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PATHOGENESISMICROBIAL

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doi:10.1016/j.m

Abbreviations

methyl-b-cyclodhaemagglutinin

agar; LAMP, ly

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Microbial Pathogenesis 44 (2008) 501–511

www.elsevier.com/locate/micpath

Cholesterol-rich domains are involved in Bordetella pertussisphagocytosis and intracellular survival in neutrophils

Yanina Lambertia, Maria Laura Perez Vidakovicsa, Ludo-W. van der Polb, MariaEugenia Rodrı́gueza,�

aCINDEFI, CONICET, School of Science, La Plata University, La Plata, ArgentinabDepartment of Neurology, University Medical Center, Utrecht, The Netherlands

Received 28 December 2007; accepted 3 January 2008

Available online 9 January 2008

Abstract

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis.

However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We

report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in

B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or

methyl-b-cyclodextrin (MbCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of

opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route

of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in

cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with

lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules

that eventually prevent cellular bactericidal activity.

r 2008 Elsevier Ltd. All rights reserved.

Keywords: Bordetella pertussis; Phagocytosis; Neutrophils; Lipid raft domains

1. Introduction

The gram-negative bacterium Bordetella pertussis is theetiologic agent of whooping cough, a re-emerging infec-tious disease. B. pertussis is a non-invasive pathogen thatproduces numerous toxins and adhesins, such as pertussistoxin (PT), adenylate cyclase-hemolysin toxin (AC-HLy),tracheal cytotoxin, dermonecrotic toxin, filamentous hae-magglutinin (FHA), pertactin, fimbriae and BrKA, allimplicated in pertussis pathogenesis [1,2]. Vaccination

e front matter r 2008 Elsevier Ltd. All rights reserved.

icpath.2008.01.002

: PMN, human polymorphonuclear leukocytes; MbCD,

extrin; CR3, complement receptor 3; FHA, filamentous

; MOI, multiplicity of infection; BGA, Bordet Gengou

sosome-associated membrane protein; MAb, monoclonal

ing author. Tel.: +54221 4833794x123;

33794x102.

ess: mer@quimica.unlp.edu.ar (M.E. Rodrı́guez).

against pertussis has resulted in reduction of B. pertussis

incidence but the circulation of these bacteria has persisted,among other reasons, as a result of waning vaccine-inducedand naturally acquired immunity. The presence of specificantibodies to some B. pertussis virulence factors isassociated with a lower likelihood of acquiring pertussis[3–6]. Phagocytosis by neutrophils is one of the main linesof host defense. Engagement of leukocyte IgG and IgAreceptors (FcgR and FcaRI, respectively) by opsonizedB. pertussis results in efficient bacterial transfer tolysosomal compartments [7]. Bactericidal activity ofphagocytes in the absence of antibodies is significantly lessefficient [7], which may contribute to bacterial persistencein human airways and host colonization. Despite itspotential importance, given the decay of pertussis-specificantibody levels after 3 or 4 years post-vaccination, theinnate interaction of phagocytes and B. pertussis isincompletely understood.

ARTICLE IN PRESSY. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511502

Several B. pertussis virulence factors facilitate interactionwith phagocytes. B. pertussis fimbriae mediate binding tothe very late antigen-5 (VLA-5) receptor on monocytes andmacrophages, and induce up-regulation of complementreceptor 3 (CR3: CD11b/CD18) [8]. CR3 expression isfurther up-regulated by PT and FHA [9,10]. In addition,CR3 serves as a docking molecule for B. pertussis bindingby FHA [10,11]. The use of CR3 on phagocytes asattachment site may enhance the odds of bacterial survival[12,13].

CR3 is functionally associated with plasma membranelipid rafts containing high concentration of cholesterol,glycosylphosphatidylinosistol (GPI)-anchored molecules,and glycosphingolipids (GSL) like asialo-ganglioside M1(asialo-GM1), which is another proposed docking moleculefor B. pertussis [14]. CR3/lipid rafts-mediated binding wasfound involved in targeting other pathogens to compart-ments that do not undergo lysosomal maturation. Studieson Leishmania [15] and Mycobacterium [16] showed theinvolvement of these domains and CR3 in microbialsurvival upon interaction with immune cells.

Non-opsonized B. pertussis failed to trigger PMN(human polymorphonuclear leukocytes) oxidative burst[7], which is consistent with the reported failure ofactivation of NADPH oxidase by independent ligation ofCR3 [17,18]. These data suggest that non-opsonizedB. pertussis succeed in interacting with immune cellswithout triggering leukocyte activation. This study aimedat further dissecting mechanisms underlying the innateinteraction of B. pertussis and neutrophils. The datapresented here indicate that membrane cholesterol-richdomains on PMN serve as docking sites that facilitatebacterial binding and phagocytosis of non-opsonizedB. pertussis. Importantly, our data not only suggest thatB. pertussis is transported to subcellular structures that donot undergo lysosomal maturation but also that forcingnon-opsonized B. pertussis to enter through a non-lipid raftpathway delivers the bacteria into compartments thatreadily fuses with lysosomes. We hypothesize that entrythrough lipid raft may contribute to enhance B. pertussis

survival into host cell.

