A PR-1-like Protein of Fusarium oxysporum Functions in
Virulence on Mammalian Hosts*□
Rafael C. Prados-Rosales‡1, Raquel Roldán-Rodríguez‡, Carolina Serena§, Manuel S. López-Berges‡, Josep Guarro§,
Álvaro Martínez-del-Pozo¶, and Antonio Di Pietro‡2
FacultaddeCienciasQuimicas,UniversidadComplutense,28040 Madrid, Spain
Background: Pathogenesis-related (PR-1-like) proteins are widely conserved in eukaryotes, but their biological function is
Results: Knockout or site-directed mutagenesis of fpr1 encoding a secreted PR-1-like protein in the fungal pathogen Fusarium
oxysporum impairs virulence on mice.
Conclusion: Secreted PR-1-like proteins are important for fungal infection of mammals.
Significance: We show the first genetic evidence for a biological function of the predicted active site of PR-1-like proteins.
The pathogenesis-related PR-1-like protein family comprises
secreted proteins from the animal, plant, and fungal kingdoms
whose biological function remains poorly understood. Here we
have characterized a PR-1-like protein, Fpr1, from Fusarium
oxysporum, an ubiquitous fungal pathogen that causes vascular
wilt disease on a wide range of plant species and can produce
life-threatening infections in immunocompromised humans.
Fpr1 is secreted and proteolytically processed by the fungus.
nodepressed mouse model, and its function depends on the
integrity of the proposed active site of PR-1-like proteins. Fpr1
belongs to a gene family that has expanded in plant pathogenic
Sordariomycetes. These results suggest that secreted PR-1-like
proteins play important roles in fungal pathogenicity.
Fungi are an extremely versatile group of organisms. Most
are saprophytes that thrive on dead organic material, but a
number of species can infect and cause disease on other organ-
penetrate a host, overcome its innate defenses, and exploit its
the severity of disease it causes on a given host.
Hosts respond to fungal infection by mounting a robust
immune response. Interestingly, plants and mammals share
common principles of innate immunity, such as the ability to
recognize pathogen-associated molecular patterns (PAMPs)
(2) or the presence of pathogenesis-related 1 (PR-1) proteins,
which have been implicated both in plant and animal immune
systems. PR-1 proteins were originally identified in tobacco as
part of the defense response to viral infection (3). PR-1 is the
most highly expressed class of PR proteins and contributes up
to 10% of total protein in infected leaves (4). PR-1-like proteins
were subsequently found in a variety of eukaryotes, including
fungi, insects, and mammals, and the term CAP protein super-
family was coined to encompass mammalian cysteine-rich
secretory proteins (CRISPs)3, Ag5-antigens from insects and
plant PR-1 proteins (5). Comparative structural analysis iden-
tified a putative active site of two histidine and two glutamate
residues that is highly conserved among the members of the
protein family (6). Despite their ubiquitous distribution, the
biochemical function and biological roles of PR-1-like proteins
have remained largely elusive (5).
gen that causes vascular wilt disease on more than a hundred
disseminated fusariosis with mostly lethal outcomes (8). The
genus Fusarium now represents the second most frequent
mold causing invasive fungal infections, and F. oxysporum,
together with Fusarium solani and Fusarium verticillioides, is
responsible for practically all cases of invasive fusariosis (8, 9).
Human pathogenic isolates of F. oxysporum have polyphyletic
origin and respond poorly to available antifungal agents (10,
11). We showed previously that a single isolate of F. oxysporum
f. sp. lycopersici can cause disease both on tomato plants and
immunodepressed mice (12) as well as on the invertebrate
* This research was supported by the Ramon y Cajal Program. This work was
also supported by Ministerio de Ciencia e Innovación (MICINN) Grant
BIO2010-15505, by MICINN ERA-NET/PathoGenoMics project TRANSPAT
Grant BIO2008-04479-E, MICINN/Plant KBBE Grant EUI2009-03942, Junta
ITN-237936), and MICINN PhD fellowships (to R. C. P. R. and M. S. L. B.).
