Impact of the Lectin Chaperone Calnexin on the Stress
Response, Virulence and Proteolytic Secretome of the
Fungal Pathogen Aspergillus fumigatus
Margaret V. Powers-Fletcher1, Kalyani Jambunathan2, Jordan L. Brewer1, Karthik Krishnan1, Xizhi Feng1,
Amit K. Galande2, David S. Askew1*
1Department of Pathology & Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 2SRI International,
Harrisonburg, Virginia, United States of America
Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system
that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER
homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin
has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under
conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and
complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but
had no impact on growth rates in either minimal or complex media at 37uC. However, the DclxA mutant was growth
impaired at temperatures above 42uC and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol.
In contrast to wild type A. fumigatus, DclxA hyphae were unable to grow when transferred to starvation medium. In
addition, depleting the medium of cations by chelation prevented DclxA from sustaining polarized hyphal growth, resulting
in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the DclxA mutant remained virulent
in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are
needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses
encountered in the host environment.
Citation: Powers-Fletcher MV, Jambunathan K, Brewer JL, Krishnan K, Feng X, et al. (2011) Impact of the Lectin Chaperone Calnexin on the Stress Response,
Virulence and Proteolytic Secretome of the Fungal Pathogen Aspergillus fumigatus. PLoS ONE 6(12): e28865. doi:10.1371/journal.pone.0028865
Editor: Martine Bassilana, Universite ´ de Nice-CNRS, France
Received August 11, 2011; Accepted November 16, 2011; Published December 7, 2011
Copyright: ? 2011 Powers-Fletcher et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grant R01AI072297 to DSA. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: David.Askew@uc.edu
Aspergillus fumigatus is the causative agent of a life-threatening
pulmonary infection that primarily affects the immunocompro-
mised patient population . Current treatment options rely on a
small armamentarium of antifungal drugs that are unable to
prevent the high mortality rates associated with this infection,
particularly in hematopoietic stem cell transplant recipients .
Exacerbating this problem are issues of drug toxicity and emerging
resistance , emphasizing the need for more information on
those aspects of fungal physiology that could be interrupted with
novel therapies to improve outcome in patients with aspergillosis.
Recent evidence has suggested that fungal pathways that
support homeostasis of the endoplasmic reticulum (ER) could
represent novel targets for antifungal therapy because of the
central role that they play in both virulence and antifungal drug
susceptibility. The ER is an interconnected network of endomem-
branes that promotes the accurate folding of proteins before
delivering them to the distal secretory pathway. Maintenance of
ER function is accomplished, in part, by a stress signaling pathway
known as the unfolded protein response (UPR). The UPR is
responsible for activating a program of gene expression to
strengthen ER folding capacity when secretion levels are high,
or when environmental conditions are not conducive to protein
folding . We have previously demonstrated that A. fumigatus
depends on the master transcriptional regulator of this pathway,
HacA, for the expression of full virulence [5,6]. This suggests that
the fungus is under ER stress in the mammalian host and needs
the UPR to sustain the infection by restoring homeostatic balance
to the secretory pathway. Similar findings were made in Alternaria
brassicicola, a necrotrophic plant pathogen that kills host cells
through the secretion of numerous enzymes and toxins. Deletion
of A. brassicicola HacA decreased the secretory capacity of the
fungus, resulting in impaired virulence and increased susceptibility
to plant antimicrobial metabolites . Notably, the rice blast
fungus Magnaporthe oryzae has been shown to rely on the ER
chaperone LHS1 for its virulence . Because LHS1 is only one
component of the entire UPR stress response [9,10], this suggests
that individual chaperones could mediate the effects of the UPR
Calnexin is an ER membrane-bound lectin chaperone that is
one of the major targets of the UPR during ER stress [11,12,13].