Fig. 1. The presence of specific antibodies facilitates PMN interaction with

opsonized GFP-expressing B. pertussis at a MOI of 50 (A), or non-opsonized G

washing steps, PMN were analyzed by flow cytometry. Untreated PMN were

times with PMN isolated from different donors, yielding essentially identical r

2. Results

2.1. Cholesterol-rich domains facilitate PMN interaction

with B. pertussis

Opsonized B. pertussis is taken up by human PMNabout 10 times more efficiently than non-opsonizedbacteria (Fig. 1). Therefore, we normalized the initial levelof bacteria associated with neutrophils by infecting PMNwith 50:1 opsonized or 500:1 non-opsonized B. pertussis

unless specified otherwise.To investigate the role of cholesterol-rich domains in the

innate interaction of PMN and non-opsonized B. pertussis,PMN were incubated with MbCD, a compound thatdisrupts cholesterol-rich domains by extracting cholesterol[19]. The experiments were performed in the presence oflovastatin to inhibit de novo cholesterol synthesis. Choles-terol depletion significantly decreased the number of non-opsonized B. pertussis associated to PMN (Fig. 2A).Similar results were obtained after incubation of PMNwith nystatin, another lipid raft disrupting drug thatbinds cholesterol (Fig. 2A). Conversely, the level ofIgG-opsonized B. pertussis associated to PMN did notchange significantly after incubation of PMN with eithernystatin or MbCD (Fig. 2A) indicating that cholesterol isnot critical for binding of IgG-opsonized bacteria to FcgR.It was previously demonstrated that uptake of

Escherichia coli DH5a does not depend on the presenceof cholesterol-rich domains [20,21]. Accordingly, PMNE. coli DH5a capture was not affected by cholesteroldepletion of the PMN plasma membrane (Fig. 2A),confirming the specific role of cholesterol-rich domainson PMN–non-opsonized B. pertussis interaction.Importantly, no significant changes in the expression

level of both FcgR and CR3 were observed after MbCDtreatment of PMN (Fig. 2B).To investigate the role of surface-exposed proteins in

lipid raft-dependent uptake of non-opsonized B. pertussis,trypsinized neutrophils lacking surface proteins wereassayed. Trypsin treatment significantly decreased thenumber of non-opsonized B. pertussis associated with

B. pertussis. PMN were incubated for 20min at 37 1C with either IgG-

FP-expressing B. pertussis at a MOI of either 50 (B) or 500 (C). After three

analyzed in parallel (unfilled curves). The experiment was repeated three

esults.

ARTICLE IN PRESSY. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511 503

PMN (Fig. 2C). Trypsinization of cholesterol-depletedPMN did not affect non-opsonized B. pertussis bindingcompared with PMN depleted of cholesterol suggestingthat protease-sensitive receptors are involved in cholester-ol-dependent uptake (Fig. 2C).

2.2. The PMN binding of B. pertussis normalizes after

replenishment of membrane cholesterol

Cholesterol-depleted PMN were incubated with FCSor water-soluble cholesterol prior to incubation withB. pertussis. Incubation with either FCS or water-solublecholesterol normalized levels of B. pertussis associated toPMN confirming that cholesterol-rich domains are essen-tial for bacterial interaction with PMN (Fig. 3).

2.3. The presence of cholesterol-rich domains in PMN

plasma membrane is critical for phagocytosis of

non-opsonized B. pertussis

Next, the relevance of the presence of cholesterol-richdomains for phagocytosis of non-opsonized B. pertussis wasinvestigated. Bacterial internalization was expressed as apercentage of surface-associated bacteria in order to allowthe evaluation of the magnitude of bacterial internalizationindependently of the different binding level under eachcondition. Treatment of neutrophils with MbCD significantlyreduced phagocytosis of non-opsonized bacteria (Fig. 4)whereas phagocytosis of IgG-opsonized bacteria was unaf-fected indicating that FcgR-mediated phagocytosis ofB. pertussis does not depend on PMN membrane cholester-ol-rich domains. Phagocytosis of non-opsonized E. coli DH5awas not affected by cholesterol depletion of PMN (Fig. 4).

2.4. Lipid raft-mediated phagocytosis of non-opsonized

B. pertussis delivers the bacteria to compartments that do not

fuse with lysosomes

Intracellular routing of non-opsonized B. pertussis

was investigated by confocal microscopy. About 70% of

Fig. 2. Cholesterol sequestration specifically affects non-opsonized

B. pertussis interaction with human PMN. (A) Neutrophils treated without

(control) or with either 10mgmL�1 of MbCD, or 35mgmL�1 of nystatin,

were incubated with non-opsonized GFP-expressing B. pertussis (MOI: 500),

IgG-opsonized GFP-expressing B. pertussis (MOI: 50), or non-opsonized

GFP-expressing E. coli DH5a (MOI: 500) for 20min at 37 1C. Cells were

washed and fixed, and PMN-associated bacteria were evaluated by

fluorescence microscopy in which at least 100 cells were counted per slide.