SThis article contains supplemental Figs. 1–7 and Methods.
stein College of Medicine, Yeshiva University, Bronx, NY 10461.
2To whom correspondence should be addressed: Departamento de
Genetica, Facultad de Ciencias and Campus de Excelencia Internacional
34-957-212072; E-mail: email@example.com.
3The abbreviations used are: CRISP, cysteine-rich secretory protein; GPI,
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 26, pp. 21970–21979, June 22, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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model host Galleria mellonella (13). In this work, we function-
ally characterized Fpr1, a secreted PR-1 like protein, from
F. oxysporum. Using a genetic approach, we established that
Fpr1 function is required for full virulence on a mammalian
host but dispensable for virulence on plants. We provide evi-
dence that Fpr1 is part of a gene family that has expanded in
F. oxysporum and other plant pathogenic Sordariomycetes.
Our results shed new light on the role of secreted PR-1-like
proteins and suggest that they are key players in fungal
Fungal Isolates and Culture Conditions—F. oxysporum f.sp.
lycopersici wild-type strain 4287 (race 2) was grown and main-
strains was described previously: MAPK mutant ?fmk1 (15), G
protein subunit ? mutant ?fgb1, and ?fmk1?fgb1 double
mutant (16). Growth conditions for microconidia production,
nucleic acid extraction, Western blot analysis, microscopic
examination, and analysis of colony phenotypes are detailed in
the supplemental Methods.
Nucleic Acid Manipulations, Construction of Plasmid Vec-
tors, and Fungal Transformation—Total RNA and genomic
DNA extraction from F. oxysporum mycelium, Southern and
Northern blot analyses, and PCR amplification were per-
formed as described (17, 18). For details on fpr1 gene clon-
ing, construction of the gene knockout vector, gene knock-
fpr1H170A,E177Aallele see supplemental Methods. Transforma-
tion of fungal protoplasts to hygromycin or phleomycin resist-
ance was performed as described (14). Gene knockout and
complementation events were confirmed by Southern and
Northern blot analysis (supplemental Fig. 2). The presence
and correct expression of the different fpr1 alleles in the com-
sequencing, as well as by Northern blot analysis.
Production and Characterization of Recombinant Fpr1
Protein—Cloning of a fpr1 cDNA clone lacking the predicted
signal peptide into the pET-28c bacterial or the pPIC9 yeast
expression vector, as well as purification of recombinant Fpr1
protein from Escherichia coli or Pichia pastoris, respectively, is
tive proteolytic activity of purified Fpr1-His6protein from
E. coli or P. pastoris culture supernatants containing Fpr1 was
done against azocaseine (19) using 25 ?g of protein in phos-
phate buffer (pH 6.0, 7.0, and 8.0) at 37 °C for periods from 30
gelatinolytic activity see the supplemental Methods. To study
proteolytic processing of Fpr1, 2 ?g of recombinant Fpr1 from
?fgb1 in 50 mM phosphate buffer (pH 7.4) in a total volume of
25 ?l and supplemented with 1 mM of different protease inhib-
itors when indicated. For Western blot analyses, protein sam-
ples were separated by electrophoresis in 14% (w/v) acrylam-
ide-SDS gels and analyzed using a polyclonal ?-Fpr1 antibody,
CD spectra were obtained on a Jasco 715 spectropolarimeter
equipped with a thermostated cell holder and a NesLab-111
circulating water bath at 0.2 nm/s scanning speed. The instru-
ment was calibrated with (?)-10-camphorsulfonic acid. CD
Mean residue weight ellipticities were expressed in units of
degree ? cm2? dmol?1.
