Microbiology (2012), 158, 1897–1907
DOI 10.1099/mic.0.058511-0
A low-molecular-mass aspartic protease inhibitor
from a novel Penicillium sp.: implications in
combating fungal infections
Vishnu Menon and Mala Rao
Correspondence
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India
Mala Rao
[email protected]
Received 17 February 2012
Revised
30 March 2012
Accepted 2 April 2012
A low-molecular-mass aspartic protease inhibitor was isolated from a novel Penicillium sp. The
inhibitor was purified to homogeneity, as shown by reversed-phase HPLC and SDS-PAGE. The
Mr of the inhibitor was 1585 and the amino acid composition showed the presence of D, D, D, E,
A, K, L, Y, H, I and W residues. The steady-state kinetic interactions of Aspergillus saitoi aspartic
protease with the inhibitor revealed the reversible, competitive, time-dependent tight-binding
nature of the inhibitor, with IC50 and Ki values of 1.8 and 0.85 mM, respectively. Fluorescence
spectroscopy and circular dichroism analysis showed that inactivation of the enzyme was due to
binding of the inhibitor to the active site. The inhibitor was found to inhibit mycelial growth and
spore germination of Aspergillus fumigatus and Aspergillus niger in vitro with MIC values of 1.65
and 0.30 mg ml”1, respectively. This study will potentially open the way towards the development
of a tight-binding peptidic inhibitor against fungal aspartic proteases to combat human fungal
infections.
INTRODUCTION
Several proteases are essential for propagation of disease,
and hence inhibition of different proteases is emerging as a
promising approach in medical applications for the
treatment of cancer, obesity, hepatitis and herpes, cardiovascular, inflammatory and neurodegenerative diseases, as
well as various infectious and parasitic diseases (Rao et al.,
1998). Aspartic proteases are a relatively small group of
proteolytic enzymes. Over the last decade, they have
received tremendous research interest as potential targets
for pharmaceutical intervention, as many have been shown
to play significant roles in physiological and pathological
processes (Dash et al., 2003). Study of the kinetic
properties of this class of enzymes has been motivated by
their pharmaceutical and commercial importance, and has
evoked considerable interest regarding the role of their
inhibitors.
Invasive aspergillosis is a major threat to the long-term
survival of immunocompromised patients (Denning &
Stevens, 1990). Risk factors for invasive aspergillosis
include prolonged and severe neutropenia, haematopoietic
stem cell and solid organ transplantation, advanced AIDS,
and chronic granulomatous disease (Segal & Walsh, 2006).
Abbreviations: CD, circular dichroism; ITS, internal transcribed spacer;
TFA, trifluoroacetate.
The GenBank/EMBL/DDBJ accession number for the ITS region of the
18S rRNA gene sequence of Penicillium sp. VM24 is JN673378.
One supplementary table and three supplementary figures are available
with the online version of this paper.
058511 G 2012 SGM
Aspergillus fumigatus is the most important airborne fungal
pathogen. Although important advances in antifungal
therapy have been achieved in the past decade, current
treatment modalities remain limited in their therapeutic
benefit (Walsh et al., 2007). Extracellular proteinases that
can hydrolyse the structural components of the lung are
thought to be possible virulence factors (Rhodes et al.,
1988; Kothary et al., 1984). A combination of various
proteolytic enzymes may contribute to the ability of A.
fumigatus to degrade host tissue for nutritional acquisition
and invasion (Dagenais & Keller, 2009). Aspartic proteases
from Aspergillus sp. have been reported to be possible
virulence factors in invasive aspergillosis (Lee & Kolattukudy,
1995; Hogan et al., 1996), although more recent studies
have challenged the importance of secreted aspartic proteases in infection and reported that they are not important
as virulence factors (Sharon et al., 2009; Hartmann et al.,
2011). A. fumigatus aspartic protease is necessary for fungal
growth in protein medium (Sriranganadane et al., 2011).
Combinations of proteolytic enzymes that include aspartic
proteases contribute to the ability of A. fumigatus to degrade
host tissue for nutritional acquisition and invasion
(Dagenais & Keller, 2009). Hence, the inhibition of aspartic
protease might provide new insight into the prevention of
invasive aspergillosis.
In the present work, fungal aspartic protease (PepA) was
used as a prototypical aspartic protease, playing an important
role as a model enzyme for the development of inhibitors for
other aspartic proteases of therapeutic significance. There are
few reports on the isolation of biological aspartic protease
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1897
V. Menon and M. Rao
inhibitors and their mechanisms of inhibition. The present
study describes for the first time the isolation and characterization of a low-molecular-mass peptidic PepA inhibitor
from a novel Penicillium sp. Evaluation of kinetic parameters
confirms that Penicillium aspartic protease inhibitor is a
tight-binding, reversible and competitive inhibitor of fungal
aspartic protease. Fluorescence microscopy and circular
dichroism (CD) analyses show that binding of the inhibitor
induces localized conformational changes in PepA. The new
inhibitor is a potential lead compound for the development
of molecules to combat human fungal infections.
