Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE: A METHOD FOR SCREENING FOR AN ANTIMICROBIAL POLYPEPTIDE
FIELD OF THE INVENTION
The present invention relates to a method for screening a polynucleotide
sequence encoding
an antimicrobial polypeptide for its antimicrobial activity.
BACKGROUND OF THE INVENTION
Various bioactive polypeptides are known to kill or inhibit the proliferation
of target cells, e.g.
antimicrobial enzymes, anti-tumor peptides and antimicrobial polypeptides.
Bioactive
polypeptides have a variety of applications for example antimicrobial
polypeptides may be
used for various medical applications, such as to combat infections. As it is
often difficult to
both express and test the activity of an antimicrobial polypeptide because its
activity may often
interfere with expression in a host cell new methods for testing the activity
of such polypeptides
are desirable.
WO 00/73433 discloses a method for screening nucleotide sequences encoding
anti microbial
peptides comprising a) ligating a plasmid with the pool of nucleotide
sequerices linked to an
inducible promoter, b) transforming host cells which are sensitive to the
peptide with the ligated
plasmids, c) screening the transformed host cells so as to select viable
cells, d) cultivating the
viable cells in the presence of inducer so as to induce expression of said
nucleotide sequence,
e) selecting cells according to the effect of the inducer on cell growth.
Reichhart J -M a t a I. (1992), Invertebrate R eproduction a nd D evelopment,
21 ( 1 ), p p. 1 5-24
discloses expression and secretion in yeast of active insect defensin, an
inducible antibacterial
peptide from the fleshfly Phormia terranovae.
Teilum K et al. (1999), Protein Expression and Purification 15, pp. 77-82
discloses that the
yield of ATP N peroxidase can be increased by using thioredoxin reductase
negative strains,
which facilitate the formation of disulfide bonds in inclusion body protein.
Kekessy DA and Piguet JD, Applied Microbiology, 20 (2), pp. 282-283 discloses
a new method
for detecting Bacteriocin production.
SUMMARY OF THE INVENTION
The present invention relates to a method for screening a polynucleotide
sequence encoding
an antimicrobial polypeptide, said method comprising the steps of:
a) introducing the polynucleotide sequence in a host cell, wherein expression
of
said polynucleotide is under control of an inducible promoter and wherein said
host cell is sensitive to the antimicrobial peptide encoded by the
polynucleotide
sequence
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b) cultivating the host cells of in the absence of an inducer capable of
inducing
expression of the antimicrobial peptide
c) cultivating the host cells in the presence of an inducer capable of
inducing
expression of the antimicrobial peptide
d) cultivating the host cells in the presence of an indicator cell, wherein
said
indicator cell is sensitive to the antimicrobial polypeptide encoded by the
polynucleotide sequence
e) selecting host cells capable of reducing the proliferation of indicator
cells
f) recovering the polynucleotide sequence encoding an antimicrobial
polypeptide
from the host cells selected in step e).
The present invention also relates to a method for screening a library of
polynucleotide
sequences encoding one or more antimicrobial polypeptide(s), comprising:
a) cultivating an indicator cell in the presence of the antimicrobial
polypeptide(s),
wherein the antimicrobial polypeptide(s) has/have been expressed by a host
which is sensitive to said antimicrobial polypeptide(s), and wherein
expression
of each of the polynucleotide sequences in the library has been under control
of an inducible promoter, and wherein the host cells comprising the library
polynucleotide sequence has been cultivated in the absence of an inducer and
subsequently in the presence of an inducer
b) selecting a host cell expressing an antimicrobial polypeptide which is
capable of
reducing the proliferation of the indicator cell
The present invention also relates to a method for testing the antimicrobial
activity of an
antimicrobial polypeptide comprising:
a) cultivating an indicator cell in the presence of the antimicrobial
polypeptide(s),
wherein the antimicrobial polypeptide(s) has/have been expressed by a host
which is sensitive to said antimicrobial polypeptide(s), and wherein
expression
of each of the polynucleotide sequences in the library has been under control
of an inducible promoter, and wherein the host cells comprising the library
polynucleotide sequence has been cultivated in the absence of an inducer and
subsequently in the presence of an inducer
Furthermore, the present invention relates to antimicrobial polypeptides
identified by these
methods.
DEFINITIONS
The term "antimicrobial polypeptide" (AMP) is in the context of t he present
invention to be
understood as a polypeptide having an antimicrobial activity.
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The term "antimicrobial activity" is in the context of the present invention
to be understood as
an activity which is capable of killing or inhibiting the proliferation of
microorganisms, including
bacteria, v iruses, a nicellular a Igae a nd p rotozoans, a nd fungi. I n t he
c ontext o f t he p resent
invention the term "antimicrobial" is intended to mean that there is a
bactericidal and/or a
bacteriostatic and/or fungicidal and/or fungistatic effect and/or a virucidal
effect, wherein the
term "bactericidal" is to be understood as capable of killing bacterial cells.
The term
"bacteriostatic" is to be understood as capable of inhibiting bacterial
proliferation, i.e. inhibiting
proliferating bacterial cells. The term "fungicidal" is to be understood as
capable of killing
fungal cells. The term "fungistatic" is to be understood as capable of
inhibiting fungal
proliferation, i.e. inhibiting proliferating fungal cells. The term
"virucidal" is to be understood as
capable of inactivating virus. The term "proliferation" or "proliferating" may
be used
interchangeably with the terms "growth" or "growing" in the present invention.
For purposes of the present invention, antimicrobial activity may be
determined according to
the procedure described by Lehrer et al., Journal of Immunological Methods,
Vol. 137 (2) pp.
167-174 (1991 ).
Polypeptides having antimicrobial activity may be capable of reducing the
number of living
cells of Escherichia eoli (DSM 1576) to 1/100 after 30 min. incubation at
20°C in an aqueous
solution of 25%(w/w); particularly in an aqueous solution of 10%(w/w); more
particularly in an
aqueous solution of 5%(wlw); even more particularly in an aqueous solution of
1 %(w/w); most
particularly in an aqueous solution of 0.5%(w/w); and in particular in an
aqueous solution of
0.1 %(w/w).
Polypeptides having antimicrobial activity may also be capable of inhibiting
the outgrowth of
Escherichia coli (DSM1576) for 24 hours at 25°C in a microbial growth
substrate, when added
in a concentration of 1000 ppm; particularly when added in a concentration of
500 ppm; more
particularly when added in a concentration of 250 ppm; even more particularly
when added in
a concentration of 100 ppm; most particularly when added in a concentration of
50 ppm; and in
particular when added in a concentration of 25 ppm.