interest were excised from the gel, subjected to tryptic digest,
and analyzed on a Voyager DE-STR MALDI-TOF mass spec-
trometer (Applied Biosystems) using ?-cyano-4-hydroxycin-
namic acid as a matrix. MALDI-MS spectra were internally
calibrated using the singly protonated trypsin autodigestion
peaks at m/z 2273.159 and 2163.056 and searched against the
F. oxysporum database downloaded from the Broad Institute
using Mascot software version 2.1 (Matrix Science) (20) (for
details, see supplemental Methods).
and transferred for 8 h either to MM, MM supplemented with
10% (v/v) bovine fetal serum (Sigma), or MM with submerged
tomato roots. Details on reverse transcription and PCR
reactions are provided in the supplemental Methods. As a con-
trol, the actin gene transcript was amplified. For quantitative
real-time RT-PCR, the wild-type strain was germinated 16 h at
28 °C in MM, transferred for 2 h to MM at 37 °C, and then
transferred for different time periods either to MM or to hepa-
rinized human whole blood (Dunn Labortechnik GmbH,
Asbach, Germany) at 37 °C. PCR products were obtained using
iQ SYBR Green Supermix (Bio-Rad) and an iCycler iQ real-
time PCR system (Bio-Rad). Transcript levels were calculated
by comparative ?Ct and normalized to act1.
assays with microconidia from different F. oxysporum strains
were performed as described (14). Ten plants were used for
each treatment. Assays for invasive growth on tomato fruits
(cultivar Daniela) were carried out as described (15). Plant
Mice were cared for in accordance with the principles out-
lined by the European Convention for the Protection of Verte-
brate Animals Used for Experimental and Other Scientific
Purposes (European Treaty Series, no. 123). Experimental con-
Faculty of Medicine, Universitat Rovira i Virgili. Infection
assays with immunodepressed OF-1 male mice (Charles River
Laboratories, Criffa S.A., Barcelona, Spain) were performed as
described (12). Briefly, groups of 10 immunosuppressed mice
were infected by injecting 0.2 ml of an inoculum of 108F. oxys-
fpr1 gene expression or determination of fungal tissue burden
in organs, randomly chosen surviving mice were sacrificed 3 or
lungs were aseptically removed and immediately frozen in liq-
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uid nitrogen for RNA extraction or weighed, homogenized in
sterile saline and 10-fold serial dilutions were spread onto
ing Units per gram of organ. Fungal colony counts were con-
verted to log10and compared using analysis of variance. Calcu-
lations were performed using SPSS for Windows version 10.0.
Sequence Alignments and Phylogenetic Analysis—Members
of the PR-1 protein family in different fungal genomes were
identified by BLASTp searches on the Web server of the Broad
W (21) and inspected manually. Selected plant PR-1 protein
alignments. A maximum likelihood tree was built from the
alignment by PhyML version 4.0 using both parsimony and
distance analysis (neighbor joining, NJ) with 1000 bootstrap
For identification of protein domains, full-length sequences
were analyzed using InterProScan. Presence of a signal peptide
was determined with SignalP version 3.0 (23) using a standard-
ized threshold value of 0.5. Putative GPI consensus sequences
were identified using the Fungal BigPi software (24).
Cloning, Mutation, and Expression Analysis of the F. oxyspo-
a F. oxysporum Expressed Sequence Tag library. One of the
sequenced clones showed homology with plant PR-1 proteins
from a ?EMBL3 genomic library of F. oxysporum, and the
sequencewasdepositedinGenBankTMUNDER ACCESSION NUMBER
GQ411527. fpr1 consists of an open reading frame encoding a
putative 259-amino acid protein with a predicted molecular
mass of 27.7 kDa and a pI of 4.9. Sequence alignment with the
fpr1 cDNA revealed the presence of a single 52-bp intron. A
BLAST search of the complete genome database of F. oxyspo-
rum produced a single high identity match (FOXG_09795),
consistent with the presence of a single hybridizing band in
Southern blot analysis.