METHODS
tricine-SDS-PAGE. Analysis of amino acids was performed by
hydrolysing 100 pM of inhibitor with 6 M HCl at 110 uC for 24 h
in vacuum-sealed tubes. The hydrolysed amino acids were derivatized
with AccQ Fluor Reagent (6-amino quinolyl-N-hydroxysuccinimide
carbamate) and run on a prepacked reversed-phase HPLC column
(3.96150 mm; AccQ.Tag). The amino acids were eluted with an
acetonitrile gradient (5–95 %, v/v) and monitored with a fluorescence
detector. To calculate the molar proportion of constituents, the peak
areas of individual amino acids were compared with standards
analysed under identical conditions. Total cysteine and tryptophan
were estimated with intact peptide according to the methods of
Cavallini et al. (1966) and Spande & Witkop (1967), respectively. The
solutions for tricine-SDS-PAGE were prepared according to Schägger
(2006). Protein concentration was determined according to the
Bradford method, using BSA as standard (Bradford, 1976).
Fungal aspartic protease (PepA) inhibition assay. Proteolytic
Materials. Acetonitrile was purchased from E-Merck, and ultra
membranes UM 10, UM 3 and UM 0.5 were from Millipore. Aspartic
protease from Aspergillus saitoi, trifluoroacetate (TFA) and haemoglobin were from Sigma-Aldrich. The Biogel P 2 column was from
Bio-Rad Laboratories. All other chemicals used were of analytical
grade.
Isolation and identification of the isolate. The organism was
isolated in the laboratory from a soil sample collected from Pashan,
Pune district, India. Soil was suspended in sterile saline and serial
dilutions of the soil sample were plated on potato dextrose agar
(PDA) and incubated at 28 uC for 5–7 days. Fungal colonies obtained
were purified by single colony plating and screened for aspartic
protease inhibitor by growing them for 120 h in liquid broth
containing soy meal (SBM) (2 %, w/v) as an inducer and other
nutrients [such as glucose (1 %, w/v), beef extract (0.75 %, w/v),
peptone (0.75 %, w/v), sodium chloride (0.3 %, w/v), magnesium
sulfate (0.1 %, w/v) and dipotassium hydrogen phosphate (0.1 %, w/
v), pH 5.0]. Samples were withdrawn every 24 h and assayed for antiPepA activity. The genomic DNA of the fungal culture producing the
inhibitor was isolated according to the method of Makimura et al.
(1994). The organism was identified based on the internal transcribed
spacer (ITS) region of the 18S rRNA gene. The BLAST program was
used for sequence searches of the National Centre for Biotechnology
Information (NCBI) GenBank database (http://blast.ncbi.nlm.nih.
gov/Blast.cgi) using BLASTN with default settings. The sequences were
downloaded from the NCBI database and aligned with the CLUSTAL W
program (http://www.genome.jp/tools/clustalw/). Sequences were
trimmed using DAMBE software. A phylogenic tree was generated via
the neighbour-joining method (Saitou & Nei, 1987) using MEGA
software (Kumar et al., 2001).
activity of PepA from A. saitoi was measured by assaying enzyme
activity using haemoglobin, as described by Dash et al. (2001a).
Enzyme (1.5 mM) and inhibitor were incubated in glycine-HCl buffer
(0.05 M, pH 3) for 10 min. The reaction was started by the addition
of 1 ml haemoglobin (5 mg ml21) and was incubated at 37 uC for
30 min. The reaction was quenched by the addition of 2 ml 10 % (w/
v) TCA acidified with 2.25 % (v/v) HCl followed by centrifugation
(10 000 g, 5 min) and filtration. The absorbance of the TCA-soluble
products in the filtrate was read at 280 nm. One unit of PepA was
defined as the amount of enzyme that produced an increase in
absorbance of 0.001 at 280 nm min21 under the conditions of the
assay. One protease inhibitor unit was defined as the amount of
inhibitor that inhibited one unit of fungal aspartic protease activity
(Dash et al., 2001a).
Assay for inhibitory activity towards trypsin, chymotrypsin,
papain and subtilisin. Inhibitory activities of the inhibitor against
other classes of proteases were also determined by assaying enzyme
activity using specific chromogenic substrates at various pHs and
temperatures. The synthetic substrates Bz-L-Arg-pNA.HCl for trypsin
and papain, Bz-Tyr-pNA for chymotrypsin and Z-Ala-Ala-Leu-pNA
for subtilisin were used (Arnon, 1970; Grant et al., 1970; Walsh,
1970).
Temperature and pH stability. For temperature stability experiments, the inhibitor (10 mM) was incubated at 25–50 uC for 6 h and
its inhibitory activity was estimated against PepA at different intervals
of time. The pH stability of the inhibitor was determined by preincubating the inhibitor (10 mM) at a range of pH in appropriate
buffers for 1 h and estimating the anti-PepA activity.
