The fatal fungal outbreak on Vancouver Island is
characterized by enhanced intracellular parasitism
driven by mitochondrial regulation
Hansong Maa, Ferry Hagenb,c, Dov J. Stekela, Simon A. Johnstona, Edward Sionovd, Rama Falkd, Itzhack Polacheckd,
Teun Boekhoutb,c, and Robin C. Maya,1
aSchool of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom;bCBS Fungal Biodiversity Centre, Royal Netherlands
Academy of Arts and Sciences, Uppsalalaan 8, NL-3584CT, Utrecht, The Netherlands;cDivision of Acute Medicine and Infectious Diseases, University Medical
Center Utrecht, NL-3508GA, Utrecht, The Netherlands; anddThe Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University
Medical Center, Ein Kerem, Jerusalem 91120, Israel
Edited by Joan Wennstrom Bennett, Rutgers, The State University of New Jersey, New Brunswick, NJ, and approved June 19, 2009 (received for review
March 19, 2009)
In 1999, the population of Vancouver Island, Canada, began to
experience an outbreak of a fatal fungal disease caused by a highly
virulent lineage of Cryptococcus gattii. This organism has recently
spread to the Canadian mainland and Pacific Northwest, but the
molecular cause of the outbreak remains unknown. Here we show
that the Vancouver Island outbreak (VIO) isolates have dramatically
increased their ability to replicate within macrophages of the mam-
malian immune system in comparison with other C. gattii strains. We
further demonstrate that such enhanced intracellular parasitism is
directly linked to virulence in a murine model of cryptococcosis,
suggesting that this phenotype may be the cause of the outbreak.
Finally, microarray studies on 24 C. gattii strains reveals that the
hypervirulence of the VIO isolates is characterized by the up-regula-
tion of a large group of genes, many of which are encoded by
mitochondrial genome or associated with mitochondrial activities.
This expression profile correlates with an unusual mitochondrial
thus demonstrate that the intracellular parasitism of macrophages is
a key driver of a human disease outbreak, a finding that has signif-
icant implications for a wide range of other human pathogens.
cryptococcus ? macrophage ? Vancouver Island outbreak ? virulence
latency. Intracellular pathogens are able to spread within the body,
triggering little or no effective immune response from the host.
Moreover, many pathogens use the host cell as a nutrient source to
support rapid replication, thus acting as genuine intracellular
parasites (1, 2). The human fungal pathogen Cryptococcus is an
intracellular parasite of macrophages (3, 4) and is believed to
exploit these host cells to traffic from the lung, the primary site of
infection, into the central nervous system (5, 6).
Therearetwopathogenicspecieswithinthe Cryptococcus genus:
C. neoformans and C. gattii. They are the causative agents of
cryptococcosis, a fatal infection of the central nervous system in
humans. Globally, the vast majority of cryptococcosis cases occur
in immunocompromised patients and are caused by C. neoformans.
In contrast, C. gattii typically infects immunocompetent individuals
but causes only 1% of cryptococcosis cases worldwide and has thus
of C. gattii infection in immunocompetent individuals on Vancou-
Columbia and the Pacific Northwest (8–10) have dramatically
increased global concern about this pathogenic species. Previous
amplified fragment length polymorphism (AFLP) and multilocus
sequence typing (MLST) studies have demonstrated that the Van-
couver Island outbreak (VIO) is mainly caused by a single, hyper-
virulent genotype of C. gattii (AFLP6A/molecular type VGIIa) (8).
Intriguingly, this genotype is not restricted to Vancouver Island but
or many pathogens, intracellular survival is critical to their
pathogenicity, because it provides a basis for dissemination and
is also shared by, for instance, the CBS6956 strain (also known as
NIH444 or ATCC32609, isolated from a patient in Seattle in 1971)
and CBS7750 (isolated from a Eucalyptus tree in San Francisco in
within the VIO lineage, potentially as a result of an unusual
same-sex mating event (12). However, the underlying molecular
cause for the hypervirulence of this lineage remains unknown.