Bacteria associated to treated PMNwas expressed as a percentage of bacteria

associated to untreated PMN arbitrary set as 100%. The data represent the

mean7S.D. of four experiments with PMN from different donors. The

number of non-opsonized B. pertussis associated to PMN either treated with

MbCD or nystatin differed significantly from the number of non-opsonized

B. pertussis found associated to untreated PMN (*Po0.05). (B) Expression

level of PMN FcgR and CR3 before (solid fill) and after (black line)

cholesterol depletion as determined by indirect immune fluorescence using

anti-CD64 (FcgRIa), anti-CD32 (FcgRIIa), anti-CD16 (FcgRIIIb), and anti-

CD11b (CR3) monoclonal antibodies. No significant differences in PMN

expression level of FcgR or CR3 were detected upon MbCD treatment.

(C) Neutrophils treated with or without 10mgmL�1 of MbCD, and further

incubated with or without 0.5% of trypsin were incubated with non-

opsonized GFP-expressing B. pertussis (MOI: 500) for 20min at 37 1C. After

three washing steps, PMN were analyzed by flow cytometry. Bacterial

association to treated PMN was expressed as a percentage of level of

bacterial association to untreated PMN (control) arbitrary set as 100%. Data

represent the mean7S.D. of six independent experiments.

ARTICLE IN PRESS

Fig. 3. PMN cholesterol replenishment restores non-opsonized B. pertussis binding. Non-opsonized GFP-expressing B. pertussis suspended in DMEM-

BSA (MOI: 500) were incubated for 20min at 37 1C with untreated PMN (A), or PMN treated with MbCD and further incubated with either DMEM-

BSA (B) or 8mgmL�1 of water-soluble cholesterol in DMEM-BSA (C). Cells were washed and fixed, and bacterial attachment was evaluated by flow

cytometry. Controls of PMN untreated, cholesterol depleted and cholesterol replenished without bacteria were analyzed in parallel (unfilled curves). The

experiment was repeated four times with PMN isolated from different donors, yielding essentially the same results.

Fig. 4. Cholesterol sequestration specifically decreases phagocytosis of

non-opsonized B. pertussis. PMN were incubated with medium alone

(control) or medium containing 10mgmL�1 of MbCD. Next, non-

opsonized B. pertussis (MOI: 500), IgG-opsonized B. pertussis (MOI: 50),

or non-opsonized E. coli DH5a (MOI: 500), all of them expressing GFP,

were incubated with PMN for 20min at 37 1C. PMN were then washed,

and split into two aliquots. One aliquot was maintained on ice (initial level

of PMN surface-associated bacteria), the other aliquot was further

incubated for 1 h at 37 1C to allow internalization. Surface-bound IgG-

opsonized B. pertussis in both aliquots was detected by addition of PE-

conjugated goat F(ab0)2 fragments of anti-human IgG antibodies. Surface-

bound non-opsonized B. pertussis in both aliquots was determined by

incubation with polyclonal rabbit anti-B. pertussis antiserum, followed by

incubation with PE-conjugated goat F(ab0)2 fragments of anti-rabbit IgG.

Similarly, surface-bound non-opsonized E. coli DH5a in both aliquots was

determined by incubation with rabbit anti-E. coli antiserum, followed by

incubation with PE-conjugated goat F(ab0)2 fragments of anti-rabbit IgG.

Phagocytosis is expressed as the percentage of the surface-associated

bacteria that was internalized. Data represent the mean7S.D. of six

independent experiments. The phagocytosis of non-opsonized B. pertussis

by PMN treated with MbCD was significantly different from the

phagocytosis of non-opsonized B. pertussis by untreated PMN (*Po0.05).

Y. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511504

non-opsonized B. pertussis co-localized with areas highlyenriched in the lipid raft marker flotillin after 20min ofincubation with PMN (Fig. 5). This percentage remainedunchanged after 1 h at 37 1C (Table 1). In contrast, minor

co-localization was observed with E. coli DH5a orIgG-opsonized B. pertussis, suggesting that the innateinteraction of B. pertussis with PMN results in internaliza-tion via cholesterol-rich domains. Accordingly, cholesteroldepletion of PMN with MbCD significantly decreased thepercentage of co-localization of non-opsonized B. pertussis

but did not modify the trafficking of IgG-opsonizedB. pertussis (Table 1 and Fig. 5). Lipid rafts-mediatedinternalization traffics pathogens to subcellular compart-ments that do not show lysosomal maturation [15,22–24].We have previously shown that non-opsonized B. pertussis

