Amylosin from Bacillus amyloliquefaciens, a K+ and Na+ channel-forming toxic peptide containing a polyene structure

ARTICLE IN PRESS

0041-0101/$ - se

doi:10.1016/j.to

Abbreviations

membrane; BSA

reference to acc

piperazine-1-eth

potential; PI, pr

imidazolo carbo�CorrespondiE-mail addre

Toxicon 49 (2007) 1158–1171

www.elsevier.com/locate/toxicon

Amylosin from Bacillus amyloliquefaciens, a K+ and Na+

channel-forming toxic peptide containing a polyene structure

Raimo Mikkolaa,�, Maria A. Anderssona, Vera Teplovaa,b, Pavel Grigorieva,c,Till Kuehnd, Sandra Lossd, Irina Tsitkoa, Camelia Apetroaiea, Nils-Erik L. Sarisa,

Pirjo Veijalainene, Mirja S. Salkinoja-Salonena

aDepartment of Applied Chemistry and Microbiology, Helsinki University, P.O.B. 56, FIN-00014, Helsinki, FinlandbInstitute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Russia

cInstitute of Cell Biophysics, Russian Academy of Sciences, Pushchino, RussiadBruker BioSpin, Fallanden, Switzerland

eFinnish Food Safety Authority Evira, Helsinki, Finland

Received 17 November 2006; received in revised form 6 February 2007; accepted 7 February 2007

Available online 25 February 2007

Abstract

Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings produce a

1197Da toxin, named amylosin. Nuclear magnetic resonance (NMR) data showed that amylosin contains a chromophoric

polyene structure and the amino acids leucine/isoleucine, proline, aspartic acid/asparagine, glutamic acid/glutamine and

tyrosine. A quantitation method for amylosin was developed using commercially available amphotericin B as a reference

compound and a known concentration of amylosin determined by NMR with the electronic reference to access in vivo

concentration (ERETIC) method. Purified amylosin inhibited motility of boar sperm cells at an exposure concentration of

135 nM and hyperpolarized their cell membrane and depolarized their mitochondria at exposure to concentration of

33–67 nM for 10min. In a 3-d exposure time only 27 nM of amylosin was needed to provoke the same toxicity functions.

Amylosin was cytotoxic to feline lung cells at concentrations of o170 nM. Purified amylosin provoked adenosine

50-triphosphate (ATP)-independent cation influx into isolated rat liver mitochondria (RLM), inducing swelling of the

mitochondria at concentrations of 200 nM K+ or 4250 nM Na+ medium. In the Kþ- or Naþ-containing medium,

amylosin uncoupled RLM, causing oxidation of pyridine nucleotides (PN), loss of the mitochondrial membrane potential,

and suppressed ATP synthesis. Purified amylosin produced cation channels in black-lipid membranes (BLMs) with a

selectivity K+4Na+ at a concentration of 26 nM, i.e. the same concentration at which amylosin was toxic to boar sperm

cells. The amylosin cation channels were cholesterol- and ATP-independent and more effective with K+ than with Na+.

e front matter r 2007 Elsevier Ltd. All rights reserved.

xicon.2007.02.010

: ADP, adenosine 50-diphosphate; TRIZMA base, 2-amino-2-(hydroxymethyl)-1, 3-propanediol; BLM, black-lipid

, bovine serum albumin; EGTA, ethylene glycol-bis(2-aminoethylether)-N, N, N0, N0-tetraacetic acid; ERETIC, electronic

ess in vivo concentrations; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HEPES, 4-(2-hydroxyethyl)

anesulfonic acid; IT-MS, iontrap mass spectrometry; DCm, mitochondrial membrane potential; DCp, plasma membrane

opidium iodide; PN, pyridine nucleotides; RLM, rat liver mitochondria; JC-1, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30-tetrabenz-

cyanine iodide

ng author. Tel.: +358 9 19159327; fax:+358 9 19159301.

ss: [email protected] (R. Mikkola).

ARTICLE IN PRESSR. Mikkola et al. / Toxicon 49 (2007) 1158–1171 1159

We propose that the toxicity of amylosin may be due its ionophoric properties, representing the first K+/Na+ channel-

forming substance reported from B. amyloliquefaciens.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Amylosin; Bacillus amyloliquefaciens; Channel; Mitochondria; Indoor air; Swelling; Ionophore; Boar sperm; Feline lung;

Uncoupling

1. Introduction

Bacillus amyloliquefaciens strains are known pro-ducers of fungicidal cyclic lipopeptides, e.g. iturin,surfactin, fengycin, bacillomycin D (Koumoutsi et al.,2004; Yoshida et al., 2001; Hiradate et al., 2002; Yuet al., 2002; Winfried et al., 1998) and fungicidalenzymes such as chitinase (Wang et al., 2002).Strains of this species were investigated for use asbiofungicides to protect agricultural crops (Yuet al., 2002; Kim and Chung, 2004) and as agentsfor plant growth promotion (Idriss et al., 2002).Apart from the chitinolytic enzymes lysing fungalcell walls, the biochemical basis of the fungicidalpotency of B. amyloliquefaciens is not clear. A totalof 10% of the genome of B. amyloliquefaciens

FZB42 is dedicated to nonribosomal peptide andpolypetide synthases (Koumoutsi et al., 2004). Sixlarge synthases were found, three of which wereconnected with the production of surfactin, fengy-cin, and bacillomycin D.

