ARTICLE IN PRESS

Toxicon 50 (2007) 971–983

[Link]/locate/toxicon

Antifungal activity of plant and bacterial ureases$

A.B. Becker-Ritta, A.H.S. Martinellia, S. Mitidierib, V. Federa,
G.E. Wassermanna, L. Santia, M.H. Vainsteina,c, J.T.A. Oliveirad,
L.M. Fiuzae, G. Pasqualia,f, C.R. Carlinia,g,
a
Graduate Program in Molecular and Cellular Biology, Center of Biotechnology,
Universidade Federal do Rio Grande do Sul—UFRGS, Brazil
b
BioPlus Biotechnological Development LTDA, Porto Alegre, Brazil
c
Department of Microbiology, UFRGS, Brazil
d
Department of Biochemistry and Molecular Biology, Universidade Federal do Ceará, Fortaleza, CE, Brazil
e
Universidade do Vale dos Sinos, São Leopoldo, RS, Brazil
f
Department of Molecular Biology and Biotechnology, UFRGS, Brazil
g
Department of Biophysics, UFRGS, Av. Bento Gonc- alves, 9500. Prédio 43.422, Porto Alegre, RS, CEP 91501-970, Brazil
Received 25 April 2007; received in revised form 10 July 2007; accepted 11 July 2007
Available online 31 July 2007

Abstract

Ureases (EC [Link]) are nickel-dependent metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon
dioxide. Produced by plants, fungi and bacteria, but not by animals, ureases share significant homology and similar
mechanisms of catalysis, although differing in quaternary structures. While fungal and plant ureases are homo-oligomeric
proteins of 90 kDa subunits, bacterial ureases are multimers of two (e.g. Helicobacter pylori) or three subunit complexes. It
has been proposed that in plants these enzymes are involved in nitrogen bioavailability and in protection against
pathogens. Previous studies by our group have shown that plant ureases, but not a bacterial (Bacillus pasteurii) urease,
display insecticidal activity. Herein we demonstrate that (Glycine max) embryo-specific soybean urease, jackbean
(Canavalia ensiformis) major urease and a recombinant H. pylori urease impair growth of selected phytopathogenic fungi
at sub-micromolar concentrations. This antifungal property of ureases is not affected by treatment of the proteins with an
irreversible inhibitor of the ureolytic activity. Scanning electron microscopy of urease-treated fungi suggests plasmolysis
and cell wall injuries. Altogether, our data indicate that ureases probably contribute to the plant arsenal of defense
compounds against predators and phytopathogens and that the urease defense mechanism is independent of ammonia
release from urea.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Antifungal protein; Plant pathogen; Urease; Soybean; Jackbean; Helicobacter pylori

$
Ethical statement: The experimental protocols and procedures used to obtain the results were approved by the Research and Ethics
Committee of the Institute of Biosciences, Federal University of Rio Grande do Sul.
Corresponding author. Department of Biophysics, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc- alves, 9500. Prédio
43.422, 91501-970 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 7606; fax: +55 51 3308 7003.
E-mail addresses: ccarlini@[Link], [Link]@[Link] (C.R. Carlini).

0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/[Link].2007.07.008
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972 A.B. Becker-Ritt et al. / Toxicon 50 (2007) 971–983

