(Received for publication, September 19, 1996, and in revised form, February 18, 1997)
From the Department of Microbiology, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands, the
§ Nestlé Research Center, Nestec Ltd.,
Vers-Chez-Les-Blanc, P.O. Box 44,
CH-1000 Lausanne 26, Switzerland, and the ¶ Centre for
Pharmacy, University of Groningen, A. Deusinglaan 2, 9713 AW Groningen, The Netherlands
A novel broad host range antimicrobial substance, Thermophilin 13, has been isolated and purified from the growth medium of Streptococcus thermophilus. Thermophilin 13 is composed of the antibacterial peptide ThmA (Mr of 5776) and the enhancing factor ThmB (Mr of 3910); the latter peptide increased the activity of ThmA ~40 ×. Both peptides are encoded by a single operon, and an equimolar ratio was optimal for Thermophilin 13 activity. Despite the antilisterial activity of Thermophilin 13, neither ThmA nor ThmB contain the YGNGV-C consensus sequence of Listeria-active peptides, and post-translational modifications comparable to that in the lantibiotics are also absent. Mass spectrometry did reveal the apparent oxidation of methionines in ThmA, which resulted in a peptide that could not be enhanced any longer by ThmB, whereas the intrinsic bactericidal activity was normal. Thermophilin 13 dissipated the membrane potential and the pH gradient in liposomes, and this activity was independent of membrane components from a sensitive strain (e.g. lipid or proteinaceous receptor). Models of possible poration complexes formed are proposed on the basis of sequence comparisons, structure predictions, and the functional analysis of Thermophilin 13.
Antibacterial membrane-acting peptides form a heterogeneous family
of structures that can be subdivided in different classes on the basis
of primary sequence, mode of synthesis (ribosomal versus
non-ribosomal), post-translational modifications, and structure (linear, cyclic, -helical, and
-sheet). The following classes can
be discriminated: (i) antibiotics (non-ribosomal synthesis, e.g. gramicidins (1)); (ii) lantibiotics
(lanthionine-containing peptides, e.g. Nisin (2)); (iii)
host defense peptides (3) of mammals (e.g. defensins), frogs
(e.g. magainins), and insects (e.g. cecropins).
Some peptides have a strong hemolytic activity in addition to
antibacterial properties, like (iv) bee venoms (e.g.
melittin (4)) and (v) bacterial cytolysins (e.g. Staphylococcus aureus
-toxin (5)). A particular class of antibacterial
peptides is formed by (vi) the bacteriocins (e.g.
lactococcins, Pediocins), which are produced by lactic acid bacteria
and preferentially inhibit species that are closely related to the
producer (6).
Besides their differences in structure and mode of membrane interaction, antibacterial membrane-acting peptides may differ in their requirement for specific lipids or proteins and/or membrane potential or pH gradient to insert properly into target membranes and/or to exhibit maximal activity. These requirements determine to a large extent the inhibitory spectrum of a particular peptide. Moreover, in case of the lantibiotics and bacteriocins, an immunity protein is synthesized that protects the producer organism (7). Bacteriocins are often considered to be different from other antibacterial membrane-acting peptides by the fact that they require a proteinaceous membrane component (i.e. receptor) for antibacterial activity, which is consistent with their narrow host range specificity (6). A property of some of the peptide bacteriocins is their ability to form poration complexes that are composed of two different peptides (8, 9). These bacteriocins have a very narrow inhibitory spectrum of activity (6). Finally, the so-called Listeria-active bacteriocins share a consensus sequence motif (YGNGV-C) at their amino terminus (9) which could be of importance for their ability to inhibit Listeria species. The "Listeria-active bacteriocins" have not been reported to form poration complexes.
In this article, we describe the properties of a broad host range pore-forming antimicrobial activity, named Thermophilin 13. The compound has structural and functional features of bacteriocins, in particular those that form poration complexes. However, none of the two peptides of Thermophilin 13 owes the antilisterial YGNGV-C consensus sequence and a proteinaceous receptor is not required for activity. We also present a model of the poration complex formed by Thermophilin 13 on the basis of structural similarities with host defense peptides and other pore-forming structures.