Initial kinetic analysis for determination of Km and Ki. For
Production and purification of the inhibitor. Penicillium sp.
VM24 was cultured in 250 ml liquid medium (as above) in 500 ml
Erlenmeyer flasks for inhibitor production at 28 uC for 96 h. The
extracellular culture filtrate obtained after centrifugation was treated
with activated charcoal (6.5 %, w/v) and centrifuged. The inhibitor
was purified from the supernatant by ultrafiltration (UM 10, UM 3,
UM 0.5), using a Biogel P 2 column and reversed-phase HPLC (Fluka
RP-C 8), pre-equilibrated with 10 % (v/v) acetonitrile (CH3CN) and
0.1 % (v/v) TFA. The fractions were eluted on a linear gradient of 0–
90 % (v/v) acetonitrile with H2O containing 0.05 % (v/v) TFA at a
flow rate of 0.5 ml min21 and monitored at a wavelength of 210 nm.
The eluted sample was lyophilized and dissolved in deionized water to
check for inhibitor activity. The active fractions were rechromatographed via reversed-phase HPLC under similar experimental
conditions.
Molecular mass and amino acid determination. The molecular
mass of the inhibitor was determined by MS (MALDI-TOF) and
1898
analysis of initial kinetics, the kinetic parameters for substrate
hydrolysis were determined by measuring the initial rate of enzyme
activity. The IC50 value was determined by non-linear regression of
the percentage inhibition data by using a four-parameter logistic
equation (see equation 1), where p is percentage inhibition and is the
relative decrease in enzyme activity due to the inhibitor concentration, [I]0 (Kulkarni & Rao, 2007). Regression analysis was performed
using the software program Microcal Origin 8E.
p5pmin+(pmax2pmin)/1+([I]0/IC50)n
(1)
The inhibition constant Ki was determined according to Dixon (1953)
and also using a Lineweaver–Burk double reciprocal plot. For the
latter, PepA (20 mM) was incubated with inhibitor at concentrations
of 0, 2, 4 and 6 mM and assayed at increasing concentrations of
haemoglobin (75–540 mM) at 37 uC for 30 min. The reciprocals of
substrate hydrolysis (1/v) for each inhibitor concentration were
plotted against the reciprocals of the substrate concentrations. In
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Microbiology 158
Aspartic protease inhibitor against fungal infections
Fig. 1. (a) Purified inhibitor was analysed for determination of Mr by MALDI-TOF (1585). (Inset) Reversed-phase HPLC of
purified inhibitor. Shown is the elution profile of the peak associated with anti-PepA activity. The peak detected showed a
retention time of 6.27 min. (b) Tricine-SDS-PAGE of the inhibitor loaded on a 16 % tryptophan, 6 % cysteine Tris-Tricine gel.
Lane 1, standard ultralow-molecular-mass markers; lane 2, HPLC-purified inhibitor. (c) Amino acid analysis of the inhibitor.
Analysis was done by hydrolysing the inhibitor with 6 M HCl, and the hydrolysed amino acids were derivatized with AccQ Fluor
Reagent and run on a prepacked reversed-phase HPLC column (AccQ.Tag). The amino acids were eluted with an acetonitrile
gradient and monitored with a Waters fluorescence detector. The asterisk indicates tryptophan as determined by N –
bromosuccinimide.
Dixon’s method, the hydrolytic activity of PepA (20 mM) was
measured in the presence of 250 and 450 mM haemoglobin at
concentrations of inhibitor ranging from 1 to 10 mM at 37 uC for
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30 min. The reciprocals of substrate hydrolysis (1/v) were plotted
against the inhibitor concentration and Ki was determined by fitting
the data using Microcal Origin 8E.