1. Introduction Winkler, 1984), while the so-called ‘‘ubiquitous’’

urease, encoded by the Eu4 gene, was found in all
Ureases (EC [Link], urea amidohydrolase) are tissues examined (Polacco and Winkler, 1984;
nickel-dependent metalloenzymes that catalyze the Stebbins et al., 1991). Although the ubiquitous
hydrolysis of urea to ammonia and carbon dioxide urease is present in seeds at 0.001–0.01% levels of
(Dixon et al., 1975), widespread in plants, fungi and that of the embryo-specific urease, this isoenzyme is
bacteria (Mobley and Hausinger, 1989). While responsible for recycling metabolically derived urea
fungal and plant ureases are homo-hexameric (Polacco et al., 1985; Stebbins et al., 1991; Witte et
proteins of 90 kDa subunits, bacterial ureases are al., 2002). It has been suggested that the embryo-
multimers of two or three subunit complexes. The specific urease does not play a urea-assimilatory role
sequence similarity of all ureases indicates that they and that it may be involved in seed chemical defense
are variants of the same ancestral protein, and are (Polacco and Holland, 1993).
likely to possess highly conserved tertiary structures Our group has shown that plant and microbial
and similar catalytic mechanisms (Mobley et al., ureases display several biological properties that are
1995; Sirko and Brodzik, 2000). Some bacterial independent of their ureolytic activity, such as the
ureases, such as those from Proteus mirabilis and activation of blood platelets, interaction with
Helicobacter pylori (Mobley et al., 1995; Olivera- glycoconjugates and insecticidal activity (Follmer
Severo et al., 2006), play an important role in the et al., 2001, 2004a). Besides these activities, jack-
pathogenesis of human and animal diseases. In bean canatoxin is also lethal to rats and mice by an
the fungi Coccidioides posadassi (Mirbod-Donovan intraperitoneal route (Carlini and Guimarães, 1981;
et al., 2006), C. immitis (Li et al., 2001), Para- Follmer et al., 2001) and inhibits the growth of
coccidioides brasiliensis (Rappleye and Goldman, phytopathogenic fungi (Oliveira et al., 1999). The
2006) and Cryptococcus neoformans (Cox et al., entomotoxic effects of jackbean ureases and of the
2000; Olszewski et al., 2004), urease production is soybean embryo-specific soybean urease persisted
also probably related to pathogenesis in humans. after treatment of the enzymes with an irreversible
Despite the abundance of ureases in plants, little inhibitor, demonstrating that protein domain(s)
has been revealed about their biological roles other than the active site might be involved (Follmer
(Polacco and Holland, 1993; Sirko and Brodzik, et al., 2004a, b). Actually, an internal entomotoxic
2000). Urease has been proposed to function peptide released upon digestion by insect cathepsins
coordinately with arginase in the utilization of seed accounts for the insecticidal activity of canatoxin
protein reserves during germination (Thompson, (Carlini et al., 1997; Ferreira-DaSilva et al., 2000).
1980). A second proposed role is the assimilation of These findings reinforce the hypothesis that ureases
urea derived from ureide metabolism (Shelp and might be involved in plant defense mechanisms
Ireland, 1985) or imported from the environment (Carlini and Grossi-de-Sá, 2002). In the present
since urea is an effective foliar fertilizer (Zonia et al., work, we evaluated the inhibitory activities of two
1995). plant ureases and a bacterial urease on mycelial
The best-studied urease derives from jackbean growth and/or spore germination of filamentous
(Canavalia ensiformis) which was crystallized by fungi, most of them important plant pathogens.
Sumner (1926), representing the first crystals of a
characterized enzyme. We described a family of 2. Material and methods
urease-related genes in jackbean (Pires-Alves et al.,
2003) and characterized an isoform of jackbean 2.1. Embryo-specific soybean urease (SBU) and jack
urease, named canatoxin (Carlini and Guimarães, bean urease (JBU)
1981; Follmer et al., 2001, 2004a). Urease activity
was found in all tissues of soybean plants (Polacco Embryo-specific urease was isolated from a local
and Havir, 1979; Das et al., 2002). Soybean commercial variety of soybeans according to Follmer
produces two urease isoenzymes (Holland et al., et al. (2004a), except for the last step in the
1987) that share 87% identity at the amino acid purification protocol aimed to separate urease iso-
level (Torisky and Polacco, 1990; Goldraij et al., forms. The jackbean (major) urease was purified from
2003). The embryo-specific urease, encoded by the C. ensiformis meal according to Follmer et al.
Eu1 gene, is synthesized only in the developing (2004a). Homogeneity of purified proteins was
embryo (Polacco and Havir, 1979; Polacco and checked by SDS-PAGE. The purified embryo-specific
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soybean urease (SBU) and jackbean urease (JBU) amount of enzyme required to release 1 mmol
were kept in 20 mM sodium phosphate (NaPB), pH NH3 min1 at 37 1C and pH 7.5 under the condi-
7.5, containing 1 mM EDTA and 1 mM b-mercap- tions described.
toethanol, at 4 1C.
2.6. Fungi
2.2. H. pylori recombinant urease (HPU)
Rhizoctonia solani, Fusarium solani, Fusarium oxy-
E. coli SE5000 cells expressing H. pylori urease sporum, Trichoderma sp., Trichoderma pseudokoningii,
(McGee et al., 1999) were kindly provided by Trichoderma viride, Penicillium sp., Colletotrichum
Dr. Harry L. Mobley, University of Michigan musae and Curvularia lunata were from fungal
Medical School, Ann Arbor, MI, USA. The crude collections maintained at the Department of Biochem-
extract obtained from an overnight culture was istry and Molecular Biology, Universidade Federal do
fractionated with ammonium sulfate at 30–70% Ceará, Fortaleza, CE, Brazil, or at Universidade do
saturation. The urease-enriched material was dia- Vale dos Sinos, São Leopoldo, RS, Brazil. Penicillium
lyzed against 20 mM NaPB, pH 7.8, 5 mM herguei, Colletotrichum gloeosporioides and Aspergillus
b-mercaptoethanol and 1 mM EDTA, and mixed glaucus were kindly provided by Dr. Valdirene
with Q-Sepharose (Amersham-Biotech Pharmacia) Gomes, Universidade Estadual do Norte Fluminense,
(1 mL resin per 10 mg protein) equilibrated with the Campos dos Goytacazes, RJ, Brazil.
same buffer. Elution of urease-enriched fractions
was achieved by adding 200 mM NaCl to the 2.7. Antifungal activity
equilibrium buffer. The material was then applied
into a Superose 6HR 10/30 column (Amersham- (1) Disc plate diffusion assay: Spores (106 in 100 mL)
Biotech Pharmacia) equilibrated in the same buffer. were spread over the surface of Petri dishes
Pooled fractions displaying urease activity were (86-mm internal diameter) containing potato-