The bacteriocin producer
Streptococcus thermophilus SFi13 as well as the indicator
micro-organisms are from the Nestlé strain collection. The
indicator strain used to assess the bacteriocin concentration
(activity) during purification was S. thermophilus SFi3.
Lactococcus, Streptococcus, and
Enterococcus strains were grown semi-anaerobically in M17
broth (Oxoid, U K) supplemented with 0.5% (w/v) glucose at 30, 42, and 30 °C, respectively. Lactobacillus, Pediococcus, Leuconostoc, and
Bifidobacteria strains were grown in MRS, 0.5% (w/v)
glucose (Sanofi Diagnostics Pasteur, France) at 30 °C except for the
thermophilic Lactobacilli (42 °C) and Bifidobacterium (37 °C). Clostridium strains
(spores and vegetative cells) were grown in RCM (Oxoid) at 30 °C
under an atmosphere of 85% (v/v) N2, 5% CO2,
10% H2. Bacillus (30 °C),
Listeria (30 °C), Micrococcus (30 °C),
Staphylococcus (30 °C), Salmonella (37 °C), and Escherichia coli strains (37 °C) were all grown in
BHI (Difco) at the temperatures indicated. E. coli BZ234
(C600 derivative allowing -complementation; Biozentrum, University
of Basel, Switzerland) was grown in Luria broth (10).
S.
thermophilus SFi13 was grown semi-anaerobically at 42 °C in 1 liter of M17 supplemented with 1% (w/v) sucrose (M17S, M17 broth
(Oxoid) supplemented with 1% (w/v) sucrose) until 2 h into the
stationary phase. Cells were removed by centrifugation (20,000 × g for 20 min), and the pH was adjusted to 1.6 by phosphoric acid. Insoluble material was removed by centrifugation (20,000 × g for 20 min at 4 °C), and the soluble fraction,
containing the antibacterial activity, was concentrated by
trichloroacetic acid precipitation (trichloroacetic acid, 10% (w/v)
final). The pellet was washed twice with cold acetone (20 °C),
resuspended in 8 ml of 0.1% trifluoroacetic acid, and heated for 20 min in boiling water. Insoluble material was removed by centrifugation; the soluble sample obtained was termed trichloroacetic acid
extract.
The bacteriocin activity was measured in an agar well assay. Briefly, 5 ml of M17S Top-Agar (0.75% (w/v)) were mixed with 20 µl of an overnight culture of the indicator strain SFi3 and poured on top of 35 ml of M17S-Agar (1.5% (w/v)). Wells of 70 µl were made using a 3.5-mm diameter punch-holer connected to a vacuum device, and serial dilutions of the sample were assayed in different wells. Consequently, the highest dilution still showing activity defines the activity of the 70-µl aliquot in terms of arbitrary units (AU).1 The amount of Thermophilin 13 in a given volume (V µl) was expressed in total units (U = (AU/70) × V). The concentration of Thermophilin 13 was defined as AU/ml = AU/70 × 1000.
Purification of Thermophilin 13 and Identification of ThmA and ThmBThe trichloroacetic acid pellet of a 1-liter culture was
dissolved in 8 ml of 200 mM Tris-HCl, pH 8.0, 6 M urea plus 2 M NaCl and used for purification
on a 20-ml Source 15-Phe resin (15-Phe) packed into a HR16/10 column
(Pharmacia Biotech Inc.). Flow rates were kept constant at 4 ml/min.
The column was first washed with 100 ml of 50 mM Tris-HCl,
pH 8.0, 2 M NaCl (buffer A) before applying a 60-ml linear
gradient of buffer A to buffer B (50 mM Tris-HCl, pH 8.0),
which was followed by 150 ml of buffer B and 100 ml of H2O.
Thermophilin 13 was eluted with 60 ml of buffer C (70% (v/v) acetonitrile, 30% (v/v) water, and 0.1% (v/v) trifluoroacetic acid).
