R E S E A R C H A R T I C L E

TheABC transporter-encoding geneAFR1 a¡ects the resistanceofCryptococcusneoformans tomicroglia-mediatedantifungalactivitybydelaying phagosomalmaturationCarlotta Francesca Orsi1, Bruna Colombari1, Andrea Ardizzoni1, Samuele Peppoloni1, Rachele Neglia1,Brunella Posteraro2, Giulia Morace3, Giovanni Fadda2 & Elisabetta Blasi1

1Dipartimento di Scienze di Sanita Pubblica, Universita di Modena e Reggio Emilia, Modena, Italy; 2Istituto di Microbiologia, Universita Cattolica del

Sacro Cuore, Roma, Italy; and 3Dipartimento di Sanita Pubblica, Universita degli Studi di Milano, Milan, Italy

Correspondence: Elisabetta Blasi,

Dipartimento di Scienze di Sanita Pubblica,

Universita di Modena e Reggio Emilia, Via

Campi 287, 41100 Modena, Italy. Tel.: 139

059 205 5468; fax: 139 059 205 5483;

e-mail: elisabetta.blasi@unimore.it

Received 25 August 2008; revised 25 October

2008; accepted 27 October 2008.

First published online 16 December 2008.

DOI:10.1111/j.1567-1364.2008.00470.x

Editor: Andrew Alspaugh

Keywords

Cryptococcus neoformans ; microglia;

phagocytosis; ABC transporter; phagosomal

maturation.

Abstract

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to

survive within phagocytes. Recently, it has been demonstrated that upregulation of

the ATP binding cassette transporter-encoding gene antifungal resistance 1 (AFR1)

is important not only for determining the resistance of C. neoformans to

fluconazole but also in influencing fungal virulence. In the present study, we

showed that the fluconazole-resistant AFR1-overexpressing mutant strain was not

sensitive to microglia-mediated anticryptococcal activity, as compared with the

fluconazole-susceptible isogenic strains, the wild type and the afr1D mutant.

Interestingly, although the three strains were phagocytosed to a similar extent,

reduced acidification and delayed maturation were observed in phagosomes

containing the AFR1-overexpressing strain with respect to the others. These

findings provide the first evidence that upregulation of the AFR1 gene affects

C. neoformans–microglia interplay, adding insights to the complexity of crypto-

coccal virulence and to its unexpected link with azole resistance.

Introduction

Cryptococcus neoformans is the aetiological agent of human

cryptococcosis, an air-borne invasive opportunistic mycosis

(Casadevall & Perfect, 1998). In immunocompromised

hosts, C. neoformans often disseminates and, because of its

marked neurotropism, it invades the central nervous system,

causing meningoencephalitis that commonly relapses

despite therapy or rather may be fatal in untreated patients

(Casadevall & Perfect, 1998). Although this fungus is known

to replicate extracellularly, recent studies have shown that

C. neoformans actually behaves as a facultative intracellular

pathogen; once ingested, it is able to survive and to replicate

within phagocytes through multiple and complex strategies

(Lee et al., 1995; Mansour & Levitz, 2002; Ma et al., 2006). In

fact, in C. neoformans-infected macrophages, accumulation

of polysaccharide-filled vesicles is observed, followed by the

formation of spacious phagosomes containing viable cryp-

tococcal cells (Tucker & Casadevall, 2002). Moreover, fungal

intracellular replication occurs and may result in lysis of the

host cell; alternatively, extrusion of the cryptococcal phago-

some has been described as a novel strategy through which

both survival of the macrophage and dissemination of the

yeast cells are allowed (Tucker & Casadevall, 2002; Alvarez &

Casadevall, 2006, 2007; Ma et al., 2006).

Microglia, the brain resident phagocytes, are in charge of

locally mediating anticryptococcal defence (Kreutzberg,

1996; Zhou et al., 2007). They respond to infection with

NFkB activation followed by the release of matrix metallo-

proteinases (Alvarez & Casadevall, 2007; Kumnok et al.,

2008), reactive oxygen species and proinflammatory cyto-

kines, such as IL1a, tumor necrosis factor a (TNFa) and

MIP-2a (Jack et al., 2005; Nimmerjahn et al., 2005; Neglia

et al., 2006; Blasi et al., 2007, 1990, 1995b). Microglia may

either allow C. neoformans intracellular survival and replica-

tion (Lee et al., 1995) or exert anticryptococcal activity,

whose levels are enhanced upon exposure to IFN-g or

chloroquine (Blasi et al., 1995b; Mazzolla et al., 1997; Sale-

ppico et al., 1999). Furthermore, C. neoformans resistance to

microglia is partially reduced by yeast cell preopsonization

FEMS Yeast Res 9 (2009) 301–310 Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

with fresh serum or by pretreatment with protease inhibi-

tors (Blasi et al., 1992, 1995a, b; 2004; Lipovsky et al., 1997).

Thus, both the early yeast-to-microglia recognition step(s)

and the postphagocytosis intracellular events seem to be

relevant in the outcome of such pathogen–host cell inter-

play. As widely described by means of other pathogen–host

cell models, the C. neoformans-containing phagosome is

expected to undergo a multistep maturation process, even-

tually allowing the formation of an acidic compartment

(Chaka et al., 1995; Levitz et al., 1999, 1987; Wozniak et al.,

2006). This process is characterized by the gradual acquisi-

tion of early and late endosomal markers, such as Rab5,

Rab7 and Rab9, and of the late lysosome-associated mem-

brane proteins LAMP1 and LAMP2 (Meresse et al., 1999;

Zerial & McBride, 2001; Barbero et al., 2002; Rink et al.,

2005; Eskelinen, 2006; Huynh et al., 2007).