The term "library" is in the context of the present invention to be understood
as a collection of
at least two different compounds, i.e. the term "library of polynucleotide
sequences" means a
collection of at least two different polynucleotide sequences. Within this
context the term
"different polynucleotide sequences" is to be understood as polynucleotide
sequences which
are different in regards to at least one nucleotide, e.g. the number of
nucleotides in the
sequences (i.e. the length of the sequence) or the identity of a nucleotide at
a given position
may be different. The term "library of indicator cells" refers to a collection
of at least two
different indicator cells, wherein the term "different indicator cells" is to
be understood as cells
which have a different genotype and/or different phenotype.
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The term "polynucleotide" or "polynucleotide sequence" is in the context of
the present
invention to be understood as a chain of two or more nucleotides including,
but not limited to,
cDNA sequences, RNA sequences, genomic DNA sequences, synthetic or semi-
synthetic
nucleotide sequences or any combination thereof.
The term "polypeptide" refers in the context of the present invention to a
peptide comprising
two or more amino acids. Thus the term includes short chains of amino acids,
such as
between 2-100 amino acids and proteins with a defined three-dimensional
structure.
The term "inducible promoter" is in the context of the present invention to be
understood as a
promoter from which transcription can be regulated, i.e. induced or repressed,
by the presence
or absence of a given compound. The term "regulator" is in this context to be
understood as a
compound which is capable of inducing or repressing transcription from an
inducible promoter,
e.g. the regulator may an "inducer", i.e. a compound capable of inducing
transcription, or it
may be a "repressor", i.e. a compound capable of repressing transcription from
the inducible
promoter.
The term "host cell" is in the context of the present invention to be
understood as any cell
which is susceptible to transformation, transfection or infection with a
nucleic acid construct.
The term "nucleic acid construct" is in the context of the present invention
to be understood as
a polynucleotide sequence comprising a polynucleotide sequence encoding a
polypeptide and
the polynucleotide sequences necessary for expression of said polypeptide,
e.g. said construct
may be a plasmid, a bacteriophage or a virus.
The term "indicator cell" is in the context of the present invention to be
understood as any cell
for which it is of interest to screen a polynucleotide sequence encoding an
antimicrobial
polypeptide by the present invention. The term "target cell" is used
interchangeably with
"indicator cell" throughout this application.
The term "sensitive" or "sensitive to" used in relation to the sensitivity of
an indicator or host
cell towards an antimicrobial polypeptide is in the context of the present
invention to be
understood as the proliferation of said indicator/host cell is reduced or said
indicator/host cell is
killed by the presence, synthesis and/or expression of an AMP. Within the
context of the
present invention the indicator cells are typically affected by the presence
of an AMP, while the
host cells are typically affected by the synthesis and/or expression of an
AMP.
The term "parent" is in the context of the present invention to be understood
as a polypeptide,
which is modified to create a protein variant. The parent polypeptide may be a
naturally
occurring (wild-type) polypeptide or it may be a variant thereof prepared by
any suitable
means. For instance, the parent polypeptide may be a variant of a naturally
occurring
polypeptide which has been modified by substitution, chemical modification,
deletion or
truncation of one or more amino acid residues, or by addition or insertion of
one or more amino
acid residues into the amino acid sequence of the naturally-occurring
polypeptide.
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The term "variant" is in the context of the present invention to be understood
as a polypeptide
which h as b een m odified a s c ompared t o a p arent p olypeptide a t o ne
or m ore a mino a cid
residues.
The term "modification(s)" or "modified" is in the context of the present
invention to be
understood as to include chemical modification of a polypeptide as well as
genetic
manipulation of the DNA encoding a polypeptide. The modifications) may be
replacements)
of the amino acid side chain(s), substitution(s), deletions) and/or insertions
in or at the amino
acids) of interest. Thus the term "modified polypeptide" is to be understood
as a polypeptide
which contains modifications) compared to a parent polypeptide.
The term "coding sequence" refers in the context of the present invention to
the polynucleotide
sequence in DNA or RNA that specifies a polypeptide sequence.
The term "upstream" refers in the context of the present invention to
polynucleotide sequences
located on the proximal site of any given point in relation to the direction
of transcription.
The term "downstream" refers in the context of the present invention to
polynucleotide
sequences located on the distal site of any given point in relation to the
direction of
transcription.
The term "disulfide bond" refers in the context of the present invention to
the covalent bond
formed between the sulphur atoms of two Cysteine residues in a polypeptide.
The term "antimicrobial polypeptide o r A MP of i nterest" refers i n t he
context of the p resent
invention to the AMP(s) which is screened for and/or tested by a method of the
present
invention.
DETAILED DESCRIPTION OF THE INVENTION
Antimicrobial polypeptides
The present invention relates to a method for screening a polynucleotide
sequence encoding
an antimicrobial polypeptide (AMP).and/or testing the antimicrobial activity
of an antimicrobial
polypeptide according t o its ability to le ill or inhibit the proliferation
of an indicator cell. T he
present invention also relates to a method for screening a library of
polynucleotide sequences
encoding one or more antimicrobial polypeptides. Because of the genetic
degeneracy the
library of polynucleotide sequences may encode only one antimicrobial
polypeptide; however it
may particularly encode two or more different antimicrobial polypeptides.
In the following reference to the AMP(s) is also to be understood as referring
to the
polynucleotide sequences) encoding said AMP(s). The AMP(s) and the
polynucleotide
sequences) encoding said AMP(s) screened for and/or tested by the present
invention may be
a variant of a parent AMP, such as a variant generated by manipulation of the
nucleotide
sequence encoding the parent AMP, and/or it may be a so-called wild-type AMP,
i.e. a
naturally occurring AMP. The AMP may also be an artificial AMP, i.e. an AMP
encoded by a
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polynucleotide sequence, wherein the polynucleotide sequence is e.g. generated
by
polynucleotide synthesis.
The AMP(s) and the polynucleotide sequences) encoding said AMP(s) screened for
and/or
tested in the present invention may be obtained from plants, invertebrates,
insects, amphibians
or mammals, or from microorganisms such as bacteria and fungi. For purposes of
the present
invention, the term "obtained from" as used herein shall mean that the
polypeptide encoded by
the nucleotide sequence is produced by a cell in which the nucleotide sequence
is naturally
present or into which the nucleotide sequence has been inserted. In a
particular embodiment,
the polypeptide is secreted extracellularly.
In a particular embodiment of the present invention the AMP(s) may be a
bacterial polypeptide.
For example, the polypeptide(s) may be a gram positive bacterial polypeptide
such as a
Bacillus polypeptide, e.g., a Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis,
Bacillus c irculans, B acillus c oagulans, B acillus I autus, B acillus 1
entus, B acillus 1 icheniformis,
Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or
Bacillus thuringiensis
polypeptide; or a Streptomyces polypeptide, e.g., a Streptomyces lividans or
Streptomyces
murinus polypeptide; or a gram negative bacterial polypeptide, e.g., an E.
coli or a
Pseudomonas sp. polypeptide. In another embodiment it may be from a gram
negative
bacteria.
