An anti- Aspergillus protein from Escherichia coli DH5α: Putative inhibitor of siderophore...

Original article

An anti-Aspergillus protein from Escherichia coli DH5a: Putativeinhibitor of siderophore biosynthesis in Aspergillus fumigatus

Meenakshi Balhara, Sonam Ruhil, Manish kumar, Sandeep Dhankhar and A. K. Chhillar

Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India

Summary An antifungal protein designated as anti-Aspergillus protein (AAP), produced by Esc-

herichia coli DH5a, was purified and characterised. It exhibited a molecular weight of

60 kDa on Sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis and

depicted 99% purity on ultra performance liquid chromatography. The purified pro-

tein manifested antimycotic potential against pathogenic isolates of Aspergillus spp.,

depicting a minimum inhibitory concentration in the range 15.62–31.25 lg ml�1

and 5.0–10.0 lg per disc, using microbroth dilution, spore germination inhibition

and disc diffusion assays respectively. In vitro toxicity tests demonstrated that it

showed no toxicity against human erythrocytes at doses up to 1000 lg ml�1.

Matrix-assisted laser desorption ionisation–Time-of-flight analysis of trypsin-digested

peptides of purified protein and subsequent Mascot search revealed that several pep-

tides of AAP have identity with bacterial siderophore biosynthetic protein, i.e. non-

ribosomal peptide synthetase enzyme, involved in critical step of fungal siderophore

biosynthesis. Siderophore-based inhibition was further corroborated by Chrome

azurol S assay. Hence, the antagonistic effect might be the result of impediment in

siderophore-mediated iron uptake and transport process which may cause critical

consequences on Aspergillus growth and virulence.

Key words: Anti-aspergillus protein (AAP), siderophore, Escherichia coli, CAS assay, non-ribosomal peptide synthe-

tase (NRPS).

Introduction

Filamentous fungi represent an increasing threat for

patients suffering from sustained immuno-suppres-

sion.1 The medically important fungi include species

of the genus Aspergillus, among which Aspergillus fu-

migatus is considered to be the most prominent organ-

ism causing aspergillosis.2 However, the antifungal

drugs commonly used to treat fungal infections are

limited. In January 2001, the echinocandins became

the first semisynthetic antifungal natural product to

be approved since polyene (Amphotericin B) was

approved forty years earlier.3 A new antifungal agent

called caspofungin was launched in 2002 and

included in the existing therapeutic options for candi-

diasis and invasive aspergillosis. It showed good

activity against Aspergillus and Candida, however, its

clinical efficacy-guided market is yet to be estab-

lished.4 Although the new generation azoles (Vorico-

nazole, Posaconazole) have been much improved as

compared with previous compounds of this class; the

concerns with respect to limited efficacy could not be

resolved.5 Several reports have underlined the

emergence of triazole resistant Aspergillus strains.6–8

Hence, the current antifungal drugs along with the

development of resistance; have considerable disad-

vantages, i.e. act on targets also found in mammalian

cells which results in toxicity or an adverse drug

Correspondence: Dr. Anil Kumar Chhillar, Centre for Biotechnology, M.D.

University, Rohtak, India.

Tel.: +91 126 227 4710. Fax: +91 126 227 4133.

E-mail: [email protected]

Submitted for publication 25 March 2013

Revised 18 July 2013

Accepted for publication 18 July 2013

© 2013 Blackwell Verlag GmbH

Mycoses, 2014, 57, 153–162 doi:10.1111/myc.12119

mycosesDiagnosis,Therapy and Prophylaxis of Fungal Diseases

interaction.9 Therefore, novel and more efficient anti-

fungal agents that are not toxic to mammalian cells

are urgently needed.

Fortunately, nature has strategically placed antimi-

crobial and antifungal peptides as a first line of

defence between the host organism and its surround-

ing environment because these peptides are able to

inhibit quickly a wide spectrum of infectious microbes

without significant toxicity to the host organism. Pro-

duction of such antifungal proteins/peptides is a wide-

spread phenomenon among all forms of life, from

multicellular organisms to bacterial cells.10 In addition

to the well-known glucanases, chitnases, thaumatin-

like proteins, defensins and ribosome-inactivating pro-

teins, there is a diversity of other antifungal proteins

such as lipid transfer proteins and protease inhibi-

tors.11 Many antifungal peptides have been reported

from bacteria also.12,13 These bacterial antifungal pep-

tides possess several interesting features such as nar-

row spectrum of activity, high potency and rapid

killing properties that have led to increased interest in

their potential application as a new class of antifungal

compounds. Therefore, bacterial species which form

part of the human microbiome as commensal could be

tested for their antimicrobial potential as demonstrated

by the fact that the probiotic bacteria (lactobacilli, bifi-

dobacteria, enterococci, non-pathogenic Escherichia coli

and yeast) are considered potential producers of

metabolites and antimicrobial agents.14 As the litera-

ture on bacterial antifungal proteins is much less as

compared with that on bacterial antifungal peptides,

the present investigation was carried out to isolate an

antifungal protein from some commensal bacterial

species capable of producing potential novel antifungal

proteins and to characterise them for further

applications.