We found that fungicidal B. amyloliquefaciens

strains were prevalent in moisture-troubled buildingswhere the occupants suffered an excess of respira-tory health problems (Andersson et al., 2002). Wereported recently that exposure to extracts of suchfungicidal B. amyloliquefaciens strains caused dis-sipation of the transmembrane potentials of mito-chondria ðDCmÞ and of plasma membranes ðDCpÞ inhuman neural cells and boar spermatozoa exposedat low concentration (0.2mgmL�1). In black-lipidmembrane (BLM) the extracts generated K+- andNa+-permanent channels (Mikkola et al., 2004).Two different substances were detected in the toxicextracts, of which one was identified as the lipopep-tide surfactin (Mikkola et al., 2004), known fromcertain strains of B. subtilis. The other substancewith a molecular mass of 1197Da, was lesshydrophobic than surfactin, contained six aminoacids (leucine, proline, serine, aspartic acid, glutamicacid, and tyrosine), but was not identical to any ofthe microbially produced (lipo)peptides described.

In the present study the structure of the 1197-Dapeptide toxin, now named amylosin, purified from

B. amyloliquefaciens was analyzed using nuclearmagnetic resonance (NMR) spectrometry. Further-more, a quantitative high-performance liquid chro-matography–ultraviolet (HPLC–UV) method wasdeveloped for amylosin and its ionophoretic proper-ties, effects on mammalian cells, and isolatedmitochondria were described.

2. Materials and methods

2.1. Animals

The experiments were performed using adult maleWistar rats (200–250 g) born and raised in theanimal care unit of the Viikki Biocenter. All studieswere performed with the permission of the AnimalEthics Committee in accordance with the animalcare policy of national and European regulations.

2.2. Chemicals

Adenosine 50-diphosphate (ADP), bovine serumalbumin (BSA), ethylene glycol-bis(2-aminoethy-lether)-N,N,N0,N0-tetraacetic acid (EGTA), carbo-nyl cyanide p-trifluoromethoxyphenylhydrazone(FCCP), 4-(2-hydroxyethyl) piperazine-1-ethanesul-fonic acid (HEPES), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA base), D-Mannitol, va-linomycin, propodium iodide (PI), amphotericin B,and phosphatidylcholine (P5638) were purchasedfrom Sigma (St. Louis, MO, USA). 5,50,6,60-tetra-chloro-1,10,3,30-tetrabenz-imidazolo carbocyanineiodide (JC-1) was from Molecular Probes Inc.(Eugene, OR, USA). Other chemicals were analy-tical grade and purchased from local sources.

2.3. Purification and analysis of amylosin from

B. amyloliquefaciens

A methanol extract was prepared from B. amylo-

liquefaciens strain 19b as described earlier (Mikkola etal., 2004). The methanol extract was diluted to 80%(v/v) methanol by adding aqueous 0.1% HCOOHand then fractionated with reversed-phase-HPLC

ARTICLE IN PRESSR. Mikkola et al. / Toxicon 49 (2007) 1158–11711160

using Smart System (Amersham Biosciences, Uppsala,Sweden). The column used was an Atlantis dC 18,4:6mm� 150mm, 3mm (Waters, Milford, MA,USA). The eluent was a gradient of A, 0.1% formicacid and B, methanol from 20% A and 80% B to 5%A and 95% B in 18min and to 100% B in 22min, at aflow rate of 300mLmin�1. For quantitation ofamylosin the gradient used was 0.1% formic acidand B, methanol from 40% A and 60% B to 100% Bin 17min and to 100% B in 25min, at a flow rate of300mLmin�1. Amphotericin B was used as a referencecompound to quantitate amylosin. For detection,absorbances at wavelengths of 215, 382, 365, and407nm were used.

2.4. Mass spectrometry

The electrospray ionization-iontrap mass spectro-metry analysis (ESI-IT-MS) was performed usingthe instrument described earlier (Mikkola et al.,2004). Purified amylosin was injected into the ESIdevice with a syringe pump at a flow rate of250 mLmin�1. The toxicity of purified amylosin withmass ion of m/z 1220 (Na+ adduct, verified withMS) was assayed for motility inhibition of boarspermatozoa as described in Andersson et al. (2004).

2.5. NMR experiments

The result of the NMR experiments using 1H,correlation spectroscopy (COSY), total correlatedspectroscopy (TOCSY), and heteronuclear multiplequantum correlation (HSQC) were recorded usingan AVANCE 600MHz Bruker spectrometer (Bru-ker Biospin, Switzerland) equipped with a TXI1-mm MicroProbe using 5-mL sample tubes. Amy-losin (400 mg) was dissolved in 5 mL dimethyl-d6sulfoxide (DMSO-d6) (66mM). The electronicreference to access in vivo concentrations (ERE-TIC) NMRmethod (Akoka et al., 1999) was used tomeasure the precise concentration of amylosin.Quinine was used as a reference compound forquantification of the amylosin with the ERETICmethod. The Perch program (version 2005.1,PERCH Solutions Ltd., Finland) was used forsimulation of the 1H-NMR spectra.

2.6. Toxicity assays and fluorescence microscopy

The boar spermatozoan motility assays andeffects on the mitochondrial and plasma membraneswere visualized with fluorescence microscopy after

staining with JC-1 or PI (viability stain), and theproliferating fetal feline lung cells were cultivatedand used for the cytotoxicity assay as describedearlier (Mikkola et al., 2004).