Specific virulence factors of C. neoformans, including

polysaccharide capsule (Bhattacharjee et al., 1984; Cherniak

& Sundstrom, 1994), melanin (Shaw & Kapica, 1972) or

mannitol production (Wong et al., 1990), superoxidase

dismutase (Jacobson et al., 1994), proteases (Brueske, 1986;

Blasi et al., 2004), phospholipase B and lysophospholipase

(Chen et al., 1997a, b), have been described; among all,

capsule formation and melanin production appear as crucial

elements to elude host defences at the brain level (Blasi et al.,

1992, 1995a). Recently, the antifungal resistance 1 (AFR1)

gene has been identified and characterized (Posteraro et al.,

2003; Sanguinetti et al., 2006): it encodes an ATP binding

cassette transporter protein, involved in the in vitro resis-

tance to fluconazole (Posteraro et al., 2003). We constructed

two C. neoformans strains, both derivatives of the flucona-

zole-susceptible clinical isolate BPY22, namely, the AFR1-

overexpressing (BPY445) and the afr1D(BPY444) strains

(Sanguinetti et al., 2006). Briefly, to generate BPY444, the

AFR1 gene was disrupted by homologous recombination,

and the absence of AFR1 transcripts in this strain was

determined by real-time reverse transcriptase-PCR (RT-

PCR) analysis. In contrast, BPY445 was generated by

random integration of the AFR1 gene, placed under the

control of the constitutively active promoter of the GPD1

gene, into the BPY22 genome; in this strain, AFR1 expres-

sion was about 12-fold increased as compared with that of

the BPY22 strain, as determined by real-time RT-PCR

analysis (Sanguinetti et al., 2006). When compared in a

mouse model of systemic infection, such laboratory mutants

not only retained the expected fluconazole-resistant and

-susceptible phenotypes, but, surprisingly, also differed in

the degree of virulence, BPY445 being consistently more

virulent than BPY444 or BPY22 (Sanguinetti et al., 2006). To

exclude the possibility that the BPY445 phenotype was

related to the genomic position of the overexpression

construct in this strain, we also analysed other independent

transformants, in which the overexpression construct was

located at different genomic sites, as assessed by Southern

blot analysis. The results obtained with these two strains

were fully consistent with those reported for BPY445, thus

confirming that the virulence phenotype of the three strains

was not linked to the site of insertion of the ectopic AFR1

gene (Sanguinetti et al., 2006).

In the present study, we investigated the role of the AFR1

phenotype in the interaction between C. neoformans and

microglia. Upon infection with the wild-type BPY22 or the

two mutants, the Afr1D and the AFR1-overexpressing

strains, microglial cells were assessed for phagocytosis,

phagosome maturation and anticryptococcal activity; NFkB

induction and cytokine response were also evaluated.

Materials and methods

Cryptococcus neoformans strains

The following strains were used: BPY22 (wild type), BPY444

(afr1D) and BPY445 (AFR1-overexpressing mutant) (Pos-

teraro et al., 2003; Sanguinetti et al., 2006). Long-term

storage was carried out in 20% glycerol at � 80 1C. Before

testing, a single colony, obtained on Sabouraud (Oxoid,

Hampshire, UK) agar from each strain, was transferred in

Sabouraud dextrose broth under shaking. When required,

staining of yeast cells was achieved by incubation with

Oregon green 488 (Levitz et al., 1999; Wozniak et al., 2006),

2 mg mL�1 in the dark at 37 1C for 1 h. After labelling, yeast

cells were washed twice with phosphate-buffered saline

(PBS), counted and suspended in Roswell Park Memorial

Institute (RPMI) medium at the desired concentration.

Microglial cell line

The previously established murine microglial cell line BV2

(Blasi et al., 1990) was maintained in RPMI 1640 medium

(Gibco, Grand Island, NY), supplemented with 10% heat-

inactivated foetal bovine serum (Defined Hyclone, Logan,

UT), gentamicin (50mg mL�1) and L-glutamine (2 mM),

hereafter referred to as complete RPMI. Cells were detached

by vigorous shaking biweekly and fresh cultures were started

at a concentration of 5� 105 mL�1, the day before the

experiment.

Reagents and chemicals

Oregon green 488 and LysoTracker DND-99 were obtained

from Molecular Probes (Eugene, OR). Uvitex 2B was

obtained from Polysciences Inc. (Warrington, PA). Poly-L-

lysine, paraformaldehyde and polyclonal antibodies against

murine Rab5, Rab7, Rab9 and LAMP2 were purchased

from Sigma (St. Louis, MO). The Chromeo 642 goat anti-

rabbit IgG, the NFkB enzyme-linked immunosorbent assay

FEMS Yeast Res 9 (2009) 301–310Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

302 C.F. Orsi et al.

(ELISA) kit and the nuclear extract kit were obtained from

Active Motif (CA).

Measurement of anticryptococcal activity

Yeast cells (105 mL�1 in complete RPMI, 100mL per well)

were plated in 96-well plates; then, BV2 cells were added

(106 mL�1 in complete RPMI, 100mL per well). After an

additional 2, 3 and 4 h of incubation, the plates were

vigorously shaken and Triton X-100 (0.1% final concentra-

tion) was added to each well. According to previously

described protocols (Blasi et al., 1995b), serial dilutions

from each well were made in saline and plated (triplicate

samples) on Sabouraud dextrose agar. The number of CFU

was determined after 48–72 h of incubation at room tem-

perature. Control cultures consisted of yeast cells incubated

in complete RPMI without effector cells. The results were

expressed as percent of anticryptococcal activity according

to the formula described elsewhere (Blasi et al., 1995b).

Phagocytosis assay

To strengthen attachment of BV2 cells to the wells, Lab-Tek

II chamber slides (Nalge Nunc International, Naperville, IL)

were pretreated with poly-L-lysine, 10 mg per well, for 30 min

and washed twice with PBS. BV2 cells (106 mL�1 in complete

RPMI, 100 mL per well) were seeded, incubated for 30 min at

37 1C and 5% CO2 and then infected with Oregon green

prelabelled yeast cells (5� 106 mL�1 in complete RPMI,

100 mL per well) and further incubated for 1.5 or 3 h. Then,

Uvitex 2B was added (20 mL per well) 15 min before each

end point (Levitz et al., 1987; Chaka et al., 1995; Lipovsky

et al., 1997). Cells were washed with PBS to remove non-

adherent cryptococci and fixed with 4% formaldehyde freshly

made from paraformaldehyde for 30 min at 4 1C. As pre-

viously described (Levitz et al., 1987; Chaka et al., 1995;

Lipovsky et al., 1997), Uvitex staining of fungi allows one to

discriminate between adherent (Uvitex accessible) and pha-

gocytized (i.e. internalized, Uvitex nonaccessible) yeast cells.