used in the experiments. Homogeneity of the dextrose-agar (PDA, Acumedia Manufacturer,
purified protein was checked by SDS-PAGE. Inc.). Sterile filter paper discs (5-mm diameter,
Whatman 3MM) loaded with about 40 mL of
2.3. Chemical modifications of ureases protein solutions (12.5 mg protein mL1 in 20 mM
NaPB, 1 mM EDTA, 1 mM b-mercaptoethanol,
Proteins were treated with 0.1 mM p-hydroxy- pH 7.5) were placed in the center onto pre-
mercuribenzoate (p-HMB, Sigma Chemical Co.) for viously inoculated PDA plates. The dishes were
24 h at 4 1C, and excess reagent was removed by inverted and then incubated at 28 1C. Antifungal
exhaustive dialysis against 10 mM NaPB, pH 7.0, activity was visualized as a zone of inhibition of
without b-mercaptoethanol (Follmer et al., 2004a). fungal growth around the paper disc. Alterna-
In the experiments using modified ureases, the last tively, to evaluate spore germination, 106 spores
change of dialysis buffer was used as control. were suspended in 40 mL of protein solutions
(12.5 mg protein mL1), incubated for 2 h at 28 1C
2.4. Protein content determination and then inoculated onto the PDA-containing
Petri dishes. The toxic effect of ureases was
The protein content of fractions was determined assessed by visual comparison of the mycelial
by the method of Bradford (1976), using bovine development of protein-treated fungi and un-
serum albumin as standard. treated controls.
(2) Turbidimetric evaluation of fungal growth:
2.5. Urease activity Spores (103 in 10 mL) were inoculated onto
96-well plates containing 110 mL potato dextrose
Aliquots of 50 mL of 100 mM urea solution were broth (PDB, Becton Dickenson Co.) buffered to
mixed with protein samples and buffered with pH 7.0 with 10 mM NaPB (to avoid precipita-
20 mM NaPB, pH 7.5, to give a final volume of tion of urease), containing 1 mM b-mercap-
500 mL. Reaction mixtures were incubated for toethanol, and incubated at 28 1C for 16 h,
30 min at 37 1C and the ammonia released was followed by addition of 50 mL of protein
determined colorimetrically (Weatherburn, 1967). solutions in the same buffer (time zero). As
One unit of urease activity was defined as the controls, 50 mL buffer alone or 9.5% v/v H2O2
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was used. The plates were incubated at 28 1C experiments were approximately 0.525, 1.62 and
without shaking and the absorbance at 430 nm 2.52 U mg1 protein min1 at pH 7.5, respectively.
(A430) was followed on a plate reader (Spectra- After treatment with r-hydroxy-mercuribenzoate,
max, Molecular Devices) at 12 h intervals up to which irreversibly blocks the enzymatic activity of
72 h (Broekaert et al., 1990). ureases by alkylating a cysteine residue near the
active site (Jabri et al., 1995), modified SBU or JBU
2.8. Determination of antifungal IC50 showed less than 5% of their initial enzymatic
activities.
Concentrations of SBU causing 50% inhibition of
growth (IC50) were calculated for some fungi. 3.2. Effect of ureases on fungal growth
Fungal growth of triplicates was measured turbidi-
metrically (as described above) in the presence of As a first approach to test the antifungal proper-
buffer (100% growth) or six different concentra- ties of ureases, the ability of embryo-specific
tions (from 0.05 to 1.15 mM) of SBU. The protein soybean urease (SBU) to inhibit hyphal growth
concentrations reducing growth to 50% of control and the germination of fungal spores was investi-
values after 72 h were taken as IC50. gated using a disc plate diffusion assay. Table 1
shows that SBU suppressed mycelial growth and/or
2.9. Scanning electron microscopy inhibited spore germination of F. solani, P. herguei,
C. lunata and three species of Trichoderma. To
Preparation of samples for scanning electron micro- obtain more quantitative data, inhibition of
scopy (SEM) analysis was done according to Faganello fungal growth was analyzed turbidimetrically in
et al. (2006). P. herguei mycelia cultured for 48 h as in microwell plates. Fig. 1 shows dose curves for the
the turbidimetric assay in the absence or presence of inhibitory effects of SBU upon the ascomycetes
0.57 mM SBU were recovered by filtration in paper phytopathogens C. musae, the etiological agent of
filters (Whatmann no. 1). The retained mycelial mass antracnose in bananas and the foliar blight on
was fixed for 4 days at 4 1C with 2% (v/v) Zoysia grass, and C. lunata, the most commonly
glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium associated organism with the crown rot disease,
cacodylate buffer at pH 7.2. Post-fixation was carried an important problem in export bananas. Table 2
out in 1% (w/v) osmium tetroxide in the same buffer. shows that SBU antifungal activity (IC50) in the
The specimens were rinsed in buffer, dehydrated in a turbidimetric assay is within the 0.1–1.0 mM
series of 30–100% acetone solutions, dried at critical range for 4 out of 7 species of the tested fungi,
point in CO2 (CPD 030 BALTEC), and coated with including against F. oxysporum, a phytopathogen
gold in a sputter-coater (SCD 050 BALTEC). The that causes wilt disease in more than a hundred
material was examined in a Jeol JSM 6060 scanning species of plants. However, no antifungal activity
electron microscope at the Centro de Microscopia was observed for T. viride or F. solani. Fig. 2
Eletrônica da Universidade Federal do Rio Grande do compares the growth of seven different fungi
Sul (CME/UFRGS, Porto Alegre, RS, Brazil). after 48 and 72 h of exposure to 0.57 mM of SBU
or JBU. C. musae and P. herquei (a maize pathogen)
2.10. Statistical analysis were affected by both ureases. SBU inhibited
also the growth of F. oxysporum, but even the
The results were subjected to analysis of variance highest concentration tested for this protein
(ANOVA) and the significance of differences among (1.15 mM) did not affect the growth of F. solani.
means was determined by the Tukey test, with Although T. viride was inhibited by SBU in the agar
pp0.05 considered statistically significant. plate tests (Table 1), it was not affected in liquid
medium assay at the maximal dose tested (1.15 mM).
3. Results On the other hand, F. solani and F. oxysporum
were inhibited only by JBU. Fig. 2 also shows
3.1. Native and modified ureases that the antifungal effect of 0.57 mM SBU was
more persistent, with little or no change at 72 h
The ureolytic specific activities of soybean em- as compared to 48 h exposition. In contrast, the
bryo-specific urease (SBU), jackbean major urease inhibitory effect of JBU was short-lasting as the
(JBU) and H. pylori urease (HPU) used in the inhibition seen at 48 h decreased or was overcome
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Table 1
Antifungal activity of soybean seed urease on mycelial growth and spore germination of filamentous fungi