phagocytosis fails to trigger PMN oxidative burst [7]. Inorder to gain a better insight into this mechanism, weexamined whether neutrophils degranulate when exposedto B. pertussis. The release of azurophil granule contentin the presence of non-opsonized or IgG-opsonizedB. pertussis was examined using the lysosomal enzymeb-glucuronidase as a marker [25]. The presence of thisenzyme in the extracellular medium reflects the fusion ofazurophil granules with nascent phagosomes [26]. Fig. 6shows that the enzyme was efficiently released solely whenneutrophils were exposed to opsonized bacteria, whereasunder non-opsonic conditions the release was not signifi-cant. Accordingly, confocal studies indicated that in theabsence of antibodies most of the B. pertussis-containingphagosomes did not co-localize with the lysosomalmembrane glycoprotein (LAMP-1) even after 1 h at37 1C unless PMN were depleted of cholesterol (Table 1and Fig. 7), suggesting that the presence of intactcholesterol-rich domains interferes with transportation ofnon-opsonized B. pertussis to lysosomal compartments(Fig. 7). Previous experiments showed that IgG-opsonizedB. pertussis is transported to LAMP-1-expressing subcel-lular fractions [7]. Accordingly, the percentage ofIgG-opsonized B. pertussis-containing phagosomes thatco-localized with LAMP-1 was high both in untreated andcholesterol-depleted PMN (Table 1 and Fig. 7), indicatingthat cholesterol-rich domains are not involved in thetrafficking of opsonized bacteria to lysosomal compart-ments. To further elucidate the localization of non-opsonized B. pertussis, we used the early endosomal

ARTICLE IN PRESS

Fig. 5. Non-opsonized B. pertussis co-localize with flotillin. Human neutrophils treated with or without MbCD were incubated for 20min at 37 1C with

non-opsonized GFP-expressing B. pertussis (MOI: 500), IgG-opsonized GFP-expressing B. pertussis (MOI: 50), or GFP-expressing E. coli DH5a (MOI:

500). After washing, PMN were fixed, and permeabilized prior to incubation with antibodies against flotillin-1. Panel a shows green fluorescent B. pertussis

co-localizating with CY-3-labeled flotillin. Panels c and d show the lack of co-localization of flotillin with E. coli DH5a and IgG-opsonized B. pertussis,

respectively. Panels b and e show the lack of co-localization of both non-opsonized and IgG-opsonized B. pertussis with flotillin after PMN treatment with

MbCD. Representative panels of one out of three independent experiments are shown.

Table 1

Intracellular trafficking of Bordetella pertussis in neutrophils

Bacterial treatment PMN

treatment

MOI (bacteria

per cell)

Flotillina LAMP-1a Transferrinb

20min 1 h 20min 1 h 20min 1 h

Non-opsonized Control 500 70710 60715 471 573 70715 60712

MbCD 500 1073 873 40715 74710 572 372

Opsonized Control 50 873 673 8278 85712 673 472

MbCD 50 572 674 7575 8079 471 271

aPercentage of B. pertussis co-localizing with flotillin, or LAMP-1, after either 20min or 1 h of incubation with PMN at 37 1C. Results shown are the

mean7S.D. of three independent experiments performed with different donors.bPMN were incubated for either 20min or 1 h with GFP-expressing B. pertussis, washed, and further incubated with Alexa transferrin-495 for another

45min. Results are expressed as the percentage of B. pertussis co-localizing with transferrin and are the mean7S.D. of three independent experiments

performed with different donors.

Y. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511 505

marker transferrin. Solely non-opsonized B. pertussis werefound co-localizing with transferrin (Table 1 and Fig. 8),indicating that these bacteria are located in endosomalvacuoles and have access to recycling endosomes after theinternalization through lipid rafts. Accordingly, cholesteroldepletion of PMN significantly decreased the percentage ofco-localization of non-opsonized B. pertussis with trans-ferrin confirming the role of cholesterol-rich domains in thetrafficking of B. pertussis under non-opsonic conditions(Table 1 and Fig. 8).

2.5. Intracellular survival of B. pertussis in PMN

Non-opsonized B. pertussis survival upon phagocytosiswas significantly higher than opsonized bacterial survival(Table 2). Treatment of PMN with MbCD led to asignificant reduction of intracellular survival of non-

opsonized B. pertussis, whereas survival of opsonizedbacteria was unaffected indicating that uptake ofB. pertussis through cholesterol-rich domains eventuallydecrease cellular bacterial killing. Control experiment withE. coli DH5a showed that the intracellular survival of thesebacteria was also not affected by cholesterol depletion(data not shown; n ¼ 3).

3. Discussion

In this study, we found evidence that interaction ofB. pertussis and molecules expressed in cholesterol-en-riched domains in the plasma membrane of PMN triggersbacterial uptake and increases the chance of its intracel-lular survival. This immune evasion mechanism maycontribute to bacterial persistence in the human airwayand host colonization.

ARTICLE IN PRESS

Fig. 6. Non-opsonized B. pertussis failed to trigger PMN b-glucuronidaserelease. Neutrophils were incubated with DMEM-BSA (control), non-

opsonized B. pertussis at a MOI of 500, or IgG-opsonized B. pertussis at a

MOI of 50. The release of b-glucuronidase is expressed as the percentage

of the total b-glucuronidase cell content. The data represent the

mean7S.D. of three experiments with PMN from different donors.

b-glucuronidase release induced by opsonized B. pertussis differed

significantly from b-glucuronidase release induced by non-opsonized

B. pertussis or control PMN (*Po0.05).

Fig. 7. Cholesterol-rich domains in PMN plasma membrane are essential

for intracellular routing of non-opsonized B. pertussis to LAMP-1

negative compartments. PMN treated without (panel a and c) or with

(panel b and d) MbCD were incubated for 20min at 37 1C with non-

opsonized B. pertussis (panel a and b) or IgG-opsonized B. pertussis (panel

c and d) expressing GFP, washed, and further incubated for 1 h at 37 1C.