Hypervirulence of VIO Strains Is Not Directly Linked to Any Known
Virulence Factors. To study the cause of hypervirulence within the
VIO isolates, we undertook a high-throughput analysis of well-
characterized virulence traits (capsule size, melanization, phospho-
lipase production, proteinase production, and other enzymatic
isolates (Table S1 and Table S2). However, there was no consistent
difference between VIO and non-VIO isolates belonging to this
genotype in any of the traits tested, suggesting that the virulence of
VIO strains does not simply result from over-expression of these
individual cryptococcal pathogenicity factors. Such a finding is
consistent with recent data suggesting that many cryptococcal
virulence genes/factors remain to be discovered (13).
VIO Strains Show Enhanced Intracellular Parasitic Capacity Compared
to Other C. gattii Strains. Given the importance of macrophage
parasitism in cryptococcal infection, we developed an in vitro
method to monitor intracellular yeast number for 64 h following
phagocytosis by murine macrophage-like cells J774 (see experi-
mental procedures). We then used intracellular proliferation rate
(IPR, which was calculated by dividing the maximum intracellular
yeast number by the initial intracellular yeast number at T0) as a
parameter to quantify the relative level of intracellular parasitism
between strains. Based on this method, we calculated average IPR
for 55 strains, including 16 C. neoformans and 39 C. gattii isolates
higher intracellular proliferation rates compared to other C. gattii
strains, including AFLP6A strains isolated from other areas of the
world (Fig. 1A). Time-lapse microscopy of J774 macrophages
infected with each of the C. gattii strains confirmed the high IPR
values observed among VIO strains (Movies S1–S3). Importantly,
Author contributions: H.M., I.P., T.B., and R.C.M. designed research; H.M., F.H., S.A.J., E.S.,
R.F., and I.P. performed research; F.H., D.J.S., I.P., and T.B. contributed new reagents/
analytic tools; H.M., F.H., D.J.S., S.A.J., I.P., T.B., and R.C.M. analyzed data; and H.M. and
R.C.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
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no. 31 www.pnas.org?cgi?doi?10.1073?pnas.0902963106
the VIO strains also show enhanced intracellular proliferation in
human primary macrophages derived from peripheral blood (Fig.
1B and Table S4). This proliferative capacity appears to reflect
specific parasitism of the live macrophage, rather than simply
enhanced utilization of nutrient sources available in the host cell,
since the VIO strains do not show significantly higher growth rates
when grown in mammalian cell lysates (Table S5). Thus the VIO
is unique to this pathogen population.
Intracellular Proliferation Rate Predicts Virulence of Cryptococcal
Strains in a Murine Model of Cryptococcosis. Several early studies
have highlighted the hypothesis that macrophages may exacerbate
associated with amelioration of disease in three murine strains as
measured by lung fungal burden (14), and decreased the dissemi-
nation of a glucosylceramide-deficient mutant of C. neoformans in
immunodeficient mice (15). We therefore tested whether high
the VIO lineage. To do this, IPR values were compared with both
published and newly-generated mouse median survival times
(ST50). Remarkably, IPR and murine ST50are highly significantly
holds true for both C. neoformans and C. gattii and is independent
of the mouse model used. In other words, the ability to survive and
proliferate inside a host macrophage contributes significantly to
cryptococcal virulence in the murine model and presumably also in
infected humans, as suggested by the human primary macrophage
data. This previously undiscovered link between intracellular pro-
why ‘‘ancestral’’ AFLP6A strains share the VIO genotype and yet
do not lead to disease outbreaks. For example, CBS6956, which is
considered as the potential origin of the AFLP6A subtype (11),
shows only a moderate IPR value of 1.35 (Fig. 1A and Table S1),
indicating that it cannot exploit the intracellular macrophage niche
as successfully as the VIO isolates.