The protein is part of an ER quality control system known as the
calnexin cycle . In metazoans, two key chaperones participate
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in the calnexin cycle; calnexin itself, a type 1 transmembrane
protein, together with calreticulin, a soluble homolog of calnexin
. However, only calnexin has been identified in fungal species
[16,17]. Functional studies have revealed that calnexin promotes
folding by binding to the N-linked glycans that are added to
nascent polypeptides as they enter the ER, thereby preventing
aggregation. This glycoprotein-calnexin interaction undergoes
cycles of release and re-binding until the glycoprotein achieves
its native conformation, after which the protein is released for
secretion into the distal secretory pathway . In this study, we
examined the contribution of calnexin to stress responses that
would be encountered by A. fumigatus in its native environment as
well as the host. Although clxA was dispensable for most aspects of
A. fumigatus physiology, it was required under conditions of
thermal, ER and nutrient stress. The virulence of the DclxA mutant
was indistinguishable from that of wild type (wt) A. fumigatus
however, indicating that clxA-dependent functions are largely
dispensable for infection of the host.
Results and Discussion
Construction of a calnexin-deficient strain of A. fumigatus
AY560606) encodes a protein with the same two sets of repeated
peptide motifs that are characteristic of the calreticulin/calnexin
family, together with a predicted membrane-spanning domain
(Figure S1). The protein is most closely related to that of other
filamentous fungi, but among yeast species it is more closely
related to the Schizosaccharomyces pombe ortholog (46% identity) than
to the Saccharomyces cerevisiae ortholog (28% identity). A calnexin
deficient strain of A. fumigatus was constructed by replacing the
gene (clxA) with a hygromycin resistance cassette (Figure 1 and S2).
The DclxA mutant was viable and grew normally on either
minimal or rich medium (data not shown). This contrasts the
essentiality of calnexin in S. pombe , but is similar to the
viability of calnexin mutants reported in A. niger , Aspergillus
oryzae  and S. cerevisiae .
Loss of calnexin alters the proteolytic secretome of A.
Previousstudies in yeast have shown that loss of calnexin function
is associated with increased
[21,22,23,24]. In this study, we used a more comprehensive
approach to determine how loss of calnexin would affect the
proteolytic secretome ofA. fumigatus, using a combinatorial library of
internallyquenched fluorogenic peptide substrates.This library
is comprised of a panel of up to eight individual fluorogenic peptides
in each well of a microtiter plate. When the substrates are cleaved, a
fluorophore is liberated from a quenching moiety, resulting in a
fluorescence signal that is proportional to the extent of cleavage. A.
fumigatus culture supernatants were used to screen the library as
described in Materials and Methods, and heat maps were generated
from the resulting data. As shown in Figure 2, loss of clxA altered the
secreted proteolytic signature of the fungus, with a remarkably high
number of substrates showing increased cleavage by DclxA
supernatants relative to wt (indicated by the red squares). However,
this was not associated with any changes in the ability of the mutant
to grow on a complex protein source such as skim milk or fetal
bovine serum (data not shown). The precise mechanism by which
loss ofcalnexin increasesthe secretionof some proteinsinbothyeast
and A. fumigatus is not yet clear. However, since part of calnexin’s
role in protein quality control is to retain incompletely folded
proteins in the ER until they achieve the appropriate conformation
, the loss of this retention function may allow for more rapid
secretion of proteins that would ordinarily take longer to traffic
through the secretory pathway. It is possible that some of these
given the large number of functional proteases identified in DclxA
supernatants in this study, it appears that redundant mechanisms of
protein folding in the ER can adequately compensate for loss of
calnexin and ensure that secreted proteases achieve the appropriate
conformation for functionality.