20 ml of 100% methanol were added to the active fractions (~40 ml
total), and the volume was reduced to 24 ml by rotary evaporation
(approximately 50% (v/v) methanol, final concentration). This sample
was applied to a 3-ml Resource RP column (RPC, Pharmacia, NL),
previously equilibrated with 50% (v/v) methanol, 50% water, 0.1%
trifluoroacetic acid (buffer E). A flow rate of 2 ml/min was kept
throughout the procedure. After washing with 100 ml of buffer E at a
flow rate of 2 ml/min, a linear gradient from buffer E to 100%
acetonitrile (in 0.1% trifluoroacetic acid) was applied in 30 min. Two
main peaks were characterized by electrospray-MS and re-chromatographed
separately after dilution (1:1) in 0.1% trifluoroacetic acid. Both
peaks were collected from two independent runs, re-analyzed by
electrospray-MS, and stored at 20 °C in their elution solvent.
Electrospray mass spectra were recorded on a R 3010 quadrupole mass spectrometer (NERMAG, Argenteuil, France) equipped with a custom-built pneumatically assisted electrospray (ion spray) ion source. The molecular weight of the peptides is determined by measurement of multiply charged ions (11). All molecular masses quoted in this paper are average, chemical atomic masses.
Inhibitory SpectrumMultiwell dishes (Falcon 3046) were filled with 6 ml of Agar medium per dish and 0.7 ml of Top-Agar were inoculated with 0.1-1% of an overnight culture or 105 spores/ml of Top-Agar. Each strain was tested with 300 AU of Thermophilin 13 which was obtained after dilution of the trichloroacetic acid extract with 100 mM potassium phosphate, pH 7.0. A control of bacteriocin activity was included for each strain using a proteinase K-treated sample (5 µg/ml at 37 °C for 20 min). Minimum inhibitory concentration values were estimated from the size of the halos obtained with the trichloroacetic acid extract; the data were calibrated on the basis of the inhibition of S. thermophilus SFi3 strain (1 AU = 11 nM). This method was validated for Clostridium botulinum, Listeria monocytogenes, Lactococcus lactis, Bacillus cereus, Bacillus subtilis and S. thermophilus, using purified Thermophilin 13 (equimolar ratio of ThmA and ThmB).
Protein DeterminationProtein concentrations were determined by the method of Lowry et al. (12), with bovine serum albumin as standard, unless indicated otherwise.
DNA PreparationsGeneral procedures for DNA isolation from E. coli, restriction analysis, ligation, electrophoresis, Southern analysis, and colony hybridization were carried out as described previously (10). For preparation of genomic DNA from S. thermophilus, cells were grown in 2 ml of M17S until the mid-log phase, treated with lysosyme (13), and resuspended in 2 ml of STE (25% (w/v) sucrose, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA). SDS and RNase A were added to final concentrations of 1% (w/v) and 50 µg/ml, respectively, and the mixture was incubated for 1 h at 37 °C. Subsequently, proteinase K was added to a final concentration of 100 µg/ml, and the mixture was incubated for 2 h at 55 °C. DNA fibers were spooled in 70% ethanol, solubilized overnight in TE, and extracted with chloroform (10).
Cloning of the Bacteriocin Encoding GenesA set of
degenerate oligonucleotides was deduced from the 48 amino acid
amino-terminal sequence of ThmA. The forward primer corresponded to
5GAYATGGGNGGNTAYGC3
(For1) and the reverse primers corresponded to 3
GTACANCCNCGNTAACG5
(Rev1),
3
GTACANCCNCGNTAGCG5
(Rev2), 3
GTACANCCNCGNTATCG5
(Rev3),
3
GTGCANCCNCGNTAACG5
(Rev4), 3
GTGCANCCNCGNTAGCG5
(Rev5), and
3
GTGCANCCNCGNTATCG5
(Rev6). Primers and chromosomal DNA were used at final concentrations of 5 µM and 1.3 µg/ml,
respectively. Other components were added as specified in the
polymerase chain reaction protocol using SuperTaq polymerase of
Boehringer Mannheim, and a 5-min denaturation cycle at 95 °C was
followed by 30 cycles of 30 s 95 °C, 30 s 42 °C, and
30 s 72 °C; elongation was achieved by a final 5-min step at
72 °C. Polymerase chain reaction fragments of the expected size (128 bp) were eluted, ligated into plasmid pGEM-T (Promega, CH), and
propagated in E. coli BZ234. The 128-bp fragment was used as
a hybridization probe (primer extension, Boehringer Mannheim) to
identify and clone a 3.8-kb EcoRI-HindIII genomic
DNA fragment that carried the genes for ThmA and ThmB. Recombinant
strains of E. coli BZ234 were identified by colony hybridization using the 128-bp probe on Zeta-probe membranes (Bio-Rad) (10).