2.7. Preparation of rat liver mitochondria (RLM)

and mitochondrial functions

Preparation of RLM was performed and theoxygen consumption and mitochondrial osmoticvolume changes were measured as described inTeplova et al. (2006). Mitochondrial swelling wasdetermined by measuring the apparent absorbancechange of mitochondrial suspensions at 540 nmusing the computer-controlled Shimadzu UV/VIS-1700 Pharmaspec spectrophotometer (ShimadzuCorp. Japan). The fluorescence changes due toredox change of the pyridine nucleotides (PN) werefollowed on a Hitachi F4000 spectrofluorometer(Hitachi Ltd., Tokyo, Japan) using an excitationwavelength of 340 nm and an emission wavelengthof 460 nm. The DCm was measured with rhodamine123 as a fluorescent probe (Emaus et al., 1986),using an excitation wavelength of 503 nm and anemission wavelength of 527 nm with the sameequipment. Details of the mitochondrial incubationmedia are given in the figure legends.

2.8. Conductance in BLM

The BLM measurements were performed asdescribed in Mikkola et al. (2004). The BLM wasformed from soybean phosphatidylcholine (20mgmL�1 n-heptane). The black membrane area neces-sary for calculation of the specific conductance wasestimated from the height of the membranecapacitance current peaks generated by the rectan-gular-shaped voltage applied to the BLM.

3. Results

3.1. HPLC purification of the 1197-Da substance

and verification of the purity and mass spectra with an

ESI-IT-MS

The 1197-Da substance was purified by HPLCfrom a methanol extract of B. amyloliquefaciens

biomass. A sperm motility test was used to indicatethe toxic fraction (peak 1 in Fig. 1A). The sperm-toxic fraction was purified and its purity verified byrerunning with HPLC and detecting with ESI-IONTRAP-MS. A single peak (peak 1 in Fig. 1B)

ARTICLE IN PRESS

4

3

2

1

0

0 5 10 15 20 25 30

min

0 5 10 15 20 25 30

min

AU

215 nm365 nm

1 1.0

0.5

0.0

AU

215 nm365 nm

1

5x105

4x105

3x105

2x105

1x105

0

250

500

750

1000

1250

1500

1750

2000

m/z

250

500

750

1000

1250

1500

1750

2000

-m/z

Inte

nsity

1220.6 6x104

5x104

4x104

3x104

2x104

1x104

0

Inte

nsity

1196.6

9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00

ppm

6.00

1

2

5.00

HH

H

H

H

H

H

H

H

H

H

H

H

Fig. 1. HPLC purification of amylosin, verification of the purity with IT-MS, and experimental and simulated polyene region 1H-NMR

spectra. (A) HPLC elution profile of methanol extract of 1117

in which peak 1 was sperm-toxic. (B) Peak 1 rerun under same HPLC

conditions as the sperm-toxic fraction in A (peak 1 from A). (C) Mass spectrum of the sperm-toxic fraction (peak 1 from A) in positive

mode obtained with ESI-IT-MS. The mass ion with m/z of 1220.6 is the Na+ adduct of amylosin. (D) The same experiment as C, but

showing the mass spectrum obtained in negative mode �m/z 1196.6 [M–H]� of amylosin. (E) 1H-NMR spectrum of amylosin in DMSO-

d6 and simulated 1H-NMR spectrum (2) of polyene structure of hexaene (1).

R. Mikkola et al. / Toxicon 49 (2007) 1158–1171 1161

absorbing at 215 and 365 nm was seen in the HPLCelution profile of the rerun toxic fraction. The toxicfraction was evaporated to dryness and dissolved in

0.1% NH4OH for the MS analysis. The massspectrum of the sprayed toxic fraction in the positivemode gave a mass ion with a mass-to-charge ratio

ARTICLE IN PRESSR. Mikkola et al. / Toxicon 49 (2007) 1158–11711162

(m/z) 1220.6 (Fig. 1C) and in the negative-modeexperiment an m/z of 1196.6 (Fig. 1D). The mass ionof m/z 1220.6 corresponds to the sodium adductidentical to the 1197-Da substance described earlier

6.0 5.0

p

8.5

p

II

8.0 7.0 6.0 5.0

ppm

6.50 6.25 6.00 5.75 5.50

ppm

131112 3 4

2157

6108

9

125

130

135

Fig. 2. COSY, TOCSY, and HSQC spectra of amylosin in DMSO-d6.

TOCSY spectrum of amylosin showing cross-peaks of NH and 1H ass

polyene and amino acid residue (Table 1) 1H–13C correlation regions. (D

assigned to a hexaene structure. (E) Amino acid residues 1Ha–13C.

in B. amyloliquefaciens extracts, showing toxicitytowards different types of mammalian cells (Mikko-la et al., 2004). We named this pure substanceamylosin.