Fungi Phylum/class/pathogenicity Antifungal activity

Mycelial growtha Spore germinationb

Aspergillus glaucus Ascomycota/Eurotiomycetes – –

Potential opportunist or pathogen, allergenic
Colletotrichum gloeosporioides Ascomycota/Sordariomycetes – –
Plant pathogen
Curvularia lunata Ascomycota/Euascomycetes 3.2070.70 +
Plant pathogen
Fusarium oxysporum Ascomycota/Euascomycetes – –
Plant pathogen
Fusarium solani Ascomycota/Euascomycetes – +
Plant pathogen
Penicillium herquei Ascomycota/Eurotiomycetes 3.8170.95 +
Plant pathogen, Saprophytic
Penicillium sp. Ascomycetes – –
Soilborne fungus, Saprophytic
Rhizoctonia solani Basidiomycota/Homobasidiomycetes – –
Plant pathogen
Trichoderma pseudokoningii Ascomycota/Sordariomycetes 4.0270.66 +
Plant pathogen
Trichoderma sp. Ascomycetes + +
Trichoderma viride Ascomycota/Sordariomycetes 2.7570.67 +
Saprophytic

Solutions of soybean seed urease (500 mg/assay) were applied onto disc papers for the assay of hyphal growth inhibition or incubated with
spore suspensions subsequently tested for germination.
a
Diameter in centimeters of inhibition zone after 48 h at 28 1C (mean7SD, N ¼ 3);
b
+, inhibition; , no inhibition.

Fig. 1. Inhibitory effect of soybean urease (SBU) on growth of C. musae (left panel) and C. lunata (right panel). Spores (103 in 10 mL) of
each fungus were inoculated onto 96-well plates containing 110 mL of potato dextrose broth buffered to pH 7.0 with 10 mM NaPB
containing 1 mM b-mercaptoethanol. After incubation at 28 1C for 16 h, 50 mL of SBU solutions in the same buffer were added to the
wells. As controls, 50 mL buffer alone or 9.5% (v/v) H2O2 was used. The plates were further incubated at 28 1C and the absorbance at
430 nm (A430) was read every 12 h up to 72 h. Data shown (mean7SD, triplicate points) are from one experiment out of at least three with
similar results. Means indicated by (*) or (**) are statistically different from fungal growth in the presence of buffer according to Tukey’s
test (po0.05 or 0.02, respectively).
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Table 2
Antifungal activity (IC50) of soybean seed urease (SBU)

Fungus SBU-IC50 (mM)

Fusarium solani NI
Colletotrichum musae 0.8170.04
Curvularia lunata 0.6170.02
Trichoderma viride NI
Penicillium herguei 0.2770.02
Fusarium oxysporum 0.6170.03

Fungal growth of triplicates was measured turbidimetrically (as

described above) in the presence of buffer (100% growth) or six
different concentrations (0.05–1.15 mM) of SBU. The protein
concentrations reducing growth to 50% of control values after
72 h were taken as IC50. Values are mean7SD of triplicate points
(N ¼ 6). NI: no-inhibition at the maximal dose tested.

by most fungi at 72 h. For C. lunata and P. herguei,

inhibition caused by 0.57 mM JBU at 48 h gives
place to an enhanced growth after 72 h. Control
experiments in which SBU and JBU were submitted
to the same incubation time (72 h) and temperature
(28 1C) in the absence of fungi indicated that the
proteins resisted these conditions without appreci-
able loss of enzymatic activity.
Fig. 3 shows that the recombinant H. pylori Fig. 2. Susceptibility of different fungi to soybean (upper panel)
and jackbean (lower panel) ureases. Spores (103 in 10 mL) were
urease also inhibited the mycelial growth of inoculated onto 96-well plates containing 110 mL of PDB buffered
P. herguei and C. lunata. Although the microbial to pH 7.0 with 10 mM NaPB, 1 mM b-mercaptoethanol,
enzyme shows 2- to 5-fold higher ureolytic activity incubated at 28 1C for 16 h, and then 50 mL of the protein
as compared to JBU or SBU, respectively, it was solutions in the same buffer were added to the wells. The plates
less effective to inhibit fungal growth when the same were incubated at 28 1C and the absorbance at 430 nm (A430) after
72 h was recorded. Data (mean7SD of triplicate points) are
dose of the three proteins was used. expressed as percentage of correspondent control growth in the
presence of buffer alone. One experiment out of at least three
with similar results is shown. Means indicated by (*) or (**) are
3.3. Antifungal effect of ureases is not related to the statistically different from fungal growth in the presence of buffer
enzymatic activity (100%) according to Tukey’s test (po0.05 or 0.02, respectively).