The predicted F. oxysporum Fpr1 protein contains three dis-
tinct regions: an N-terminal signal peptide of 19 amino acids, a
proline-rich region of unknown function, and a C-terminal
domain with homology to the PR-1-like, SCP, or CAP protein
family PF00188 (supplemental Fig. 1A). This family includes
glioma-associated protein GliPR (26), mammalian CRISP pro-
teins (27), allergens of insect venoms (28), and snake or lizard
venoms (29). Fpr1 contains two conserved histidine (H170,
stitute the proposed active site (30) (supplemental Fig. 1, A and
B). On the basis of these data, we conclude that F. oxysporum
fpr1 encodes a fungal homologue of PR-1-like pathogenesis-
F. oxysporum mutants lacking a functional copy of fpr1 were
generated by targeted gene disruption. For complementation
experiments, a 2.7-kb DNA fragment encompassing either the
wild-type fpr1 allele or a fpr1H170A,E177Aallele in which two
conserved residues at the predicted active site had been
replaced by site-directed mutagenesis with alanines was intro-
duced into the ?fpr1–1 mutant (supplemental Fig. 2).
Northern blot analysis detected a single fpr1 transcript in
F. oxysporum mycelium grown either in liquid or solid MM
(Fig. 1). High concentrations of glucose (1% w/v) resulted in
detected in the ?fpr1 mutant but was restored in the comple-
mented ?fpr1?fpr1 and ?fpr1?fpr1H170A,E177Astrains. A
mutant lacking the MAPK Fmk1 (15) had drastically reduced
fpr1 transcript levels both in liquid and on solid medium,
whereas the ?fmk1?fgb1 mutant lacking both Fmk1 and the
heterotrimeric G? subunit Fgb1 (16) had further reduced tran-
Fmk1 MAPK cascade and repressed by glucose.
fpr1 or fpr1H170A,E177AcDNA in E. coli resulted in the presence
side-induced cells that was absent from uninduced cells (Fig.
shown). The apparent mass of affinity-purified Fpr1 and
Fpr1H170A,E177Aprotein deduced from SDS-PAGE was 40 kDa,
which is significantly higher than predicted. By contrast,
MALDI-MS analysis of the purified recombinant protein
detected a major peak with a mass of 30 kDa, in line with the
predicted mass of His-tagged Fpr1.
Recombinant Fpr1 protein was also obtained from the
methylotrophic yeast P. pastoris. After induction with 0.5%
methanol, culture supernatants of fpr1-expressing P. pastoris
transformants contained a major protein band that was absent
in the control strain transformed with the empty pPIC9 vector
(Fig. 2B). Similar to E. coli-produced Fpr1, the recombinant
protein band with an apparent mass of ?80 kDa, which is con-
sistent with the expected mobility of a putative Fpr1
homodimer (Fig. 3A). Analysis of MALDI MS spectra of the
high and low molecular weight bands confirmed that both cor-
respond to Fpr1 (supplemental Fig. 3). Western blot analysis of
FIGURE 1. Expression of fpr1 is repressed by glucose and stimulated by
the pathogenicity MAP kinase Fmk1. Shown is the Northern analysis for
fpr1 transcript accumulation in fungal cultures grown either on liquid mini-
mal medium supplemented with 0.1% (w/v) glucose (G) or with 1% w/v glu-
cose where indicated (A) or on solid minimal medium (B). Total RNA was
extracted, fractionated in an agarose gel, blotted onto a nylon membrane,
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unboiled Fpr1 and Fpr1H170A,E177Aprotein with a polyclonal
?-Fpr1 antibody detected the presence of the dimer in both
protein species, suggesting that the H170 and E177 are not
essential for dimerization (Fig. 3B). Comparison of circular
dichroism spectra of wild-type and mutant Fpr1 showed that
the percentage of secondary structural elements was un-
changed by the mutation, indicating that the Fpr1H170A,E177A
protein is folded correctly (supplemental Fig. 4).