When visualized by epifluorescence microscopy, all the yeast

cells appeared as green (independently of the localization),

while the noninternalized yeast cells were blue stained. Finally,

merging of Oregon green and Uvitex images allowed one to

definitively exclude the extracellular yeast cells. A minimum

of 200 microglial cells per group were scored and any cell

containing one or more particles was counted as phagocytic.

Phagolysosome acidification assay

Visualization of the acid yeast-containing vesicles was per-

formed as previously described (Via et al., 1998). Briefly,

Lab-Tek II chamber slides were prepared as in Phagocytosis

assay; then, the BV2 cells were exposed to the acidotropic

dye LysoTracker DND-99 (75 nM) (Binker et al., 2007) and

infected as described above. Samples were then fixed with

4% formaldehyde and immediately examined by fluores-

cence microscopy. Acidification control groups consisted of

uninfected cells. A minimum of 200 yeast-containing pha-

gosomes were scored; the percent of acid phagolysosomes

was evaluated as the ratio between the number of Lyso-

Tracker-labelled phagosomes and the total number of yeast-

containing phagosomes.

Colocalization experiments

The procedure for immunofluorescence labelling of phago-

some membrane markers was adapted from a previously

described method (Barnewall et al., 1997). Briefly, Lab-Tek

II chamber slides were prepared as in Phagocytosis assay;

then, the BV2 cells were infected with BPY444 or BPY445

strain. At 3 h postinfection, BV2 cells were washed, fixed

with 4% formaldehyde and permeabilized for 10 min with

0.02% Triton X-100; then, primary antibodies (anti-Rab5,

Rab7, Rab9 or LAMP2) were added to each well for 1 h at

room temperature. After two washes with PBS, the second-

ary antibody was added for an additional 1 h. Samples were

washed and then observed by epifluorescence microscopy.

Determination of NFkB activation

BV2 cells (106 mL�1) were infected with BPY22, BPY444 or

BPY445 (107 mL�1; 106 mL�1) for 30 min in 25-cm2 cell

culture flasks. In parallel, BV2 cells were exposed to lipopo-

lysaccharide (1 mg mL�1) as positive controls. Then, cell and

nuclear extracts were obtained using a nuclear extract kit.

Briefly, cells were collected in the PBS/phosphatase inhibitor

solution and suspended in lysis buffer containing DTT

10 mM and a cocktail of protease inhibitors. Solubilized

proteins were then separated from cell debris by centrifuga-

tion (20 min at 14 000 g). For each sample, the protein

concentration in cytoplasmatic and nuclear fractions was

determined by Bradford assay and adjusted to the same

levels. NFkB activation was measured by the NFkB ELISA

kit, according to the manufacturer’s recommendations. The

levels of NFkB activation were measured by a Sunrise

spectrophotometer (Tecan, Salzburg, Austria) at OD450 nm.

Each sample was run in triplicate.

Cytokine measurement

BV2 cells (106 mL�1) were infected with BPY22, BPY444 or

BPY445 (107 mL�1) strain for 6 and 24 h in 24-well plates. In

parallel, BV2 cell treatment with lipopolysaccharide

(1mg mL�1) was included in the assay as positive control.

Then, the supernatants were collected and frozen at

� 80 1C. MIP-1a and TNFa levels were measured by sand-

wich ELISA according to the manufacturer’s protocol (R&D

Systems). The reaction was read as OD using a Microplate

FEMS Yeast Res 9 (2009) 301–310 Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

303Phagosomal maturation altered by the C. neoformans AFR1 gene

Reader (Sunrise). Experiments were repeated three to five

times and each sample was run in triplicate. Results were

expressed as pg mL�1.

Epifluorescence microscopy

Images were generated and captured with a Nikon Eclipse

90i system, equipped with Nomarski differential interfer-

ence contrast (DIC) optics. The overlapping signals of

Oregon green-labelled yeasts (green fluorescence) with

either the Uvitex 2B (blue fluorescence) or the red fluores-

cence of the LysoTracker or of the secondary antibody

Chromeo 642 were always interpreted as colocalization. At

each time point, samples were photographed with a DS-5Mc

Nikon digital camera and the photographs were then

analysed by the NIKON software program.

Statistical analysis

Statistical analysis was performed by one-way ANOVA with a

Bonferroni correction post-test or by the Student’s t-test.

The results reported in the figures and the table are the

mean� SD of replicates from a representative experiment

out of three to five performed.

Results

The AFR1-overexpressing phenotype enhances C.neoformans resistance to microglia-mediatedantifungal activity

In the present study, we compared three C. neoformans

strains, differing in the AFR1 phenotype, for resistance to

microglia-mediated defences. In particular, the BV2 cells

were infected with the wild-type fluconazole-susceptible

clinical isolate BPY22 or with its derivatives, the AFR1-

overexpressing BPY445 (fluconazole resistant) and the

Afr1D BPY444 (fluconazole susceptible) mutant strains;

then, the anticryptococcal activity was measured as detailed

above. Significant levels of antifungal activity were observed

against both BPY22 and BPY444 strains at 2, 3 and 4 h

postinfection; in contrast, BPY445 remained unaffected by

BV2 cells, irrespective of the time points tested (Fig. 1).

The AFR1-phenotype does not alter C.neoformans susceptibility to phagocytosis bymicroglia

With the purpose of detailing the molecular events involved

in C. neoformans–microglia interaction, fluorescence studies

were performed. In particular, before infection, yeast cells

were labelled with Oregon green 488 and subsequently

incubated with BV2 cells for different times (0.5, 1.5 and 3 h;

E : T = 1 : 5; E : T, effector : target ratio). Then, the cells were

incubated with Uvitex 2B for 15 min, washed and fixed. A

representative experiment is depicted in Fig. 2 (upper part).