Material and methods

Fungal isolates

Different fungal strains of three pathogenic species of

Aspergillus, i.e. A. fumigatus (Vallabhbhai Patel Chest

Institute: VPCI 190/96), A. flavus (VPCI 223/96) and

A. niger (VPCI 56/96) were obtained from mycology

department (VPCI, Delhi, India). These strains were

subcultured on Sabouraud dextrose (SD) agar plates in

a Biochemical Oxygen Demand (BOD) incubator at

37 °C. A homogeneous spore suspension was prepared

from 96 h culture in phosphate buffer saline (PBS), pH

7.4 with spore count adjusted to 1 9 108 spores ml�1

using haemocytometer spore count method.

Bacterial strains

The following bacterial strains were purchased from

Institute of Microbial Technology, Chandigarh (India):

E. coli DH5a (MTCC 1652), E. coli (MTCC 1674), E.

coli (MTCC 10312), Streptococcus thermophilus (MTCC

1938), Streptococcus lactis ssp. lactis (MTCC 460),

Staphylococcus epidermidis (MTCC 3615), S. epidermidis

(MTCC 6810) and S. epidermidis (MTCC 9041). All

these strains were routinely cultured in Luria Bertani

(LB) broth medium at 37 °C in an incubator shaker.

Extraction of proteins

The aforementioned bacterial strains were cultured in

LB broth at 100 r.p.m and 37 °C in a BOD incubator

shaker (Hicon, New Delhi) for 48 h. Bacterial cultures

were centrifuged at 7155 g for 30 min in a refriger-

ated centrifuge. The supernatant was separated from

pellet. The ammonium sulphate precipitation (70%)

along with dialysis against distilled water at 4 °C was

carried out. The dialysed concentrate was used as

secretory protein. The pellet was subjected to sonica-

tion (Bio age sonicator, Mohali, India) in a sonication

buffer (50 mmol l�1 Tris–HCl, 50 mmol l�1 EDTA,

5 mmol l�1 DTT, 1 mmol l�1 PMSF) at 20 s burst at

200 W and 10 s cool period. The sonicate was centri-

fuged at 16 099 g for 1 h, the supernatant obtained

was precipitated with ammonium sulphate, dialysed

and used as cytosolic or lysate protein. The concentra-

tion (lg ml�1) of secretory and cytosolic protein frac-

tions was quantified by Bradford method. All the

chemicals used in the study were purchased from Hi-

media Laboratories (India).

Susceptibility testing

The anti-aspergillus activity of bacterial proteins was

evaluated using microbroth dilution assay (MDA), per

cent spore germination inhibition assay (PSGI) and

disc diffusion assay (DDA) as described by.15

Microbroth dilution assay

The spore suspension (1 9 108 spores ml�1) obtained

from freshly revived fungal plates of various Aspergil-

lus species were treated with different concentrations

of bacterial proteins (3.90–1000.00 lg ml�1) in 96-

well microtitre plates. The plates were examined after

24 h of incubation in a BOD incubator at 37 °C for

any visible growth of fungal mycelia. The protein

concentration at which the wells appeared macro-

scopically clear without any visible growth of fungi


Mycoses, 2014, 57, 153–162154

M. Balhara et al.

was considered as minimum inhibitory concentration

(MIC).

Percentage spore germination inhibition assay (PSGI)

In this assay, the wells of 96-well culture plate

impregnated with 90 ll of SD broth and serially diluted

with bacterial proteins (3.90–1000.00 lg ml�1),

were inoculated with 10 ll of spore suspension

(1 9 104 spores ml�1) containing 100 � 5 spores. The

numbers of germinated and non-germinated spores were

counted after incubation at 37 °C for 16 h in an

inverted microscope (Labomed, Fremont, CA, USA). PSGI

was calculated using the following formula: PSGI =(100 � No. of spores germinated in drug-treated well/

No. of spores germinated in control well) 9 100.