Multiple sequence alignments, data base
searching, and structure prediction methods were used from the GCG
package (Genetics Computer Group, Inc.), PCGENE and the EMBL worldwide
web servers (PredictProtein@EMBL-Heidelberg.DE). Hairpin turns were
accepted when the conformational potential for each residue of the
tetrapeptide to be in turn <pt> is higher than <p> and <p
>
(14), and the probability of forming a
-turn by a tetrapeptide was
p(t)10
4 > 2 (15).
Right-side out membrane vesicles of S. thermophilus and their fusion to cytochrome c oxidase containing liposomes (COVs) were performed in 50 mM potassium phosphate, pH 7.0, as described previously (16).
Measurement of Membrane Potential (The membrane potential () was estimated from the
distribution of tetraphenylphosphonium ion (TPP+) using an
ion-selective electrode (17). COVs or hybrid membranes were diluted
into 50 mM potassium phosphate, pH 6.3, plus 5 mM MgSO4, (saturated with oxygen) to a final
concentration of 153 nM in cytochrome c oxidase.
A proton motive force was generated in COVs or hybrid membranes by
adding the electron donor system ascorbate (10 mM), TMPD
(200 µM) plus cytochrome c (10 µM), and oxygen (18). The
was calculated after
correction for probe binding to the membrane (19) and using a specific
internal volume of 1.5 µl/mg lipids (20). The pH gradient was
monitored by the fluorescent pH indicator pyranine (21). Pyranine was
entrapped in the COVs by freeze/thaw/sonication as described previously (18), and external pyranine was removed by gel filtration on Sephadex
G25. Pyranine-containing COVs were diluted into 50 mM potassium phosphate, pH 6.3, plus 5 mM MgSO4
(saturated with oxygen), to a final concentration of 46 nM
in cytochrome c oxidase, and
pH was estimated from the
changes in pyranine fluorescence.
Cytochrome c
oxidase activity was measured in liposomes by monitoring the decrease
in absorbance at 550-540 nm (-peak) (22) in the presence and
absence of Thermophilin 13. The reaction was performed in 50 mM potassium phosphate, pH 6.3, plus 5 mM
MgSO4 (saturated with oxygen), at a final concentration of
16 nM in cytochrome c oxidase and in the
presence of 60 nM valinomycin plus 60 nM
nigericin.
S. thermophilus
SFi13 produced 570 AU/ml of Thermophilin 13 when grown in M17S and
harvested after 2 h in the stationary phase. Clarification of the
medium supernatant by phosphoric acid resulted in the removal of
exo-polysaccharides as well as 99% (w/w) of proteins (Table
I); the increase in activity after trichloroacetic acid
precipitation could be assigned to the decrease in pH as the activity
of Thermophilin 13 increases 4-fold when the pH is lowered from 8.0 to
2.0 (data not shown). Also, variations in activity throughout the
purification can be ascribed to a loss of Thermophilin 13, variations
in pH, or changes in aggregation states (i.e.
solubilization) of the bacteriocin. Thermophilin 13 was purified
further on a Source 15-Phe resin, which eliminated 93% of the
remaining contaminants (Table I). An activity of 113 AU/ml was obtained
after the Resource-RPC for the fractions eluting between 17.5 and 19 min; a much higher activity was obtained when these fractions were
mixed with those eluting between 21 and 24 min. Both sets of fractions
were re-chromatographed independently on the Resource-RPC and analyzed
by electrospray MS (Fig. 1). A Mr
of 5776 was determined for the fractions eluting between 17.5 and 19 min, and two molecular weights were found for the fractions eluting
between 21 and 24 min (Fig. 1). These latter two compounds were named
ThmB (Mr of 3910) and ThmB
(Mr of 3892). As shown in Table I, the
Resource-RPC eliminated 94% of the remaining contaminating proteins
but resulted in a decrease in total activity from 220 to 27 kU. The
apparent loss in activity is partly due to the fact that only peak
fractions, which were pure by electrospray-MS criteria, were used in
the calculation of the activity. It should also be stressed that the
concentration of the purified peptides was estimated from their
absorbance at 280 nm, using extinction coefficients of 1490 and 5500 M
1 cm
1 for Tyr and Trp,
respectively (23), rather than the Lowry assay (12) which was used
throughout the purification procedure.