The lower antifungal activity of HPU suggested 3.4. Scanning electron microscopy analysis
that ammonia release is not important for the
protein’s fungicidal/fungistactic property. To assess Marked reduction in the turbidity of the growing
the involvement of the enzymatic activity of SBU cultures was seen under the present experimental
and JBU in their antifungal property, p-hydroxy- conditions. In control P. herguei, hyphae appeared
mercuribenzoate inactivated enzymes (mSBU and linear, elongated and with smooth surfaces (Fig. 5).
mJBU, respectively) were tested on P. herguei In contrast, fungi treated with SBU or JBU showed
(highly sensitive) and C. lunata (moderately sensi- vegetative hyphae appearing branched, disorga-
tive) development. Fig. 4 shows that modified nized and collapsed, some with distorted ends.
JBU, although devoid of ureolytic activity, was Other important alterations of P. herguei in the
fungal growth. The same result was obtained with presence of ureases include cell enlargement (dis-
modified SBU (not shown), indicating that the tended balloon-shaped cells), irregularities of the
ureolytic activity of the proteins does not contribute hyphal surface, abnormal hyphal outgrowth and
to their antifungal effect. ruptures of the cell wall, suggestive of plasmolysis.
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proteins, chitinases, chitin-binding proteins, defen-

sins, defensin-like proteins, ribosome-inactivating
proteins, lipid transfer protein-like proteins, pro-
tease inhibitors and lectins, among others (Broe-
kaert et al., 1997; De Lucca, 2000, 2005;
Selitrennikoff, 2001).
To our knowledge, there are no reports so far on
the isolation of antifungal proteins from plants of
the Canavalia genus. The glucose–mannose lectins
from C. ensiformis (concanavalin A) and from
the closely related C. gladiata are devoid of
antifungal activity (Wang and Ng, 2007; Wong
and Ng, 2005). An endochitinase (Schlesier et al.,
1998) and an inactivated chitinase-like protein
(concanavalin B, (Hennig et al., 1995) were isolated
from C. ensiformis, but they were not assayed for
antifungal properties.
In soybeans, volatiles derived from the enzymatic
action of lipoxygenase are able to inhibit the growth
of Aspergillus flavus and the production of aflatox-
ins (Boue et al., 2005). Glysojanin, a protein
(25 kDa) homologous to chitin synthase isolated
from the black soybean Glycine soja, showed potent
antifungal activity against the fungi F. oxysporum
and Mycosphaerella arachidicola (Ngai and Ng,
Fig. 3. Effect of H. pylori urease (HPU) on fungal growth. 2003).
Spores (103 in 10 mL) of P. herguei (panel A) or C. lunata Regarding H. pylori, it has been shown that HP
(panel B) were inoculated onto 96-well plates containing 110 mL (2–20), a cecropin-like peptide derived from the N-
of PDB buffered to pH 7.0 with 10 mM NaPB, 1 mM b-
mercaptoethanol, incubated at 28 1C for 16 h, and then 50 mL of
terminal region of the ribosomal protein L1,
HPU solution in the same buffer was added to the wells. As possesses antimicrobial activity and exerts antifun-
control, 50 mL buffer alone or 9.5% (v/v) H2O2 was used. The gal effects by damaging the plasma membranes of
plates were incubated at 28 1C and the absorbance at 430 nm Candida albicans (Ribeiro and Medina-Acosta,
(A430) was read every 12 h up to 60 h. Data shown (mean7SD of 2003).
triplicate points) are from one experiment out of at least three
with similar results. Means indicated by (*) or (**) are
In this work, we showed that the major jackbean
statistically different from fungal growth in the presence of urease and the soybean seed urease were able to
buffer according to Tukey’s test (po0.05 or 0.02, respectively). inhibit the vegetative growth and/or germination of
several filamentous fungi at sub-micromolar con-
centrations. Although less active, the two-chain
4. Discussion urease of H. pylori was also detrimental to fungal
growth. Canatoxin, an isoform of jackbean urease,
Antifungal peptides and proteins are among the was previously shown to inhibit the growth of the
arsenal of compounds plants produce to combat phytopathogenic fungi Macrophamina phaseolina,
phytopathogenic fungi (Broekaert et al., 1997; Sclerotium rofstii and Colletotrichum gloesporioides
Selitrennikoff, 2001; Thomma et al., 2002; De using 1 mg purified protein in a disc plate diffusion
Lucca et al., 2005). Transgenic plants expressing assay (Oliveira et al., 1999).
antifungal proteins are expected to be resistant to Little is known about the physiological role of
the devastating damage caused by fungal infections ureases, especially in plants. A perception shared by
(De Lucca, 2000, 2005). Several families of anti- many is that urease plays no major role in plants
fungal plant proteins have been isolated from because its substrate, urea, is not a major plant
different organs (seeds, leaves and flowers) and metabolite (Polacco and Holland, 1993).
intercellular fluids. Major groups of plants proteins The distribution and accumulation pattern of ureases
with antifungal activity comprise thaumatin-like in leguminous seeds during embryo maturation are
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Fig. 4. Effect of p-hydroxymercuribenzoate modified jackbean urease (mJBU) on fungal growth. Spores (103 in 10 mL) of P. herguei
(panel A) or C. lunata (panels B–D) were inoculated onto 96-well plates containing 110 mL of PDB buffered to pH 7.0 with 10 mM NaPB,
1 mM b-mercaptoethanol, incubated at 28 1C for 16 h, and then 50 mL of native or modified JBU solution was added to the wells. As
control, 50 mL buffer or 9.5% v/v H2O2 was used. The plates were incubated at 28 1C and the absorbance at 430 nm (A430) was read every
12 h up to 60 h. Data shown (mean7SD of triplicate points) are from one experiment out of at least three with similar results. Means
indicated by (*) or (**) are statistically different from fungal growth in the presence of buffer according to Tukey’s test (po0.05 or 0.02,
respectively).