The protein concentration which inhibits the germi-

nation of spores in the range 90%–100% was repre-

sented as MIC90.

Disc diffusion assay

The DDA was performed in SD agar plates inoculated

with 1 9 108 spores ml�1. Sterilised discs (5 mm in

diameter) of Whatman filter paper no. 1 were impreg-

nated with bacterial protein (25 lg to 1.50 lg/disc).The plates were incubated at 37 °C for 24 h for zone

of inhibition if any. The protein concentration which

causes a minimum 6.00 mm zone of inhibition around

the discs was considered as MIC as per our standar-

dised protocols that much protein concentration which

led to 6 mm zone of inhibition maintained the inhibi-

tory potential even after we double the incubation

period.

Purification of active molecule

The most active bacterial protein (E. coli DH5a cyto-

solic protein) was purified to homogeneity using anion

exchange, gel filtration and ultra performance liquid

chromatography (UPLC).

Anion exchange and gel filtration chromatography

The crude cytosolic protein (20 mg) dissolved in equil-

ibration buffer (Tris-HCl, pH 7.4), was run through

prepacked anion exchange (Hi Prep 16/10 Q-XL

sepharose) column of an Akta purifier system (GE

Healthcare, Bjorkgatan, Sweden), preequilibrated with

the same buffer. The fractions were eluted out using a

salt gradient from 0.0 to 1.0 mol l�1 NaCl as elution

buffer, with a flow rate of 0.1 ml min�1. The fractions

obtained were quantified by Bradford method and fur-

ther analysed for antifungal activity. The active anion

exchange fraction (6.00 mg of protein) was further

subfractionated by gel filtration chromatography using

sephadex G100 column (1.5 cm 9 75.0 cm). The

column had been preequilibrated with standard

molecular markers from GE Healthcare including

phosphorylase b (94 kDa), bovine serum albumin

(67 kDa), ovalbumin (43 kDa), carbonic anhydrase

(30 kDa), soybean trypsin inhibitor (20 kDa) and a-lactalbumin

(14.4 kDa). The subfractions were eluted out with

Tris-HCl buffer, with a flow rate of 1.0 ml/min�1.

Ultra performance liquid chromatography

The reverse phase column C4 (BEH C4 100 mm

Waters) of Acquity UPLC system was used to check

the purity and resolve the components of active gel fil-

tration chromatographic subfraction. All the chro-

matographic solvents were of UPLC grades and

purchased from WATERS. These solvents were filtered

through 0.22 lm filters (Millipore) and degassed. The

mobile phase used for the separation of sample compo-

nents was acetonitrile: 0.1% formic acid in a ratio of

70:30.

Sodium dodecyl sulphate–polyacrylamide gel electrophore-

sis (SDS-PAGE)

The molecular mass of purified protein was determined

by SDS-PAGE conducted according to the method of

Laemmli & Favre [16]. After electrophoresis, the gel

was visualised by silver staining.17

Biochemical characterization of purified protein

Anti-aspergillus potential of purified protein

The antifungal potential of purified protein was evalu-

ated against various Aspergillus species using MDA,

PSGI and DDA assays as described earlier. The anti-

fungal activity of purified protein anti-Aspergillus pro-

tein (AAP) was also determined according to CLSI

document M38-A2 18 which recommends the use of

RPMI 1640 supplemented with 20 g l�1 glucose as

test media.

Toxicological study of purified protein

Haemolytic activity of purified bacterial protein was

investigated against human RBCs (Red blood cells) in

96-well microtitre plates.15,19 Fresh blood samples

from healthy individuals were used in this study. A

2% erythrocyte suspension obtained after centrifuga-

tion at 252 g for 15 min was incubated with bacterial

protein in the concentration range 1000.0–3.90 lg ml�1 at 37 °C for 1 h. After incubation cells

were centrifuged at 2795 g for 10 min and the


Mycoses, 2014, 57, 153–162 155

Siderophore biosynthesis in Aspergillus fumigatus

absorbance of supernatant recovered was taken at

450 nm (UV Vis Spect Lambda Bio 20; Perkin Elmer

spectrophotometer, Milton Freewater, OR, USA). PBS

was used as negative control for background lysis,

whereas lysis buffer was used as positive control for

100% lysis. A protein concentration which causes less

than 5% lysis was considered as non-toxic.

In haemagglutinating assay, a serial twofold dilution

of purified protein (a concentration range of 3.90–1000 lg ml�1) was mixed with 50 ll of 2% human

erythrocyte suspension. The results were examined

after 1 h when the control without antifungal protein

has fully sedimented to observe if human RBCs has

agglutinated in response to the antifungal protein.