Calnexin facilitates growth under conditions of thermal
In nature, A. fumigatus resides in composting material, an
environment that undergoes wide fluctuations in temperature
because of intense microbial activity . A. fumigatus has
responded to the thermal selection pressure in this environmental
niche by evolving mechanisms of thermotolerance that allow the
fungus to thrive at temperatures up 60u C, with an optimum
between 37uC and 42uC [27,28]. As shown in Figure 3, the DclxA
mutant grew normally at temperatures up to 37uC, but showed a
45% reduction in growth rate at 42uC indicating a role for
calnexin in the thermotolerance of this fungus. We have previously
shown that loss of UPR signaling by deletion of hacA creates a cell
wall defect that reduces thermotolerance by increasing hyphal
fragility at higher temperatures, resulting in tip lysis . The DclxA
mutant showed no evidence of hyphal tip fragility at temperatures
up to 50uC however (data not shown), indicating that A. fumigatus
can maintain cell wall integrity at high temperatures in the
absence of calnexin. Moreover, although calnexin has been
previously implicated in cell wall synthesis in S. cerevisiae , the
A. fumigatus DclxA mutant showed wt sensitivity to multiple cell wall
stressors, including calcofluor white, nikkomycin, Congo red or
caspofungin (Figure S3 and S4). While these data do not eliminate
the possibility that calnexin contributes to cell wall homeostasis in
Figure 1. Deletion of calnexin from A. fumigatus. The clxA gene
was deleted by replacing the coding region with the hygromycin
resistance cassette (HYG). Southern blot analysis of SacI-digested
genomic DNA using a probe located with the clxA coding region
identified the predicted 2.7 kb fragment in wt A. fumigatus, which was
not present in the DclxA mutant. The portion of the calnexin gene that
was used to generate the complemented strain (C9) contains a single
internal SacI site, so the 3.7 kb band evident with this probe reflects a
single ectopic integration of the reintroduced clxA gene.
Calnexin in A. fumigatus Growth & Virulence
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A. fumigatus, they suggest that any change in cell wall composition
caused by loss of calnexin is relatively minor and unlikely to
account for the heightened thermosensitivity of the DclxA mutant.
Since protein folding is temperature dependent , we speculate
that the chaperone function of calnexin promotes the folding of
one or more client proteins that are needed for optimal growth at
elevated temperatures. Alternatively, calnexin could increase
overall fitness by preventing the toxic accumulation of misfolded
proteins in the ER that may arise as a result of thermal stress.
Calnexin facilitates growth under conditions of acute
protein folding stress
Calnexin mRNA levels are increased by the UPR under
conditions of acute ER stress, suggesting a role for this chaperone
in the response to unfolded proteins [11,12,13,31]. To test this, we
compared growth in the presence of dithiothreitol (DTT), a
reducing agent that induces the UPR by disrupting the disulfide
bonds that are necessary for protein folding . As shown in
Figure 4, the DclxA mutant was unable to grow in concentrations
of DTT that had minimal effects on the growth of the wt and
complemented strains. This is similar to what has been described
in a calnexin mutant of A. oryzae , and supports a role for
calnexin under conditions that induce acute ER stress. However,
this finding contrasted the effects of the ER stress-inducing agents
tunicamycin (TM) and brefeldin A (BFA), neither of which showed
differential activity against the A. fumigatus DclxA mutant (data not
shown). The inability of calnexin deletion to alter sensitivity to TM
or BFA is likely due to the different mechanisms by which these
agents induce ER stress. For example, TM increases the level of
misfolded proteins in the ER by interfering with the N-linked
glycosylation that is necessary for accurate protein folding .
Since calnexin binds N-linked glycans on nascent polypeptides as
they begin to fold in the ER, TM may mask the effects of calnexin
by interfering with the assembly of the glycan precursor on these
proteins. Similarly, BFA induces ER stress by interfering with ER-
Golgi transport , which is a relatively late step in the secretory
pathway where calnexin function may have less influence. The
ability of the DclxA mutant to grow normally in the presence of
TM and BFA contrasts the DhacA mutant, which is highly sensitive
to these compounds . This reflects the dominant role that HacA
plays as the master regulator of the UPR, as opposed to the more
specialized role of calnexin as one of several ER chaperones that
are downstream of UPR signaling.
Calnexin facilitates growth under starvation conditions
An increasing body of evidence suggests that A. fumigatus is
under nutrient stress in the host environment and must undergo
metabolic changes to adapt to these conditions . To determine
whether calnexin contributes to this process, the DclxA mutant was
tested for its ability to grow under nutrient limiting conditions.