A PR-1-like protein of the CRISP subfamily from the cone
snail Conus textile, Tex31, was suggested previously to exhibit
substrate-specific serine protease activity (31). Incubation of
protease substrate azocasein buffered at different pHs in the
absence or presence of metal ions (CaCl2, MgCl2, ZnCl2) or
with a synthetic substrate containing the reported cleavage site
of Tex31 (H-LVKA-pNA) failed to detect protease activity
under any of the conditions tested (results not shown). Gelati-
nolytic activity zymograms of P. pastoris culture supernatants
from the fpr1-expressing transformant and the control strain
revealed similar clearing bands, suggesting that they originate
from P. pastoris proteases (data not shown).
F. oxysporum—Sequence analysis of Fpr1 predicted the pres-
and culture supernatant were subjected to Western blot analy-
sis with ?-Fpr1 antibody. No signal was detected in the cell
lysates, whereas culture supernatants contained a major hy-
bridizing band of ?30 kDa (Fig. 4A). The signal intensity was
higher in culture supernatants from MM (containing 0.1% w/v
glucose) than in those from potato dextrose broth (2% glucose)
no hybridizing signal was detected in supernatants of the two
ground hybridization in the mutants indicates that the poly-
clonal ?-Fpr1 antibody exhibits a high specificity toward the
Fpr1 protein. Supernatants of the ?fmk1 and ?fmk1?fgb1
mutants contained significantly lower amounts of Fpr1 than
those of the wild-type strain, confirming the results from
Northern blot analyses (see Fig. 1). Interestingly, supernatants
apparent mass of 40 kDa, similar to that of recombinant Fpr1.
Inspection of the Western blot analyses revealed at least four
hybridizing bands with approximate molecular masses of 26,
absent in the ?fpr1 mutants, suggesting that they represent
different isoforms of Fpr1.
FIGURE 2. Heterologous expression of Fpr1 in E. coli and P. pastoris.
A, purification of recombinant Fpr1–6xHis protein from E. coli. Lanes 1 and 2,
crude cell lysate of bacterial strain BL21 transformed with plasmid pet28c
containing the fpr1 cDNA, either before (lane 1) or after induction with 1 mM
IPTG (lane 2). Lanes 3–7, different fractions eluted from a Ni2?NTA column.
5), and 250 mM imidazol (lanes 6 and 7). B, expression of Fpr1 in P. pastoris.
Culture supernatants of yeast strain GS115 transformed either with the
or silver staining (B). Relative positions of molecular weight markers (in kDa)
are indicated at the left.
FIGURE 3. Fpr1 forms a dimer in solution. A, 10-?g recombinant Fpr1 pro-
tein obtained from E. coli or P. pastoris was subjected to SDS-PAGE either
with Coomassie Blue. B, Western blot analysis of recombinant Fpr1 or
FprH170A,E177Aprotein obtained from E. coli, subjected to SDS-PAGE either
without (NB) or with 5 min of boiling (B) prior to loading and hybridized with
?-Fpr1 antiserum. m and d indicate positions of hybridizing bands corre-
sponding to the monomeric and dimeric form of Fpr1, respectively. Relative
positions of molecular weight markers (in kDa) are indicated at the left.
supernatant of the F. oxysporum wild-type strain. Upper panel, samples sepa-
rated by SDS-PAGE and stained with Coomassie Blue. Lower panel, immuno-
blot analysis with ?-Fpr1 antiserum. B, Western analysis of culture superna-
(MM) or presence of tomato roots (Root) or on potato dextrose broth (PDB).
Relative positions of molecular weight markers are indicated at the left.