Stability of purified protein

The stability of protein at temperatures ranging from

4 to 100 °C was determined by incubating purified

protein AAP in 10 mmol l�1 Tris-HCl buffer (pH 7.5)

for 20 min at different temperatures. The solution of

protein was cooled to 4 °C and examined for anti-

aspergillus activities. The stability of AAP under acidic

and alkaline conditions was tested by using citrate

phosphate buffer (pH 2.5–8.0) and Tris-HCl buffer (pH

7.5–10.5). AAP was incubated in each buffer at vari-

ous pH values at 4 °C for 20 min. The anti-aspergillus

activity was assayed after the pH of each AAP solution

was readjusted to 7.5. The effect of various hydrolytic

enzymes on activity of purified protein was determined

by incubating the concentrated purified fraction with

a-amylase, pronase E, lipase, trypsin and proteinase K

at a final concentration of 1.0 mg ml�1 at 37 °C for

1 h. The effect of organic solvents (methanol, ethanol,

formaldehyde and acetone) up to a concentration of

20% v/v was examined after incubation at 37 °C for

1 h along with evaporation of solvent using speed vac

system. The biological activity after treatment was

evaluated by MDA. The effect of detergents (SDS,

PMSF and EDTA in 1 mmol l�1 concentration) was

also determined on activity of purified protein fraction.

MALDI-TOF analysis and Mascot search

In gel digestion of single band recovered during SDS-

PAGE analysis of purified antifungal protein was car-

ried out20 and subjected to Matrix-assisted laser

desorption ionisation–Time-of-flight (MALDI-TOF) at

Genbio (New Delhi). The peptide-mass fingerprint

obtained by mass spectrometry was searched in Mas-

cot search of Matrix Science to find out the peptide

matches present in the MSDB protein database.

Chrome azurol sulphonate (CAS) agar plate assay

The CAS assay, i.e. universal assay of Schwyn & Nei-

lands [21] was used. Cultures were spot inoculated

onto the blue agar and incubated at 37 °C for 12 h.

The results were inferred as positive for siderophore

activity if there was colour change due to removal of

the ferric ion from its intense blue complex to the sid-

erophore. The mean diameter of orange zones around

the growth signified the amount of siderophores

released.

Results

Screening of bacteria’s for antagonistic potential

The cell free supernatants (secretory) and cellular pro-

teins (cytosolic) of chosen bacterial strains were

Table 1 Screening of bacterial secretory & lysate proteins against Aspergillus spp. by microbroth dilution assay.

MTCC No. Bacterial strain

Average inhibitory concentration (MIC90) in lg ml�1

BSP (1000 to 3.9 lg ml�1) BLP (1000 to 3.9 lg ml�1)

A. fm A. fl A. ng A. fm A. fl A. ng

1938 Streptococcus thermophilus 125.00 250.00 – 250.00 500.00 –

460 Streptococcus lactis 125.00 250.00 500.00 – – –

1652 Escherichia coli DH5a 62.50 125.00 125.00 62.50 62.50 125.00

1674 E. coli 125.00 125.00 250.00 250.00 250.00 500.00

10312 E. coli 500.00 – – 500.00 – –

6810 Staphylococcus epidermidis – – – – – –

3615 S. epidermidis – – – – – –

9041 S. epidermidis 250.00 250.00 500.00 500.00 – –

MIC of Amphotericin B: 1.95 lg ml�1

BSP, Bacterial supernatant proteins; BLP, Bacterial lysate proteins; A. fm, Aspergillus fumigatus; A. fl, Aspergillus flavus; A. ng, Aspergillus

niger; –, means no activity up to tested concentration, i.e. 1000 lg ml�1

Bold values signify the most active protein fraction.


Mycoses, 2014, 57, 153–162156

M. Balhara et al.

screened for antifungal potential if any against Asper-

gillus spp. (Table 1) using different assays (MDA, DDA,

PSGI). The preliminary screening for susceptibility

against fungal pathogens revealed that cytosolic pro-

teins of E. coli DH5a were endowed with utmost anti-

fungal potential against all the pathogenic isolates of

A. fumigatus, A. flavus and A. niger depicting an aver-

age inhibitory concentration (MIC90) of 62.50, 62.50

and 125.00 lg ml�1 respectively. In DDA, a positive

and low concentration of 25.00 lg per disc of various

bacterial proteins used in this study showed that lysate

proteins of E. coli DH5a possessed potent antifungal

activity against pathogenic strains of Aspergillus spp.