Cells were fixed and permeabilized prior to incubation with antibodies

against LAMP-1. Panel a shows green fluorescent B. pertussis inside PMN.

No co-localization with CY-3-labeled LAMP-1 is observed. Panels b–d

show co-localization of B. pertussis and LAMP-1, reflected by yellow

areas. Representative panels of one out of three independent experiments

are shown.

Y. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511506

Previous studies showed that opsonization of B. pertussis

with IgA or IgG antibodies efficiently triggers binding toPMN, phagocytosis, oxidative burst and subsequent

bacterial killing [7]. These studies underlined the impor-tance of specific antibodies for host defense. The innateinteraction of B. pertussis with phagocytes has not beenelucidated completely. CR3 is probably the main receptoremployed by B. pertussis to bind to phagocytes [27].Binding to CR3 may be advantageous for bacteria, sinceligation of this receptor does not activate professionalphagocytes. Accordingly, we found that non-opsonizedB. pertussis resides in phagosomes, which do not fuse withazurophil granules as determined by the b-glucuronidaserelease. Neutrophils contain at least two types of secretorygranules that are secreted during phagocytosis, theazurophil and the specific granules [25]. Once neutrophilsphagocyte particles, azurophil granules fuse with nascentphagosomes [28,29]. The release of the azurophil granulemarker b-glucuronidase in the extracellular medium wasdetected only in the presence of opsonic antibodies,indicating that when neutrophils ingest non-opsonizedB. pertussis the biogenesis of phagolysosomes does nottake place. Accordingly, experimentally induced uptake ofB. pertussis via CR3 resulted in significantly higher survivalrates as compared to bacteria targeted to FcR [13].Engagement of specific membrane receptors on phagocytesmay crucially determine chances of intracellular survival,since the signaling pathways triggered by membranemolecules may determine intracellular routing, i.e. trans-port to lysosomes, or subcellular structures withoutlysosomal maturation. FcR engagement triggers signalingvia the immune receptor tyrosine-based activation motif(ITAM) in the intracellular tail of the receptor or viaassociated signaling chains, which results in efficientintracellular routing to lysosomes. The signaling cascadestriggered by the innate interaction of B. pertussis withPMN membrane structures remain to be dissected.Bacterial virulence factors including fimbriae, PT andFHA up-regulate CR3 expression, suggesting that virulentnon-opsonized B. pertussis may stimulate its own attach-ment to immune cells via CR3 [8–10]. The results from thisstudy indicate that, in addition to the presence of dockingmolecules such as CR3, the presence of cholesterol-richdomains ensures transport to subcellular structureswithout lysosomal maturation. Treatment of PMN withcholesterol sequestering agents that disrupt lipids raftssignificantly decreased phagocytosis, and intracellularsurvival of non-opsonized B. pertussis. Uptake ofIgG-opsonized B. pertussis and E.coli DH5a were notaffected by PMN treatment with MbCD, indicating thatcholesterol depletion does not generally interfere withphagocyte function. Trypsin treatment of PMN furtherdemonstrated that protease-sensitive receptors are involvedin non-opsonized B. pertussis interaction with lipid rafts.Importantly, the lack of changes in PMN CR3 and FcgRexpression, particularly FcgRI, further suggests that underour experimental conditions PMN were not activatedby MbCD treatment. Cholesterol replenishment resultedin a restoration of the level of attachment confirming thatthe associated reduction in B. pertussis adhesion and

ARTICLE IN PRESS

Fig. 8. Non-opsonized B. pertussis in PMN are accessible to early endosomes. PMN treated without (panel a and c) or with (panel b) MbCD were

incubated for 20min at 37 1C with either non-opsonized GFP-expressing B. pertussis at a MOI of 500 (panel a and b) or IgG-opsonized GFP-expressing

B. pertussis at a MOI of 50 (panel c), and further incubated for 1 h at 37 1C to allow bacterial internalization. PMN were analyzed by confocal microscopy

after 45min of PMN incubation with Alexa transferrin-594 (10 mgmL�1). Panel a shows co-localization of B. pertussis and transferrin-594 as reflected by

yellow areas. Panels b and c shows green fluorescent B. pertussis inside PMN. No co-localization with transferrin-594 is observed in these panels.

Representative panels of one out of three independent experiments are shown.