suggestive of an important physiological role. Both it can be concluded that the internal fragment of
jackbean urease isoforms and the embryo-specific plant ureases displaying entomotoxic activity is not
soybean urease present entomotoxic activity, but involved in the fungicidal property of plant and
not the tri-chain bacterial enzyme from Bacillus H. pylori enzymes. Confirming this hypothesis, we
pasteurii (Follmer et al., 2004a, b). The entomotoxi- observed that a recombinant insecticidal peptide
city relies on an internal fragment of the proteins derived from an isoform of jackbean urease
released upon hydrolysis by insect midgut proteases (jaburetox-2Ec) did not affect the growth of
(Ferreira-DaSilva et al., 2000). The lack of insecti- different fungi (unpublished data).
cidal properties of B. pasteurii urease is probably The antifungal activity of ureases is not related to
related to the absence of part of this entomotoxic their enzymatic activity. This was shown by the
peptide, whose amino acid sequence corresponds to lower antifungal effect of the more enzymatically
a gap between the C-terminal of B chain and the active H. pylori enzyme, as well as by the persistence
N-terminal of the C chain of the bacterial enzyme of the antifungal properties of plant ureases
(Follmer et al., 2004a). Here we show that H. pylori irreversibly inactivated by p-hydroxy-mercuribenzo-
urease has fungicidal activity. This two-chain ate. Canatoxin is devoid of chitinase and proteinase
bacterial urease lacks the same part of the protein inhibitory activities (Carlini et al., 1997), which
molecule as the B. pasteurii enzyme when both are could account for the antifungal property of the
compared to one-chain plant ureases (Fig. 6). Thus, jackbean ureases. Preliminary results showed that
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A.B. Becker-Ritt et al. / Toxicon 50 (2007) 971–983 979

Fig. 5. Scanning electron microscopy of P. herguei. The fungi were fixed after 48 h of culture at 28 1C in the absence (panels C and F) or
presence of 0.57 mM of soybean urease (panels A and D) or jackbean urease (panels B and E). White bars: 5 mm.

JBU is also able to inhibit the growth of the The antifungal activity of plant defensins, such as
oomycete Pythium oligandrum whose cell walls lack Psd1 and Psd2, isolated from Pisum sativum
chitin, reinforcing that the antifungal property of (Almeida et al., 2000) appears to require specific
ureases probably does not involve a chitin-binding binding to membrane targets, particularly glyco-
or chitinase activity. sphingo lipids, resulting in fungal growth arrest
The antifungal properties of ureases affected (Thevissen et al., 2004, 2005). Carbohydrate-
fungi pertaining to Ascomycota and Basidiomycota. binding properties are also a common feature of
On the other hand, not all fungi tested were H. pylori urease (Icatlo et al., 2000; Aspholm-Hurtig
affected under our experimental conditions, with et al., 2004), jackbean ureases (Follmer et al., 2001)
contrasting responses seen even at genus level. We and some antifungal plant lectins (Lungu
have no clues yet to the factors that dictate(s) the et al., 1990), such as that of the stinging needle
sensitivity of different fungi to urease antifungal agglutinin (Does et al., 1999) or from the legume
activity. Luetzelburigia auriculata (Melo et al., 2005). Thus,
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M65260 MKLSPREVEKLGLHNAGYLAQKRLARGVRLNYTEAVALIASQIMEYARDGEKTVAQLMCL 60
AY230157 MKLSPREVEKLGLHNAGYLAQKRLARGLRLNYTEAVALIATQIMEFARDGEKTVAQLMCI 60
Hpylori MKLTPKELDKLMLHYAGELAKKRKEKGIKLNYVEAVRLISAHIMEEARRGKKTAAELMQE 60
***:*:*::** ** ** **:** :*::***.*** **:::*** ** *:**.*:**

M65260 GQHLLGRRQVLPAVPHLLNAVQVEATFPDGTKLVTVHDPISRENGELQEALFGSLLPVPS 120

AY230157 GKHLLGRRQVLPEVQHLLNAVQVEATFPDGTKLVTVHDPISCEHGDLGQALFGSFLPVPS 120
Hpylori GRTLLKPDDVMDGVASMIHEVGIEAMFPDGTKLVTVHTPIEAN----------------- 103
*: ** :*: * ::: * :** *********** **. :

M65260 LDKFAETKEDNRIPGEILCEDECLTLNIGRKAVILKVTSKGDRPIQVGSHYHFIEVNPYL 180

AY230157 LDKFAENKEDNRIPGEIIYGDGSLVLNPGKNAVILKVVSNGDRPIQVGSHYHFIEVNPYL 180
Hpylori ---------GKLVPGELFLKNEDITINEGKKAVSVKVKNVGDRPVQIGSHFHFFEVNRCL 154
.: :***:: : :.:* *::** :** . ****:*:***:**:*** *

M65260 TFDRRKAYGMRLNIAAGTAVRFEPGDCKSVTLVSIEGNKVIRGGNAIADGPVNETNLEAA 240

AY230157 TFDRRKAYGMRLNIAAGNATRFEPGECKSVVLVSIGGNKVIRGGNNIADGPVNDSNCRAA 240
Hpylori DFDREKTFGKRLDIASGTAVRFEPGEEKSVELIDIGGNRRIFGFNALVDRQADNESKKIA 214
***.*::* **:**:*.*.*****: *** *:.* **: * * * :.* .:: . . *

M65260 MHAVRSKGFGHEEEKDASEGFTKEDPNCPFNTFIHRKEYANKYGPTTGDKIRLGDTNLLA 300

AY230157 MKAVVTRGFGHVEEENAREGVTGED--YSLTTVISREEYAHKYGPTTGDKIRLGDTDLFA 298
Hpylori LHRAKERGFHGAKSDDNYVKTIKEM------KKISRKEYASMYGPTTGDKVRLGDTDLIA 268
:: . :** :..: * . * *:*** ********:*****:*:*