In another embodiment the AMP(s) and/or the polynucleotide sequences) encoding
said
AMP(s) screened for and/or tested by the present invention may be a fungal
polypeptide, and
particularly a yeast polypeptide such as a polypeptide obtained from Candida,
Kluyveromyces,
Pichia, Saccharomyces, e.g. S, carlsbergensis, S. cerevisiae, S, diastaticus,
S. douglasii, S.
kluyveri, S. norbensis or S, oviformis, Schizosaccharomyces, or Yarrowia
polypeptide; or more
particularly a filamentous fungal polypeptide such as an Acremonium,
Aspergillus,
Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,
Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Piromyces,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or
Trichoderma
polypeptide.
In another particular embodiment, the polypeptide may be obtained from
Aspergillus aculeatus,
Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus
nidulans,
Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides, Fusarium
cerealis, Fusarium
crookwellense, F usarium c ulmorum, F usarium graminearum, F usarium g
raminum, F usarium
heterosporum, Fusarium longypes, Fusarium negundi, Fusarium oxysporum,
Fusarium
reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium
sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium
trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora
thermophila, Neurospora crassa, Penicillium purpurogenum, Trichoderma
harzianum,
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Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma wide
polypeptide.
Other examples of fungi where the AMP(s) may be obtained from include
Pseudoplectania,
e.g. P. vogesiaca or P. nigrella, Plectania, e.g. P. melaena, P. melastoma or
P. nannfeldtii,
Umula, e.g. U. helvelloides or Galiella, e.g. G. rufa.
It will be understood that for the aforementioned species, it encompasses both
the perfect and
imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless
of the species
name by which they are known. Those skilled in the art will readily recognize
the identity of
appropriate equivalents.
Strains of these species are readily accessible to the public in a number of
culture collections,
such as the American Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor
Schimmelcultures
(CBS), and Agricultural Research Service Patent Culture Collection, Northern
Regional
Research Center (NRRL).
Furthermore, the antimicrobial polypeptide(s) may be identified and obtained
from other
sources including microorganisms isolated from nature (e.g., soil, composts,
water, etc.).
Techniques for isolating microorganisms from natural habitats are well known
in the art. The
polynucleotide sequences) may then be derived by similarly screening a genomic
or a cDNA
library of another microorganism.
AMPs' often exert their antimicrobial effect on the target cell by
interacting/binding/sequestering essential molecular targets in said cell. The
antimicrobial
polypeptide(s) (AMP) and the polynucleotide sequences) encoding said AMP(s)
screened for
and/or tested by the present invention may be a membrane-active antimicrobial
polypeptide, or
an antimicrobial polypeptide affecting/interacting with intracellular targets,
e.g.
interacting/binding with molecules involved in signal transduction and/or
interacting/binding to
cell DNA. The antimicrobial polypeptide(s) may act on cell membranes of the
indicator cell, e.g.
through non-specific binding to the membrane, usually in a membrane-parallel
orientation,
interacting only with one face of the bilayer.
The AMP(s) and the polynucleotide sequences) encoding said AMP(s) screened
and/or tested
by a method of the present invention may be an enzyme or a short peptide (less
than 100
amino acid residues).
Examples of antimicrobial enzymes include a muramidase, a lysozyme, a
protease, a lipase, a
phospholipase, a chitinase, a glucanase, a cellulase, a peroxidase, or a
laccase. Alternatively,
a consortium of enzymes synthesizing conventional antibiotics, e.g.
polyketides or penicillins,
can be employed.
The AMP(s) and the polynucleotide sequences) encoding said AMP(s) screened
and/or tested
by a method of the present invention may also be a relatively short
polypeptide, consisting of
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less than 100 amino acid residues, typically 5-95 residues, such as between 5-
90, 5-80, 5-70,
5-60, 5-50, 5-40, 5-30, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 20-
90, 20-80, 20-70,
20-60, 20-50, 20-40, 30-90, 30-80, 30-70, 30-60, 30-50, 40-90, 40-80, 40-70,
40-60, 50-90, 50-
80, 50-70, 60-90 or 60-80 residues. In a particular embodiment of the present
invention the
AMP(s) and the polynucleotide sequences) encoding said AMP(s) to be screened
for and/or
tested has a low cytotoxicity against normal mammalian cells, including human
cells.
Many known short AMPs are cationic and/or hydrophobic polypeptides. Thus it
may typically
contain several arginine and lysine residues and/or not contain any or very
few glutamic acid
or aspartic acid residues, and it may often contain a large proportion of
hydrophobic residues,
such Alanine, Valine, Leucine, Isoleucine, Methionine, Proline, Phenylalanine
and Tryptophan.
Furthermore, many known short AMPs generally have an amphiphilic structure,
with one
surface being positive and the other hydrophobic.
Antimicrobial polypeptides typically has a structure belonging to one of five
major classes:
alpha-helical, cystine-rich (defensin-like), beta-sheet, an unusual
composition of regular amino
acids, and containing uncommon modified amino acids.
Examples of AMPs with an alpha-helical structure, which may be used as parent
AMPs and/or
tested by the method of the present invention include Magainin 1 and 2 which
may be obtained
from frog skin; Cecropin A, B and P1; CAP18; Andropin which may be obtained
from insect
hemolymph (e.g. Drosophila melanogaster); Clavanin A which may be obtained
from tunicate
leukocytes (Styela clava) or Clavanin AI<; Styelin D which may be obtained
from tunicate
leukocytes (Styela clava) and StyelinC; and Buforin II which may be obtained
from Asian toad
(Bufo bufo garagrizans). Examples of cystine-rich polypeptides include alpha-
Defensin HNP-1
(human neutrophil peptide) HNP-2 and HNP-3; beta-Defensin-12, Drosomycin,
gamma1-
purothionin, and Insect defensin A. Another example is Novispirin G10 which is
an alpha-
helical antimicrobial polypeptide that does not contain any disulfide bonds.
Novispirin G10
(SEQ ID No. 17 in WO 02/00839) is obtained by rational design based on
homology to SMAP-
29, an ovine cathelicidin peptide.
Examples of beta-sheet polypeptides include Lactoferricin B, Tachy-plesin I,
and Protegrin
which may be obtained from porcine neutrophils, e.g. Protegrin PG1-5. Examples
of
polypeptides with an unusual composition include Indolicidin; PR-39 which may
be obtained
from Porcine leukocytes; Bactenicin BacS which may be obtained from sheep
leukocytes (Ovis
cries) o r from b ovine I ekocytes (Bos taurus) a nd B ac7 w hich m ay b a
obtained from s heep
leukocytes (Ovis cries) or from bovine lekocytes (Bos taurus); and Histatin 5
which may be
obtained from human saliva or a variant of Histatin 5, such as d-histatin5 (d-
h5) which
corresponds to the 14 C-terminal amino acid residues of histatin5, or dhvar1
which is a variant
of d-h5. Examples of polypeptides with unusual amino acids include Nisin,
Gramicidin A, and
Alamethicin.