Unlike wt A. fumigatus hyphae, which can support a limited amount
of growth in the absence of extracellular nutrients , the DclxA
mutant was unable to grow under these conditions, suggesting a
role for calnexin in the adaptive response to acute starvation stress
(Figure 5). This phenotype is reminiscent of the A. fumigatus Datg1
mutant, which is deficient in a serine kinase required for
autophagy . Autophagy is a catabolic pathway that employs
Figure 2. Loss of calnexin alters proteolytic secretion. Secreted
protease activity in culture supernatants was profiled using a library of
fluorescence resonance energy transfer (FRET) labeled peptide sub-
strates, as described in Materials and Methods. Equimolar mixtures of
up to 8 individual FRET peptides in each well of duplicate microtiter
plates were incubated with culture supernatants from the indicated
strains and heat maps were generated from the average fluorescent
signals generated by substrate cleavage, with each square correspond-
ing to a single assay well. Panel 1: wt A. fumigatus. Panel 2: DclxA
mutant. Panel 3: Relative change in substrate specificity profile
expressed as the difference of normalized fold change values in panels
1 and 2 (wt minus DclxA). Wells containing substrates with greater
cleavage in DclxA supernatants relative to wt supernatants are shown in
red. The range of fold change values used to generate the wt and DclxA
heat maps is 1–55 and the range of values used to generate the
subtracted heat map (difference) is 225 to +25.
Figure 3. Calnexin is required for thermotolerant growth. Equal
numbers of conidia were plated onto the center of an IMA plate and
incubated for 3 days at the indicated temperatures. Values represent
the average radial growth rate (mm/h) from three individual
experiments 6 SD. *Statistically significant by Student’s T-test
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a complex membrane trafficking system to degrade intracellular
constituents into usable nutrients during periods of nutrient
deprivation . Although the exact role that calnexin plays in
the response to starvation is presently unknown, it is intriguing to
speculate that autophagy components are important clients of the
calnexin cycle in A. fumigatus.
Calnexin is required for growth in cation-depleted
Sequestration of iron or zinc isa major mechanism through which
the host inhibits microbial growth, and the ability of A. fumigatus to
adapt to iron or zinc limitation is an established virulence
mechanism for this fungus [37,38]. To determine whether calnexin
impacts this adaptiveresponse, we compared the ability of conidiato
germinate inmedium that was depleted of metalions bythe addition
of the chelating agent ethylenediaminetetraacetic acid (EDTA). The
germination of A. fumigatus conidia begins with a period of isotropic
expansion (swelling), followed by the elaboration of a germ tube and
the establishmentofpolarized hyphal growth. The germination rates
of wt and DclxA conidia were indistinguishable in the absence of
EDTA (data not shown). In the presence of EDTA, wt conidia were
able to germinate into hyphae within two days and had already
started to branch (Figure 6A). This contrasted the DclxA mutant,
which had only formed small germlings at the same time point,
indicating a delay in germination (Figure 6A, day 2). Unlike wt, the
DclxA germlings were unable to sustain polarized growth upon
further incubation, resulting in abnormally swollen hyphae with
irregular morphology (Figure 6B). Supplementation with an excess
of Zn2+or Fe2+fully rescued the ability of DclxA conidia to elaborate
hyphae at this concentration of EDTA (Figure 6C and data not
shown), consistent with metal ion deficiency as the cause of this
phenotype. The increased sensitivity of DclxA to EDTA may reflect
the existence of specific calnexin client proteins involved in metal ion
homeostasis, such as membrane transporters or zinc-finger tran-
scription factors. Alternatively, since many ER functions are metal
ion-dependent, it may be more difficult for a metal ion-depleted ER
induces the UPR is consistent with this latter possibility .
Calnexin is dispensable for A. fumigatus virulence
The virulence of the DclxA mutant was tested in two distinct
mouse models of invasive aspergillosis that differ in the extent of
immunosuppression; a corticosteroid model that involves transient
immunosuppression with a single-dose of triamcinolone acetonide
and a neutropenic model that involves a prolonged immunosup-
pression regimen that pairs neutrophil depletion with corticoste-
roid-induced immunosuppression. As shown in Figure 7, the loss
of clxA has little-to-no effect on the virulence of A. fumigatus in the
corticosteroid model. Similar results were obtained using the
neutropenic model (Figure S5). Histopathologic analysis of lung
tissue in the neutropenic model confirmed that fungal growth and
inflammation were comparable in both wt- and DclxA-infected
mice (Figure S6). Combined, these results indicate that calnexin
functions are dispensable for surviving the major environmental
stresses that are encountered in the mammalian host. This finding
contrasts the situation in the plant fungal pathogen M. oryzae,
where calnexin is required for the elaboration of a specialized
infection structure called an appressorium that is essential for
virulence . A. fumigatus does not form these structures however,
which may account for the different requirements for calnexin in
the pathogenicity of these diverse fungal pathogens.