Identification of Genes Associated with Enhanced Intracellular Para-
sitism. Statistical analysis confirmed that the high IPR values
exhibited by the VIO isolates did not result from increased expres-
sion of any of the known virulence factors analyzed above (Table
S2B) nor was it due to better utilization of macrophage nutrients
(Table S5). Therefore to investigate the molecular basis of en-
hanced intracellular proliferation, and thus hypervirulence, in the
VIO isolates, we used custom-designed C. gattii whole-genome
tiling microarrays to conduct transcriptional profiling of 24 C. gattii
strains (21 AFLP6 and 3 AFLP4 isolates) recovered from within
J774 macrophages. These strains are genetically very similar but
strain were isolated from intracellular cryptococcal cells 24 h after
infection and competitively hybridized against a pooled sample
point, the intracellular number reaches the peak for the majority of
strains. Linear regression identified 1,367 target loci in the genome
within the C. gattii species (n ? 39). Black bars represent
VIO isolates, which proliferate far better than other C.
gattii strains (gray bars). The only exception was A1M-
R272, which belongs to the minor (AFLP6B) group of the
outbreak and has previously been shown to be less viru-
lent than other VIO strains (12). An asterisk (*) denotes
strains used for the microarray study. (B) IPR values of 8
(HPBMCs) correlate significantly with those observed in
J774 (P ? 0.00044, Spearman’s test, n ? 8). The IPR values
obtained with HPBMC are generally lower than those
seen in J774 macrophages, which may be a result of the
higher cryptococcal expulsion rates observed in primary
cells, as previously reported (36).
(A) Significant inter-strain variation in IPR occurs
Ma et al. PNAS ?
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with IPR values. Two hundred twenty-four of these have predicted
function annotations, most of which can be categorized into one of
five groups: carbohydrate metabolism, stress response, vesicle/
vacuole fusion and transport, protein degradation and synthesis,
and nucleotide metabolism (Table S3). Interestingly, although our
phenotypic analysis demonstrated that no single known virulence
factor was responsible for the hypervirulence of the VIO isolates,
genes on the mating type locus,) showed a significant expression
correlation with IPR, suggesting that they may synergistically
our uncharacterised candidate genes were also recently identified
by Liu et al. as influencing melanin and capsule production and,
thus, cryptococcal infectivity (e.g., COP9 signalosome complex and
ubiquitin carboxyl-terminal hydrolase) (13).
Due to poor annotation of the C. gattii genome, BLAST search-
ing did not identify the function of most of the candidate genes we
identified (Table S3). We therefore mapped the genomic distribu-
tion of all of the microarray hits by localizing them on the 28
supercontigs of A1M-R265 genome. The distribution was homog-
enous across all supercontigs, with the remarkable exception of
supercontig 25, corresponding to the mitochondrial genome
(mtDNA), which was 10-fold overrepresented (Fig. 3A, P ? 10?24,
?2test). Quantitative PCR demonstrated that the observed over-
in mtDNA copy number, because AFLP6 strains showing very
different IPR values nonetheless have similar mtDNA/genomic
DNA ratios (Table 1). In addition, mtDNA copy number does not
change significantly following intracellular replication, eliminating
this as a possible explanation for the overrepresentation of mito-
chondrial hits (Table 1). Furthermore, the expression of many
nuclear-encoded proteins that function in mitochondria is also
up-regulated in the VIO strains (e.g., respiratory genes and mito-
chondrial proteins, Table S3), which is particularly relevant given
published in ref. 12 (?) and newly generated (?)] and intracellular proliferation
sion, n ? 18). For the unpublished mouse survival assays, experiments were
conducted as described in the Materials and Methods section. Published mouse
survival data are taken from Fraser et al. (12). Note that strain WM276 has been
reasons that are not currently understood (12, 45). For strains that did not cause
mortality of 50% or more within the 45-day timeframe of the experiment, we
arbitrarily assigned an ST50of 55 days.
A significant correlation between mouse survival data [both previously
Large Ribosomal protein
Small Ribosomal protein
using the ?2test. Supercontig 25 is 10-fold over-represented. (B) The predicted genome structure of A1M-R265 supercontig 25. The genome is 34.7 kb in size and, as
with other cryptococcal mitochondria, encodes 17 genes important for mitochondrial function and protein synthesis. The ORFs were predicted using Open Reading
Frame Finder at NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) in combination with alignment to the H99 mitochondrial sequence (available at http://
www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db ? nucleotide&val ? AY101381). The supercontig represents the whole mitochondrial genome of A1M-R265. Sections
with light blue color are either introns or intergenic spaces, both of which are substantially expanded relative to C. neoformans H99.
www.pnas.org?cgi?doi?10.1073?pnas.0902963106Ma et al.
system infection (16, 17). Taken together, we propose that mito-
chondrial function is critical for the virulence of this lineage.