Table 2

Cholesterol-rich domains of PMN plasma membrane facilitate uptake and intracelullar survival of non-opsonized B. pertussis

Bacterial treatment PMN

treatment

MOI

(bacteria

per cell)

PMN-associated bacteria

(bacteria per cell)

aPercentage of

phagocytosis

bBacterial survival upon

phagocytosis (% of

internalized bacteria)

Non-opsonized Control 500 2573 7774 (1971) 4.8

MbCD 500 7.370.5 5774 (4.170.3) 1.3

Opsonized Control 50 3574 8672 (30.070.7) 0.05

MbCD 50 3775 8775 (3272) 0.05

aThe number of internalized bacteria per PMN is depicted in brackets. Data are mean7S.D. of three independent experiments.bMean percentage of the internalized bacteria still alive after polymyxin B treatment.

Y. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511 507

phagocytosis upon cholesterol depletion is not due to ageneral disruption of cell membrane integrity, but the resultof a specific disruption of a cholesterol-mediated entryemployed by the bacteria.

Lipid rafts are plasma membrane domains rich incholesterol, glycolipids, and GPI-anchored molecules.CR3 was previously found associated with several GPI-anchored receptors [30,31]. In our hands, CR3 expressionwas unaffected by the cholesterol sequestering drugs,suggesting that cholesterol-rich domains may function asplatforms that cluster docking molecules such as CR3,asialo-GM1 [14] and GSL which would facilitate interac-tion with microbial adhesins. Such clustering may beimportant for triggering of relevant signaling cascades.Previous studies showing the central role of cholesterol onCD47 signaling and CR3-mediated B. pertussis binding tohuman monocytes seem to support this hypothesis [32,33].

Confocal microscopy showed that non-opsonized B.

pertussis was routed to LAMP-1 negative, flotillin-contain-ing intracellular compartments. Additionally, we foundthat B. pertussis vacuoles remain accessible to thetransferrin recycling pathway of the neutrophils furtherindicating that B. pertussis entry trough lipid rafts preventsthe normal maturation of their phagosomes. Lipid raft-mediated phagocytosis of pathogens generally favorsintracellular microbial survival in macrophages. Theincorporation of cholesterol-rich domains into the phago-cytic cup critically determines the routing of phagosomes

containing microorganisms such as Brucella [24,34],Mycobacterium [16], Shigella [35], Dr-positive E. coli [36],and Chlamydia [37]. The fact that non-opsonized B.

pertussis co-localize with LAMP-1 only after PMN weredepleted of cholesterol suggest that B. pertussis employssimilar mechanisms as these intracellular pathogens toenhance the odds of intracellular survival. Accordingly,intracellular survival of non-opsonized bacteria, signifi-cantly higher than survival of opsonized B. pertussis uponphagocytosis, was drastically reduced in cholesterol-de-pleted PMN.The data presented here provide new insights into the

intracellular fate of B. pertussis upon innate interactionwith professional phagocytes. Taken together, our resultsshow that in the absence of specific antibodies PMNuptake of B. pertussis is not only less efficient but cruciallydependent on a lipid raft-mediated trafficking thateventually leads to the failure of lysosomal maturationand bacterial clearance.

4. Materials and methods

4.1. Bacterial strains and growth conditions

B. pertussis strain B213, a streptomycin-resistant deri-vate of Tohama I, was transformed with plasmid pCW505[38] (kindly supplied by Dr. Weiss, Cincinnati, OH, USA)which induces cytoplasmic expression of GFP without

ARTICLE IN PRESSY. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511508

affecting growth, or antigen expression [38]. Bacteria werestored at �70 1C and recovered by growth on BordetGengou agar (BGA) plates supplemented with 15%defibrinated sheep blood at 35 1C for three days. Virulentbacteria were subsequently plated on BGA, culturedovernight, and used in phagocytosis experiments.

E. coli DH5a transformed with plasmid PML2 whichinduces cytoplasmic expression of GFP (a generous giftfrom Dr. Lagares, Institute of Biochemistry and MolecularBiology, La Plata, Argentina) was grown in Luria Bertani(LB) medium at 37 1C overnight, and used in phagocytosisexperiments.

4.2. Antibodies

The following antibodies were used: anti-hFcgRI (CD64)monoclonal antibody (mAb) 22 (mIgG1), anti-hFcgRII(CD32) mAb AT10 (mIgG1), anti-hFcgRIII (CD16) mAb3G8 (mIgG1) (all from Medarex, Annandale, NJ, USA),mAb anti-CD11b (Becton Dickinson, San Jose, CA, USA),mAb against human lysosome-associated membrane pro-tein LAMP-1 (Pharmingen, San Diego, CA, USA), andpolyclonal rabbit antibody against human flotillin-1 (SantaCruz Biotechnology, Santa Cruz, CA, USA).

IgG fractions from pooled sera of pertussis patients withhigh titers against B. pertussis, as measured by ELISA [39],were isolated as previously described [7]. Polyclonal rabbitanti-B. pertussis antiserum was obtained as describedelsewhere [13].

4.3. Receptor expression

The expression level of PMN FcgR and CR3 wasassayed by indirect immune fluorescence using a panel ofmAbs, followed by FITC-labeled F(ab0)2 fraction of goatanti-mouse immunoglobulin antiserum (Tago, Burlingame,CA, USA). Fluorescence was quantitated by FACStar flowcytometer (Becton Dickinson, Lincoln Park, NJ, USA). Toavoid cytophilic binding of mAb to FcgR, all incubationswere done in the presence of 25% heat-inactivated humanserum. Additionally, isotype controls were run in parallel.