M65260 EIEKDYALYGDECVFGGGKVIRDGMGQSCGHPPAISLDTVITNAVIIDYTGIIKADIGIK 360

AY230157 EIEKDFALYGDECVFGGGKVLRDGMGQSCGDPPAISLDTVITNAVIIDYSGIIKADIGIK 358
Hpylori EVEHDYTIYGEELKFGGGKTLREGMSQSN-NPSKEELDLIITNALIVDYTGIYKADIGIK 327
*:*:*:::**:* *****.:*:**.** .*. .** :****:*:**:** *******

M65260 DGLIASIGKAGNPDIMNGVFSNMIIGANTEVIAGEGLIVTAGAIDCHVHYICPQLVYEAI 420

AY230157 DGLIVSIGKAGNPDIMDDVFFNMIIGANTEVIAGEGLIVTAGAIDCHVHYICPQLVDEAI 418
Hpylori DGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAGGIDTHIHFISPQQIPTAF 387
** *..***.** *: :.* *: :*. **.:**********.** *:*:*.** : *:

M65260 SSGITTLVGGGTGPAAGTRATTCTPSPTQMRLMLQSTDYLPLNFGFTGKGSSSKPDELHE 480

AY230157 SSGITTLVGGGTGPTAGTRATTCTPAPSQMKLMLQSTDDLPLNFGFTGKGSSSKPDELHD 478
Hpylori ASGVTTMIGGGTGPADGTNATTITPGRRNLKWMLRAAEEYSMNLGFLAKGNASNDASLAD 447
:**:**::******: **.*** **. ::: **:::: .:*:** .**.:*: .* :

M65260 IIKAGAMGLKLHEDWGSTPAAIDNCLTIAEHHDIQINIHTDTLNEAGFVEHSIAAFKGRT 540

AY230157 IIKAGAMGLKLHEDWGSTPAAIDSCLTVADQYDIQINIHTDTLNEAGFVEHSIAAFKGRT 538
Hpylori QIEAGAIGFKIHEDWGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMAAIAGRT 507
*:***:*:*:*****:**:**: .* :*:::*:*: ********** **.::**: ***

M65260 IHTYHSEGAGGGHAPDIIKVCGIKNVLPSSTNPTRPLTSNTIDEHLDMLMVCHHLDREIP 600

AY230157 IHTYHSEGAGGGHAPDIIKVCGMKNVLPSSTNPTRPLTLNTIDEHLDMLMVCHHLNREIP 598
Hpylori MHTFHTEGAGGGHAPDIIKVAGEHNILPASTNPTIPFTVNTEAEHMDMLMVCHHLDKSIK 567
:**:*:**************.* :*:**:***** *:* ** **:*********::.*

M65260 EDLAFAHSRIRKKTIAAEDVLNDIGAISIISSDSQAMGRVGEVISRTWQTADPMKAQTGP 660

AY230157 EDLAFACSRIREGTIAAEDILHDIGAISIISSDSQAMGRVGEVISRTWQTANKMKVQRGP 658
Hpylori EDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTWQTADKNKKEFGR 627
**: ** **** ****** *:*:* :** **************:******: * : *

M65260 LKCDSSDNDNFRIRRYIAKYTINPAIANGFSQYVGSVEVGKLADLVMWKPSFFGTKPEMV 720

AY230157 LQPGESDNDNFRIKRYIAKYTINPAIANGFSQYVGSVEVGKLADLVMWKPSFFGAKPEMV 718
Hpylori LKEEKGDNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMI 687
*: ..*******:**::*********:*:*:*********:****:*.*:***.**:*:

M65260 IKGGMVAWADIGDPNASIPTPEPVKMRPMYGTLGKAGGALSIAFVSKAALDQRVNVLYGL 780

AY230157 IKGGVVAWADMGDPNASIPTPEPVKMRPMFGTLGKAGGALSIAFVSKAAVDQRVHALYGL 778
Hpylori IKGGFIALSQMGDANASIPTPQPVYYREMFAHHGKAKYDANITFVSQAAYDKGIKEELGL 747
****.:* :::**.*******:** * *:. *** .*:***:** *: :: **

M65260 NKRVEAVSNVRKLTKLDMKLNDALPEITVDPESYTVKADGKLLCVSEATTVPLSRNYFLF 840

AY230157 NKRVEAVGNVRKLTKLDMKLNDSLPQITVDPDNYTVTADGEVLTSFATTFVPLSRNYFLF 838
Hpylori ERQVLPVKNCRNITKKDMQFNDTTAHIEVNPETYHVFVDGKEVTLNQSIK---------- 797
:::* .* * *::** **::**: ..* *:*:.* * .**: : :

