial period after infection, whereas its ability to activate complement

ap-pearedto facilitate bacterial growthin alveoliatlater times. Recovery ofPLY( +) and PLY(-) bacteria from

CS-defi-cient mice. Complement playsacriticalrole in the initial

clear-ance ofpneumococci from the lung by generating neutrophil chemotaxins(primarily CSa)andby opsonizing bacteria(C3a and

CSa)

(26). Theabilityofpneumolysintoactivate

comple-menthas beenspeculated tosubvert this essential host defense

by diverting complementaway from thebacteria, thereby

com-promising complement-mediated opsonization and phagocyto-sis. Therefore, we hypothesized that addition ofpneumolysin

possessing

intactcomplement-activating propertiestobacterial inoculamightincrease the recovery of PLY( -) bacteria at 6 h

by interfering withcomplement-mediatedclearanceby

neutro-phil phagocytosis.Wefurther investigated this hypothesis using mice witha

specific

congenital complement deficiency,which have reduced pulmonary granulocyte recruitment in response

toS. pneumoniae(19). Congenicdeficient () and C5-sufficient (C5+) mice were infected e.t. with PLY(+) and

PLY( -) bacteria,andsamples of alveolar lavage, lung homog-enates, and blood were obtained for culture and biochemical studies.

As observed with Swissmice, PLY(+) bacteria produced greater albumin influx into alveolar lavage of C5- mice at 3 and 6 h than did PLY(-) (data not shown). Similarly, total numbersofPLY( +)bacteriawereincreased lungs at 3 and 24

h after infection, compared with PLY(-) bacteria (Fig. 7).

However,severalimportantdifferences were observed with e.t. infectioninC5- mice compared with C5+ mice. First, the total numbers of PLY(-) and PLY(+) bacteria in lungs were at least twofold greateratalltimes aftere.t.infection of C5- mice

comparedwithC5+ mice (Fig. 7). Second, earlier and higher

gradebacteremiawasseen inboth PLY(-_{) and PLY(}+) bacte-ria in C5- _{mice compared with complement-sufficient mice}

(data

not

shown).

Third, the net clearance of PLY( -) and

especially

PLY(+)mice at 24 h was substantiallyreduced in

C5- mice

(Fig.

7).

G1)

U)

C)

I)

0

-0

a

so

V

a) a)

0) 0)

t.o

-)

0 0

7

6

5 a

7

6

A.

o PLY(-)

* + rPLY

I0

_I

O~~~~

A\!

0 6 12 18 24

Time, h

B.

o PLY(-) * + rPLY

l0

0

0

Q

III

0 6 12 18 24

Time, h

Figure6. Effect ofmodifiedpneumolysins on recovery of PLY(-) bacteria from murine lungs. Total numbers of bacteria recovered from murinelungsweredetermined at theindicated times aftere.t. instillation of inocula of PLY( -) bacteria, either alone (o) or supplemented with

modifiedrecombinant pneumolysins (+rPLY, 1

Og/ml,

*). Pneumoly-sins weregeneticallymodified to have(A) reduced ability to activate complement or (B) reduced cytolytic activity. Each datum point

repre-sentsthegeometricmean±SE of total CFU per lung for four animals.

8

s0

Q)

0

C.) 7

a)

on 0

to 6

0

5

0 6 12 18 24

Time, h

Figure 7. Recovery of pneumococci fromC5-deficientandC5-sufficient

mice. Total bacterial CFU were determined at the indicated times after e.t. instillationof PLY(-) bacteria (o,

u)

orPLY(+) pneumococci

(e,

m)in congenicC5-deficient(o, *)andC5-sufficient (a,*) mice. Each datumpointrepresents the geometric meantSE of total CFU per lung for four animals. Statistical comparisons as are described for Fig. 1.

Finally, the total number of PLY(-) bacteria appreciably increased from 3-6 h in C5- murine lungs (Fig. 7, open

cir-cles),in contrasttothenetclearance of PLY( -) bacteria after 3 hconsistentlyobserved in C5+ mice (Fig. 7, open squares).

Inparticular, netclearance of PLY(-) bacteria from alveolar

lavage at 6 h was markedly reduced in C5- mice (data not

shown).Thus,asimilar increase in numbers of PLY(-) pneu-mococci at 6 h was seen when the complement system was

manipulated, either by genetic deficiency of C5orby addition of recombinant pneumolysins with intact complement-activating

function. Furthermore, inhibition of complement appeared to

predominantlydecrease thenetclearance of pneumococci from alveoli.