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Other examples of AMPs and t he polynucleotide sequences) encoding said AMP(s)
which
may be used in the present invention include an antifungal protein (AFP), such
as an AFP
obtained from Aspergillus, e.g. A. niger or A. giganteus; Defencin hBD3 which
may be
obtained from human epithelia; Heliomycin which may be obtained from insect
hemolymph;
Enterocin L50B which may be obtained from Lactobacillus; Thanatin which may be
obtained
from insect hemolymph; Indolicidin which may be obtained from bovine
neutrophils; Tritrpticin
which may be obtained from porcine neutrophils; Temporin B which may be
obtained from frog
skin (e.g. Rana temporaria); Dermaseptin which may be obtained from frog skin;
Apidaecin
which m ay b a o btained from frog s kin o r h oneybee; S MAP29 which m ay b a
o btained from
sheep leukocytes (Ovis cries); LL-37 which may be obtained from human or
rabbit neutrophils;
BPI which may be obtained from human white blood cells; or Plectasin which may
be obtained
from Pseudoplectania nigrella (e.g. SEQ ID NO:2 in PA 2001 01732).
In a particular embodiment of the invention the AMP screened for and/or tested
by a method of
the present invention is free of any protecting scaffold proteins.
Library of polynucleotide sequences
In one embodiment of the present invention the polynucleotide sequence
screened in the
present invention may be a library of polynucleotide sequences, such as a
library containing
two or more different polynucleotide sequences, such as more than 5 different
polynucleotide
sequences, or more than 10 different polynucleotide sequences, or more than 25
different
polynucleotide sequences, or more than 50 different polynucleotide sequences,
or more than
100 different polynucleotide sequences, or more than 300 different
polynucleotide sequences,
or more than 500 different polynucleotide sequences, or more than 1000
different
polynucleotide sequences or more than 5000 different polynucleotide sequences
or more than
10.000 different polynucleotide sequences, e.g. between 2-10.000 different
polynucleotide
sequence or between 2-5.000 different polynucleotide sequences or between 2-
500 different
polynucleotide sequences or between 50-500 different polynucleotide sequences.
The library of polynucleotide sequences encoding antimicrobial peptides may be
obtained from
naturally occurring genomic DNA, cDNA derived from naturally occurring
organism or it may be
chemically synthesized. Said genomic DNA or cDNA may be derived from any
organism, such
as one of those described above.
The commercial utility of antimicrobial polypeptides generally depends on
their potency,
species specificity and ability to perform under the appropriate conditions.
Often these
conditions are quite different from those under which the polypeptide
originally evolved. Most
antimicrobial polypeptides have, for example, not been evolved to
simultaneously target a
broad range of different microbes, to work in a physiological salt range, to
evade the human
immune system or resist the clearing capacity of the mammalian circulatory
system. Therefore
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it may be desirable to alter one or more properties of a known AMP to improve
its performance
under a given commercial condition.
Thus in one embodiment of the present invention the library of polynucleotide
sequences may
be a library of polynucleotide sequences encoding variants of a parent AMP.
Said variants may
be created by any method known within the art of manipulating polynucleotide
sequences, e.g.
it may be created by random mutagenesis, by site-directed mutagenesis or by
gene shuffling.
The sequences to be shuffled may be related sequences from different organisms
(so-called
"family shuffling"), or they may include a parent sequence and a variant
thereof.
In a particular embodiment of the invention random mutagenesis is achieved by
shuffling of
homologous DNA sequences in vitro such as described by Stammer (Stammer, 1994.
Proc.
Natl. Acad. Sci. USA, 91:10747-10751; Stammer, 1994. Nature 370:389-391)
and/or Crameri
(Crameri A., et al., 1997. Nature Bio-technology 15:436-438). The method
relates to shuffling
homologous DNA sequences by using in vitro P CR techniques. T he above method
is also
described in WO 95/22625 in relation to a method for shuffling homologous DNA
sequences.
An important step in the method is to cleave the homologous template double-
stranded
polynucleotide into random fragments of a desired size followed by
homologously
reassembling the fragments into full-length genes.
In another particular embodiment of the invention random mutagenesis is
achieved by the
method d ascribed i n WO 9 8/41653, w hich d iscloses a m ethod o f D NA s
huffling i n w hich a
library of recombined homologous polynucleotides is constructed from a number
of different
input DNA templates and primers by induced template shifts during in vitro DNA
synthesis. In
this context especially the special version of this in vitro recombination
through induced
template shifts during DNA synthesis, described in WO 98/41653, may
particularly be used.
Here, small (>5 nucleotides) random DNA primers are employed to randomly
initiate DNA
synthesis on the mutant DNA templates that are to be combined.
Especially if the AMP is a short polypeptide, such as between 20-100 amino
acid residues,
special attention has to be taken into consideration when using each of the
above methods for
generation and combination of sequence diversity. Since most shuffling methods
rely on a
substantial number of identical by (20-100 bp) flanking the mutations that has
to be
recombined, the mutations in small polynucleotide sequences are technically
difficult to
combine by the above described methods.
Accordingly, other formats of directed evolution may be employed on small
polynucleotide
sequences. In a particular embodiment involving the combination of variants of
a given peptide
of less than approximately 50 amino acids, one degenerate DNA primer
harbouring all the
desired mutations may be synthesized. In a given position in this degenerate
primer, both the
wildtype (wt) (naturally occurring) nucleotide as well as the mutant
nucleotide should be
present. The frequency o f w t-to-mutant n ucleotides m ay b a a djusted a s c
onsidered o ptimal
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and rules and considerations to determine the optimal frequency are known in
the art. By
including all desired mutations in one primer, the desired sequence-space
could be completely
sampled. This method allows for the sampling and combination of all desired
mutations
irrespectively of how close they would be in the primary gene sequence.
If polypeptides of more than approximately 50 amino acids are employed, two or
more
separate and degenerate primers may have to be used. This is due to the
constraints generally
experienced w hen s ynthesizing D NA p rimers; o nly D NA p rimers o f I ess t
han a pproximately
180-200 nucleotides may routinely be synthesized.
In another embodiment where polypeptides of more than approximately 50 amino
acids are
employed, the sequence diversity (the individual mutants) to be combined can
individually be
harboured in small oligonucleotides of 20-30 base pairs of length. In this
approach, a specific
DNA oligonucleotide is employed for each mutation that should be included in
the library. The
mutations may in particular be located in the middle of the small
oligonucleotide to optimize
annealing. Spiking in several or numerous of these small oligonucleotides in a
PCR reaction
using the wt polypeptide sequence as backbone for the amplification, would
allow for the
combination of the desired mutations. By varying the amount of the individual
oligonucleotides
to be combined, desired ratios of individual variants to wt's can be created.