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V. Menon and M. Rao
Table 1. Purification of the inhibitor
Purification step
Centrifugation
Activated charcoal
Ultrafiltration 10 kDa cut-off
Ultrafiltration 3 kDa cut-off
Ultrafiltration 0.5 kDa cut-off
Biogel P-2 column
Reversed-phase HPLC
Volume (ml)
Total activity
(units)
Total protein
(mg)
Specific activity
(units mg”1)
Purification
(fold)
Yield (%)
650
600
575
550
525
12
3.5
4083
3498
2942
2648
2458
1821
735
607.61
280
86.25
48.03
35.62
10.38
2.14
6.72
12.49
34.11
55.13
69.03
176.75
350
1
1.86
5.08
8.12
10.28
26.32
52.12
100
85.67
72.05
64.85
60.19
44.59
18.14
Initial apparent inhibition constants. Inhibition studies were
performed by adding 100 ml of the enzyme (0.05 mM) to 300 ml of
250 mM haemoglobin solution in standard buffer containing varying
concentrations of the inhibitor (1–10 mM) at 37 uC for 30 min. The
product was estimated as described above. Relative enzyme activity R
was calculated from the ratio of product amounts obtained in the
presence and absence of inhibitors as R512[P]/[P]0. Relative
inhibition was fitted by a non-linear least-squares regression to
equation 2, where [I]0 is the total concentration of inhibitor and Kapp
(the fitting parameter) is the apparent inhibition constant:
R5[I]0/(Kapp+[I]0)
(2)
Inhibitor progress curve analysis. The reaction time course in the
presence of inhibitor was fitted to equation 3, which is a modification
of the standard kinetic model:
p5p0+Vst+(V02Vs)[12exp(2Kappt)]/Kapp
(3)
Instrumental offset p0 is treated as an adjustable parameter to account
for the possibility of systematic errors in measuring the degree of
product conversion. Each individual progress curve was fitted
separately. The local fitting parameters were initial velocity V0,
steady-state velocity Vs, steady-state velocity at time t Vst, apparent
first-order rate constant Kapp and instrumental offset p0. Equation 4
was used as the theoretical model and is applicable to a pure tightbinding inhibitor:
v5v0([E]02[I]02Kapp+{([E]02[I]02Kapp)2+4[E]0Kapp}2K
(4)
To fit data to this equation, the modified Marquardt–Levenberg leastsquare fitting equation was used. The rate constants were obtained by
regression analysis of these data using the software Origin 8E.
Another approach used to calculate the apparent Ki* value for the
inhibitor using equation 5:
IC505Et/(2+Ki*)
(5)
Et is the total enzyme concentration and Ki* is the apparent enzyme–
inhibitor dissociation constant. For competitive inhibition, the true Ki
was obtained by dividing Ki* by (1+S/Km) (Dixon, 1953).
Fluorescence
analysis
of
PepA–inhibitor
interactions.
Fluorescence measurements were performed on a Cary Varian eclipse
fluorescence spectrophotometer connected to a Cary Varian temperature controller. Protein fluorescence was excited at 295 nm, and the
emission was recorded from 300 to 400 nm at 25 uC. Slit widths on
both excitation and emission were set at 5 nm, and the spectra were
obtained at 1 nm min21. For inhibitor binding studies, PepA
(20 mM) was dissolved in 0.05 M HCl. Titration of the enzyme with
inhibitor was performed by the addition of different concentrations of
1900
the inhibitor (0, 2.5, 5 and 10 mM) to a fixed concentration of enzyme
solution. For each inhibitor concentration on the titration curve, a
new enzyme solution was used. All the data on the titration curve
were corrected for dilutions, and the graphs were smoothed.
Corrections for the inner filter effect were performed as described
by equation 6:
Fe5F antilog[(Aex+Aem)/2]
(6)
Fc and F are the corrected and measured fluorescence intensities,
respectively, and Aex and Aem are the absorbances of the solution at
the excitation and emission wavelengths, respectively (Lakowicz,
1983). Background buffer spectra were subtracted to remove the
contribution from Raman scattering.
Secondary structural analysis of PepA–inhibitor complexes.
CD spectra were recorded on a Jasco-J715 spectropolarimeter at
ambient temperature using a cell of 1 mm path length. Replicate
scans were obtained at 0.1 nm resolution, 0.1 nm bandwidth and a
scan speed of 50 nm min21. Spectra were the averages of six scans
with the baseline subtracted spanning from 260 to 200 nm in 0.1 nm
increments. The CD spectrum of PepA (25 mM) was recorded in
0.05 M glycine-HCl buffer (pH 3) in the absence/presence of
inhibitor or pepstatin (5 mM each). Secondary structure content of
the PepA and the PepA–inhibitor complex was calculated using the
algorithm of the K2d program (Andrade et al., 1993; Merelo et al.,
1994).
Antifungal activity assay. A. fumigatus NCIM 902 and Aspergillus
niger were obtained from our in-house culture collection unit, the
National Collection of Industrial Micro-organisms, Pune, India.
Antifungal activity was assayed essentially via (i) spore suspension
and (ii) microspectrometric assays. Fungal spores were harvested
from the freshly grown fungal culture and suspended in sterile water.
The concentration of the spore suspension was adjusted to 1.06106.