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We next tested whether the 40-kDa band corresponds to an
Fpr1 precursor which is processed into the major 30-kDa form
recombinant Fpr1 protein from E. coli was incubated with cul-
ture supernatant from the ?fpr1#1 or the ?fgb1 strains. Super-
natant of ?fpr1#1 was used instead of wild-type supernatant to
circumvent hybridization interference from the native Fpr1
protein. As shown in Fig. 5B, the 40-kDa Fpr1 band was con-
verted into the 30-kDa form upon incubation with culture
supernatant of the ?fpr1#1 strain but not that of the ?fgb1
strain. Similar results were obtained with recombinant Fpr1
from P. pastoris (data not shown). Treatment with NaOH had
in O-glycosylation (Fig. 5B). However, addition of a protease
inhibitor mixture completely abolished the mass shift of
recombinant Fpr1 incubated with ?fpr1#1 culture supernatant
(Fig. 5C, left panel). To gain insight into the nature of the pro-
teolytic enzyme(s) responsible for Fpr1 processing, different
protease inhibitors were added, including leupeptin (inhibits
serine and cysteine proteases), PMSF (serine proteases), EDTA
(metalloproteases), and pepstatin A (aspartyl proteases). All
inhibitors except EDTA prevented the size shift of Fpr1 to dif-
ferent extents (Fig. 5C, right panel). This suggests that multiple
proteolytic enzymes other than metalloproteases contribute to
processing of secreted Fpr1 by F. oxysporum.
Fpr1 Is Dispensable for Vegetative Growth, Development and
Virulence on Tomato Plants—Hyphal growth and conidiation
of the ?fpr1 mutants was indistinguishable from the wild-type
strain either on minimal or rich medium, in liquid or solid cul-
ture, as well as under conditions of osmotic (0.8 M NaCl), oxi-
dative (10 ?g ml?1menadione), high temperature (37 °C), or
cell wall stress (20 ?g ml?1Congo Red or 20 ?g ml?1Calco-
as efficiently as the wild type in colonization and maceration of
of the wild type, ?fpr1#1, or ?fpr1#1?fpr1 strains caused sim-
ilar extent of vascular wilt symptoms and plant mortality (sup-
plemental Fig. 5B). In different virulence-related phenotypic
assays, the ?fpr1 mutant was indistinguishable from of the
wild-type strain, including secretion of pectinolytic enzymes,
penetration of cellophane membranes, vegetative hyphal
these results suggest that Fpr1 is dispensable for vegetative
growth, different stress responses, and virulence of F. oxyspo-
rum on the plant host.
Fpr1 Is Essential for Efficient Dissemination and Virulence in
a Mouse Model—Mortality rates of immunodepressed mice
?fpr1#3 were significantly lower (p ? 0.05) than in mice
infected with the wild-type strain (Fig. 6). Complementation of
the ?fpr1#1 mutant with the native fpr1 allele restored viru-
in lung, liver, spleen, and kidney of surviving mice sacrificed 7
days after challenge was significantly (p ? 0.05) lower in mice
infected with the ?fpr1#1 and ?fpr1#3 mutants than in those
infected with the wild-type or the ?fpr1#1?fpr1 strain (Fig. 7).
These results demonstrate that Fpr1 function is required for
full virulence of F. oxysporum on a mammalian host.
Expression of fpr1 Is Induced during Fungal Growth in
Human Blood and in a Mammalian Host—Transfer of F. oxys-
porum germlings to whole human blood resulted in rapid acti-
vation of fpr1 expression (Fig. 8A). Within 60 min, transcript
Induced transcript levels of fpr1 were also observed in kidney
ture supernatants of different F. oxysporum strains with ?-Fpr1 antiserum.
of recombinant Fpr1 protein incubated with culture supernatant of strain
absence or presence of a protease inhibitor (PI) cocktail or of the indicated
protease inhibitors. Relative positions of molecular weight markers are indi-
cated at the left.
FIGURE 6. Fpr1 function is required for virulence of F. oxysporum on
immunodepressed mice. Groups of ten mice were infected by lateral tail
vein injection with 2 ? 107 microconidia of the following strains: wild type
?fpr1#1 mutant complemented either with a wild-type fpr1 allele (●) or the
fpr1H170A,E177Aallele (ƒ). Percent survival was plotted for 14 days. The data
shown are from one representative experiment.
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