As approximately
10 base pairs are required on each side of the sequence mismatch, this method
cannot
efficiently combine mutations that are immediately adjacent.
In a particular embodiment doped oligonucleotides may be used as primers for
the PCR.
Doped oligonucleotides contain mixed bases in un-equal representation to
encode the
template with different amino acid residues with a specific distribution.
The present invention is not limited to screening libraries of polynucleotide
sequences
encoding variants of existing or already characterized antimicrobial
polypeptides. It may also
be used to screen for polynucleotide sequences encoding new and/or unknown
antimicrobial
polypeptides. For example the library of polynucleotide sequences may comprise
polynucleotide sequences obtained from a single microorganism or it may
comprise
polynucleotide sequences obtained from two or more different microorganisms.
Examples of
organisms from which the polynucleotide sequences) encoding an AMP may be
obtained are
given above.
Host cells
The host cell according to the definition may be any cell susceptible to
transformation,
transfection or infection with a nucleic acid construct. One advantage of the
present invention
is that the host cell is sensitive to the AMP activity, but that the host cell
is first allowed to
proliferate in the absence of the AMP (as expression of the AMP is not
induced) and then
subsequently the host cells are induced to express the AMP. By allowing t he
host cells to
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proliferate before induction of AMP expression it is possible to have enough
host cells to
produce enough AMP to affect an indicator cells. If on the other hand the host
cells were not
allowed to proliferate before induction of AMP expression it is most likely
that the expressed
AMP would kill or inhibit the proliferation of the host cells before they were
able to express
enough amounts of AMP to affect an indicator cell.
The sensitivity of the host cell towards the AMP may be tested by growing the
host cells into
which a nucleic acid construct comprising the polynucleotide sequence encoding
the AMP on
for example solid media in the presence and absence, respectively of the
inducer and then
compare if cell growth of the host cells are inhibited or reduce when the
inducer is present as
compared when the inducer is absent ( as described in example 2 and 3). I f
cell g rowth is
inhibited or reduced the host cells are sensitive to the peptide when they are
expressing it.
Another way of testing this is as described in example 13. The host cells
expressing the
antimicrobial polypeptide are grown in liquid media in the presence and
absence, respectively
of the inducer. The growth curves of the cultures are monitored for a period
of time, such as
overnight by measuring the OD and the percentage of growth inhibition is
calculated. If the
growth of the host cells is reduced or inhibited the host cells are sensitive
to the AMP when
they are expressing it.
It is an advantage if the host cell does not itself contain and/or express
polynucleotide
sequences) encoding antimicrobial polypeptide(s) as this may interfere with
the screening
method. This cell characteristic may either be a natural feature of the cell
or it may be obtained
by deletion of such sequences as described e.g. in Christiansen et al. (1997)
or Stoss et al.
( 1997).
The host cell may be a unicellular microorganism, such as a prokaryote, or a
non-unicellular
microorganism, such as a eukaryote.
Examples of unicellular cells are bacterial cells such as gram positive
bacteria including, but
not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus
brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus
lautus, Bacillus lentus,
Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Baeillus subtilis, and
Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or
Streptomyces
murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a
particular
embodiment, the bacterial host cell is Baeillus subtilis
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or
fungal cell.
In a particular embodiment, the host cell may be a fungal cell. "Fungi" as
used herein includes
the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as
defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th
edition, 1995, CAB
International, University Press, Cambridge, UK) as well as the Oomycota (as
cited in
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Hawksworth et al., 1995, supra, page 171 ) and all mitosporic fungi
(Hawksworth et al., 1995,
supra).
In a more particular embodiment, the fungal host cell is a yeast cell. "Yeast"
as used herein
includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and
yeast
belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may
change in the future, for the purposes of this invention, yeast shall be
defined as described in
Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport,
R.R., eds, Soc.
App. Bacteriol. Symposium Series No. 9, 1980).
Examples of yeast host cell include Candida, Hansenula, Kluyveromyces, e.g. K.
lactis, Pichia,
Saccharomyces, e.g. S, carlsbergensis, S, cerevisiae, S. diastaticus, S.
douglasii, S. kluyveri,
S. norbensis or S. oviformis, Schizosaccharomyces, or Yarrowia cell, e.g.
Y.lipolytica.
In a nother a mbodiment the fungal h ost cell m ay b a a f ilamentous fungal c
ell. " Filamentous
fungi" include all filamentous forms of the subdivision Eumycota and Oomycota
(as defined by
Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a
mycelial wall
composed of chitin, cellulose, glucan, chitosan, mannan, and other complex
polysaccharides.
Vegetative growth is by hyphal elongation and carbon catabolism is obligating
aerobic. In
contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by
budding of a
unicellular thallus and carbon catabolism may be fermentative.
Examples o f filamentous fungal h ost c ell i nclude a c ell o f A cremonium,
A spergillus, a .g. A .
awamori, A. foetidus, A. japonicus, A. nidulans, A. niger or A. oryzae,
Fusarium, e.g. F.
bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F.
graminum, F.
heterosporum, F. negundi, F. oxysporum, F. reticulatum, F. roseum, F.
sambucinum, F.
sarcochroum, F. sporotrichioides, F. sulphureum, F. torulosum, F.
trichothecioides, or F.
venenatum, Humicola, e.g. H. insolens, H. lanuginose, Mucor, e.g. M, miehei,
Myceliophthora,
e.g. M, thermophila, Neurospora, e.g. N. crassa, Penicillium, e.g. P.
purpurogenum, Thielavia,
e.g. T. terrestris, Tolypocladium, or Trichoderma, e.g. T. harzianum, T.
leoningii, T.
longibrachiatum, T, reesei, or T, viride.
In another embodiment of the invention the host cell is a bacterial cell or a
eukaryotic cell.
Further the bacterial cell may particularly be an ElectroMAX DH10B cell
(GibcoBRL/Life
technologies, UK) or of the genus E. coli, e.g. SJ2 E. coli of Diderichsen et
al. (1990) or E.coli
with the genotype: F-mcrA ~(mrr-hsdRMS-mcrBC) ~801acZ~M15 ~IacX74 deoR recA1
araD139 ~(araA-leu)7697 galU galK rpsL endA1 nupG, also known as the
commercially
available TOP10 cells from Invitrogen. Other particular host cells may be
strains of Bacillus,
such as Bacillus subtilis or Bacillus sp. A particular useful eukaryotic cell
is a yeast, e.g. S.
cerevisae.