Fig. 2a shows the infected microglial cells as observed by DIC;

in Fig. 2b, the total yeast cells are detectable by Oregon green;

Fig. 2c shows the extracellularly localized yeast cells that,

being Uvitex accessible, were blue stained; in Fig. 2d, the

arrowheads indicate the intracellular yeast cells (retaining

the green color), while the arrows show the extracellularly

localized fungi that appeared as blue-green by merging. By

this methodology, we performed a time-dependent phagocy-

tosis assay comparing the three strains. As shown in Fig. 2

(lower part), the percent of phagocytic cells increased with

time and remained consistently comparable against either

BPY22, BPY444 or BPY445 strains. The phagocytosis index

was also comparable among groups and showed the expected

time-dependent increase (data not shown).

The AFR1-overexpressing phenotype delays C.neoformans phagolysosomal maturation withinmicroglia

To investigate the events following C. neoformans internali-

zation by microglia, the fate of the yeast-containing phago-

somes was evaluated in terms of acidification and

appearance of endolysosomal markers. In particular,

BPY22, BPY444 or BPY445 yeast cells, preincubated with

Oregon green 488, were exposed to BV2 cells (E : T = 1 : 5)

and LysoTracker was then added to the samples. Subse-

quently, the cultures were stained with Uvitex 2B, washed,

fixed, permeabilized and observed by epifluorescence micro-

scopy. As shown in Fig. 3, the percent of C. neoformans-

containing acid phagolysosomes varied with the strains; in

particular, at 1.5 h postinfection, the percent was 33.5% and

27% in BV2 cells that had ingested BPY444 or BPY22,

–5

0

5

10

15

20

2 3 4

% A

ntic

rypt

ococ

cal a

ctiv

ity

BPY22 BPY444 BPY445

Time (h)

∗ ∗ ∗

Fig. 1. Susceptibility of the BPY22, BPY444 and BPY445 strains to

antifungal activity by microglia. Yeast cells were exposed to BV2 cells at

E : T = 10 : 1. After 2, 3 and 4 h, the percent of anticryptococcal activity

was determined as detailed in Materials and methods. The values shown

are the mean� SD of triplicates obtained in a representative experiment.

Data relative to BPY444 or BPY445 were compared with those obtained

with BPY22. The asterisks indicate statistically significant differences

(Po 0.01).

FEMS Yeast Res 9 (2009) 301–310Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

304 C.F. Orsi et al.

respectively; in contrast, a significantly lower percent of acid

vacuoles (12%) was found in BV2 cells exposed to BPY445.

After 3 h, the percent of acid phagolysosomes reached

similar levels among the three strains.

To further dissect the phenomenon, in parallel groups,

BV2 cells infected with BPY444 or BPY445 were investigated

for the appearance of endolysosomal markers (Rab5, Rab7,

Rab8 and LAMP2). Figure 4 shows fields representative of

the various conditions. In particular, BV2 cells and the yeast

cells were observed by DIC (a and f); the total yeast cells

were detectable by Oregon green staining (b and g); the

extracellularly localized yeast cells were evidenced by Uvitex

2B (c and h); the anti-LAMP2 antibody staining identified a

positive intracellular compartment, corresponding to a

vacuole containing the internalized BPY444 yeast cell (d),

while no anti-LAMP2 antibody staining was evident in the

BPY445-infected cells (i). These differences were confirmed

by the merged images (e and l). Unrelated control antibodies

did not label any phagosome-like structure (data not

shown). A quantitative analysis of such data was performed

and the results were expressed as percent of immunostained

C. neoformans containing-vesicles with respect to the total

phagocytosis events (Fig. 5). When directly compared,

BPY444 and BPY445 showed a different degree of colocali-

zation with most of the antibody-stained compartments. In

particular, Rab5-, Rab7- and even more LAMP2-positive

phagosomes were significantly less present in BPY445- than

0

2

4

6

8

10

12

14

16

18

% P

hago

cytic

cel

ls

BPY22

BPY 444

BPY 445

DIC

(a) (b) (c) (d)

1.5 3Time (h)

MergeUvitex 2BOregon green 488

Fig. 2. Susceptibility of the BPY22, BPY444 and BPY445 strains to phagocytosis by microglia. Upper: epifluorescence microscopy on microglia infected

with BPY444 strain. Oregon green 488 prelabelled yeast cells were exposed to BV2 cells (E : T = 1 : 5) for 3 h. Then, yeast cells were counterstained with

Uvitex 2B. Samples were washed, fixed and analysed by epifluorescence microscopy. (a) The DIC image of infected cultures. (b) The Oregon green image

showing total yeast cells. (c) The Uvitex 2B image revealing blue extracellularly localized yeast cells. (d) The merge image indicating extracellularly

localized yeast cells (arrows) and internalized ones (arrowheads). Lower: phagocytosis of the BPY22, BPY444 and BPY445 strains by microglia. Oregon

green 488 prelabelled yeast cells were exposed to BV2 cells (E : T = 1 : 5) for 1.5 and 3 h. At each end point, Uvitex 2B was added for 15 min; the cultures

were then washed, fixed and analysed by epifluorescence microscopy. The percent of phagocytic cells was calculated as detailed in Materials and

methods. The values shown are the mean� SD of triplicate readings obtained in a representative experiment.

0

5

10

15

20

25

30

35

% A

cid

phag

olys

osom

es

BPY 22 BPY444 BPY445

1.5 3Time (h)

∗

Fig. 3. Acidification of phagolysosomes containing BPY22, BPY444 and

BPY445 yeast cells. Oregon green 488 prelabelled yeast cells were

exposed to BV2 cells (E : T = 1 : 5); then LysoTraker dye was added. After

counterstaining with Uvitex 2B, samples were fixed and then visualized

by epifluorescence microscopy. The results, expressed as percent of acid

phagolysosomes, were calculated by evaluating the number of red-

stained vacuoles among 200 yeast-containing vacuoles. The values

shown are the mean� SD of triplicates obtained in a representative

experiment. Results of BPY444 or BPY445 were compared with those

obtained with BPY22. The asterisk indicates statistically significant

differences (Po 0.05).

FEMS Yeast Res 9 (2009) 301–310 Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

305Phagosomal maturation altered by the C. neoformans AFR1 gene

in BPY444-infected microglial cells (18%, 17% and 13% vs.

42%, 35% and 53%). In contrast, no differences were

observed in terms of staining with the anti-Rab9 antibody.