A. fumigatus is normally found in compost, a harsh environment
that challenges the fungus to tolerate wide fluctuations in
temperature, nutrient availability and the toxic effects of
compounds released by competing microbes. Our data reveal
that calnexin protects A. fumigatus from the adverse effects of high
temperature, nutrient deprivation and toxins that disrupt ER
homeostasis. This suggests that calnexin has important functions
that contribute to the ability of A. fumigatus to thrive in its
ecological niche of decaying organic debris. However, our data
demonstrate that calnexin is dispensable for infection of a
mammalian host, suggesting that redundant pathways of ER
homeostasis are sufficient to support the virulence of this organism.
Further work is needed to identify these pathways and determine
how they cooperate with calnexin to meet the challenge of protein
folding in a fungus that is highly adapted for secretion.
Figure 4. Calnexin promotes growth under conditions of acute
ER stress. Equal numbers of conidia were added to individual wells of
a 24-well plate containing liquid AMM and the indicated concentrations
of DTT. Plates were incubated at 37uC for 3 days, after which the
mycelial biomass that was adhered to the plate surface was stained
with methylene blue and photographed.
Figure 5. Calnexin is required under nutrient starvation
conditions. Agar plugs containing hyphae from overnight cultures
of conidia plated onto YG plates were transferred to starvation medium
(1% agarose in sterile distilled water) and colony diameters were
measured after 7 days at 37uC. Values represent the average of 9
biological replicates 6 SD. *Statistically significant by Student’s T-test
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Materials and Methods
Strains and culture conditions
Strains were maintained on Aspergillus Minimal Medium (AMM)
 containing 0.01 M ammonium tartrate as the nitrogen
source. The wild type strain used was H237, a clinical isolate.
Unless otherwise noted, all experiments were conducted at 37uC
to recapitulate in vivo growth conditions. Thermotolerance was
assessed by inoculating 5,000 conidia into the center of a plate of
rich medium (Inhibitory Mold Agar, IMA) and radial growth was
monitored for 3 days at different temperatures. For analysis of
DTT susceptibility, 5,000 conidia were inoculated into each well
Figure 6. Calnexin promotes growth in cation-depleted medium. Equal numbers of conidia from the indicated strains were inoculated onto
glass coverslips in liquid AMM containing 1 mM EDTA (AMM-EDTA) and incubated at 37uC. (A) Impaired germination of DclxA conidia in EDTA:
coverslips were removed after 2 and 6 days incubation and fungal morphology was photographed by differential interference contrast microscopy.
(B) Abnormal morphology of the DclxA mutant after prolonged incubation in EDTA: A high power image of the abnormally swollen conidia and
hyphae of the DclxA mutant after 6 days of incubation in AMM-EDTA at 37uC is shown. (C) Supplementation with zinc rescues the growth of DclxA in
AMM-EDTA: The DclxA conidia were inoculated into AMM-EDTA medium supplemented with 500 mM ZnS04and cultured for 2 days at 37uC.
Figure 7. Calnexin is dispensable for A. fumigatus virulence. Groups of 8 CF-1 outbred mice were immunosuppressed with a single dose of
triamcinolone acetonide on day 21 and infected intranasally with conidia from the indicated strains on day 0. Mortality was monitored for 7 days.
The virulence of the DclxA mutant was statistically indistinguishable from that of wt.