For s ome p olypeptides i t may b a i mportant for t heir a ctivity t hat a d
isulfide b and i s formed
between two cysteine residues in the polypeptide. In some host cells, e.g.
yeast, formation of
13
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WO 2004/033715 PCT/DK2003/000669
disulfide bonds often takes place as a natural part of the expression of
polypeptides. However,
for other types of host cells it doesn't. Thus in a particular embodiment the
redox state of the
host cell may be such that it allows disulfide bond formation. This is
particularly important if the
activity of the antimicrobial polypeptide is affected by the presence of
disulfide bonds) in said
polypeptide. The host cell may be such that it naturally allows disulfide bond
formation or it
may harbour one or more mutations that allow disulfide bond formation. For
example the host
cell may harbour a mutation in the thioredoxin reductase gene (trx8) and/or in
the glutathione
reductase gene (gor). In particular the host cell may be a K-12 derivative of
E.coli harbouring
mutations in both the thioredoxin reductase gene and the glutathione reductase
gene, such as
the commercially available E.coli origami cells from Novagen.
Expression of a polynucleotide sequence in a host cell
For expression of a polynucleotide sequence in a host cell, a nucleic acid
construct is generally
generated comprising the polynucleotide sequence encoding a polypeptide
together with
polynucleotide sequences capable of facilitating expression of the
polynucleotide in the host
cell. T he p olynucleotide s equences facilitating expression o f a p
olynucleotide s equence a re
often known collectively as an expression vector. For expression of the
polynucleotide
sequence, i.e. production of the polypeptide, the expression vector comprising
the
polynucleotide sequence encoding a polypeptide is introduced into a host cell
which is then
cultured under conditions facilitating expression of the polypeptide. Methods
for cloning of
polynucleotide sequences and introducing expression vectors into host cells
are well-known in
the art.
In the present invention a polynucleotide sequence or a library of
polynucleotide sequences
encoding the antimicrobial polypeptide(s) is expressed in a host cell.
Expression of said
polynucleotide sequences) should be under control of an inducible promoter.
The polynucleotide sequences) may in the present invention be expressed
intracellularly in
the host cell, e.g. in the cytoplasm or other intracellular compartments, such
as vacuoles or the
endoplasmic reticulum (ER), or it may be expressed in the periplasm or
secreted by the host
cell.
Expression vectors
The expression vector may be any vector, e.g. a plasmid or a virus, which may
conveniently be
subjected to recombinant DNA procedures and which can bring about expression
of the
antimicrobial polypeptide encoded by the polynucleotide sequence in the host
cell. The choice
of the vector will typically depend on the compatibility of the vector with
the host cell into which
the vector is to be introduced. The vectors may be linear or closed circular
plasmids.
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WO 2004/033715 PCT/DK2003/000669
The vector may be an autonomously replicating vector, i.e., a vector which
exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or an
artificial
chromosome.
The vector may contain any means for assuring self-replication. Alternatively,
the vector may
be one which, when introduced into the host cell, is integrated into the
genome and replicated
together with the chromosomes) into which it has been integrated. Furthermore,
a single
vector or plasmid or two or more vectors or plasmids which together contain
the total DNA to
be introduced into the genome of the host cell, or a transposon may be used.
The expression vector used in the present invention may in a particular
embodiment comprise
one or more selectable markers which permit easy selection of
transformeditransfected cells.
A selectable marker is typically a gene of which the product provides for
biocide or viral
resistance, resistance to heavy metals, prototrophy to auxotrophs, and the
like.
Examples of bacterial selectable markers are the dal genes from Baeillus
subtilis or Bacillus
licheniformis, or markers which confer antibiotic resistance such as
ampicillin, kanamycin,
chloramphenicol or tetracycline resistance.
Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1,
and URA3.
Selectable m arkers for use i n a f ilamentous fungal h ost cell i nclude, b
ut a re n of I invited t o,
amdS (acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin
acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate
reductase), pyre
(orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC
(anthranilate
synthase), as well as equivalents thereof.
Particularly for use in an Aspergillus cell are the amdS and pyre genes of
Aspergillus nidulans
or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
The vector used in the present invention may contain an element that permits
stable
integration of the vector into the host cell's genome or autonomous
replication of the vector in
the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the
nucleotide sequence
encoding the polypeptide or any other element of the vector for stable
integration of the vector
into the genome by homologous or non-homologous recombination. Alternatively,
the vector
may contain additional nucleotide sequences for directing integration by
homologous
recombination into the genome of the host cell. The additional nucleotide
sequences enable
the vector to be integrated into the host cell genome at a precise location in
the chromosome.
To increase the likelihood of integration at a precise location, the
integrational elements should
particularly contain a sufficient number of nucleotides, such as 100 to 1,500
base pairs,
particularly 400 to 1,500 base pairs, and most particularly 800 to 1,500 base
pairs, which are
highly homologous with the corresponding target sequence to enhance the
probability of
CA 02500757 2005-03-31
WO 2004/033715 PCT/DK2003/000669
homologous recombination. The integrational elements may be any sequence that
is
homologous with the target sequence in the genome of the host cell.
Furthermore, the
integrational elements may be non-encoding or encoding nucleotide sequences.
On the other
hand, the vector may be integrated into the genome of the host cell by non-
homologous
recombination.
For autonomous replication, the vector may further comprise an origin of
replication enabling
the vector to replicate autonomously in the host cell in question. Examples of
bacterial origins
of replication are the origins of replication of plasmids pBR322, pUC19,
pACYC177, and
pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAMf31
(pAMbeta1 ) permitting replication in Bacillus. Examples of origins of
replication for use in a
yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1
and C EN3, a nd the c ombination o f A RS4 a nd C EN6. The o rigin o f
replication m ay b a o ne
having a mutation which makes its ability to function temperature-sensitive in
the host cell
(see, e.g., Ehrlich, 1978, Proceedings of the National Aeademy of Sciences USA
75: 1433).
More than one copy of a polynucleotide sequence of the present invention may
be inserted
into the host cell to increase production of the gene product. An increase in
the copy number of
the n ucleotide sequence c an b a o btained b y i ntegrating a t I east o ne
additional c opy o f t he
sequence into the host cell genome or by including an amplifiable selectable
marker gene with
the nucleotide sequence where cells containing amplified copies of the
selectable marker
gene, and thereby additional copies of the nucleotide sequence, can be
selected for by
cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the
expression
vectors are well known in the art (see, e.g., Sambrook et al., 1989, supra).