4.4. Cells

Peripheral blood PMN were isolated from heparinizedvenous blood using Ficoll-Histopaque (Sigma, St. Louis,MO, USA) gradient centrifugation. Polymorphic leuko-cytes were harvested and remaining erythrocytes removedby hypotonic lysis. PMN purity determined by cytospinpreparations exceeded 95%, and cell viability was 499%as determined by trypan blue exclusion. Prior to functionalassays, PMN were washed twice with DMEM supplemen-ted with 0.2% of BSA (Sigma), resuspended, and usedimmediately. All experiments described in this study werecarried out with freshly isolated PMN lacking FcgRI(CD64) expression, as monitored by FACS analysis withanti-FcgRI mAb 22 [40].

4.5. Cholesterol sequestration of PMN

Cholesterol sequestration was achieved by incubatingPMN with 10mgmL�1 of MbCD (Sigma) (15min at37 1C), or 35 mgmL�1 of nystatin (Sigma) (30min at 37 1C)in serum-free DMEM plus BSA (0.2%) and lovastatin(5 mgmL�1) (Sigma) (DMEM-BSA-L). Cells were thenwashed, suspended in DMEM-BSA-L, and used immedi-ately. No decrease in PMN viability was detected aftertreatment. In selected experiments, PMN cholesterolreplenishment was performed by incubation of MbCD-treated PMN with 8mgmL�1 of water-soluble cholesterol(Sigma) for 30min at 37 1C or with FBS (20%) for 3 h at37 1C in DMEM-BSA.Total cellular cholesterol in treated and untreated PMN

was checked using the Amplex Red cholesterol assay kit(Molecular Probes).

4.6. Quatification of B. pertussis PMN association

B. pertussis cell association was determined by quantita-tive determination of bacteria associated with the infectedcell. GFP-expressing B. pertussis was incubated for 20minat 37 1C with human neutrophils at a multiplicity ofinfection (MOI) of either 50 or 500 bacteria per cell. Afterthree washing steps to remove non-attached bacteria, thesamples were fixed using paraformaldehyde and analyzedby both flow cytometry and fluorescence microscopy usinga DMLB microscope coupled to a DC 100 camera (LeicaMicroscopy Systems Ltd., Heerbrugg, Switzerland).In selected experiments, GFP-expressing B. pertussis

were opsonized with human IgG (200 mgmL�1) for 30minat 37 1C prior to incubation with PMN at a MOI of 50.Additionally, GFP-expressing E. coli DH5a PMN associa-tion was determined as described above at a MOI of 500.In some experinents, PMN were incubated with trypsin

(0.5%) for 30min at 37 1C in DMEM plus BSA (0.2%)prior to incubation with bacteria.

4.7. Quantification of phagocytosis

Phagocytosis of B. pertussis was evaluated as in Ref. [42]with a few modifications. Briefly, bacteria incubated withPMN for 20min at 37 1C to allow bacterial interaction withPMN as described above were extensively washed at 4 1Cto remove non-attached bacteria. An aliquot was main-tained on ice to determine PMN surface-associatedbacteria at this time point, while another aliquot wasfurther incubated for 1 h at 37 1C. Phagocytosis wasstopped by placing PMN on ice. PMN surface-boundbacteria in both samples were detected by a two-steplabeling procedure. For this purpose, PMN were incubatedwith polyclonal rabbit anti-B. pertussis antiserum (30minat 4 1C), followed by incubation with PE-conjugated goatF(ab0)2 fragments of anti-rabbit immunoglobulin (Mole-cular Probes, OR, USA) for another 30min at 4 1C. Toavoid eventual cytophilic binding of antibodies to FcgR, all

ARTICLE IN PRESSY. Lamberti et al. / Microbial Pathogenesis 44 (2008) 501–511 509

incubations were done in the presence of 25% heat-inactivated human serum. After washing, samples wereanalyzed by flow cytometry. Ten thousand cells wereanalyzed per sample. Green and red fluorescence intensitiesof the cells maintained at 37 1C for 20min served as controlfor PMN surface-bound bacteria at that incubation time.The decrease in red fluorescence after 1 h incubation at37 1C reflects the level of phagocytosis of the surface-associated bacteria. Phagocytosis rates were calculatedfrom the drop in mean red fluorescence intensity ofgreen-positive cells as described [7]. Phagocytosis wasexpressed as a percentage and was calculated as follows:100� (1�PE2/PE1), where PE1 is the mean PE-fluores-cence of the FITC-positive cells maintained at 37 1C for20min and PE2 is the mean PE-fluorescence of the FITC-positive cells maintained at 37 1C for an additional 1 h.

In selected experiments, B. pertussis was opsonized withhuman IgG (200 mgmL�1) prior to incubation with PMNat a MOI of 50. Phagocytosis assays were performed asoutlined above, albeit that in this experimental set-upattached bacteria were detected by incubation with PE-conjugated goat F(ab0)2 fragments of anti-human IgG(from Southern Biotechnology, Birmingham, UK).