4.0 3.0 2.0

6.0

5.0

4.0

3.0

2.0

1.0

pm

8.0 7.5

pm

5.0

4.0

3.0

2.0

1.0

IVIII

4.0 3.0 2.0 1.0

140120100806040200

a b

d

c

e

4.75 4.50 4.25 4.00

60

55

50

45

ppm

(A) COSY spectrum of amylosin showing the polyene region. (B)

igned to amino acids. (C) HSQC spectrum of amylosin showing

) Enlarged HSQC spectra of amylosin showing the polyene region

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3.2. NMR analysis of amylosin chromophore group

Amylosin appeared as a yellow color containing achromophoric group. The 1H-NMR spectra ofamylosin showed proton chemical shift signals atan olefinic region of 5.3–6.4 ppm, indicating apolyene structure (Fig. 1E). Thirteen correlationsbetween the 1H chemical shift region of 5.4–6.5 ppmand the 13C region of 126–137 ppm were found in aHSQC experiment (Figs. 2D and E, Table 1). TheseHSQC data corresponded to the conjugated double-bound system of hexaene. The COSY spectrum ofamylosin (Fig. 2A) showed in the polyene region(5.4–6.5 ppm) proton chemical shift patterns similarto those in the published NMR data for thecorresponding structures. A simulated 1H-NMRspectrum of hexaene (Fig. 1E, 1) using the

Table 1

HSQC data of amylosin polyene 1H–13C region and amino acids1Ha–13C

1H (ppm) 13C (ppm) Amino acid

residues

1Ha (ppm) 13C (ppm)

1a 5.5 126.6 ab Glx/Asx (I) 4.8 48.8

2 5.4 126.6 b Glx/Glx (II) 4.6 50.3

3 5.5 136.4 c Leu/Ileu (III) 3.9 46.4

4 5.4 136.4 d Tyr (IV) 4.3 54.7

5 5.9 127.3 e Pro (V) 4.2 59.0

6 6.2 131.0

7 6.4 128.3

8 6.4 132.3

9 6.5 130.9

10 6.3 131.9

11 6.4 135.4

12 6.3 135.4

13 6.5 135.5

aIndex numbers refer to Fig. 2D.bIndex letters refer to Fig. 2E.

Table 2

TOCSY cross-peaks of amylosin amino acid residues NH–Ha–e spin sy

Amino acid residue Proton chemical shifts (ppm)

NH Ha Hb

(I) Glu/Gln or 8.4 4.7 2.6

Asp/Asn 8.4 4.7 2.6, 2.2

(I) Glu/Gln or 8.2 4.5 2.4

Asp/Asn 8.2 4.5 2.4, 2.2

(III) Leu/Ileu 7.7 3.9 2.2

(IV) Tyr 7.6 4.4 2.9

(V) Proline 4.2 2.0

Perch program showed 1H chemical shift patterns(Fig. 1E, 2) similar to those in the experimentallyobtained spectrum of amylosin in the region(5.4–6.5 ppm) (Fig. 1E).

3.3. NMR analysis of amylosin amino acids

In the TOCSY spectrum (Fig. 2B) of amylosin, four(I–IV) spin systems were assigned to amino acidresidues. In the COSY spectrum the cross-peaks offour amino acid NH and Ha protons were assigned.The proline spin system was assigned both to COSYand TOCSY (Table 2). The proton doublet signals 6.6and 7.0ppm had cross-peaks in TOCSY and COSYand were assigned to the tyrosine aromatic rings Hd

and He, respectively. The 7.4- and 6.8-ppm cross-peaksin COSY and TOCSY could be assigned to asparagineNHd or glutamine NHe. The characteristic peptideregions of the cross-peaks between Ha–e and 13C werefound in amylosin using the HSQC (Fig. 2D, Ha–13Cdata are shown in Table 1). Considering the NMR dataobtained from COSY, TOCSY, and HSQC wepropose that amylosin contains five amino acid residuesGlx/Asx (I), Glx/Asx (II), Leu/Ileu (III), Tyr (IV), andPro (V) (Tables 1 and 2). In conclusion, the NMR datashowed that amylosin consisted of a polyene chromo-phore similar to the hexaene and amino acids residuesof Leu/Ileu, Tyr, Pro, Glu/Gln, or Asn/Asp.

3.4. Quantitation of amylosin

The concentration of amylosin in DMSO-d6could be measured applying the ERETIC methodin NMR with quinine (30mM) as a referencecompound. The data obtained on the exact con-centration of amylosin and the structural informa-tion on the chromophoric group obtained with

stems

Hg Hd He

2.2

6.8 (Gln/Asn) 7.4 (Gln/Asn)

2.2

6.8 (Gln/Asn) 7.4 (Gln/Asn)

1.3 1.2

2.6 6.6 7.0

1.8 3.4

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1.0

0.5

0.0

AU

0 5 10 15 20

min

382 nm 1

2

Fig. 3. Amylosin quantitation with HPLC–UV using amphoter-

icin B as reference compound. HPLC elution profiles showing

amphotericin B (peak 1) and amylosin (peak 2) absorption at

382 nm in 1 mg per injection.

R. Mikkola et al. / Toxicon 49 (2007) 1158–11711164

NMR were used to develop an HPLC–UV quanti-fication method with amphotericin B as a referencecompound. The retention time of a referencecompound amphotericin B was too short in theHPLC conditions used to purify amylosin andtherefore improved HPLC method was developed.In HPLC the retention time (16.55min) of ampho-tericin B was shorter than that of amylosin(22.15min) and the UV absorbances at 215, 365,and 407 nm were different for amylosin andamphotericin B. Amylosin absorbed at 215 nm fivetimes more intensively than amphotericin B,whereas at 382 nm the extinction coefficients ofamylosin and amphotericin B were similar for theinjected amount of 1 mg (Fig. 3). Based on the UVabsorbance and the structural properties of amylo-sin, an HPLC–UV method was developed toquantify amylosin using the macrolide polyeneamphotericin B as a reference compound.