Mitochondrial Morphology Changes in VIO Strains Following Phago-
cytosis. Given the overrepresentation of mitochondrial genes in the
VIO strains, we analyzed the morphology of cryptococcal mito-
chondria both before and after phagocytosis. Surprisingly, we
observed a striking difference in mitochondrial morphology be-
tween the VIO and non-VIO strains following intracellular para-
sitism. During in vitro growth either at 25 °C with shaking in YPD
or 37 °C in DMEM ? 5% CO2without shaking, more than 95% of
cryptococcal cells have mitochondria with morphologies that we
termed either ‘‘diffuse’’ or ‘‘globular’’ (Fig. 4A), regardless of the
strains developed a tubular mitochondrial morphology (Fig. 4C)
that was rarely exhibited by non-VIO isolates (P ? 0.0001, ?2test
for VIO versus non-VIO strains). Remarkably, the percentage of
cryptococcal cells exhibiting tubular mitochondria shows a strong,
a relationship that raises the possibility of accurately predicting the
virulence of cryptococcal genotypes based on a simple, 1-step
observation of mitochondrial morphology.
Mitochondrial tubular morphology is generally thought to result
from mitochondrial fusion, a phenomenon that allows mitochon-
from the detrimental effect of mtDNA mutations by allowing
functional complementation of mtDNA gene products (19). More-
over, mitochondrial fusion has been found to protect cells from cell
death (20, 21). It therefore appears likely that the altered mito-
Table 1. Real-time PCR to quantify mtDNA copy number per cell
RT-cycle difference between mitochondrial and
Intracellular yeast cellsYeast cells grown in YPD at 25°C
9.75 ? 1.01
8.74 ? 0.38
9.44 ? 0.19
10.71 ? 0.63
9.77 ? 0.61
10.68 ? 1.04
10.14 ? 1.59
10.44 ? 0.53
Cryptococcal mtDNA copy number (ranging from 400–1,600 copies per cell)
does not vary among different AFLP6 strains (2 VIO isolates and 2 other AFLP6
strains) or change following intracellular replication within host macrophages.
Percentage of tubular
characteristic mitochondrial morphology after intracel-
lular growth. (A) Representative images of the 3 differ-
intracellular yeast cells with tubular mitochondria in 6
AFLP6 strains (3 VIO and 3 non-VIO isolates). For each
strain, a random selection of cryptococcal cells were
ogies. Mitochondria with a tubular morphology were
found only rarely in strains with low IPR values (non-VIO
strains) or in strains (both VIO and non-VIO) that had
been grown extracellularly. (B) The percentage of intra-
cellular yeast exhibiting a tubular mitochondrial mor-
0.00021, linear regression). (C) A Z-projection confocal
image showing the tubular mitochondrial morphology
of a VIO strain. C. gattii strain ENV152 (IPR ? 2.28),
isolated 24 h of growth within J774 macrophages and
labeled with MitoTracker.
Vancouver Island Outbreak isolates acquire a
Ma et al. PNAS ?
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chondrial gene expression and morphology seen in the VIO strains
is a protective response that facilitates rapid intracellular growth
and thus enhanced virulence.
Our study demonstrates a link between the intracellular para-
sitism of phagocytes and the virulence of a facultative intracel-
lular pathogen in a murine model of infection. Moreover, we
propose that a recent change in mitochondrial regulation within
the C. gattii lineage has led to an increased intracellular prolif-
erative capacity resulting in the hypervirulent phenotype that
underlies the VIO. This change leads to a quantitative linear
relationship between intracellular proliferation rate, mitochon-
drial gene expression, mitochondrial morphology, and virulence.
The mitochondrion, as an essential organelle, has been linked to
various cellular activities, such as intermediary metabolism and
respiration, cell signaling, iron metabolism, apoptosis, and aging.