4.8. Exocytosis of the lysosomal enzyme b-glucuronidase

Neutrophils in suspension (5� 106 cellsmL�1) wereincubated with non-opsonized or IgG-opsonized B. per-

tussis for 1 h at 37 1C and pelleted. The supernatants werecentrifuged at 10,000� g for 10min to eliminate bacteria.The b-glucuronidase activity was measured at 405 nm incells pellet lysed with 0.2% Triton X-100, and in the cellsupernatants as described [41].

4.9. Killing assays

Non-opsonized or IgG-opsonized B. pertussis wereallowed to attach to human PMN at 37 1C for 20min.Non-adherent bacteria were removed by washing at 4 1C.Next, PMN were incubated at 37 1C for 1 h to allowphagocytosis. The cells were then incubated for 1 h at 37 1Cin DMEM supplemented with 400 mgmL�1 of polymyxin Bsulfate (Sigma), an antibiotic that cannot penetratemammalian cells [43], to kill the remaining extracellularbacteria. Next, serial dilutions of samples were preparedand plated in duplicate on BGA to determine the numberof surviving bacteria. The percentage of survival wascalculated using the number of bacteria associated withneutrophils and the phagocytosis index as follows:

�

N: number of viable bacteria per cell after incubationwith polymyxin B, � A: number of bacteria associated with PMN after 20min

at 37 1C (determined by fluorescent microscopy),

� B: phagocytosis index (determined in parallel by flow

cytometry as described above), 1�PE2/PE1,

� % bacterial survival ¼ N� 100/A�B.

Control experiments to assess efficacy of antibioticbactericidal activity were performed in parallel. Briefly,samples of 5� 108 bacteria were incubated with antibioticsand plated on BG agar after 1 h at 37 1C. This resulted in99.999% decrease in colony forming units (CFU).

4.10. Confocal microscopy

Co-localization studies were performed as describedbefore [7]. Briefly, aliquots of neutrophils incubated withGFP-expressing bacteria at 37 1C for 20min were washedat 4 1C to remove non-attached bacteria, and furtherincubated for either 20min or 1 h at 37 1C for co-localization studies of bacteria and LAMP-1, or bacteriaand flotillin. After fixation with paraformaldehyde, PMNwere washed twice with PBS, and incubated for 10min atroom temperature with PBS containing 50mM NH4Cl.After two washing steps, cells were incubated for 30minwith PBS containing 0.1% saponin (Sigma), and 0.5%BSA. Next, cells were incubated 1 h at 4 1C with eithermouse anti-human LAMP-1 monoclonal antibodies orrabbit anti-human flotillin-1 antibodies in the presence of0.1% saponin and 0.5% BSA. After three washing steps,PMN were incubated with CY3-conjugated F(ab0)2fragment of goat anti-mouse IgG or CY3-conjugatedsheep anti-rabbit IgG (Jackson InmunoResearch, WestGrove, USA) for 30min. To avoid cytophilic binding ofantibodies to FcgR, all incubations were done in thepresence of 25% heat-inactivated human serum. Addition-ally, isotype controls were run in parallel. Finally, cellswere spun on microscope slides. Microscopic analyses wereperformed using a confocal laser-scanning microscope(Olympus FV 300, Japan). The percentage of bacterium-containing phagosomes that co-localized with a givenmarker was calculated by analyzing at least 50 phagosomesper donor.

4.11. Transferrin uptake

Non-opsonized or IgG-opsonized GFP-expressingB. pertussis were allowed to attach to human PMN at37 1C for 20min. After washing at 4 1C to remove non-attached bacteria, PMN were further incubated for another20min or 1 h at 37 1C to allow phagocytosis. Neutrophilswere then depleted of transferrin by incubation in DMEMcontaining 1% BSA for 1 h at 37 1C, and further incubatedfor 10min at 4 1C with 10 mgmL�1 of Alexa transferrin-594(Molecular Probes) in an excess of BSA (1%) to saturatenon-specific endocytosis. Next, cells were incubated for5min at 37 1C to allow internalization of the ligand,washed with DMEM containing 1% of BSA, and furtherincubated for another 45min at 37 1C. Finally, the cellswere fixed and spun on microscope slides. Microscopicanalyses were performed using a confocal laser-scanningmicroscope (Olympus FV 300, Japan). At least 50 bacteriaper donor were analyzed for co-localization with transfer-rin in each experiment.

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4.12. Statistics

Student’s test (confidence level: 95%) or analysis ofvariance (ANOVA) was used for statistical data evalua-tion. The significance of the differences between the meanvalues of the data evaluated by ANOVA was determinedwith the least significant difference (LSD) test at aconfidence level of 95%. Results are shown as means andstandard deviations (S.D.).

Acknowledgments

This study was partially supported the SECyT (PICT 14522). M.E.R. is a member of the Scientific Career ofCONICET. M.L.P.V. and Y.L. are doctoral fellows ofCONICET.

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