Discussion

Our studiesprovidein vivo evidence for theimportantrole of

pneumolysinin theearly pathogenesis of pneumococcal pneu-monia. Although pneumolysin's cytotoxicity to lungcells and its ability to disrupt the alveolar-capillary barrier has been

previouslyestablishedinvitro (7), the relevance of these obser-vationstothepathogenesisof pneumococcalpneumoniainvivo

hasremainedunknownfor severalreasons.First,because

pneu-molysin is an intracellular protein that is only released upon

autolysisof the bacterium(27),itwas notcertain that

replicat-ing intraalveolar bacteria release sufficiently high

concentra-tions ofpneumolysin to produce acute lung injury. Second, pneumolysin is a member ofa family of related "thiol-acti-vated" bacterial toxins that are oxygen labile (27), and thus

mightbeinactivated eitherby

oxidizing

conditions within the

pulmonary alveolusorby hydrogen peroxide generated bythe

pneumococcusitself.Finally, pneumolysinand other

thiol-acti-vatedtoxins can be inhibited by nanomolar concentrations of cholesterol(17),present in thelung in alveolar surfactant, cel-lular membranes, and serum lipoprotein. Thus, despite extensive biochemical and molecular characterization of its properties in vitro, the contribution of pneumolysin to the early pathogenesis of pneumococcal pneumonia remained unknown.

Using a technique of direct e.t. instillation of bacterial inoc-ula (23), which delivers a known quantity of bacteria at a discrete time, we were able to quantify accurately changes in bacterialnumbers in lung samples at early times after infection. Comparison of infection of Swiss mice with genetically

con-structed pneumolysin-deficient mutant and wild-type strains of type 2 S. pneumoniae confirmed that PLY(+) bacteria were more virulent than PLY(-) pneumococci after both systemic and mucosal infection, as previously described (12). Impor-tantly, we observed that mice had a marked intrinsic resistance to infection by e.t. compared with i.p. route, similar to that notedduring infection of BALB/c mice with these same strains (12). These results demonstrate that the mouse lung serves as

amajor natural line of defense against disseminated pneumo-coccal infection.

Supporting our observations of lung cell injury by pneumo-lysin in vitro (7), we demonstrated that pneumopneumo-lysin released by bacteria during pneumococcal pneumonia in vivo was cyto-toxic to the alveolar-capillary barrier. The alveolar epithelium is the principal barrier to colloid and crystalloid flux into the alveolus, and injury to the epithelium permits leakage of serum proteinsinto the alveoli (28). Infection with PLY( -) bacteria producedminimal increases in concentrations of albumin recov-ered by alveolar lavage, similartothoseseenfrom instillation ofPBS alone. In contrast, infection with PLY(+) bacteria pro-duced substantialincreases in alveolar albumin concentrations, indicating that pneumolysin is released in sufficient quantities

andremainssufficiently cytotoxic,despitepotentialinhibitors,

toproduceacutelung injuryin vivo.

Pneumolysin alsoproducedspecificpathogenic effects

dur-ingthe early hours of S. pneumoniae infection, including in-creased intraalveolar replication of bacteria, penetration of pneumococci from alveoli into the interstitium of the lung, and dissemination of bacteria from lung into the bloodstream. These effects couldnotbe attributedto anintrinsic defect in growth ofthePLY( -)strain,which grewaswellasthePLY(+) strain in vitro in a variety of culturemedia, including homogenized murinelung.Facilitation of dissemination ofpneumococcifrom alveolar to vascular compartments presumably is related to

pneumolysin's ability to disrupt the alveolar epithelium and endothelium (6, 7), permitting bacterial penetration into and multiplication within thepulmonaryinterstitium. Pneumococci

most likely move from the interstitium into the bloodstream vialymphatic drainage, althoughdirect dissemination into the vascular space followingendothelialinjury by

pneumolysin

is also possible. Pneumolysinmay also contribute to the earlier andhigh-gradebacteremiaseenwith thePLY( +)strain

through

its ability to inhibit the clearance of pneumococci from the vascular space (12).