To 1 ml of the freshly prepared spore suspension, 1 ml half-strength
modified Mueller–Hinton (MH) agar (per litre: 2 g beef extract,
17.5 g acid hydrolysate of casein, 1.5 g starch and 25 g agar; Mueller
& Hinton, 1941) (pH 5.5±0.1) was added and was immediately
overlaid on Petri dishes containing MH agar. To allow for spore
germination and initial vegetative growth, plates were incubated at
28 uC for 24–48 h. At this time, wells were bored on the agar surface,
and different concentrations of the inhibitor were applied. The plates
were incubated at 28 uC and photographed after 24–72 h. All test
solutions were filtered through a 0.22 mm pore-size membrane prior
to application. A microspectrometric antifungal assay was performed
for quantitative demonstration of antifungal activity (Broekaert et al.,
1990). Briefly, routine tests were performed with 20 ml of filtersterilized (0.22 mm pore-size) test solution and 80 ml fungal spore
suspension (106 spores ml21) in half-strength MH broth. The control
microculture contained 20 ml sterile distilled water and 80 ml fungal
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Microbiology 158
Aspartic protease inhibitor against fungal infections
RESULTS
(a)
70
Isolation and identification of the isolate
60
Inhibion (%)
50
40
30
20
10
0
0
0.5
1.0
1.5 2.0 2.5 3.0
Inhibitor concn (µM)
3.5
4.0
4.5
(b)
8
7
Slope
1.65
1.64
1.63
1.62
1.61
1.60
1.59
1.58
1.57
–4 –3 –2 –1 0 1 2 3 4 5 6
1/v (mM-1 min)
Inhibitor concn (µM)
6
5
Purification and biochemical characterization of
the inhibitor
4
3
2
1
0
–500 –400 –300 –200 –100
0 100 200 300 400 500 600
_
1/[S] (µM 1)
Fig. 2. (a) PepA activity (20 mM) was determined in the presence
of increasing concentrations of inhibitor. The sigmoidal curve
indicates the best fit for the percentage inhibition data obtained
(mean of triplicate determinations), and the IC50 value was
calculated based on the equation of Cha et al. (1975). (b)
Estimation of the kinetic constant by Lineweaver–Burk analysis.
Enzyme activity was assayed at increasing concentrations of the
substrate at inhibitor concentrations of 0 (&), 2 ($), 4 (m) and
6 mM (.). The reciprocal of substrate hydrolysis (1/v) for each
inhibitor concentration was plotted against the reciprocal of the
substrate concentration. The straight lines indicate the best fits for
the data obtained by non-linear regression and analysed by the
Lineweaver–Burk reciprocal equation for competitive inhibition.
spore suspension. Unless stated otherwise, incubation conditions for
the experiments were 28 uC for 48 h. Antifungal activity is expressed
in terms of percentage inhibition as defined by Cammue et al. (1992).
The MICs for A. fumigatus and A. niger were determined by a broth
dilution method (Amsterdam, 1991). Serial dilutions of the inhibitor
were made in half-strength MH broth in microtitre plates. Each well
was inoculated with 10 ml of the test organism at 105 spores ml21.
The MIC was determined after overnight incubation of the plates and
was taken as the lowest concentration of the inhibitor at which
growth was inhibited.
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Sixty-eight fungal cultures were isolated on PDA plates
from soil samples. Only one fungal isolate was found to
produce an extracellular aspartic protease inhibitor. The
isolated organism was identified on the basis of morphological and 18S rRNA gene sequence similarity. The ITS
region of the 18S rRNA gene of Penicillium sp. VM24 was
sequenced and comprised 433 bp, and BLAST search analysis
showed 94 % homology to various Penicillium species. The
neighbour-joining phylogenetic tree based on ITS
sequences also confirmed that the isolate belongs to the
genus Penicillium (Fig. S1 available with the online version
of this paper). Based on BLAST search analysis, the isolate
showed only 94 % similarity to the type strain of
Penicillium pinophilum and of other Penicillium sp., and
hence the new isolate may represent a novel species of the
genus Penicillium. The isolate grew at pH 4–6 with an
optimum at pH 5 and at an optimum temperature of
28 uC.
Penicillium sp. VM24 produced the inhibitor in medium
containing peptone beef extract with SBM as inducer at
28 uC and pH 5. The extracellular culture filtrate was
subjected to activated charcoal treatment, ultrafiltration
and gel filtration to remove high-molecular-mass impurities and salts. The concentrated inhibitor sample was
further purified to homogeneity by reversed-phase HPLC,
and the anti-PepA activity was associated with a single peak
(Fig. 1a, inset). The purified inhibitor showed specific
activity of 350 U mg21 and a 52-fold increase in
purification with a yield of 18 % (Table 1). The inhibitor
was stable over a broad range of pH (2–6) and temperature
(25–40 uC). The inhibitor was found to be specific for
fungal aspartic protease (PepA) and exhibited no inhibitory activity against pepsin and other classes of proteases
such as trypsin, chymotrypsin, papain and subtilisin (data
not shown). The Mr of the inhibitor as determined by
MALDI-TOF MS was 1585 (Fig. 1a). The inhibitor also
showed a single homogeneous band with an Mr of 1580 on
tricine-SDS-PAGE (Fig. 1b). The amino acid composition
of the inhibitor revealed the presence of D, D, D, E, A, K, L,
Y, H, I and W residues (Fig. 1c).