Moreover, it was observed that secretory proteins of

Streptococus thermophilus, E. coli (MTCC 1674) and

Streptococcus lactis were moderately active against A.

fumigatus at a preset concentration of 25.00 lg per

disc (Additional file 1: Table S1). Hence, the lysate

proteins of E. coli DH5a were subjected to further puri-

fication process to identify the active protein moiety.

Purification of antifungal protein

Anion exchange chromatography of crude cytosolic

protein of E. coli DH5a resulted in three unadsorbed

fractions, F1–F3 and three adsorbed fractions, F4–F6(Fig. 1A). The antifungal activity resided in unad-

sorbed fraction F2 which on SDS-PAGE analysis

resulted into three major bands in the molecular

weight range 28–66 kDa after staining with silver

stain. Fraction F2 was further resolved on gel filtration

column into three subfractions SF1, SF2 and SF3

(Fig. 1B). The subfraction SF2 was endowed with anti-

fungal activity and depicted a single band on SDS-

PAGE as well as on native PAGE at a molecular

weight of 60 kDa on the gel stained with silver stain

(Fig. 2). It demonstrated similar molecular mass of

60 kDa in gel filtration which suggests that AAP is a

monomer. The yield of antifungal protein in different

purification steps is summarised in Table 2. The purity

of the active subfraction obtained from gel filtration

chromatography (i.e. SF2) was further checked by

UPLC. UPLC profile of SF2 showed a single major peak

at a retention time of 2.630 min (Fig. 3). The purified

protein molecule of E. coli DH5a was named as AAP.

The AAP recovered from UPLC showed purity up to

99.14% and it was found to be active against tested

Aspergillus spp.

Inhibitory spectrum of purified protein

The susceptibilities of various pathogenic isolates of A.

fumigatus, A. flavus and A. niger to growth inhibition

by the dialysed concentrate containing the purified

protein was analysed by MDA, PSGI and DDA assays.

The MIC90 values of antifungal activity of AAP against

these fungi are represented in Fig. 4A, B and C. The

degree of inhibition was maximal against A. fumigatus

and A. flavus depicting a MIC of 15.62 lg ml�1 via

MDA and PSGI, whereas the susceptibility of A. niger

was low against the antifungal protein (MIC90 being

31.25 lg ml�1). The inhibitory concentrations of anti-

fungal protein which developed significant zone of

inhibitions (at least 6 mm in diameter excluding the

disc) led to approximate values of MICs about 5.0 lgper disc against A. fumigatus, A. flavus and 10.0 lgper disc against A. niger. However, the zone of inhibi-

tion increased with increase in dose of antifungal pro-

tein (Fig. 5). A similar dose-dependent inhibitory

behaviour was revealed in PSGI as shown in Fig. 6.

(a) (b)mAU500

450

400

Abs

orba

nce

at 2

80 n

m

Abs

orba

nce

at 2

80 n

m

NaC

l Gra

dien

t350

300F1

F4

F6

F2

F3

250

200

150

100Injection Valve Load

1

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

Fraction Off

220 min

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 5152 Waste32

Gradient Length 150.0 ml, Target 100% B50

Fraction Number

0.0 mol l–1

0.5 mol l–1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

00 10

SF1

SF2

SF3

20 30

Elution Volume (ml)

40

1.0 mol l–1F5

Figure 1 (A) Anion exchange chromatogram of crude cytosolic protein of Escherichia coli DH5a on Q-XL sepharose column of Akta

purifier system, F1–F3: Unadsorbed fractions, F4–F6: Adsorbed fractions, F2 – Fraction with antimycotic activity having a MIC of

31.25 lg ml�1 against Aspergillus fumigatus in MDA assay (B) Gel filtration chromatograph of fraction F2 on sephadex G100 column;

subfraction (SF2) endowed with antimycotic activity.


Mycoses, 2014, 57, 153–162 157


The MIC values of AAP were similar against all the

three tested Aspergillus spp., being in the range 15.62–31.25 lg ml�1 using NCCLS (CLSI) standards.

Toxicological studies and stability parameters of

purified protein

In vitro cytotoxicity of the purified protein was deter-

mined against healthy human erythrocytes by haemo-

lytic and haemagglutination assays. The purified

protein AAP was found to have negligible cytotoxicity

against human RBCs (Red blood cells). The effect of

various concentrations of purified protein on erythro-

cyte lysis is depicted in Fig. 7. It was non-toxic in the

concentration range 3.90–1000 lg ml�1 (the highest

concentration tested). The extended exposure of ery-

throcytes to 1000 lg ml�1 of AAP up to 24 hs also

did not show any toxicity to erythrocytes. Amphoteri-

cin B causes 100% haemolysis at a concentration of

37.50 lg ml�1 only. Hence, AAP was manifold less

toxic against human red blood cells. The purified pro-

tein also was found to have no haemagglutinating

activity to a dose of 1000 lg ml�1, however, exposure

to higher doses, i.e. 2000, 4000 lg ml�1 causes slight

haemagglutination.