The AFR1 phenotype of C. neoformans does notinfluence the response of microglia to infection

To investigate the impact of C. neoformans AFR1 phenotype

on the response of microglia to infection, BV2 cells were

exposed or not to each of the three yeast strains and then

assessed for levels of NFkB activation and cytokine response.

In particular, BV2 cells were infected for 30 min with BPY22,

BPY444 or BPY445 strain (E : T = 1 : 10 or 1 : 1) or exposed

to lipopolysaccharide (1mg mL�1). As shown by the result of

a representative experiment (Fig. 6), we found no induction

of NFkB in BV2 cells when exposed to BPY22, BPY444 or

BPY445 yeast cells; comparably low levels were observed in

cell extracts and in nuclear extracts. In contrast, as expected

DIC

Oregongreen 488

Uvitex 2B

LAMP2

Merge

BPY444 BPY445

(a)

(b) (g)

(h)(c)

(d) (i)

(e)

(f)

(j)

Fig. 4. Colocalization of BPY444 (a–e) or

BPY445 (f–l) with LAMP2. Oregon green 488

prelabelled yeast cells were exposed to BV2 cells

at E : T = 1 : 5. Three hours later, samples were

counterstained with Uvitex 2B; then, the

cultures were washed, fixed, permeabilized and

processed for immunostaining, as detailed in

Materials and methods. Samples were visualized

by epifluorescence microscopy. (a) and (f) DIC

images, (b) and (g) the Oregon green image

showing total yeast cells, (c) and (h) the Uvitex 2B

image showing the blue-stained extracellularly

localized yeast cells, (d) and (i) LAMP2 staining,

(e) and (j) the merge image showing the LAMP2-

positive compartment that was detected only in

BV2 cells infected with BPY444.

FEMS Yeast Res 9 (2009) 301–310Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

306 C.F. Orsi et al.

(Blasi et al., 2007), the NFkB levels significantly augmented

in the lipopolysaccharide-treated cells. Furthermore, the

production of MIP-1a and TNFa was measured in BV2 cells

infected with BPY22, BPY444 or BPY445 strains. As positive

control, cells were exposed to lipopolysaccharides. As de-

picted in Table 1, we found that microglia constitutively

produced high levels of MIP-1a; such levels were further

enhanced by lipopolysaccharides. In contrast, BPY22,

BPY444 or BPY445 caused a significant and comparable

inhibition of MIP-1a production, at both 6 and 24 h

incubation time. In parallel, no appreciable levels of TNFa

were observed either after 6 or 24 h incubation time (data

not shown). Finally, the expression of different cell surface

receptors was evaluated in BV2 cells upon infection with

BPY22, BPY444 or BPY445 strains. We found that microglial

cells constitutively expressed all the markers, namely CD54,

CD16/CD32, CD71, CD86, TLR4, F4/80, CD4 and CD11b/

18, and no significant modulation occurred following infec-

tion with either one of the strains employed in the present

study (data not shown).

Discussion

Macrophages play a key role in host defence against virtually

all pathogenic microorganisms. In particular, microglia, the

resident brain macrophages, directly act as a local defence

barrier against the neurotropic yeast C. neoformans (Blasi

et al., 1990, 1992, 1995a, 2004; Mazzolla et al., 1997; Barluzzi

et al., 2000). Phagocytosis and killing occur to a different

extent, depending on the activation state of the effector cell

and on the opsonization of the fungal target or its exposure

to protease inhibitors. Furthermore, we recently demon-

strated that the AFR1 gene of C. neoformans, initially

identified as responsible for fluconazole resistance (Poster-

aro et al., 2003), is critical not only in determining failure of

therapy but also in enhancing the virulence of the yeast

in vivo, that paralleled with increased fungal resistance to

bone marrow-derived macrophages in vitro (Sanguinetti

et al., 2006). Here, we provide evidence that C. neoformans

interaction with microglia strictly depends on the AFR1

phenotype. When exposed to such effector cells, in fact,

BPY22, BPY444 or BPY445 are ingested to a similar extent,

irrespective of the levels of AFR1 expression. Interestingly,

only the wild-type BPY22 and the knockout BPY444 strains

are significantly growth inhibited, while the BPY445 AFR1-

overexpressing strain remains unaffected.

0

10

20

30

40

50

60

% C

oloc

aliz

atio

n

BPY 444 BPY 445

∗ ∗∗

Rab5 Rab7 Rab9 LAMP2

Staining with antibodies against:

Fig. 5. Percent of colocalization of BPY444 and BPY445 yeast cells with

Rab5, Rab7, Rab9 and LAMP2 markers. Oregon green 488 prelabelled

yeast cells were exposed to BV2 cells (E : T = 1 : 5). Three hours later,

samples were counterstained with Uvitex 2B for 15 min; then, the

cultures were washed, fixed, permeabilized and processed for immuno-

staining, as detailed in Materials and methods. Samples were visualized

by epifluorescence microscopy. The results were calculated by evaluating

the number of antibody-stained vacuoles among 200 yeast-containing

vacuoles. The values shown are the mean� SD of triplicate readings

obtained in a representative experiment. Results obtained with BPY444

and BPY445 were compared. The asterisks indicate statistically signifi-

cant differences (Po 0.01).

0

0.3

0.6

0.9

1.2

1.5

1.8

Med LPS BPY22 BPY444 BPY445 BPY22 BPY444 BPY445

NF

kB a

ctiv

atio

n (O

D45

0 nm

)

Cell extractNuclear extract

E : T=1 : 10 E : T=1 : 1

Fig. 6. Levels of NFkB activation in microglia

infected with the BPY22, BPY444 and BPY445

strains. BV2 and yeast cells (E : T = 1 : 10 or 1 : 1)

were cocultured for 30 min. Then, cell and

nuclear extracts were prepared and processed

for analysis of NFkB activation, as detailed in

Materials and methods. Results, reported as

OD450 nm, are from a representative experiment

out of three performed.