|
Antibacterial Properties of ThmA and ThmB on S. thermophilus SFi3
As already suggested by the experiments presented above,
fractions containing ThmB seem to enhance the activity of ThmA. The enhancing properties of ThmB are clearly shown in Fig. 2
(left panel), i.e. the diameter of inhibition of
growth by ThmA is increased in the presence of ThmB (top left
panel). The activity of 2.2 µM ThmA was 20 AU
(top left panel), whereas the activity of 1.1 µM ThmA plus 1.1 µM ThmB was 400 AU. In
other words, the activity of ThmA is enhanced 40-fold in the presence
of an equimolar concentration of ThmB. The antibacterial activity at
different concentrations and ratios is presented in Fig.
3. Surfaces of the inhibition zones were plotted as a
function of ThmA and ThmB concentration in the range of 0 to 2.2 µM (0-155 pmol/well). The largest inhibition zones were
observed at the diagonal connecting equimolar concentration of ThmA and
ThmB (Fig. 3A), and the minimum inhibitory concentration value of Thermophilin 13 was estimated to be 11 nM for
S. thermophilus SFi3 as indicator strain (Fig.
3C). In Fig. 3B, the antibacterial activity of
Thermophilin 13 is shown at high amounts of ThmA relative to ThmB. It
appeared that 1100 nM ThmA has the same activity as 27 nM of ThmA plus 27 nM ThmB (Fig.
3B). Fig. 3C shows that ThmB has no intrinsic
activity even at high concentrations (2200 nM), and Fig.
3D shows that a large excess of ThmB over ThmA inhibits the
activity of the latter peptide.
Inhibitory Spectrum of Thermophilin 13
Cells or spores were mixed with Top-Agar and poured on top of agar medium to obtain a homogeneous lawn. Prior to growth, wells were made in which diluted, neutralized trichloroacetic acid extract or purified ThmA and/or ThmB were introduced; proteinase K-treated samples were used as negative controls. The results obtained with the neutralized extract were similar to those of ThmA plus ThmB, indicating that a single bacteriocin is produced by S. thermophilus SFi13 under the conditions tested. Diameters of inhibition surrounding the wells were measured after overnight growth. As for other peptide bacteriocins, a narrow inhibitory spectrum was expected for Thermophilin 13 (6, 9). Thermophilin 13, however, exerted a broad host range activity among Gram-positive bacteria (Table II). Not only lactic acid bacteria were affected, but also L. monocytogenes and spore-forming micro-organisms like C. botulinum and B. cereus. Interestingly, growth from spores as well as from vegetative cells was inhibited. Bacterial genera most related to the producer (e.g. streptococci, enterococci, lactococci, lactobacilli) were inhibited to the same extent as more distantly related organisms (e.g. bacilli, clostridiae). All Gram-negative bacteria tested (E. coli, Pseudomonas and Salmonella species) were resistant. ThmA alone had an intrinsic activity that was enhanced by ThmB on all strains tested.