Fig. 6. Alignment of primary sequences of jackbean major urease (accession code M65260), embryo-specific urease (AY230157) and
H. pylori urease (subunit A-AAO34402; subunit B-AAO34403). Amino acids participating in the ureolytic active site are shadowed in
gray. The Cys residues modified by p-hydroximercurybenzoate (Jabri et al., 1995) are shown in a box.
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the sugar composition of fungal cells might be Aspholm-Hurtig, M., Dailide, G., Lahmann, M., Kalia, A., Ilver,
implicated in the difference of their susceptibility to D., Roche, N., Vikstrom, S., Sjostrom, R., Linden, S.,
ureases. Backstrom, A., Lundberg, C., Arnqvist, A., Mahdavi, J.,
Nilsson, U.J., Velapatino, B., Gilman, R.H., Gerhard, M.,
The effect of ureases on the morphology of Alarcon, T., Lopez-Brea, M., Nakazawa, T., Fox, J.G.,
P. herguei, a maize pathogen, was investigated. Correa, P., Dominguez-Bello, M.G., Perez-Perez, G.I., Blaser,
Scanning electron microscopy of urease-treated M.J., Normark, S., Carlstedt, I., Oscarson, S., Teneberg, S.,
fungi revealed that cell wall is damaged, suggesting Berg, D.E., Boren, T., 2004. Functional adaptation of BabA,
that ureases may interfere with the osmotic balance, the H. pylori ABO blood group antigen binding adhesin.
Science 305, 519–522.
as described for micafungin on Aspergillus fumiga- Boue, S.A., Shih, B.Y., Carter-Wientjes, C.H., Cleveland, T.E.,
tus (Nishiyama et al., 2005). 2005. Effect of soybean lipoxygenase on volatile generation
Most fungi are able to hydrolyze urea into and inhibition of Aspergillus flavus mycelial growth. J. Agric.
ammonia. In some fungi, urea is hydrolyzed by Food Chem. 53, 4778–4783.
urease, a nickel-containing enzyme homologous to Bradford, M.M., 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
the plant and bacterial enzymes studied in this principle of protein–dye binding. Anal. Biochem. 72, 248–254.
work. In other fungi, urea is first carboxylated to Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A., Vanderley-
yield allophanate by an ATP-hydrolyzing urease or den, J., 1990. An automated quantitative assay for fungal
urea amidolyase (Nishiya and Imanaka, 1993; growth-inhibition. FEMS Microbiol. Lett. 69, 55–59.
Sumrada et al., 1982). We are presently testing Broekaert, W.F., Cammue, B.P.A., DeBolle, M.F.C., Thevissen,
K., DeSamblanx, G.W., Osborn, R.W., 1997. Antimicrobial
fungal ureases for the ureolysis-independent proper- peptides from plants. Crit. Rev. Plant Sci. 16, 297–323.
ties displayed by plant and microbial ureases. Carlini, C.R., Grossi-de-Sá, M.F., 2002. Plant toxic proteins with
In conclusion, the antifungal properties of plant insecticidal properties. A review on their potentialities as
and bacterial ureases we described here may shed bioinsecticides. Toxicon 40, 1515–1539.
some light on the physiological roles of these Carlini, C.R., Guimarães, J.A., 1981. Isolation and characteriza-
tion of a toxic protein from Canavalia ensiformis (jack bean)
proteins. The data presented further reinforce the seeds, distinct from concanavalin A. Toxicon 19, 667–675.
possibility of a protective role of plant ureases Carlini, C.R., Oliveira, A.E., Azambuja, P., Xavier-Filho, J.,
besides phytophagous insects also against phyto- Wells, M.A., 1997. Biological effects of canatoxin in different
pathogenic fungi. insect models: evidence for a proteolytic activation of the
toxin by insect cathepsin-like enzymes. J. Econ. Entomol. 90,
340–348.
Acknowledgments Cox, G.M., Mukherjee, J., Cole, G.T., Casadevall, A., Perfect,
J.R., 2000. Urease as a virulence factor in experimental
The authors wish to thank Dra Valdirene Gomes cryptococcosis. Infect. Immun. 68, 443–448.
(UENF, Brazil) for kindly providing some of the Das, N., Kayastha, A.M., Srivastava, P.K., 2002. Purification
fungi used in this work, and Dr. Osana Cunha de and characterization of urease from dehusked pigeonpea
(Cajanus cajan L.) seeds. Phytochemistry 61, 513–521.
Lima (Department of Micology, Fundac- ão Oswal- De Lucca, A.J., 2000. Antifungal peptides: potential candidates
do Cruz, Instituto Manguinhos, Rio de Janeiro, for the treatment of fungal infections. Exp. Opin. Invest.
Brazil) for helpful discussions on the microscopy Drugs 9, 273–299.
analyses. We are grateful to Dr. Joseph C. Polacco, De Lucca, A.J., Cleveland, T.E., Wedge, D.E., 2005. Plant-
University of Missouri, Columbia, MO, USA, for derived antifungal proteins and peptides. Can. J. Microbiol.
51, 1001–1014.
suggestions and critical reading of the manuscript. Dixon, N.E., Gazzola, C., Watters, J.J., Blakeley, R.L., Zerner,
This work was supported by CNPq (Conselho B., 1975. Inhibition of jack bean urease (EC [Link]) by
Nacional de Desenvolvimento Cientı́fico e Tecno- acetohydroxamic acid and by phosphoramidate. An equiva-
lógico), CAPES (Coordenac- ão de Aperfeic- oamento lent weight for urease. J. Am. Chem. Soc. 97, 4130–4131.
de Pessoal de Nı́vel Superior) and FAPERGS Does, M.P., Houterman, P.M., Dekker, H.L., Cornelissen,
B.J.C., 1999. Processing, targeting, and antifungal activity
(Fundac- ão de Amparo a Pesquisa do Estado do of stinging nettle agglutinin in transgenic tobacco. Plant
Rio Grande do Sul). Physiol. 120, 421–431.
Faganello, J., Arruda, W., Schrank, A., Vainstein, M.H., 2006.
An alternative method to prepare samples of the pathogenic
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