Ourstudies alsoprovideevidence that

pneumolysin's

cyto-toxic andcomplement-activating properties

independently

con-tributetoenhancedbacterialgrowth

during early

pneumococcal pneumonia. When inocula of thePLY(-) strainwere

supple-mented withpurifiedrecombinant

pneumolysins,

pneumococcal

growth at3h after infection wasfacilitatedby pneumolysin's

*

* C5-, PLY(+)

oC5-, PLY(-)

cytolytic activity. However, compared with studies using

PLY(+)bacteria, fewer viable PLY(-) bacteriawere

recov-ered fromlungsat6 and24 hafterinstillation ofpneumococci

with addedrecombinant pneumolysin, despiteasimilardegree of initial alveolarinjury.Thesedisparitiesmayreflect the

differ-encebetween the addition ofasingle dose ofexogenous

pneu-molysintothePLY(-) strain andtheongoing release of

pneu-molysin in vivo from the PLY ( +)strain.The latter may

facili-tate bacterial growth at 6 and 24 h by continued alveolar damage, interference with complement-mediated clearance, or

other mechanismsnotyetdelineated.

Flooding of alveoli with serum anderythrocytes following pneumolysin-mediated injurymay support intraalveolar replica-tion of bacteria by providing necessary nutrients and

antioxi-dants. Acute hemorrhagic edema may also decrease bacterial elimination (29).Potentially, influx ofserumalbuminmay

neu-tralize intraalveolar freefatty acids,which aretheprincipal anti-pneumococcal factor inalveolarlining fluids of the murine lung (30). The cytotoxicity of pneumolysin formonocytes(5) and neutrophils (4) in vitro may also increase recovery of viable pneumococci by inhibiting bacterial elimination by these

im-mune cells.However,weobservedthattheratesof netclearance of pneumococci during the period of neutrophil influx were

essentially identical for boththePLY(+) andPLY( -)strains,

suggesting that pneumolysin's effectonimmune cells makes a

minor contribution atmost to the early pathogenesis of

pneu-monia.

Pneumolysin activates the classical complement system in

vitro(10). However, this functionhas notpreviouslybeen dem-onstratedtoaffect pneumococcal growthoreliminationin vivo.

By supplementing inoculaof PLY(-) bacteria with modified recombinant pneumolysins possessing either cytolytic or

com-plement-activating properties,wedemonstrated that intact

com-plement-activating activity correlated with enhanced alveolar bacterialgrowth at6 hafter e.t. infection. Similarly, numbers of PLY(-) bacteria were increased from 3 to 6 h in mice

with genetic complement deficiency compared with congenic complement-sufficient mice. In both sets of experiments, the net clearance of PLY(-) bacteria from alveolar lavage was

predominantly affected. Taken together, these studies suggest that theabilityofpneumolysintoactivatecomplement indepen-dently contributes to the intraalveolar replication of

pneumo-cocci during the early period of neutrophil influx in

pneumococ-calpneumonia. These observationsareconsistent withamodel of ineffectiveactivation ofthe complementsystemby

pneumo-lysin, whichmay consumecomplement factorsanddivert

com-plement opsoninsaway from bacterial cells.

In summary, wehavedemonstrated in vivo that

pneumoly-sin'scytotoxicandcomplement-activating properties contribute independentlytofacilitate intraalveolar replicationand dissemi-nation ofpneumococci into lung tissue and the bloodstream

during the initial hours of experimental pneumonia. The im-portant role ofpneumolysin intheearly pathogenesis of pneu-mococcalpneumoniasuggests thatimmunization against

pneu-molysin mightdecrease acutelunginjuryand bacteremiaduring pulmonary infection. Neutralizing antibodies to pneumolysin

are produced during pneumococcal infection in humans (31, 32), and immunization with pneumolysin has been shown to

reduceordelaydeath in animals ( 14). Furthermore,as aprotein toxin produced by essentiallyallclinical isolates ofS.

pneumo-niae,pneumolysinisapromisingcandidate foruseina

protein-conjugatepneumococcal vaccine (33).

Acknowledgments

We thank Claire Pomeroy and Dennis Niewoehner for their critical reviewsof thismanuscript.

This workwassupported bythe U.S.Departmentof Veterans Affairs ResearchService (grantto E.N.Janoff),NationalInstitutes of Health grantsR29-AI34051 (J.B.Rubins)andR29-AI31373(E.N.Janoff),and

the Medical Research Council and theRoyal Society (T.J.Mitchell).Dr. Mitchell isaRoyal SocietyResearch Fellow.

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