|
Amino-terminal
sequences of ThmA and ThmB were determined (initial yield of 1150 and
1160 pmol, respectively). Oligonucleotide primers were designed on the
basis of the amino-terminal sequence of ThmA (48 residues):
YSGKDXLKDMGGYALAGAGSGALXGAPAGXVGALPGAFVGAHVGAIAG. The sequences DMGGYA and HVGAIA were used to design the forward (For1)
and six different reverse primers (Rev1-6), respectively. The expected
128-bp fragment was obtained in a polymerase chain reaction with the
primers For1 and Rev1 using SFi13 genomic DNA as template. The 128-bp
probe hybridized to a 4.2-kb HindIII and a 3.8 HindIII-EcoRI fragment in the producer strain
SFi13; these fragments were not detected by hybridization in the
non-producer sensitive SFi3 strain. Subsequently, SFi13 chromosomal
HindIII-EcoRI DNA fragments of 3.6-4 kb were
ligated into pUC19, and the recombinant plasmids were transformed to
E. coli BZ234. Colony hybridization identified 16 putative
positive colonies, among a total of 250, containing a 3.7-kb
HindIII-EcoRI insert that was sequenced and searched for open reading frames (ORFs) (Fig. 4). Two
ORFs corresponding to ThmA and ThmB were found. Both peptides are
synthesized with a signal sequence typical of lactic acid bacteria
peptide bacteriocins, i.e. the processing site is preceded
by a double glycine motif. The calculated molecular masses of ThmA and
ThmB are 5776.7 and 3910.5 Da, respectively. The translation of ThmB is
most likely coupled to that of a 52-amino acid hydrophobic peptide,
termed ORFC; no homologue of ORFC was found in the data bases. Putative promoter elements, ribosome binding sites, and a rho-independent terminator structure were found in the regions flanking the ORFs (Fig.
4).
Chemical Modifications of ThmA and ThmB
Genetic analysis of
ThmA and ThmB showed the presence of two cysteines in each peptide.
Since Thermophilin 13 was fully active in the presence of 50 mM dithiothreitol, using the agar well assay, disulfide
bonds are most likely not required for activity. The calculated average
molecular masses of ThmA (5776.7 Da) and ThmB (3910.5) are consistent
with the masses obtained after purification on Resource-RPC,
i.e. 5776 ± 1 and 3910 ± 1 Da, respectively
(Table III). However, a peak shoulder was noticed upon
re-chromatography of ThmB on Resource-RPC (Fig. 1). MS analysis
revealed a mass of 3892 Da in the shoulder (Table III), which is ~18
Da lower than that calculated from the translated nucleotide sequence
of ThmB. This reduction in mass also occurred in the synthesized
peptide ThmB upon prolonged storage but did not affect the enhancing
properties of ThmB (purified or synthesized). Electrospray-MS also
revealed chemical modifications in ThmA; the modified product was
termed ThmA. Comparison of the MS spectra of ThmA and ThmA
shows two additional peaks for each multiply protonated species, leading to
triplets for the ions with 3, 4, and 5 charges
(Mr + 3H+, Mr + 4H+, or Mr + 5H+)
(Table III). These data indicate that ThmA
has gained one or two times
16 Da (average of 3 × 5, 4 × 4, and 5 × 3 Da), which corresponds to the gain of one or two oxygen atoms and suggests that
ThmA becomes oxidized upon storage. Although ThmA
was as active as
ThmA (Fig. 2, right panel, top part), the activity of ThmA
was no longer enhanced by ThmB (Fig. 2, right panel, lower part). Oxidation of ThmA to ThmA
could be prevented by storage at
20 °C. Neither ThmA nor ThmB contain post-translational
modifications common to those of lantibiotics or cystibiotics like
Pediocin PA-I (6). In the experiments described below, Thermophilin 13 refers to an equimolar ratio between ThmA (non-oxidized) and ThmB (both
forms).
|
The
poration complex bacteriocins have a narrow host range specificity and
require a "receptor" for pore-forming activity (7). The poration
complex bacteriocins function in membrane vesicles derived from
sensitive species but not in (proteo)liposomes, with the exception of
Lactococcin G, which is only active in whole cells (24). Thermophilin
13, on the other hand, affects a large variety of organisms (Table II),
suggesting that it might not require a specific factor to exert its
bactericidal activity. To investigate whether Thermophilin 13 exerted a
pore-forming activity in target membranes, we studied the ability of
the bacteriocin to dissipate the membrane potential () in
cytochrome c oxidase containing liposomes (COVs) fused with
membranes of the sensitive S. thermophilus strain SFi3. In
the presence of the electron donor system potassium
ascorbate/TMPD/cytochrome c, a
(inside negative) and
a
pH (inside alkaline) was generated. To clamp the
pH to 0, nigericin was present in the measurements of
. Increasing amounts
of purified Thermophilin 13 dissipated the
progressively (Fig.