The anti-aspergillus potential of purified protein after

treatment with wide range of temperatures (4–100 °C)and various pH conditions (2.5–10.5) is represented in

Fig. 8A and B. Its antifungal activity was preserved up

to a thermal treatment of 70 °C. However, the activity

of AAP decreased with further rise in temperature,

decreasing by 40% after incubation at 80 °C and the

antifungal potential was totally abrogated following

60 kDa

P F2 C M kDa

97

66

42

28

20

14

M - Molecular Marker; C - Crude Cytosolic ProteinF2 - Anion Exchange Fraction; P - Purified Protein (SF2)

Figure 2 Sodium dodecyl sulphate–polyacrylamide gel electro-

phoresis results of Escherichia coli DH5a-purified antifungal

protein.

Table 2 Summarised purification steps of anti-Aspergillus protein

(AAP) from crude cytosolic fraction of Escherichia coli DH5aagainst Aspergillus fumigatus.

Purification step

Vol.

(ml)

Protein yield

(mg ml�1)

MIC

(lg ml�1)

Purification

(factor)

Crude cytosolic

fraction

50 1.00 62.50 1

70% NH4SO4

precipitation

15 2.50 62.50 1

Anion exchange

fraction F2

10 0.60 31.25 2

Gel filtration fraction

SF2 (purified protein

AAP)

7 0.34 15.62 4

0.010

0.008

0.006

0.004

AU

0.002

0.000

0.00 0.50 1.00 1.50

1 2.630 26915 100.00 9943

Height% AreaAreaRT

2.63

0

2.00 2.50 3.00Min

3.50 4.00 4.50 5.00 5.50 6.00Figure 3 UPLC analysis of purified pro-

tein anti-Aspergillus protein fraction SF2

depicting a retention time (RT) of

2.630 min.


Mycoses, 2014, 57, 153–162158

M. Balhara et al.

treatment at 90 °C. The purified protein completely

retained its biological activity in the pH range 6–10,showing maximum activity at pH 7.5. However, the

activity was reduced at pH values below 4.5, depicting

complete loss after treatment at pH 3.5 for 20 min.

There was absolute detriment of antimycotic activity

after treatment with proteinase K and pronase E, con-

firming the proteinaceous nature of antifungal mole-

cule. Treatment with trypsin led to partial inactivation

of AAP, retaining 65% of activity which connotes the

presence of distinct activity domains. However, AAP

was resistant to treatment with a-amylase and lipase,

retaining its antifungal potential. The biological activ-

ity remained intact after exposure to EDTA, PMSF and

SDS at 1 mmol l�1 concentration for 1 h. The

(a) (b) (c)

I II I II I II

250.0125

62.5

31.2

125.0

62.5

31.2

15.6

15.6

7.8

7.8

3.9

3.9

1.9

µg ml–1

250.0

125.0

62.5

31.2

15.6

7.8

3.9

µg ml–1µg ml–1

Figure 4 (A) Growth inhibition of Asper-

gillus fumigatus by purified protein AAP:

(Lane I: 1st four wells media only, 5–7wells showing normal growth of fungus),

(Lane II: Wells treated with different con-

centrations of AAP depicting a MIC of

15.6 lg ml�1) (B) Growth inhibition of

A.flavus by purified protein AAP: (Lane I:

Wells showing normal growth of fungus),

(Lane II: Wells treated with different con-

centrations of AAP depicting a MIC of

15.6 lg ml�1) (C) Growth inhibition of

A.niger by purified protein AAP: (Lane I:

1st three wells media only, 4–7 wells

showing normal growth of fungus), (Lane

II: Wells treated with different concentra-

tions of AAP depicting a MIC of

31.2 lg ml�1).

MIC of 5 µg disc–1C

EA

D

B

Figure 5 Inhibitory effect of purified protein AAP on growth of

Aspergillus fumigatus using DDA: A-20 lg AAP, B-10 lg AAP,

C-5 lg AAP, D-2.50 lg AAP, E-2.5 lg Amphotericin B.