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of a 24-well plate containing liquid AMM supplemented with
different concentrations of DTT. Plates were incubated at 37uC
for 3 days without shaking. The medium was aspirated, and the
hyphae adhering to the base of the well were stained with 0.5%
(w/v) methylene blue for 1 hour at 37uC. After removing the
methylene blue solution the adherent hyphae were rinsed with
sterile water and dried prior to photographing. Sensitivity to
tunicamycin (10–100 mg/ml) and brefeldin A (5–15 mg/ml) was
determined by spotting conidia onto the center of a plate of AMM
containing the drug and monitoring radial growth for 2–4 days at
37uC. Caspofungin susceptibility was determined using the Etest
antifungal susceptibility kit (AB BIODISK) according to the
manufacturer’s instructions, with the following modifications. One
million conidia were spread evenly onto the surface of a 150 mm
plate of IMA agar using a glass rod. The inoculated agar surface
was allowed to dry for approximately one hour before Etest strips
containing caspofungin were applied. The plates were incubated at
37uC for 24 hours. The minimal inhibitory concentration was
read as the lowest drug concentrations at which the border of the
elliptical inhibition zone intercepted the scale on the antifungal
strip. Sensitivity to Congo red (CR, 25–150 mg/ml), nikkomycin
(50–250 mg/ml), or calcofluor white (CFW, 5–35 mg/ml) was
determined by spotting 2,000 conidia onto the center of a plate of
IMA containing the compound and monitoring radial growth for
24 hours (CR and CFW) or 36 hours (Nikkomycin) at 37uC.
Growth under starvation conditions was determined as
previously described . Briefly, 200 conidia were spread onto
the surface of a YG plate (0.5% yeast extract and 2% glucose) and
incubated overnight at 37uC. Hyphal plugs containing individual
colonies were obtained using the tip of a sterile glass pasture
pipette and transferred onto the center of a plate of water/agarose
medium (1% agarose in sterile deionized distilled water) and the
extent of radial growth was monitored after 7 days of incubation at
For analysis of growth under conditions of metal ion depletion,
36104conidia were inoculated onto sterile glass coverslips
submerged in 3 mL of AMM containing 1 mM EDTA and
incubated at 37u. Coverslips were removed after 2 and 6 days
incubation, rinsed with sterile water, and photographed by
differential interference contrast microscopy.
Deletion and reconstitution of the A. fumigatus clxA gene
PCR primers used in the study are listed in Table 1. Total RNA
was extracted from overnight cultures of wt A. fumigatus by
crushing the mycelium in liquid nitrogen and resuspending in TRI
reagent LS (Molecular Research Center, Cincinnati, OH). The
RNA was then reverse-transcribed using the Superscript II reverse
transcriptase first-strand synthesis system (Invitrogen) and PCR
amplified using primers 639 and 640.
The calnexin gene was replaced with the hygromycin resistance
gene using the split-marker method . The first two-thirds of
the hygromycin resistance cassette were amplified from pAN7-1
using primers 398 and 395, creating PCR product #1. The second
two-thirds of hygromycin were then amplified with primers 396
and 399, creating PCR Product #2. The left arm of the clxA gene
was amplified from wt DNA using primers 632 and 633, and the
right arm was amplified with primers 634 and 635, generating
PCR products #3 and #4, respectively. PCR products #1 and
#3 were then combined in an overlap PCR reaction with primers
632 and 395 to generate PCR product #5 and PCR products #2
and #4 were combined in an overlap reaction with primers 396
and 635 to generate PCR product #6. PCR products #5 and #6
were then cloned into pCR-Blunt II-TOPO (Invitrogen) to create
plasmids p569 and p568, respectively. The p569 and p568
plasmids were linearized with NsiI and EcoRI, respectively, and
transformed into A. fumigatus protoplasts as previously described
. Loss of the clxA gene was confirmed by Southern blot analysis
of genomic DNA isolated from hygromycin resistant monoconidial
isolates using an internal probe that was PCR-amplified from wt
genomic DNA using primers 636 and 637 (Figure 1), as well as an
upstream probe that was PCR-amplified from wt genomic DNA
using primers 632 and 633, corresponding to the left arm of the
calnexin-deletion cassette (Figure S2).