5A). To establish the influence of membrane
proteins and/or lipids of the target organism on this putative
pore-forming activity, the bacteriocin was also tested on COVs solely
composed of E. coli lipids plus egg phosphatidylcholine at a
molar ratio of 3 to 1. Thermophilin 13 also dissipated the
in
COVs (Fig. 5B). ThmA alone had little effect, and ThmB alone
had no effect on the
both in the hybrid membranes and COVs (data
not shown). To investigate the activity of Thermophilin 13 in the
absence of a membrane potential, the effect of Thermophilin 13 on the pH gradient in COVs was analyzed. Valinomycin was used to keep
at zero, and the COVs were loaded with the pH indicator pyranine. It
appeared that Thermophilin 13 was able to dissipate the
pH at
nanomolar concentrations, whereas similar concentrations of ThmA and
ThmB alone had virtually no effect (Fig. 5C). Nisin had only
little effect on the
pH in the COVs at micromolar
concentrations.
Effect of Thermophilin 13 on the Cytochrome c Oxidase Activity
It was possible that Thermophilin 13 dissipated the
and
pH by inhibiting cytochrome c oxidase
activity. Up to a concentration of 100 nM, we observed no
effect on cytochrome c oxidase activity reconstituted in
liposomes; the measurements were performed in the presence of nigericin
plus valinomycin to prevent increases in oxidase activity due to
respiratory control. Overall, these results suggest that Thermophilin
13 is able to dissipate
in the absence of
pH and to dissipate
pH in the absence of
, without the need for a specific
proteinaceous receptor or lipids from a sensitive host strain.
In this paper, we describe a novel antimicrobial compound, Thermophilin 13, which differs from other known bacteriocins in its structural and/or functional properties. (i) Thermophilin 13 exerts an activity in COVs which so far has only been observed for bacteriocins of the lantibiotic type; (ii) Thermophilin 13 has antilisterial activity but lacks the YGNGV-C motif, typical of the Listeria-active peptides; (iii) Thermophilin 13 forms a poration complex but in contrast to other "two-component bacteriocins" it has a broad host range activity.
The experiments in COVs, prepared from E. coli/egg phosphatidylcholine lipids (Fig. 5), indicate that Thermophilin 13 does not need a specific component (proteinaceous or lipid) in the membrane for activity. It should be stressed that, although a receptor has not yet been identified for any lactic acid bacterial bacteriocin, the "non-lantibiotics" require an "additional factor" in the target membrane to exert pore-forming activity (6, 25). In fact, only lantibiotics, and bacteriocins thought to contain lanthionines (e.g. Plantaricin C), have so far been shown to exert pore-forming activity in COVs (2, 26, 27). Furthermore, and in contrast to the lantibiotics (2), Thermophilin 13 does not require a threshold membrane potential to dissipate the pH gradient (Fig. 5C) or a threshold pH gradient to dissipate the membrane potential (Fig. 5B).
The experiments also established that Thermophilin 13 forms a poration
complex that is composed of an equimolar ratio of the two peptides ThmA
and ThmB (Fig. 3A). ThmA alone has antibacterial activity
against S. thermophilus, C. botulinum, L. monocytogenes, and B. cereus, which is enhanced
~40-fold when an equal amount of ThmB is present. ThmB, by itself, is
not bactericidal, and an excess of this peptide inhibits the activity
of ThmA (Fig. 3D), possibly because it destabilizes the pore
leading to dysfunctional oligomeric structures. By comparison, Lactacin
F (28), and Plantaricin S (29) are also composed of an active and
enhancing peptide, whereas the two peptides of Lactococcin G have
virtually no activity when tested separately, i.e. 5 × 105 times less than when used in combination (24). These
considerations have led us to subdivide the poration complex
bacteriocins into two classes: type E, for Enhancing, i.e.
when one of the peptides only functions as an enhancer as for
Thermophilin 13, Lactacin F and Plantaricin S, and type S, for Synergy,
i.e. when activity is believed to require the combination of
both peptides, e.g. Lactococcin G (24) and Plantaricin A
(30). Interestingly, this functional classification is substantiated by
similarities in primary sequence and predicted secondary structure of
the peptides (Fig. 6). Type E peptides are characterized
by several G(A/G)G repeats and are predicted to form an amino-terminal
amphipathic -helix followed by a hydrophobic anchor. On the basis of
these criteria, Lactococcin M (31) and Curvaticin FS47 (32) were classified as type E peptides (Fig. 6). The amino-terminal amphipathic
-helix of ThmA could be stabilized by a salt bridge between
Asp5 and Lys8 and by hydrogen bonding between
the amino- and carboxyl-terminal parts (Fig.