Nevertheless, its role in modulating virulence of pathogens is
unclear. Indeed such a role has been reported only once before, in
Heterobasidion annosum, which, like Cryptococcus, is also a basid-
in emerging pathogens because their genomes are present at high
copy number and show a mutation rate much higher than that of
nuclear DNA (23–25). Within the Cryptococcus genus, mitochon-
drial genomes show conserved gene synteny but very different sizes
[e.g., 34.7 kb for C. gattii (Fig. 3B), 32 kb for C. neoformans var.
that they are under intense selection. This is further supported by
along with the fact that most of the mitochondrial genetic variation
between WM276 (an AFLP4/VGI strain with a low IPR value) and
A1M-R265 (a VIO strain with high IPR) lies in coding regions,
whilst the intergenic regions are highly conserved.
Mitochondria are dynamic organelles that frequently divide and
fuse with each other and it is thought that such behaviors are
coordinated with their metabolic function (19). In yeast and mam-
mals, several factors including Drp1/Dnm1 and Mfn/Fzo1 are
known to regulate mitochondrial morphology by controlling mem-
brane fission or fusion. Interestingly, we find that FZO1 is up-
regulated in the VIO strains (Table S3) and might therefore
contribute to the observed tubular morphology in VIO strains.
However, a recent study demonstrated that most fundamental
mitochondrial functions, including metabolism and oxidative phos-
phorylation, are also necessary for the maintenance of tubular
of up-regulation of many candidate genes listed in Table S3. Early
studies on Cryptococcus have demonstrated the importance of
mitochondria in responding to hypoxic conditions and oxidative
stress (30, 31). We therefore propose that, after being engulfed by
macrophages, the VIO strains are able to promote mitochondrial
fusion to form long tubular mitochondria to more efficiently repair
mtDNA damage caused by the oxidative species and hypoxic
conditions present within the macrophage phagosome.
Since mitochondria are essential for cryptococcal viability (32),
attempts to make petite (respiratory) mutants have not been suc-
with those from less virulent strains (33–35). Therefore, we cannot
distinguish whether it is the mitochondrion itself or the regulation
of mitochondrial activity via nuclear encoded proteins that is
important for the virulence of the VIO lineage. In either scenario,
however, mitochondrially-regulated intracellular replication capac-
ity may be a widespread phenomenon in other eukaryotic patho-
gens and hence an improved appreciation of this process is likely to
have significant implications for our understanding of disease
epidemics caused by a range of otherwise unrelated pathogens.
Materials and Methods
Yeast Strains and Growth Conditions. All cryptococcal strains used are listed in
(2% glucose, 1% peptone, and 1% yeast extract) with moderate shaking (240
rpm) at 25 °C. Antibiotics were added to the media at the following concentra-
PBS twice, and resuspended in PBS.
Intracellular Proliferation Measurement. Aproliferationassaywasdevelopedto
monitor intracellular proliferation rate of individual strains for a 64-h period
following phagocytosis. For this assay, J774 macrophage cells or human primary
blood-derived macrophage cells [HPBMCs, which were isolated and activated as
previously described (36)] were exposed to cryptococci opsonised with 18B7
remove as many extracellular yeast cells as possible and 1 mL fresh serum-free
dH2O was added into wells to lyse macrophage cells. After 30 min, the intracel-
well to collect the remaining yeast cells. The intracellular yeast was then mixed
with Trypan Blue at a 1:1 ratio and the live yeast cells were counted. For the
subsequent 5 time points T16, T24, T40, T48, and T64, both extracellular and intra-
cellular cryptococci were collected and independently counted by hemocytom-
eter in the same manner. For each strain tested, each time course was repeated
at least 3 times on different occasions and using different batches of macro-
phages. Intracellular proliferation rate was calculated by dividing the maximum
intracellular yeast number (which is T24for most of the strains) by the initial
intracellular yeast number at T0. We confirmed that Trypan Blue stains 100% of
sensitive in detecting the clustered yeast population or yeast cells undergoing
Mitochondrial Staining and Microscopy. Yeast cells, grown overnight in YPD
medium at 25 °C at 240 rpm, or grown at 37 °C in DMEM in a 5% CO2incubator
harvested, washed with PBS twice and resuspended in PBS containing the Mito-
Tracker Red CMXRos (Invitrogen) at a final concentration of 40 nM. Cells were
incubated for 30 min at 37 °C. After staining, cells were washed 3 times and
resuspended in PBS. For each condition, more than 60 yeast cells for each of the
tested strains were chosen randomly and analyzed. For quantifying different
mitochondrial morphologies, images were collected on a Nikon Eclipse E300
microscope using 60? oil immersion 1.40NA plan APO objective lens. Both
fluorescence images and brightfield images were collected simultaneously. Im-
ages were captured with identical settings on a Hammatsu Orca C4745–12NRB
with a 0.5? camera lens using Openlab (version 5.5.0; Improvision). All Images
were processed identically in Photoshop CS2 (Adobe) and mitochondrial mor-
phologies were analyzed and counted blind. For confocal microscopy, images
were collected on a Nikon Eclipse E600 confocal microscope with BioRad Radi-
lens. Images were processed using ZeissSharp2000 software (version 6.0).