The molecular mass of the inhibitor was determined to be
1585 Da. The peptide or isomers would have a molecular
mass of 1404 Da. The difference in the molecular mass may
be due to the presence of multiple groups (e.g. acetyl,
carboxyl, methyl, nitro) attached to the side chains of the
amino acids. Pepstatin, a biological aspartic protease
inhibitor from Streptomyces sp., is known to contain a
statin group (Umezawa, 1976). Several other synthetic
peptide inhibitors are known to have similar groups,
increasing their potency (Nguyen et al., 2008). However,
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V. Menon and M. Rao
Table 2. Kinetic rate constants for the inhibitor obtained by various methods
Method of analysis
Inhibitor curve analysis
Lineweaver–Burk plot
Dixon plot
Substrate kinetics
Inhibitor progress curve
Dissociation constant analysis
Cha analysisD
Kinetic rate constant
Value obtained
IC50
Km
Ki
Vm
Ki
Km
Vm
Ki
Ki*
Vm
kA
kD
Ki*
Ki
Ki*
1.82 mM
180 mM
2.54 mM
0.61 mM min21
1.2 mM
168 mM
0.59 mM min21
0.85 mM
0.71 mM
0.65 mM min21
6.2561023 s–1
11.8461023 s–1
1.48 mM
1.25 mM
1.05 mM
DCha et al. (1975).
structural studies of the inhibitor will provide further
insight into the side groups and differences in molecular
mass.
with inhibitor concentration. The rate constants were
obtained by fitting the data to equations 7–9:
V05Vm[S]0/([S]0+Km)
(7)
Kinetic analysis of the inhibition of PepA by the
inhibitor
Vs5Vm[S]0/{[S]0+Km(1+[I]0/Ki)}
(8)
Kapp5kD+kA[I]0/(1+[S]0/Km)
(9)
Kinetic analysis indicated an IC50 value of 1.82 mM (Fig.
2a). Inhibition of PepA followed a hyperbolic pattern with
increasing concentrations of the inhibitor. Initial kinetic
assessments via Lineweaver–Burk analysis revealed that the
aspartic protease was competitively inhibited by the
inhibitor (Fig. 2b). The Km value of the enzyme was
180 mM. The secondary plot of the slope versus inhibitor
concentration gave a Ki value of 2.24 mM (Fig. 2b, inset),
while with the Dixon plot a Ki value of 1.2 mM was
obtained.
Progress curve analysis
Progress of the reaction was analysed by two different
methods, one based on the assumption of rapid equilibrium and the other on the assumption of slow equilibrium.
The results of the analysis based on the assumption of rapid
equilibrium are shown in Fig. S2(a), as the data did not fit
the other model. The progress curves obtained at 2, 4, 6, 8
and 10 mM inhibitor were fitted individually to equations
7–9. The best-fit values of the individual parameters obtained are given in Table S1. The results corroborate the
one-step inhibition mechanism, wherein the enzyme–inhibitor complex is formed rapidly. The onset of inhibition is
rapid and the binding indicates a tight binding mechanism.
The rate constants are in agreement with the model scheme
outlined in Fig. S3 and rule out the possibility of a slow
onset of inhibition. The initial velocity V0 obtained is constant and the apparent rate constant Kapp increases linearly
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The parameters listed in Table 2 favour the one-step
mechanism, because the initial velocity does not decrease
with the concentration of the inhibitor, as predicted by
equation 7. Also, the increase of the apparent rate constant
with [I]0 is linear. The non-linear least-squares fit of V0, Vs
and Kapp to equations 7–9 is shown in Fig. S2(b, c). The
parameters obtained with the mechanism as outlined by
Cha et al. (1975) corroborate well with the abovementioned mechanism, yielding a Ki* of 1.075 mM and a
Ki of 1.1245 mM.
Fluorometric analysis of PepA–inhibitor
interactions
The localized changes induced in PepA due to interactions
with the inhibitor were investigated by fluorescence spectroscopy. The conformational changes induced in PepA
upon binding of inhibitor were monitored by exploiting
the intrinsic fluorescence by excitation of the p–p* transition in the tryptophan residues. The fluorescence emission spectra of aspartic protease exhibited an emission
maximum (lmax) at 340 nm as a result of the radiative
decay of the p–p* transition from the tryptophan residues,
confirming the hydrophilic nature of the tryptophan
environment (Fig. 3a). Titration of the native enzyme
with increasing concentrations of inhibitor resulted in a
concentration-dependent quenching of the tryptophanyl
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Microbiology 158
Aspartic protease inhibitor against fungal infections
(a)
0 µM
2.5 µM
400
5 µM
10 µM
Fluoresence intensity (a.u.)