3.5. Amylosin-induced channel activity in BLM

The ionophoretic properties and ion selectivity ofamylosin in BLM were measured in NaCl and KClmedia. The starting concentrations of NaCl and KClwere 50mM at the cis and trans sides. During theexperiment the cis-side concentrations of the electro-lytes were increased in 100-mM steps to a finalconcentration of 500mM. Zero-current potentialsacross the membrane generated by the ionic concen-tration gradient were measured for both saltsseparately. The amylosin cation-over-anion perme-

ability ratios were K=Cl ¼ 11 and Na=Cl ¼ 7,calculated using the Goldman–Hodgkin–Katz equa-tion (Mikkola et al., 2004) and data shown in Fig. 4C.For single-channel measurements, amylosin dissolvedin methanol was added (o30 ngmL�1,o26 nM) tothe electrolyte solution in the cis chamber withconstant stirring at room temperature. Fig. 4A showsthat amylosin in a potassium medium induced higherconductivity and longer opening time of singlechannels than in a sodium medium. Fig. 4B showsthe conductance across the membrane as a functionof the concentration of amylosin added to the cis sideof the membrane in KCl medium. The conductancebegan to rise when exposed to concentration of30ngmL�1 (26 nM) of amylosin, while at a fivetimes higher concentration it had already increased100-fold. The steep dependence of bilayer conduc-tance on amylosin concentration in Fig. 4B suggeststhat at least four amylosin molecules forms thechannels. The amplitude histogram of the potassiumchannels is shown in Fig. 4D where max of thehistogram is at 60pS. We conclude that amylosininduced permeabilization of the BLM to cations byforming channels with selectivity of K+4Na+.

3.6. Toxicity of amylosin to feline lung cells and boar

spermatozoa

Feline fetal lung cells were selected as a target,due to complaints of respiratory illness by occu-pants of the buildings in which the toxinogenicB. amyloliquefaciens strains originated. Completecell death, with extensive cell lysis, of the feline fetallung cells was observed after exposure for 24 h toamylosin at a concentration of o170 nM (Table 3).For inhibition of motility in boar spermatozoa, thetoxicity endpoint concentrations were 135 nM after10min of exposure and 27 nM after 3 d (Table 3),i.e. the toxicity endpoint concentration decreased bya factor of 5 when the exposure time was extendedfrom 10min to 3 d. This indicates that penetrationof amylosin into the cell increased with prolongedexposure time. This may be due to the moderatelylipophilic nature of amylosin seen also as retentiononto the C18 column at methanol concentrationsbelow 80% (v/v) in the HPLC eluent (Fig. 1A). Theresults thus show that amylosin inhibited lung cellproliferation as well as sperm motility at exposureconcentrations of p30 nM.

The effects of amylosin on the cellular electrictransmembrane potentials were assessed in boarspermatozoa. In sperm cells the mitochondria are

ARTICLE IN PRESS

Table 3

Toxicity of amylosin to boar spermatozoa and feline lung cells

Toxicity parameters Toxicity endpoints (nM)

Exposure time

Sperm cells Feline lung cells

10min 3 d 1 d

Damage to the permeability barrier of the plasma membrane towards PI 44200 4840 ND

Inhibition of motility450% of sperm cells 135 27 ND

Hyperpolarization of Dcp 33–67 13–27 ND

Partial (50%) depolarization of Dcm 67 27 ND

Inhibition of motility all cells 270 54 ND

Complete depolarization of Dcp and Dcm 270–540 54–108 ND

Swelling of the sperm head 270–540 64–128 ND

Inhibition of proliferation o170

The toxicity endpoints recorded were permeability of the plasma membrane to PI, decrease and inhibition of motility, changes in Dcp and

Dcm indicated by emission shifts of the JC-1 stain from yellow to green, and inhibition of growth. ND, not determined.

4 pA

5 s

KCl

NaCl

CaCl2

25

20

15

10

5

0

-5

-0.1 0.0 0.1 0.2 0.3 0.4 0.5

Log [Co/Ci]

Mem

bra

ne p

ote

ntial (m

V)

NaCl

KCl

106

105

104

103

102

101

100

15 150 1500

Concentration (ng/mL)

Conducta

nce (

nS

/cm

2)

5

4

3

2

1

0

0 25 50 75 100 125 300

pS

Rel. u

nits

Fig. 4. Amylosin-induced single-channel currents recorded in 1M salts and 100mV membrane voltage, concentration dependence and

cation/anion selectivity in BLM. (A) The conductance was measured at a voltage of 100mV using 1M salts. Channel conductances were

40 pS for KCl and 12.5 pS for NaCl. (B) Potassium-specific membrane conductance as function of amylosin concentration. Conditions:

0.5M KCl, pH 7.0, transmembrane voltage 80mV. (C) Zero-current potentials of BLM generated by salt concentration difference across

the membrane: KCl (’) lines are potentials for potassium concentration differences and NaCl (m) for sodium. (D) Amplitude histogram

of the potassium channels of amylosin.

R. Mikkola et al. / Toxicon 49 (2007) 1158–1171 1165

located in the mid-piece, distant from the main partof the cytoplasm which is located in the head,allowing us to observe Dcp and Dcm separately.