tail veins of male albino BALB/c mice (weight, 20–23 g) by administration of a
total killing within 5 to 50 days. For each experiment 10 mice were used, main-
tained in separate cages. The number of surviving animals in each group was
used. All procedures, care and treatment of mice were in accordance with the
principles of humane treatment outlined by the Guide for the Care and Use of
Laboratory Animals of the Hebrew University, and were approved by the Com-
mittee for Ethical Conduct in the Care and Use of Laboratory Animals (approval
RNA Isolation from Intracellular Cryptococci 24 h After Infection. The phago-
cytosis assay was carried out as described earlier. After 24 h, extracellular cryp-
tococci were removed with several prewarmed PBS washes and macrophages
were lysed with 10 mL ice-cold H2O for 20 min before being scraped from T75
tissue flasks. The whole mixture was then centrifuged at 1,500 rpm at 4 °C for 5
www.pnas.org?cgi?doi?10.1073?pnas.0902963106Ma et al.
min and the resulting pellet was washed twice with ice-cold H2O. Subsequently,
5 min to remove macrophage RNA. At this SDS concentration, the macrophages
Yeast RNA was isolated using Microto-Midi Total RNA Purification System (In-
vitrogen) in accordance with the manufacturer’s instructions.
Microarray Experiment. An Agilent printed whole genome tiling array with
242,003 probes generated against 2 genomes of C. gattii, A1M-R265 (Broad
(OGT). Average interprobe distance was 140 nucleotides, evenly distributed
repeat regions, etc.) were then filled using suboptimal probes (700 probes in
total) in the same manner. There are 20,212 probes (8.4%) that map onto both
experiment, RNA samples from each strain were labeled with Cy3, whilst the
control, consisting of a pooled sample containing equal quantities of RNA sam-
ples from all 24 strains (reference) was labeled with Cy5. See SI Materials and
Methods for more experimental details.
Microarray Data Analysis. Data were background subtracted using Bayesian
method, normalized using Loess normalization (38), and analyzed using the
statistical package R (http://www.r-project.org/) based on linear regression
Data were corrected for false-discovery rate (39) before candidates showing q
genomic DNA generated from newly isolated intracellular cryptococci to check
For the reaction, 4 strains (ENV152, A1M-R271, CBS7750, and CBS8684) were
of the candidates and primers are listed in the table below.
Candidate loci Primer 5?
Nuclear locus GGTCGAATTGTTCTCAGG
Mitochondriallocus TTCGTCTTGCTGGTCGACTT TGGGAGTTGTTGATCGTTG
Phenotypic Analysis. Proteinase and phospholipase activities were measured as
C. gattii strains were tested with API-ZYM as described in (44).
Unless otherwise stated, statistical tests were performed by linear regression
ACKNOWLEDGMENTS. We thank Arturo Casadevall (Albert Einstein College of
Harrison, Andrew Rogers and Volker Brenner at Oxford Gene Technology for
assistance with the microarray analysis and James Kronstad and the Broad Insti-
tute for access to the unpublished WM276 and A1M-R265 genome sequences.
This work was made possible with financial support from the Medical Research
(Heredity Fieldwork and Training Grants) and the Society of General Microbiol-
research visit to the CBS to conduct the phenotypic analysis reported here.
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