Inducible promoters and inducers
The expression vector used to express the polynucleotide sequence encoding an
antimicrobial
polypeptide according to the present invention should comprise an inducible
promoter so that
expression of said AMP may be controlled by a regulator. It is an advantage if
the promoter
allows tight regulation of the synthesis of the encoded AMP, i.e. that
leakiness from the
promoter is kept at a minimum. In addition, control of the transcription of
the encoded AMP
may particularly be significant as particularly short polypeptides are often
inherently unstable
and easily degraded in the cytoplasm of microorganisms. The inducible promoter
may be
regulated by more than one regulator. For example the promoter may be
positively and
negatively regulated, respectively, by two different compounds, e.g. in the
presence of an
inducer, expression from the promoter may be turned on; while in the absence
of said inducer
only very low levels of expression occur from the promoter. The uninduced
(i.e. in the absence
of the first inducer) levels may then be further repressed by the presence of
a repressor. By
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WO 2004/033715 PCT/DK2003/000669
varying the activity of the two regulators, protein expression levels may be
manipulated to
optimize expression of potentially toxic or essential genes.
One example of an inducible promoter and inducers is the Lac promoter as
described in
Taguchi S., Nakagawa K., Maeno M. and Momose H.; "In Vivo Monitoring System
for
Structure-Function Relationship Analysis of the antibacterial peptide
Apidaecin"; Applied and
Environmental Microbiology, 1994, pp. 3566-3572, which may be regulated by
presence of the
inducer lactose or by the synthetic non-digestible lactose derivative IPTG.
Other examples
include the trp promoters induced by tryptophan or gal promoters induced by
galactose for E.
coli, gall promoter for S. cerevisiae, AOX1 promoter for Pichia pastoris, pMT
(metallothionein)
promoter for Drosophila, MMTV LTR, pVgRXR or pIND promoters for mammalian
expression.
It is an advantage to use an inducer which is not metabolized or digested in
the host cell as
this may keep the inducer concentration constant during the screening
procedure.
Furthermore, it may be an advantage to select/use a promoter for which the
corresponding
inducer is able to permeate the cell membranes) and gain access to the
promoter.
In a particular embodiment of the invention the promoter may be the pBAD
promoter as used
in the examples, vide infra, which is induced by the digestible inducer
arabinose. If the pBAD
promoter is used the host cells ability to digest arabinose may be eliminated
by deleting
suitable genes from the host cell genome so as to achieve the above mentioned
advantage of
having a constant level of inducer.
The pBAD promoter is an example of a promoter which is regulated by two
regulators as this is
both positively and negatively regulated by AraC and cAMP-CRP. In the presence
of
arabinose, expression from the promoter is turned on, while in the absence of
arabinose, only
very low levels of expression occur from the promoter.
Uninduced levels may be even further repressed by culturing the cells in the
presence of
glucose. Glucose acts by lowering cAMP levels, which in turn decreases the
binding of cAMP
CRP to the promoter region of pBAD. As cAMP levels are lowered,
transcriptional activation is
decreased. This is an advantage if the antimicrobial polypeptide of interest
is extremely growth
inhibitive or toxic to the host. In conclusion, by varying the activity of the
two regulators, protein
expression levels can be manipulated to optimize expression of potentially
toxic or essential
genes.
Other reaulatory seauences
The nucleic acid construct used for expressing the polynucleotide sequence
encoding the
antimicrobial polypeptide of the present invention may comprise, besides the
promoter and the
polynucleotide sequence encoding said AMP, other polynucleotide sequences of
importance
for the expression of said AMP(s). For example the nucleic acid construct may
further
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WO 2004/033715 PCT/DK2003/000669
comprise a signal peptide coding region, a transcription terminator sequence,
a leader
sequence, a polyadenylation sequence and/or a propeptide coding region.
A signal peptide coding region is a polynucleotide sequence located upstream
of the 5' end of
the coding sequence, which is both transcribed and translated and where the
translated signal
peptide directs th'e encoded polypeptide into the host cell's secretory
pathway. The 5' end of
the coding sequence may inherently contain a signal peptide coding region
naturally linked in
translation r eading frame with t he s egment o f t he c oding r egion t hat a
ncodes t he s ecreted
polypeptide. Alternatively, the 5' end of the coding sequence may contain a
signal peptide
coding region which is foreign to the coding sequence. The foreign signal
peptide coding
region may be required where the coding sequence does not naturally contain a
signal peptide
coding region. Alternatively, the foreign signal peptide coding region may
simply replace the
natural signal peptide coding region in order to enhance secretion of the
polypeptide. However,
any signal peptide coding region which directs the expressed polypeptide into
the secretory
pathway of a host cell of choice may be used in the present invention.
Examples of signal peptide coding regions for bacterial host cells are the
signal peptide coding
regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase,
Bacillus
stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus
lichenif~rmis beta
lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM),
and Baeillus
subtilis prsA. Further signal peptides are described by Simonen and Palva,
1993,
Microbiological Reviews 57: 109-137.
Another example is the gill signal peptide, as used in the examples, which
directs expression
to the periplasm.
Examples of signal peptide coding regions for filamentous fungal host cells
are the signal
peptide coding regions obtained from the genes for Aspergillus oryzae TAKA
amylase,
Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic
proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
Useful signal peptide coding regions for yeast host cells are obtained from
the genes for
Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
Other useful
signal peptide coding regions are described by Romanos et al., 1992, supra.
A transcription terminator sequence is a polynucleotide sequence recognized by
a host cell to
terminate transcription. The terminator sequence is generally located
downstream of the 3'
terminus of the coding sequence.
Examples of terminators for filamentous fungal host cells include terminators
obtained from the
genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,
Aspergillus
nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and
Fusarium oxysporum
trypsin-like protease.
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Examples of terminators for yeast host cells include terminators obtained from
the genes for
Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1
), and
Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other
useful
terminators for yeast host cells are described by Romanos et al., 1992, supra.
A leader sequence is in the context of the present invention to be understood
as a
polynucleotide sequence which is transcribed but not translated and which
contains the
ribosome-binding site. The leader sequence is linked to the 5' terminus of the
coding
sequence.
Examples of leader sequences for filamentous fungal host cells include those
obtained from
the genes f or Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose
phosphate
isomerase.
Examples of leader sequences for yeast host cells include those obtained from
the genes for
Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae 3-
phosphoglycerate
kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae
alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
A polyadenylation sequence is a polynucleotide sequence linked to the 3' end
of the coding
sequence and which, when transcribed, is recognized by the host cell as a
signal to add
polyadenosine residues to the transcribed mRNA.
Examples of polyadenylation sequences for filamentous fungal host cells
include those
obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger
glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum
trypsin-like
protease, and Aspergillus niger alpha-glucosidase.
Useful polyadenylation sequences for yeast host cells are described by Guo and
Sherman,
1995, Molecular Cellular Biology 15: 5983-5990.