Changes in Dcp and Dcm were made visible byJC-1 fluorescence, in which shifts from yellow togreen at the Dc dissipation. Damage to the plasma

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membrane permeability barrier was monitoredusing PI staining. The result of sperm cell exposureto amylosin after 3 d is shown in Fig. 5. Exposure to13–27 nM of amylosin increased the Dcp of spermcells (hyperpolarization) and decreased the Dcm

(depolarization) (Fig. 5B). Exposure to 27 nM ofamylosin decreased the motility of the spermatozoaover 50%. Depolarization of the mitochondria andhyperpolarization of the plasma membrane contin-ued to increase until complete inhibition of motilityoccurred at 54 nM of amylosin (Fig. 5C). Swellingof the sperm head was observed at 108 nM ofamylosin (Fig. 5D). None of these exposuresdamaged the permeability barrier of the plasmamembrane towards PI; such damage was onlyobserved at very high exposures, e.g. 860 nM.

The results presented in Table 3 and Fig. 5 showthat amylosin inhibited sperm motility and dis-turbed Dcp and Dcm in cells where the plasmamembrane permeability barrier to PI was intact.

Fig. 5. Effects of amylosin on boar sperm cells Dcp and Dcm stained

exposed to the vehicle (methanol) only, while the mitochondrial membra

potential ðDcpÞ is low (green). (B) Exposure to 32 ng (27 nM) of amylos

spots), whereas the mitochondria are partially depolarized. These sperm

exposed sperm cells. (C) The sperm cell Dcm depolarized and Dcp hyper

(54 nM) of amylosin. (D) Sperm cells exposed to 128ng (108 nM) of am

swollen, and sperm cells completely immotile.

The increase of conductivity in BLM was observedin the presence of 426 nM of amylosin (Fig. 4B).This coincides with the sperm cell data, in which thetoxicity threshold was 25 nM, and with the felinelung cell data, in which toxic effects were seen atconcentrations o170 nM. The cation selectivity andsingle-channel activity presented in Fig. 4 indicatethat the toxicity observed in boar sperm cells and infeline fetal lung cells (Table 3) was due to thechannel-forming properties of amylosin.

3.7. Effects of amylosin on mitochondrial Kþ uptake,

swelling, PN oxidation, and Dcm

A K+-selective electrode was used for investigat-ing the effects of amylosin on K+ transport inRLM. Addition of amylosin to energized RLMrespiring on glutamate plus malate as substratesinduced a decrease in [K+] in the external mediumdue to K+ influx into the mitochondrial matrix

with JC-1 and exposed for three days. (A) The sperm cells were

ne potential ðDcmÞ is high (yellow and red) and plasma membrane

in hyperpolarized the plasma membrane of the sperm cells (yellow

cells exhibited a decrease in motility compared with the methanol-

polarized more when sperm cells lost motility at exposure to 64 ng

ylosin. The sperm cell Dcm’s are depolarized, sperm cell heads are

ARTICLE IN PRESSR. Mikkola et al. / Toxicon 49 (2007) 1158–1171 1167

(Figs. 6A and B). No effect on [K+] was seen in thecontrol with the vehicle only (methanol). Collapseof the Dcm by the protonophorous uncouplerFCCP induced a substantial efflux of K+ fromRLM in the presence of amylosin, but only a minorefflux in the presence of methanol.

Fig. 7 shows the decrease in absorbance at 540 nmðA540Þ due to the swelling of mitochondria asso-ciated with cation uptake, depending on theconcentration of amylosin in the KCl and NaClmedia. Amylosin already induced a decrease in A540

at 170 nM in KCl medium, but in NaCl mediumhigher concentrations of amylosin ð4250 nMÞ were

1000

900

800

700

600

500

K+ c

oncentr

ation (

µM)

0 2 3 4 5 6 7 8

Time (min)

Amylosin

FCCP

FCCP

Methanol

Amylosinor methanol

- ATP

1

Fig. 6. Amylosin-induced influx of potassium in energized RLM. The c

selective electrode in the external medium containing 250mM sucrose,

HEPES (pH adjusted to 7.4 with Trizma base), and 1mgmL�1 RLM

5mLmL�1, and FCCP 1 mM are indicated by arrows. (A) Without AT

1.8

1.7

1.6

1.5

1.4

1.3

1.2

0 1 2 3 4 5 6 7 8

Time (min)

A 5

40 n

m

KCl

Amylosin, methanol,valinomycin

methenol

200nm/mL

300nm/mL

400nm/mL

500nm/mL

valinomycin 25 ng/mL

Fig. 7. Amylosin-induced energy-dependent swelling of RLM in KCl m

decrease in absorbance at 540 nm occurred. Initially, mitochondria w

medium containing 120mM KCl, 2 mM KH2PO4, 5mM glutamate p

base), or in NaCl medium (B) Similar to above, but KCl was rep

concentrations by addition of amylosin (from 200 ngmL�1 (170 nM) t

(23 nM) are indicated by arrows.

needed and the swelling was less extensive. Forcomparison, Fig. 7 also shows the swelling inducedby the K+-specific ionophore valinomycin and acontrol trace with the solvent only (methanol). Inthe choline chloride medium A540 did not changeafter addition of up to 420 nM of amylosin (thehighest concentration tested, data not shown).Amylosin thus increased mitochondrial membranepermeability not only to K+, as did valinomycin,but at 1.5 times higher concentrations also to Na+.The amylosin-induced K+/Na+ fluxes in intactmitochondria are in accordance with the formationof K+/Na+ channels in BLM (Fig. 4).