To construct the clxA complementation plasmid, the clxA gene
including 472 bp upstream of the ATG was PCR-amplified from
wt genomic DNA using primers 641 and 672 and cloned into
pCR-Blunt II-TOPO (Invitrogen) to create p575. A phleomycin
resistance cassette was then excised from plasmid 565 and inserted
into p575 to create p612. Plasmid 612 was linearized with XbaI,
transformed into DclxA protoplasts, and stable integrants were
selected on plates containing phleomycin. Ectopic reconstitution of
the clxA gene was confirmed by genomic Southern blot analysis of
phleomycin-resistant monoconidial isolates using the internal clxA
probe shown in Figure 1 and the upstream probe shown in Figure
Analysis of protease secretion by substrate specificity
Conidia were inoculated to a concentration of 16105conidia/
ml in 60 mL of AMM supplemented with 10% heat-inactivated
human AB serum (Innovative Research). After incubating at 37uC
for 72 h at 150 rpm, the mycelium was removed by filtration and
each culture supernatant was diluted 1:50 in sterile-filtered
HEPES buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2,
pH 8.0). These dilutions were found to give comparable
fluorescence intensity values in preliminary experiments. A FRET
peptide library comprised of 512 microtiter plate wells, each
containing an equimolar mixture of up to 8 individual peptides,
was obtained (Mimotopes, Clayton, Australia) . Each well
(50 nmol of peptide) was dissolved in 100 mL of 50% acetonitrile
in ultrapure water. This solution (5 mL/well) was transferred to
low volume black microtiter plates (Molecular Devices, Sunnyvale,
CA) containing 20 mL of HEPES buffer. Diluted fungal culture
Table 1. PCR primers used in this study.
Primer Sequence (59-39)
M13-derived sequences used for overlap PCR are underlined.
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supernatant (20 mL per well) was added to each well. Time-
resolved fluorescence data were obtained on an Analyst HT
instrument (Molecular Devices) using excitation and emission
filters of 320 nm and 420 nm, respectively. The fluorescence
intensity fold change after 5 hr at room temperature was
calculated as Ffinal/Finitialand each data set was normalized to
the highest global signal intensity. No fluorescence was observed in
control culture medium lacking fungal supernatant. Heat maps
were generated from these data in which each square corresponds
to a single assay well (Heatmap Builder, Ashley Lab, Stanford)
Animal models of invasive aspergillosis
For the corticosteroid immunosuppression model, groups of 8
CF-1 outbred female mice were given a single dose of the synthetic
corticosteroid triamcinolone acetonide (40 mg/kg of body weight
injected subcutaneously) on day 21. On day 0, the mice were
anaesthetized with 3.5% isofluorane and inoculated intranasally
with a 20 mL saline suspension containing 26105conidia from wt
or the DclxA mutant, or with 20 ml of a 0.9% sodium chloride
solution for a mock infection control (4 mice). Mortality was
monitored for 7 days, and statistical significance was assessed by
the log-rank test using Sigma Stat 3.5.
For the neutropenic model, groups of 12 CF-1 outbred female
mice were immunosuppressed by intraperitoneal injection of
cyclophosphamide (150 mg/kg) on days 22 and +3, as well as
subcutaneous injections of triamcinolone acetonide (40 mg/kg) on
days 21 and +6. Mice were inoculated with 26105conidia and
mortality was monitored for 14 days. Statistical significance was
assessed using the Sigma Stat 3.5 log-rank test.
For histopathologic analysis, CF-1 outbred female mice were
immunosuppressed according to the neutropenic model described
above, infected with 26105conidia, and sacrificed on day +3. The
lungs were fixed by inflation with 4% phosphate-buffered
paraformaldehyde, dehydrated and embedded in paraffin, sec-
tioned at 5 mm, and stained with hematoxylin and eosin (HE) or
Grocott methenamine silver (GMS). Microscopic examinations
were performed on an Olympus BH-2 microscope and imaging
system using Spot software version 4.6.
Animal ethics statement
Animal experiments were carried out in strict accordance with
the Guide for the Care and Use of Laboratory Animals, the Public
Health Service Policy on the Humane Care and Use of
Laboratory Animals and all U.S. Animal Welfare Act Regulations.
The experiments were approved by the Institutional Animal Care
and Use Committee of the University of Cincinnati (protocol #
06-01-03-02). All efforts were made to minimize animal suffering.
Genbank accession numbers
A. fumigatus clxA gene (XM_746454.1), A. fumigatus clxA mRNA
orthologs. The A. fumigatus calnexin protein (Af; XP_751547) is
compared to orthologs from A. niger (An; AJ299945), S. pombe (Sp;
P26581), H. sapiens (Hs; P27824), and S. cerevisiae (Sc; P27825).