350
300
250
200
150
100
50
0
300
320
360
340
Wavelength (nm)
380
400
native enzyme to 225 nm (Fig. 3b). This shift reveals a
subtle change in the secondary structure of the enzyme
upon inhibitor binding. Furthermore, to elucidate the
mechanism of inactivation of PepA by the newly isolated
inhibitor, we analysed the interactions of a representative
competitive inhibitor, pepstatin (Richards et al., 1989), on
the secondary structure of PepA. Binding of pepstatin to
PepA exhibited a similar pattern of negative ellipticity in
the far-UV region, indicating that the newly isolated
inhibitor causes similar structural changes making the
bound enzyme distinctly different from the unliganded
enzyme. Comparative analysis of the CD spectra of PepA
upon binding of the inhibitor or the known active sitebased inhibitor pepstatin showed that they were similar,
suggesting binding of the newly isolated inhibitor to the
active site of PepA.
(b)
10
Antifungal activity of the inhibitor
Ellipcity (millidegrees)
5
0
–5
–10
–15
–20
200
210
220
230
240
Wavelength (nm)
250
260
Fig. 3. (a) Protein fluorescence was excited at 295 nm, and
emission was monitored from 300 to 400 nm at 25 6C. Titration
was performed by the addition of different concentrations of the
inhibitor to a fixed concentration of enzyme. (b) Far-UV CD spectra
of the unligated PepA and its complexes with pepstatin and
inhibitor. PepA (25 mM) was dissolved in the buffer and the CD
spectra were recorded in the absence (&) or in the presence of
the inhibitor ($, 5 mM) or pepstatin (*, 5 mM) from 260 to 200 nm
at 25 6C (the last two lines largely overlap). Each spectrum
represents the average of six scans.
fluorescence. However, the lmax of the fluorescence profile
indicated no blue or red shift, suggesting that there were no
gross conformational changes in the 3D structure of the
enzyme due to inhibitor binding.
CD analysis of PepA–inhibitor complexes
To elucidate the effects of the inhibitor on the secondary
structure of PepA, the CD spectra of the enzyme–inhibitor
complex were analysed. The a-helix, b-sheet and b-turn
content of PepA was 2.5, 59 and 18 %, respectively. The CD
spectrum of the PepA–inhibitor complex showed a
pronounced shift in the negative band at 220 nm for the
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The inhibitory activity of the purified inhibitor against
mycelial growth and spore germination of A. fumigatus and
A. niger was assessed in various standard biological assays.
A. fumigatus and A. niger were grown in MH medium
(pH 5.5) at 28 uC and produced 80 IU extracellular PepA
(data not shown). The antifungal activity of the inhibitor
was indicated by the zone of inhibition that developed
around the wells against the vegetative growth after spore
germination (Fig. 4a). Fungal growth inhibition was
monitored in a microscopic assay, wherein the spores were
cultured in the presence of varied concentrations of the
inhibitor. The morphological differences observed in
mycelial growth after 48 h are shown in Fig. 4(b). In the
presence of the inhibitor, germination of A. niger spores
was delayed, whereas in A. fumigatus, the rate of mycelial
growth was reduced. As observed from the micrographs,
lysis was not observed in mycelia in the presence of
inhibitor. After 24 h, the concentration of inhibitor
required for 50 % inhibition (IC50) of fungal growth was
0.58 mg ml21 for A. niger and 2.34 mg ml21 for A.
fumigatus, whereas the MICs were 0.30 and 1.65 mg ml21,
respectively. The time-dependent dose–response curves
revealed that the extent of growth inhibition tended to
decrease with the increase in incubation time. The timedependent decrease in the potency of the inhibitor was less
pronounced in A. niger than in A. fumigatus (Fig. 5).
DISCUSSION
The data reported here showed that the newly isolated
inhibitor from Penicillium sp. VM24 is a tight-binding
inhibitor of PepA. Penicillium sp. grows well when it
produces the inhibitor. Inhibitor production was growthassociated, and the highest inhibitor yield was obtained
with maximum cell growth. During initial kinetic analysis
the inhibitor showed competitive inhibition against the
enzyme in vitro. The 1 : 1 molar ratio of the interaction of
the inhibitor with the target enzymes classified it in the
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1903
V. Menon and M. Rao
Fig. 4. (a) Fungal spores were allowed to
germinate on MH agar and grown for 24 h
before the test solution was added.
Subsequently, wells were bored on the agar,
30 ml aliquots of test solutions were added to
the wells, and the fungi were allowed to grow
for 12 h. The test solution (30 ml) contained 0
(1), 1 (2), 2 (3) and 3 mg (4) of the inhibitor.
Fungal strains tested were A. fumigatus (I) and
A. niger (II). (b) Morphological changes
induced in mycelia of the fungal strains in the
presence of inhibitor. Fungal spores were
germinated in half-strength MH broth in the
absence (panels in row I) or presence (panels
in row II) of the inhibitor, and growth was
observed after 24 h. The fungal strains tested
were A. fumigatus (A) and A. niger (B).