FEMS Yeast Res 9 (2009) 301–310 Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

307Phagosomal maturation altered by the C. neoformans AFR1 gene

These findings closely recall those previously reported

(Sanguinetti et al., 2006) showing that BPY445 displays

enhanced intracellular survival when compared with strains

BPY22 and BPY444 in bone marrow-derived macrophages;

yet, in those cells such a phenomenon is evident at 28 h,

while in microglia 2–4 h are sufficient to discriminate

between resistant and susceptible C. neoformans strains. At

the moment, it remains to be elucidated whether the

anatomical origin and/or the activation state of the two

effector populations may account for the diverse timing

observed. In any case, taken together, our present and

previous findings provide the first evidence on the relevance

of AFR1 phenotype in the intracellular fate of C. neoformans.

In this respect, the fact that two other independent transfor-

mants, having differently located the overexpressing con-

struct, exhibit behavior similar to BPY445 (Sanguinetti

et al., 2006; data not shown) argues against the relevance of

the insertion site in the phenomenon observed.

Because of the marked neurotropism of C. neoformans

and the clinical relevance of cryptococcal meningoencepha-

litis (Casadevall & Perfect, 1998), we further focused our

studies on the outcome of microglia–C. neoformans infec-

tion. As shown here, the early events occurring in such

yeast-to-effector cell interplay are not critical, because

phagocytosis as well as binding (data not shown) are

comparable with respect to each of the three strains. In

contrast, the results by epifluorescence microscopy demon-

strate that phagocytic vacuole acidification is significantly

reduced in BV2 cells engulfed with BPY445 in contrast to

BPY22- or BPY444-infected cells. In line with these findings,

the pathway of phagolysosome maturation happens to

depend on AFR1 phenotype, as demonstrated by the fact

that the early endosome marker Rab5 is detectable in

4 40% but in o 20% of the microglia infected with

BPY444 and BPY445, respectively. A similar trend is also

observed with the late marker Rab7, while no differences

occur when assessing another late maker, Rab9. The im-

paired maturation of the BPY445-containing phagolyso-

somes has been further confirmed by staining with the

anti-LAMP2 antibody, known to identify a very late marker

of phagolysosome development (Eskelinen, 2006; Huynh

et al., 2007). Our data show that LAMP2 is detected in

4 50% of the BPY444-containing phagolysosomes while

o 15% positivity is observed with BPY445. Besides provid-

ing a direct explanation for the lack of growth inhibition

observed against BPY445, these findings imply that the

differences in handling the three strains by microglia strictly

depend on the levels of maturation and acidification of the

phagocytic vacuole. Because the protein encoded by the

AFR1 gene is an efflux pump, it is reasonable to speculate

that such a phenotypical trait of C. neoformans, being

associated with reduced acidification of the phagocytosis

vacuole, is responsible for the enhanced intracellular survi-

val of the AFR1-overexpressing strain. Thus, a novel me-

chanism may be added to the ones already established by

which C. neoformans may alter phagosomal homoeostasis,

such as altered permeabilization, intraphagosomal growth

and eventually extrusion (Chaka et al., 1995; Levitz et al.,

1999; Tucker & Casadevall, 2002; Alvarez & Casadevall,

2006, 2007). On the other hand, we tend to exclude any

involvement of other cryptococcal virulence determinants in

the phenomenon described above, because no differences

have been observed between the AFR1-overexpressing

BPY445 and the parental BPY22 in terms of polysaccharide

capsule formation or other well-recognized parameters of

virulence (Sanguinetti et al., 2006).

In the present report, the consequences of C. neoformans–

microglia interaction have also been assessed in terms of

molecular and functional changes occurring in immune

cells, namely NFkB activation, cytokine secretion and

expression of surface markers. NFkB is known to be a central

mediator of gene transcription for a variety of cellular

responses (Li & Verma, 2002); in particular, it represents a

pivotal signal driving microglia response against infectious

agents (Neglia et al., 2006; Blasi et al., 2007). Unexpectedly,

the present results indicate that BPY22, BPY444 or BPY445

infection does not allow NFkB activation in microglial cells.

These findings are in line with (1) no changes in the

expression of surface markers by the microglial infected cells

and (2) no induction of secretory response, in terms of

TNFa production. Furthermore, the constitutively high

levels of MIP-1a production, observed in uninfected control

cells, are significantly impaired by C. neoformans infection;

the extent of the phenomenon is similar when comparing

BPY22, BPY444 or BPY445. Such inhibitory effects are in

line with the previous studies investigating the influence of

cryptococcal capsule in vitro (Barluzzi et al., 1998) or the

Table 1. Levels of MIP-1a in BV2 cells infected with BPY22, BPY 444 or

BPY445 strains

Treatment

Levels of MIP-1a

6 h 24 h

Medium 2139.0� 80.2 848.0� 80.0

Lipopolysaccharide 6579.0� 110.5� 5960.0� 118.7�

BPY22 958.0� 34.8� 327.0� 56.5�

BPY444 918.0� 28.9� 367.0� 70.0�

BPY445 960.0� 65.0� 558.0� 81.0��

BV2 cells were infected with BPY22, BPY444 or BPY445 yeast cells for 6

or 24 h. In parallel, lipopolysaccharide treatment (1 mg mL�1) was in-

cluded as positive control. Then, the supernatants were harvested and

MIP-1a levels were evaluated, as detailed in Materials and methods. The

values, expressed as picograms per millilitre, are the mean� SD of

triplicate determinations obtained in a representative experiment out of

four performed.�Po 0.001;��P = 0.02 untreated cells vs. treated cells.

FEMS Yeast Res 9 (2009) 301–310Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

308 C.F. Orsi et al.

role of melanin production by an in vivo model of menin-

goencephalitis (Huffnagle et al., 1995). As TNFa and MIP-

1a are crucial regulatory elements in initiating host immune

reaction (Biron et al., 1998; Maurer & von Stebut, 2004), the

present in vitro data suggest that MIP-1a production is

downregulated while TNFa is not even involved in the early

microglia–C. neoformans interplay. In any case, C. neofor-

mans immunosuppressive effects do not depend on the

AFR1 phenotype.

Although aware of the intrinsic limitations of an in vitro

model, here we provide new insights on the role of the AFR1

gene in the resistance of C. neoformans to microglia-

mediated antifungal activity, especially highlighting the

potential innate function of the AFR1 protein that has been

only lately co-opted for antifungal resistance.