7A). By contrast to other type E peptides,
three
-turns are predicted in the sequence of ThmB, at distances
that allow the intervening sequences to span the membrane as
antiparallel
-sheets (Fig. 6); the presence of helix-breaking
residues (serines and asparagines) and the prediction of two
amphipathic strands further support a
-sheet conformation for ThmB.
Type S peptides are characterized by an amphipathic
-helix composed
of charged residues at one face of the helix and highly hydrophobic
residues at the opposite face (Fig. 6). On the basis of structural
similarities with Lactococcin G (8) and Plantaricin A (30), Plantaricin E, F, J, and K (33) have been placed into class S peptides (Fig. 6)
even though experimental evidence that these bacteriocins form similar
poration complexes is lacking.
High probabilities of hairpin turns are not only found in ThmB but also
in ThmA, in the Listeria-active peptides (see Ref. 6 for
review), and downstream of the amphipathic -helix of the partially
sequenced peptide Curvaticin FS47 (32) (Fig. 6). Since Thermophilin 13 and Curvaticin FS47 have been shown to exert antilisterial activity, we
speculate that the hairpin turn might be one of the critical elements
of the GG-processed peptides to exert antilisterial activity. In this
respect, it is worth emphasizing that the membranes of
Listeria species differ significantly from that of lactic
acid bacteria, not only in lipid conjugates (e.g. lipoteichoic acids), apolar lipids (e.g. isoprenoid
quinones), but also in fatty acids (34).
The sequence analysis has been used to derive a structure model for the
poration complexes formed by ThmA (plus ThmB). The intrinsic activity
observed for ThmA and ThmA can be explained by oligomerization of the
peptide to form the structure ((A)n) (Fig. 7).
The MS analysis of ThmA
strongly suggests oxidation of methionines to
methoxides (Met10, Met54, and/or
Met57; yellow circles in Fig. 7), which would
not dramatically influence the pore-structure as shown in Fig. 7,
(A)n, but would disturb the interactions between
ThmA and ThmB (Fig. 7 (AB)n). The enhancing properties of ThmB can be explained by stabilization of ThmA through subunit/subunit interactions and/or participation of ThmB to the pore
hydrophilicity (Ser6, His10, Asn41,
and Lys37 in Fig. 7). The proposed model of the poration
complex formed by ThmA and ThmB reminds us of the acetylcholine
receptor channel (35, 36). Finally, The spontaneous loss of 18 Da in
ThmB is consistent with the cyclization of the amino-terminal
glutamine.
In conclusion, Thermophilin 13 shares functional properties of
lantibiotics and structural properties of, what we propose to name,
type E poration complexes. The prediction of -turns in putative loop
regions of ThmA, YGNGV-C peptides, and Curvaticin FS47 suggests a
structural basis for the interaction of these peptides with the
membranes of Listeria species. Our model of the poration
complex is consistent with the difference in enhancing properties of
ThmB on ThmA and ThmA
. Finally, the ionophoric activity of
Thermophilin 13 in the absence of a membrane potential, and without the
need for a receptor, makes it an extremely potent compound that differs
in its mode of action from Nisin and other bacteriocins described so
far.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U93029[GenBank].
We are grateful to Dr. Bruno Suri and Dr. Rene Knecht (Ciba Geigy AG, Basel, CH) for amino acid analysis and protein sequencing. We thank Dr. Yves Lemoine (ESBS, Strasbourg, FR) and Dr. A. P. Bruins (Groningen, NL) for their support. We also thank Dr. Christin Choma (Groningen, NL) for peptide synthesis and helpful discussions.