Black boxes denote identical amino acids, whereas grey boxes
denote similar amino acids. The sequence was aligned using
DNAMAN software (Lynnon Corp, Canada) using default
parameters. Results were exported in CLUSTALW format for
shading using BOXSHADE 3.21 (http://www.ch.embnet.org/
Multiple sequence alignment of calnexin
software/BOX_form.html). The two sets of repeated peptide
motifs (1–4) that are characteristic of the calreticulin/calnexin
family are shown by the brackets. The asterisk denotes the
transmembrane domain of A. fumigatus calnexin predicted by
TMHMMServer v. 2.0 (http://www.cbs.dtu.dk/services/
Southern blot analysis of BamHI-digested genomic DNA using a
flanking probe located upstream of the clxA gene was used to
confirm calnexin gene deletion. Replacement of the clxA gene with
the hygromycin resistance cassette introduced a BamHI site that
reduced a 5.6 kb wt fragment to the expected 3.7 kb. Two closely
migrating bands above 5.6 kb were also evident in the DclxA
mutant, indicating the presence of at least two ectopic integrations
of the disruption cassette. The complemented strain (C9) contains
a single ectopic integration of the clxA gene, which is evident by the
unique 5.0 kb band that is smaller than the wt 5.6 kb band
because it lacks the flanking BamHI sites.
Deletion of calnexin from A. fumigatus.
to caspofungin. Caspofungin sensitivity was determined using
the Etest method. Etest strips containing caspofungin were applied
to IMA plates inoculated with equal amounts of conidia. The
plates were incubated at 37uC for 24 hours. The lowest drug
concentrations at which the border of the elliptical zone of
inhibition intercepted the scale on the antifungal strip (MIC) was
indistinguishable between the strains, indicating that loss of
calnexin did not alter caspofungin sensitivity. In addition, fungal
growth was evident within the zone of inhibition in all three
strains, consistent with the known fungistatic effects of this drug
against A. fumigatus.
Loss of calnexin does not increase sensitivity
stress conditions. Sensitivity to Congo red (CR), nikkomycin,
or calcofluor white (CFW) was determined by spotting equal
amounts of conidia onto the center of a plate of IMA containing
each compound at the indicated concentrations and monitoring
radial growth for 24 hours (CR and CFW) or 36 hours
(Nikkomycin) at 37uC. The DclxA strain phenocopies wt at all
concentrations of each cell wall stress-inducing agent.
Calnexin is not required under cell wall
virulence. Groups of 12 CF-1 outbred mice were immunosup-
pressed with cyclophosphamide and triamcinolone acetonide and
inoculated with 26105conidia as described in Materials and
Methods. Pulmonary fungal infections were confirmed in all mice
that died by plating lung tissue for fungal growth. One of the four
mock-infected control mice died on day +10 of a bacterial
infection. The virulence of the DclxA mutant was statistically
indistinguishable from that of wt.
Calnexin is dispensable for A. fumigatus
the neutropenic immunosuppression model, mice were infected
with 26105conidia in a separate experiment and sacrificed on day
+3, as described in Materials and Methods. The lungs were
sectioned at 5 mm and stained with hematoxylin and eosin (HE) or
Grocott methenamine silver (GMS). Comparable levels of fungal
growth and inflammation were observed in both wt- and DclxA-
infected mice, resulting in similar amounts of bronchiolar erosion
and migration of the hyphae across the airway wall. Microscopic
examinations were performed on an Olympus BH-2 microscope
Histopathology of infected lung tissue. Using
Calnexin in A. fumigatus Growth & Virulence
PLoS ONE | www.plosone.org7December 2011 | Volume 6 | Issue 12 | e28865
and imaging system using Spot software version 4.6. Scale bar
represents 100 mm.
The authors thank Stephanie White for technical assistance and Jay Card
for photography and illustration.
Conceived and designed the experiments: MVP-F KJ AKG DSA.
Performed the experiments: MVP-F KJ JLB KK XF. Analyzed the data:
MVP-F KJ JLB KK XF AKG DSA. Wrote the paper: MVP-F DSA.
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PLoS ONE | www.plosone.org8 December 2011 | Volume 6 | Issue 12 | e28865