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%0 1.9 3.9

A. flavus

A. fumigatus

A. niger

7.8 15.6Conc. of protein in µg ml–1

Per

cent

age

spor

e ge

rmin

atio

nin

hibi

tion

31.2 62.5 125 250 500

Figure 6 Dose–response curve showing inhibitory effect of puri-

fied protein AAP against Aspergillus fumigatus, A.flavus and

A.niger using PSGI assay.

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%0 0.3 0.6 0.95

Log conc. of protein in µg ml–1

Ampho B

AAP

1.2 1.6 1.8 2.1 2.4 2.7 3 33 36

Per

cent

age

of h

aem

olys

is o

f e

ryth

rocy

tes

Figure 7 Toxicity of purified protein AAP and Amphotericin B

(Ampho.B) against human erythrocytes using haemolytic Assay.


Mycoses, 2014, 57, 153–162 159


antimycotic activity was also unaffected in the pres-

ence of organic solvents throughout the duration of

assay. The stability parameters of purified protein AAP

are summarised in Table S2.

Proteomic and bioinformatics analysis

The mass spectrum obtained after MALDI-TOF analysis

of trypsin digested peptides of purified protein is repre-

sented in Fig. 9. The mass spectrometric data when

subjected to Mascot algorithm revealed that 11 pep-

tides had significant sequence similarity with

siderophore biosynthetic protein (Non-ribosomal pep-

tide synthetase) from E. coli. In CD (conserved domain)

domain search at NCBI blast, five peptides having

potential match against the siderophore biosynthetic

protein, majority in the C-terminal region, i.e. 1961–1973, 2999–3015, 2612–2630, 1747–1766, repre-

sents the conserved domain of non-ribosomal peptide

synthetase active site, the enzyme involved in initial

step of siderophore (non-ribosomally synthesised pep-

tides) biosynthesis (Table 3). The inhibitory effect of

purified protein AAP was further evaluated by addi-

tion of 10 mmol l�1 ferric chloride to medium; the

activity was drastically eliminated which supplements

that AAP protein plays an important role in disruption

of iron uptake and transport process in fungus.

Siderophore detection and analysis

Mass spectrometric data disclosed similarity with sid-

erophore biosynthetic protein which prompted us to

analyse the siderophore production in A. fumigatus

using CAS agar plates which produced orange halo

confirming siderophore release. However, inoculation

of spot along with purified protein AAP resulted in loss

of this characteristic which correlates its role in inhibi-

tion of siderophore biosynthesis in fungus. The mean

diameter of orange zone decreases with increase in

concentration of AAP, depicting complete loss at

MIC75 which suggests a dose-dependent inhibitory

behaviour (Fig 10). The purified protein itself does not

give the change in colouration, depicting that activity

(a) (b)

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

0 4 20 30 40 50 60

Temperature in °C

Rel

ativ

e P

erce

ntag

e A

ctiv

ity

70 80 90

Activity

III

I

II

IV

V

100 100

Figure 8 (A) Effect of exposure to varying temperatures (4–100°C) on antifungal potential against A. fumigatus of purified protein anti-

Aspergillus protein (AAP) (B) Effect of various pH treatments on antifungal activity of purified protein AAP (10 µg per disc) against A.

fumigatus: I: pH 3.5, II: pH 5.5, III: pH 7.5, IV: pH 8.5, V: pH 9.5.

6000

605.819607.811

Inte

ns.[a

.u.]

4000

2000768.458

882.620

1077.445

1639.435

1839.533

2235.762

2399.552 2705.553

2584.6582932.757

0

500 1000 1500 2000 2500 3000 m/z

Figure 9 Mass spectrum of trypsin digested peptides of purified

protein anti-Aspergillus protein using MALDI-TOF.


Mycoses, 2014, 57, 153–162160

M. Balhara et al.

is not due to bacterial siderophore, but some other

iron uptake and transport regulated protein.

Discussion and future prospects

There is currently a lack of reliable diagnostic tools

and effective treatment options for invasive aspergillo-

sis, resulting in a high mortality rate despite

therapy.22 The major concerns associated with avail-

able antifungal drugs are toxicity and the development

of resistance in pathogens against almost all the che-

motherapeutic agents23 which persuade a need to

identify other less toxic natural products, particularly

antimicrobial proteins or peptides like inhibitory sub-

stances. As fungi are more similar to mammalian cells

on cellular level as compared to bacteria, therapeutics

targeting fungal specific pathways is an urgent medi-

cal need. In the current investigation, an antifungal

protein AAP has been purified and partially character-

ised from commensal E. coli DH5a strain (MTCC

1652), depicting significant growth inhibition against

A. fumigatus, A. flavus and A. niger (MIC90 from 15.62

to 31.25 lg ml�1). The high toxicity of previously

reported antifungal peptides and proteins from micro-

bial sources has impeded their role as potential candi-

dates for future antifungal therapy.24,25 Therefore, the

feature which paramounts the usefulness of this iso-

lated protein was its ground level cytotoxicity against

human erythrocytes (causing less than 5% lysis up to

a dose of 1000 lg ml�1).