Acknowledgements

We wish to acknowledge Mr A. Martino for excellent

informatic support and Prof. A. Todaro for sharing experi-

ence and providing access to the epifluorescence Eclipse 90i

Nikon microscope (recently founded by CaRiMo). Very

special thanks are also forwarded to Prof. M. Sanguinetti

(Universita Cattolica S. Cuore, Roma) for helpful sugges-

tions and a critical review of the manuscript. This work was

partially supported by the Italian grant MIUR-Programmi

di Ricerca cofinanziati 2005, no. 2005068754.

References

Alvarez M & Casadevall A (2006) Phagosome extrusion and host-

cell survival after Cryptococcus neoformans phagocytosis by

macrophages. Curr Biol 16: 2161–2165.

Alvarez M & Casadevall A (2007) Cell-to-cell spread and massive

vacuole formation after Cryptococcus neoformans infection of

murine macrophages. BMC Immunol 8: 16.

Barbero P, Bittova L & Pfeffer SR (2002) Visualization of Rab9-

mediated vesicle transport from endosomes to the trans-Golgi

in living cells. J Cell Biol 156: 511–518.

Barluzzi R, Brozzetti A, Delfino D, Bistoni F & Blasi E (1998) Role

of the capsule in microglial cell-Cryptococcus neoformans

interaction: impairment of antifungal activity but not of

secretory functions. Med Mycol 36: 189–197.

Barluzzi R, Brozzetti A, Mariucci G, Tantucci M, Neglia RG,

Bistoni F & Blasi E (2000) Establishment of protective

immunity against cerebral cryptococcosis by means of an

avirulent, non melanogenic Cryptococcus neoformans strain.

J Neuroimmunol 109: 75–86.

Barnewall RE, Rikihisa Y & Lee EH (1997) Ehrlichia chaffeensis

inclusions are early endosomes which selectively accumulate

transferrin receptor. Infect Immun 65: 1455–1461.

Bhattacharjee AK, Bennett JE & Glaudemans CP (1984) Capsular

polysaccharides of Cryptococcus neoformans. Rev Infect Dis 6:

619–624.

Binker MG, Cosen-Binker LI, Terebiznik MR et al. (2007)

Arrested maturation of Neisseria-containing phagosomes in

the absence of the lysosome-associated membrane proteins,

LAMP-1 and LAMP-2. Cell Microbiol 9: 2153–2166.

Biron CA, Cousens LP, Ruzek MC, Su HC & Salazar-Mather TP

(1998) Early cytokine responses to viral infections and their

roles in shaping endogenous cellular immunity. Adv Exp Med

Biol 452: 143–149.

Blasi E, Barluzzi R, Bocchini V, Mazzolla R & Bistoni F (1990)

Immortalization of murine microglial cells by a v-raf/v-myc

carrying retrovirus. J Neuroimmunol 27: 229–237.

Blasi E, Barluzzi R, Mazzolla R, Mosci P & Bistoni F (1992)

Experimental model of intracerebral infection with

Cryptococcus neoformans: roles of phagocytes and

opsonization. Infect Immun 60: 3682–3688.

Blasi E, Barluzzi R, Mazzolla R, Pitzurra L, Puliti M, Saleppico S &

Bistoni F (1995a) Biomolecular events involved in

anticryptococcal resistance in the brain. Infect Immun 63:

1218–1222.

Blasi E, Barluzzi R, Mazzolla R, Tancini B, Saleppico S, Puliti M,

Pitzurra L & Bistoni F (1995b) Role of nitric oxide and

melanogenesis in the accomplishment of anticryptococcal

activity by the BV-2 microglial cell line. J Neuroimmunol 58:

111–116.

Blasi E, Colombari B, Orsi CF, Pinti M, Troiano L, Cossarizza A,

Esposito R, Peppoloni S, Mussini C & Neglia R (2004) The

human immunodeficiency virus (HIV) protease inhibitor

indinavir directly affects the opportunistic fungal pathogen

Cryptococcus neoformans. FEMS Immunol Med Mic 42:

187–195.

Blasi E, Ardizzoni A, Colombari B, Neglia R, Baschieri C,

Peppoloni S & Cinco M (2007) NF-kB activation and p38

phosphorylation in microglial cells infected with Leptospira or

exposed to partially purified leptospiral lipoproteins. Microb

Pathogenesis 42: 80–87.

Brueske CH (1986) Proteolytic activity of a clinical isolate of

Cryptococcus neoformans. J Clin Microbiol 23: 631–633.

Casadevall A & Perfect J (1998) Cryptococcus neoformans. ASM

Press, Washington, DC, 541pp (hardcover).

Chaka W, Scharringa J, Verheul AF, Verhoef J, Van Strijp AG &

Hoepelman IM (1995) Quantitative analysis of phagocytosis

and killing of Cryptococcus neoformans by human peripheral

blood mononuclear cells by flow cytometry. Clin Diagn Lab

Immun 2: 753–759.

Chen SC, Muller M, Zhou JZ, Wright LC & Sorrell TC (1997a)

Phospholipase activity in Cryptococcus neoformans: a new

virulence factor? J Infect Dis 175: 414–420.

Chen SC, Wright LC, Santangelo RT, Muller M, Moran VR,

Kuchel PW & Sorrell TC (1997b) Identification of extracellular

phospholipase B, lysophospholipase, and acyltransferase

produced by Cryptococcus neoformans. Infect Immun 65:

405–411.

Cherniak R & Sundstrom JB (1994) Polysaccharide antigens of

the capsule of Cryptococcus neoformans. Infect Immun 62:

1507–1512.

FEMS Yeast Res 9 (2009) 301–310 Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

309Phagosomal maturation altered by the C. neoformans AFR1 gene

Eskelinen EL (2006) Roles of LAMP-1 and LAMP-2 in lysosome

biogenesis and autophagy. Mol Aspects Med 27: 495–502.

Huffnagle GB, Chen GH, Curtis JL, McDonald RA, Strieter RM &

Toews GB (1995) Down-regulation of the afferent phase of T

cell-mediated pulmonary inflammation and immunity by a

high melanin-producing strain of Cryptococcus neoformans. J

Immunol 155: 3507–3516.