K+ c

oncentr

ation (

µM)

Amylosin

FCCP

FCCP

Methanol

Amylosinor methanol

+ ATP1000

900

800

700

600

5000 2 3 4 5 6 7 8

Time (min)

1

hanges in potassium concentrations were determined with a K+-

0.7mM KCl, 2mM H3PO4, 5mM glutamate plus malate, 10mM

. The additions of amylosin 500 ngmL�1 (425 nM), methanol

P. (B) Incubation medium was supplemented with 1mM ATP.

0 1 2 3 4 5 6 7 8

Time (min)

1.8

1.7

1.6

1.5

1.4

1.3

1.2

A 5

40 n

m

Amylosin, methanol

methenol

200nm/mL300nm/mL400nm/mL500nm/mL

NaCl

edium and NaCl medium. (A) Swelling in RLM was measured as

ere incubated for 2min at room temperature in KCl incubation

lus malate and 10mM HEPES (pH adjusted to 7.4 with Trizma

laced by NaCl and KH2PO4 was replaced by NaH2PO4. The

o 500ngmL�1 (425 nM), methanol, and valinomycin 25 ngmL�1

ARTICLE IN PRESS

200

150

100

50

0 1 2 3 4 5 6 7 8

Time (min)

PN

flu

ore

scence (

arb

.un.)

FCCP

FCCP

FCCPADPCholinCl

NaCl

KCl

Amylosin 350

300

250

200

150

0 1 2 3 4 5 6 7 8

Time (min)

Amylosin ADP FCCP

KCl

NaCl

cholinCl

Rhodam

in123 flu

ore

scence (

arb

. un.)

Fig. 8. Cation-selectivity of amylosin, effect on redox state of PN, and the membrane potential of isolated RLM. (A) The redox state of

PN was estimated using PN fluorescence. (B) The fluorescence of rhodamine 123 (0.1mM) was used to record the mitochondrial membrane

potential ðDCmÞ. The increase in rhodamine 123 fluorescence shows the decrease in DCm. The experiments were done with RLM

(1mgmL�1) incubated at 25 1C in various media containing KCl, NaCl, and choline chloride as explained in legend to Fig. 7, with

glutamate plus malate as the respiring substrate. The additions of 240 ngmL�1 (200 nM) of amylosin, 160mM of ADP, and 1mM of FCCP

are indicated by arrows.

R. Mikkola et al. / Toxicon 49 (2007) 1158–11711168

Amylosin also affected the redox state of PN(Fig. 8A) and the Dcm (Fig. 8B) in RLM in acation-specific way. In the KCl medium, amylosincaused oxidation of PN and strong inhibition ofoxidative phosphorylation, indicating uncoupling(Fig. 8B). In the NaCl medium, the rate ofoxidation of reduced PN after addition of amylosinwas about half of that in a KCl medium (Fig. 8A)and oxidative phosphorylation was only slightlyinhibited. In the choline chloride medium nosignificant change in the mitochondrial functionswas observed after addition of amylosin. Amylosindid not change the steady state of PN before or afteraddition of ADP which induced the oxidativephosphorylation cycle with initial oxidation of PN,followed by complete reduction (Fig. 8B). Amylosineffectively lowered the DCm (shown as increasedrhodamine 123 fluorescence) and inhibited oxidativephosphorylation in the KCl medium (Fig. 8B), withminor effects in the NaCl medium and none incholine chloride medium. All the data obtained withisolated RLM demonstrated that amylosin affectedmitochondrial functions in the presence of K+ andNa+, with the cation selectivity K+4Na+, but notwhen choline was used as the cation.

4. Discussion

Here we present ESI-IT-MS and NMR charac-terization of the structure and biological properties

of the purified 1197-Da substance, previouslydescribed from fungicidal strains of B. amylolique-

faciens (Mikkola et al., 2004). We show here thatthe purified 1197-Da substance, now named amy-losin, is responsible for the formation of K+- andNa+-permeant channels in BLM and for thedissipation of DCm and DCp in mammalian cellsexposed to extracts prepared from the B. amyloli-

quefaciens strains. This paper further describes thetoxic properties of amylosin: permeabilization ofmitochondria to K+ and Na+, induction ofswelling in mitochondria, collapse of DCm, anduncoupling of oxidative phosphorylation. We de-signed a chemical method to quantitate amylosinand found that this substance caused dissipationof DCm and DCp in boar spermatozoa and death offeline lung cells at exposure concentrations of10–100 nM, which is similar to that in which K+-and Na+-permeant channels were formed in BLM.It is thus likely that the toxic effects were linked tothe ion channel-forming property of amylosin.

The 1H-NMR spectrum of amylosin revealed achemical shift in the olefinic region of 5.3–6.4 ppm.The COSY spectrum of amylosin resembled thecorresponding NMR spectra published for macro-lide polyenes such as nystatins (Volpon andLancelin, 2002; Bruheim et al., 2004), amphotericinA (Sowinski et al., 1985), vacidin A (Sowinskiet al., 1989), filipin III (Rochet and Lancelin,1997), phenelfamycin A (Hochlowski et al., 1988),

ARTICLE IN PRESSR. Mikkola et al. / Toxicon 49 (2007) 1158–1171 1169

efrotomycin (Dewey et al., 1985), and retinoids(Tatariunas and Matsumoto, 2000). The HSQCspectrum data of amylosin showed correlationsbetween the olefinic methine proton (1H) andcarbon (13C). The published HSQC spectra forfilipin III (Rochet and Lancelin, 1997) and nystatin(Bruheim et al., 2004) show 1H–13C correlation datain the olefinic region similar to those of amylosin.The HSQC spectrum data of amylosin corre-sponded to the published 13C NMR data for theolefinic carbons of the macrolide polyenes (Deweyet al., 1985; Sowinski et al., 1985, 1989). Based onthe NMR results and comparison of the simulated1H-NMR spectrum of hexaene structure with the1H-NMR spectrum obtained for amylosin, theamylosin chromophore group absorbing at 365 nm(yellow color) may be a polyene structure.