‘tight-binding inhibitor’ group (Williams & Morrison,
1979). As a rule, when comparing Ki values for tight-binding
inhibitors, it is essential to examine the mathematical
methods used for their estimation, as different methods even
when applied to the same data can yield Ki estimates
differing by several orders of magnitude (Szedlacsek et al.,
1991; Reich, 1992). The Lineweaver–Burk reciprocal plot
shows that the inhibitor is a competitive inhibitor of the
fungal aspartic protease. In the region of Ki, the kA value is
high, while the kD value is low. This indicates that the
inhibitor binds rapidly to the enzyme but that the rate of
dissociation is slow. This suggests fast inactivation of the
enzyme in the presence of the inhibitor. Tight-binding
inhibitors combine at the active site and rapidly cause the
enzyme to lose activity.
Tight-binding inhibitors induce conformational changes
that cause the enzyme to clamp down on the inhibitor,
resulting in the formation of a stable enzyme–inhibitor
complex. On the basis of our fluorescence studies, we
propose that the rapid fluorescence loss was due to the
formation of a reversible enzyme–inhibitor complex.
Fluorescence quenching of PepA by the inhibitor revealed
that binding of the inhibitor reduces the quantum yield of
tryptophan emission. The kinetically observable formation
1904
of an enzyme–inhibitor complex does not involve a major
alteration in the 3D structure of the enzymes, as reflected in
the absence of any shift in tryptophanyl fluorescence. Any
changes in the environment of individual tryptophan residues may result in an alternation of fluorescence characteristics such as emission wavelength, quantum yield and
susceptibility to quenching (Pawagi & Deber, 1990). Fluorescence quenching can also result from energy transfer to
an acceptor molecule with an overlapping absorption spectrum (Cheung, 1991). As the inhibitor shows no absorption
in the region 300–450 nm (data not shown), we ruled out
fluorescence quenching due to energy transfer between the
inhibitor and the tryptophan residues. The effect of inhibitor
concentration on fluorescence quenching of the enzymes
was also consistent with a 1 : 1 molar ratio. These findings
indicated that the polarity of the tryptophan environment was negligibly altered after binding of the inhibitor,
suggesting minimal conformational changes in the tertiary
structure of the fungal aspartic protease. Our interpretation
of the changes observed in the secondary structure of
the fungal aspartic protease due to binding of the classical
inhibitor pepstatin to the active site correlates with the
similar pattern of changes observed due to the binding of the
newly isolated peptidic inhibitor. Our fluorescence spectroscopy and CD analyses suggest that the peptidic inhibitor
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Microbiology 158
Aspartic protease inhibitor against fungal infections
Fig. 5. Time-dependent dose–response growth inhibition curves. Growth inhibition of A. fumigatus (a) and A. niger (b) at
different concentrations of the inhibitor was recorded after 24 (&), 48 ($) and 72 h (m). Antifungal activity of the inhibitor was
estimated in terms of percentage inhibition, and IC50 values were calculated from the curves.
binds to the active site of the fungal aspartic protease and
causes inactivation.
Extracellular proteases of eukaryotic microbial pathogens
have received tremendous research interest as potential
drug targets for pharmaceutical intervention (Dash et al.,
2003; Dos Santos, 2010). Analysis of proteolytic enzymes of
pathogenic micro-organisms might lead to the design of
inhibitors to control these pathogens (Dos Santos, 2011).
To colonize a host, fungal micro-organisms have evolved
strategies to invade tissues, optimize growth and propagate.
To gain entrance, fungi generally secrete a cocktail of
hydrolytic enzymes, including proteases, elastinolases and
collagenases (Monod et al., 2002; Dagenais & Keller, 2009).
However, there is a lacuna in the literature on inhibitors of
aspartic proteases that exhibit antifungal activity; such
literature could provide further insight into our understanding of host–pathogen interactions. Here the newly
isolated inhibitor was analysed for its efficacy as an
antifungal agent against Aspergillus spp. The inhibitor
was found to be active against A. fumigatus and A. niger,
and its IC50 values indicated exceptionally high potency.
Our results documented that the specific activity of the
inhibitor was decreased when the incubation time for
fungal growth was increased. A possible explanation for
this phenomenon is that the germlings at the early stages of
growth were more affected than mycelium development at
later stages. At high concentrations the inhibitor was found
to inhibit spore germination, and at lower concentrations delayed growth of the hyphae, which subsequently
http://mic.sgmjournals.org
exhibited abnormal morphology. Inhibition of fungal
growth by the inhibitor is correlated with its anti-proteolytic
activity, leading to depletion of nutrients required for
growth and propagation. As protease inhibitors play an
important role in protection of the host from pathogen
attack by virtue of anti-nutritional interactions (Dash et al.,
2001b), our results suggest therapeutic application of the
inhibitor in opportunistic fungal infections to circumvent
host invasion.
ACKNOWLEDGEMENTS
M. R. and V. M. acknowledge financial support and a senior research
fellowship, respectively, from the CSIR Emeritus Scheme, Government
of India.
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