Huynh KK, Eskelinen EL, Scott CC, Malevanets A, Saftig P &

Grinstein S (2007) LAMP proteins are required for fusion of

lysosomes with phagosomes. EMBO J 26: 313–324.

Jack CS, Arbour N, Manusow J, Montgrain V, Blain M, McCrea E,

Shapiro A & Antel JP (2005) TLR signaling tailors innate

immune responses in human microglia and astrocytes.

J Immunol 175: 4320–4330.

Jacobson ES, Jenkins ND & Todd JM (1994) Relationship

between superoxide dismutase and melanin in a pathogenic

fungus. Infect Immun 62: 4085–4086.

Kreutzberg GW (1996) Microglia: a sensor for pathological

events in the CNS. Trends Neurosci 19: 312–318.

Kumnok J, Ulrich R, Wewetzer K, Rohn K, Hansmann F,

Baumgartner W & Alldinger S (2008) Differential

transcription of matrix-metalloproteinase genes in primary

mouse astrocytes and microglia infected with Theiler’s murine

encephalomyelitis virus. J Neurovirol 14: 205–217.

Lee SC, Kress Y, Zhao ML, Dickson DW & Casadevall A (1995)

Cryptococcus neoformans survive and replicate in human

microglia. Lab Invest 73: 871–879.

Levitz SM, DiBenedetto DJ & Diamond RD (1987) A rapid

fluorescent assay to distinguish attached from phagocytized

yeast particles. J Immunol Methods 101: 37–42.

Levitz SM, Nong SH, Seetoo KF, Harrison TS, Speizer RA &

Simons ER (1999) Cryptococcus neoformans resides in an acidic

phagolysosome of human macrophages. Infect Immun 67:

885–890.

Li Q & Verma IM (2002) NF-kappaB regulation in the immune

system. Nat Rev Immunol 2: 725–734.

Lipovsky MM, Gekker G, Anderson WR, Molitor TW, Peterson

PK & Hoepelman AI (1997) Phagocytosis of nonopsonized

Cryptococcus neoformans by swine microglia involves CD14

receptors. Clin Immunol Immunop 84: 208–211.

Ma H, Croudace JE, Lammas DA & May RC (2006) Expulsion of

live pathogenic yeast by macrophages. Curr Biol 16:

2156–2160.

Mansour MK & Levitz SM (2002) Interactions of fungi with

phagocytes. Curr Opin Microbiol 5: 359–365.

Maurer M & von Stebut E (2004) Macrophage inflammatory

protein-1. Int J Biochem Cell Biol 36: 1882–1886.

Mazzolla R, Barluzzi R, Brozzetti A, Boelaert JR, Luna T,

Saleppico S, Bistoni F & Blasi E (1997) Enhanced resistance to

Cryptococcus neoformans infection induced by chloroquine in a

murine model of meningoencephalitis. Antimicrob Agents Ch

41: 802–807.

Meresse S, Steele-Mortimer O, Finlay BB & Gorvel JP (1999) The

Rab7 GTPase controls the maturation of Salmonella

typhimurium-containing vacuoles in HeLa cells. EMBO J 18:

4394–4403.

Neglia R, Colombari B, Peppoloni S, Orsi C, Tavanti A, Senesi S &

Blasi E (2006) Adaptive response of microglial cells to in vitro

infection by Candida albicans isolates with different genomic

backgrounds. Microb Pathog 41: 251–256.

Nimmerjahn A, Kirchhoff F & Helmchen F (2005) Resting

microglial cells are highly dynamic surveillants of brain

parenchyma in vivo. Science 308: 1314–1318.

Posteraro B, Sanguinetti M, Sanglard D, La Sorda M, Boccia S,

Romano L, Morace G & Fadda G (2003) Identification and

characterization of a Cryptococcus neoformans ATP binding

cassette (ABC) transporter-encoding gene, CnAFR1, involved

in the resistance to fluconazole. Mol Microbiol 47: 357–371.

Rink J, Ghigo E, Kalaidzidis Y & Zerial M (2005) Rab conversion

as a mechanism of progression from early to late endosomes.

Cell 122: 735–749.

Saleppico S, Boelaert JR, Omodeo Sale F, Mazzolla R, Morucci P,

Bistoni F & Blasi E (1999) Differential effects of iron load on

basal and interferon-gamma plus lipopolysaccharide enhance

anticryptococcal activity by the murine microglial cell line BV-

2. J Neuroimmunol 93: 102–107.

Sanguinetti M, Posteraro B, La Sorda M, Torelli R, Fiori B,

Santangelo R, Delogu G & Fadda G (2006) Role of AFR1, an

ABC transporter-encoding gene, in the in vivo response to

fluconazole and virulence of Cryptococcus neoformans. Infect

Immun 74: 1352–1359.

Shaw CE & Kapica L (1972) Production of diagnostic pigment by

phenoloxidase activity of Cryptococcus neoformans. Appl

Microbiol 24: 824–830.

Tucker SC & Casadevall A (2002) Replication of Cryptococcus

neoformans in macrophages is accompanied by phagosomal

permeabilization and accumulation of vesicles containing

polysaccharide in the cytoplasm. P Natl Acad Sci USA 99:

3165–3170.

Via LE, Fratti RA, McFalone M, Pagan-Ramos E, Deretic D &

Deretic V (1998) Effects of cytokines on mycobacterial

phagosome maturation. J Cell Sci 111: 897–905.

Wong B, Perfect JR, Beggs S & Wright KA (1990) Production of

the hexitol D-mannitol by Cryptococcus neoformans in vitro and

in rabbits with experimental meningitis. Infect Immun 58:

1664–1670.

Wozniak KL, Vyas JM & Levitz SM (2006) In vivo role of dendritic

cells in a murine model of pulmonary cryptococcosis. Infect

Immun 74: 3817–3824.

Zerial M & McBride H (2001) Rab proteins as membrane

organizers. Nat Rev Mol Cell Biol 2: 107–117.

Zhou Q, Gault RA, Kozel TR & Murphy WJ (2007) Protection

from direct cerebral cryptococcus infection by interferon-

gamma-dependent activation of microglial cells. J Immunol

178: 5753–5761.

FEMS Yeast Res 9 (2009) 301–310Journal compilation c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original Italian government works

310 C.F. Orsi et al.