Matrix-assisted laser desorption ionisation–Time-of-

flight results revealed similarity with siderophore bio-

synthetic protein and further bioinformatics search

exposed the presence of a conserved condensation

domain present in non-ribosomal peptide synthetase

enzymes. Siderophores are non-ribosomally synthesised

peptides which play crucial role in iron uptake and

transport in iron-starving conditions as found in mam-

malian hosts during pathogenesis.26 Several studies

have demonstrated the requirement of siderophores for

growth and virulence in A. fumigatus.27,28 The role of

Table 3 Analysis and sequence similarity of peptides matched with the siderophore biosynthetic protein from Escherichia coli after tryp-

sin digestion of purified protein AAP by Mascot algorithm.

Observed Mr (expt) Mr (calc) ppm Start–end Miss Peptide sequence

599.0845 598.0772 598.3122 �0.2350 790–793 1 R.RHMR.Q

607.8108 606.8035 607.2384 �0.4349 989–993 0 R.CTDGR.Y

1639.4345 1638.4273 1638.8412 �0.4140 1961–1973 1 K.AWNQLIARHDMLR.M + Oxdn (M)

2211.6876 2210.6804 2210.9693 �0.2890 2999–3015 1 R.MVLIDPVCRQDFCCENR.A

2225.6906 2224.6833 2225.0614 �0.3781 2612–2630 1 R.QSYHFTDVSAQFLNDARAR.F

2249.7461 2248.7389 2248.9664 �0.2275 1747–1766 0 R.GMGALSDAEGCWHLEQAVMR.G + 2 Oxdn M

2399.5519 2398.5446 2398.1409 0.4037 2944–2963 1 K.SPQRFATLDQMIDEYVGCIR.R

2501.7249 2500.7176 2501.1935 �0.4759 694–716 0 R.QFELDLAANNGTQHTVFSGPEAR.L

2584.6583 2583.6510 2583.2526 0.3984 1006–1028 0 R.ESLLTELASYCEGFQAIPDTIAR.A

2705.5532 2704.5459 2704.3568 0.1890 483–508 1 R.LATDYAGALRENADASSLAFTALHAR.R

2932.7572 2931.7499 2931.5011 0.2488 299–326 1 R.KVGYTAPSVAGQQAVIEEALMLAAIDDR.Q

Identified protein: Siderophore biosynthetic protein [E. coli O26:H11 str.CVM10026] accession no. -gi|388374259, molecular mass:

351235 D, score: 54, expect: 15, matches: 11.

Peptides highlighted in Table 3 represents conserved domain, i.e. condensation domain (accession: c|17111; PSSM Id: 214272) found

in many multidomain enzymes which synthesise non-ribosomal peptide antibiotics.

CONTROL

0.125 MIC

0.75 MIC

0.250 MIC

0.50 MIC

Figure 10 Effect of AAP on siderophore biosynthesis in Aspergil-

lus fumigatus using CAS agar plate assay: Treated with different

concentrations of AAP; from 0.125 MIC to 0.750 MIC depicting

dose-dependent inhibitory (diameter of orange zone decreases)

mechanism MIC of AAP: 5 lg per disc.


Mycoses, 2014, 57, 153–162 161


AAP in disruption of siderophore biosynthesis has

been substantiated by CAS assay which revealed inhi-

bition in siderophore synthesis as its possible mecha-

nism for action. Further studies related to its mode of

action may lead to novel targets in fungal proteome as

the absence of such siderophore biosynthetic pathways

in mammals holds much promise to siderophore bio-

synthesis as basis of antifungal therapy.

Acknowledgements

This work has been supported by University Grants

Commission (UGC) India, Haryana State Council for

Science and Technology (HSCST, India).

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Supporting information

Additional supporting information may be found in

the online version of this article.

Table S1. Antifungal evaluation of bacterial pro-

teins using agar disc diffusion assay at specific concen-

tration of 25.00 µg per disc.

Table S2. Effect of hydrolytic enzymes, surfactants

and organic solvents on biological activity of purified

protein AAP against Aspergillus fumigatus.


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