The NMR data of amylosin described heresupport the previously reported (Mikkola et al.,2004) amino acid composition of HCl-hydrolyzedamylosin containing leucine, proline, serine, aspar-tic acid, glutamic acid, and tyrosine, except that noserine was assigned in NMR. In addition, we alsoassigned asparagine NHd- or glutamine NHe-spe-cific cross-peaks in TOCSY. Due to acid hydrolysisthe amino acids asparagine and glutamine maybecome deaminated to aspartic acid and glutamicacid, respectively. Amylosin gave a negative ninhy-drin reaction (Mikkola et al., 2004) and therefore itis either linear with a blocked N-terminus or a cycliccompound. The strains of B. amyloliquefaciens

produce iturins (Yoshida et al., 2001; Hiradate etal., 2002; Yu et al., 2002), bacillomycin D andfengycin (Koumoutsi et al., 2004). The iturins andbacillomycin D contain seven amino acids andfengycins contain ten amino acids (Volpon et al.,1999) including some of the same acids were alsofound in amylosin. The published 1H-NMR spectraof the lipopeptides bacillomycin L (Volpon et al.,1999) and surfactin (Peypoux et al., 1991) inDMSO-d6 showed that there is no chemical shiftof protons in the region 5.4–6.5 ppm such as thatfound in the 1H-NMR spectrum of amylosin. Basedon these data, we conclude that amylosin is relatedmost closely to the iturinic group of lipopeptides,but contains a chromophore polyene structureinstead of fatty acid chain.

Amylosin had an antifungal activity similar(Mikkola et al., 2004) but the channel formingproperties are different than other polyene anti-biotics. Many macrolide polyenes, including am-photericin B, require ergosterol or cholesterol to

form channels to the membranes. However, theinner RLM mitochondrial membrane, which in thispaper became permeabilized towards K+ and Na+

by amylosin exposure, contains no cholesterol(Colbeau et al., 1971) and neither did the lipidsused for BLM. A similar situation has beendescribed for the RLM membrane-active lienomy-cin (Zieniawa et al., 1977) and the Na+/K+/H+

permeability-inducing vacidin (Cybulska et al.,1992) which are macrolide polyenes. Thus, theamylosin channel-forming properties are sterol-independent and distinct from those of the chan-nel-forming peptides or polyenes described earlier.

Using a K+-specific electrode, we showed thatamylosin induced a respiration-driven influx of K+

in the KCl medium and an efflux of K+ when DCm

was collapsed by the addition of FCCP. Themitochondrial adenosine 50-triphosphate (ATP)-sensitive K+ channels (KATP) open when the ATPconcentration is decreased. This occurs when anuncoupler is added, while an addition of ATPresults in inhibition of these channels (Garlid andPaucek, 2003). The amylosin-induced channel is notrelated to the KATP channel, since addition of ATPdid not inhibit it (Fig. 6).

The amylosin-induced effects, i.e. mitochondrialswelling, depletion of ATP, oxidation of PN, andcollapse of DCm, already occurred at low concen-trations ðo200 nMÞ of amylosin in the K+ medium,at higher concentrations in an Na+ medium and notat all when choline was used as the cation (thispaper and Mikkola et al., 2004). Amylosin thusinduced K+/Na+-mediated mitochondrial dysfunc-tion, similar to that described as promoting celldeath by release of apoptosis-inducing factors fromthe mitochondria (Lemasters et al., 2002).

A low concentration of amylosin (30 nM, Table 1)caused hyperpolarization of the boar sperm plasmamembrane, likely resulting from an efflux of K+

from the cytosol where its concentration was higherthan in the external medium. Amylosin thus is notonly mitochondrio-toxic, but also disturbs ionicgradients in the plasma membranes, explaining therapid lethal effect on feline lung cells described hereand the observed disruption of transmembraneelectric potentials of excitable cells such as neuronalcells (Mikkola et al., 2004).

A total of 40% of the B. amyloliquefaciens strainsisolated from indoor dusts of moisture-troubledbuildings were putative amylosin producers (Mik-kola et al., 2004). The amylosin content wasestimated as 1% of the dry weight in biomass

ARTICLE IN PRESSR. Mikkola et al. / Toxicon 49 (2007) 1158–11711170

cultivated on laboratory media (Mikkola et al.,2004). The species B. amyloliquefaciens has GRAS(generally recognized as safe) status (Food andDrug Administration, 1999) and is widely used inindustry (Fogarty and Kelly, 1990; Beynon andBeaumont, 1998; Outtrup and Jorgensen, 2002).The results described in the present paper call forsafety reassessment of the B. amyloliquefaciens

strains used as production organisms for sensitiveapplications such as enzymes or amino acidsintended for human consumption or as biopesti-cides for crop protection.

Acknowledgments

This study was supported by the Academy ofFinland (Grant 50733), the grant for Center ofExcellence Microbial Resources, and grants fromthe Magnus Ehrnrooth Foundation, the FinnishMedical Society, and the Russian Foundation forBasic Research. We thank Drs. Manfred Spraul,Detlef Moskau, Alexandre Schefer, Li-Hong Tsengand Larus Einarsson from Bruker BioSpin fortechnical help and resources in NMR.

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