A propeptide region is a part of the coding sequence which encodes an amino
acid sequence
positioned at the amino terminus of a polypeptide. A polypeptide comprising a
propeptide
region is known as a propolypeptide or a proenzyme (or a zymogen in some
cases). A
propolypeptide is generally inactive and can be converted to a mature active
polypeptide by
catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
The propeptide
coding region may be obtained from the genes for Bacillus subtilis alkaline
protease (aprE),
Bacillus subtilis neutral protease (nprTj, Saccharomyces cerevisiae alpha-
factor, Rhizomucor
miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO
95133836).If both a
signal peptide coding region and a propeptide region are present at the amino
terminus of a
polypeptide, the propeptide region may be positioned next to the amino
terminus of the mature
polypeptide and the signal peptide region may be positioned next to the amino
terminus of the
propeptide region. In this context the term "mature polypeptide" refers to the
functional active
polypeptide without the propeptide sequence.
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WO 2004/033715 PCT/DK2003/000669
Introduction of nucleic acid constructs in a host cell
The polynucleotide sequence encoding the antimicrobial polypeptide in the
present invention
may be introduced into the host cell by any method and methods for introducing
nucleic acid
constructs into host cells are well known to a person skilled in the art.
For example a nucleic acid construct may be introduced into a bacterial host
cell may by
protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General
Genetics
168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961,
Journal of
Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of
Molecular
Biology 56: 209-221 ), electroporation (see, e.g., Shigekawa and Dower, 1988,
Biotechniques
6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169:
5771-5278).
Fungal cells may be transformed by a process involving protoplast formation,
transformation of
the protoplasts, and regeneration of the cell wall in a manner known per se.
Suitable
procedures for transformation of Aspergillus host cells are described in EP
238 023 and Yelton
et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-
1474. Suitable
methods for transforming Fusarium species are described by Malardier et al.,
1989, Gene 78:
147-156 a nd WO 9 6/00787. Y east m ay b a t ransformed a sing t he p
rocedures d escribed b y
Becker and Guarente, In Abelson, J.N. and Simon, M.L, editors, Guide to Yeast
Genetics and
Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic
Press, Inc.,
New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et
al., 1978,
Proceedings of the Nafional Academy of Sciences USA 75: 1920.
Indicator cells
The indicator cell used in the present invention may be any cell which it is
interesting to test for
antimicrobial polypeptide activity against. Thus the indicator cell may be any
cell which is
sensitive to an antimicrobial polypeptide activity. One advantage of the
present invention is
that the antimicrobial activity is tested on indicator cells thus one need
only to introduce the
polynuleotide sequence encoding said AMP activity into a single type of host
cells but is able
to test the AMP activity on a number of different indicator cells. Thus it is
possible to test the
antimicrobial activity towards several indicator cells, i.e. a library of
indicator cells without
having to introduce the polynucleotide sequence encoding said AMP(s) into more
than one
host cell. It is thereby possible to reduce the amount of work which is
associated with
introducing a polynucleotide sequence into host cells.
The indicator cell may be a prokaryotic cell, e.g. a bacterium, or it may be a
eukaryotic cell,
such as a fungal cell, a plant cell, an insect cell or a mammalian cell.
Examples of fungal cells
include both filamentous fungi and yeast.
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For example an indicator cell may be any of those described above.
Examples of useful bacterial indicator cells include Bacillus, e.g. B,
subtilis or the
chloramphenicol-resistant B, subtilis (1315-1), Bordetella, e.g. B,
bronchiseptica, Burkholderia,
e.g. B. cepacia, Coagulase-negative Staphylococcus, Staphylococcus carnosus,
Citrobacter,
e.g. C. freundii, Enterococcus, e.g. E. hirae or E. spec., Escherichia, e.g.
E. coli such as the
TOP10 E.coli cells from Invitrogen, Klebsiella, e.g. K. pneumonia,
Micrococcus, e.g. M. luteus,
Mycobacterium, e.g. M. smegmatis, Pseudomonas, e.g. P, aeruginosa,
Staphylococcus, e.g.
S. aureus, S, epidermidis or S. simulans or Stenotrophomonas, e.g. S.
maltophila.
Examples of yeasts which may be used as indicator cells include Saccharomyces,
e.g. S.
cerevisiae, Candida, e.g. C. albicans or Pityrosporum.
Examples of filamentous fungi which may be used as indicator cells include
Epdidermophyton,
e.g. E. floccosum, Trichophyton, e.g. T. mentagrophytes or Aspergillus, e.g.
A. niger,
Fusarium, e.g. F.longypes.
In a particular embodiment the methods of the present invention may be used to
test a library
of indicator cells, i.e whether two or more indicator cells are sensitive
towards a particular
antimicrobial p olypeptide o r t owards a I ibrary o f p olynucleotide s
equences a ncoding o ne o r
more, e.g. a library of antimicrobial polypeptides. In particular more than 5
indicator cells, such
as more than 10 indicator cells, or more than 25 indicator cells, or more than
50 indicator cells,
or more than 100 indicator cells or more than 200 indicator cells, or more
than 300 indicator
cells or more than 500 indicator cells or more than 1000 indicator cells or
more than 5000
indicator cells may be used in a method of the present invention. For example
2-5000 indicator
cells, a .g. 2-1000 i ndicator cells o r 2-500 i ndicator cells o r 2-100 i
ndicator cells o r 5- 1 000
indicator cells or 5-500 indicator cells or 5-100 indicator cells or 10-1000
indicator cells or 10-
500 indicator cells or 10-100 indicator cells or 50-500 indicator cells may be
tested in a method
of the present invention. Typically if a library of indicator cells are used
in the present invention
the indicator cells are cultured separately in the presence of the
antimicrobial polypeptide(s) or
the host cells expressing the antimicrobial polypeptide(s).
Screening process
As mentioned above the present invention relates to a method for screening a
polynucleotide
sequence encoding an antimicrobial polypeptide, said method comprising the
steps of:
a) introducing the polynucleotide sequence in a host cell, wherein expression
of said
polynucleotide is under control of an inducible promoter and wherein said host
cell is
sensitive to the antimicrobial peptide encoded by the polynucleotide sequence
b) cultivating the host cells of in the absence of an inducer capable of
inducing expression
of the antimicrobial peptide
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c) cultivating the host cells in the presence of an inducer capable of
inducing expression
of the antimicrobial peptide
d) cultivating the host cells in the presence of an indicator cell, wherein
said indicator cell
is sensitive to the antimicrobial polypeptide encoded by the polynucleotide
sequence
e) selecting host cells capable of reducing the proliferation of indicator
cells
f) recovering the polynucleotide sequence encoding an antimicrobial
polypeptide from the
host cells selected in step e).
Prior to step a) of the screening process certain preparatory steps may be
necessary. An
indicator cell for which an antimicrobial polypeptide with a killing and/or
cell proliferation
inhibiting effect it is desired to identify should be found, examples of
useful indicator cells are
given above. A suitable host cell and an expression vector compatible with
said host cell
should be chosen, examples of suitable host cells and vectors are given above.
In a particular
embodiment it may be a host cell, which allows formation of disulfide bond,
e.g. it may be a
host cell harbouring a mutation which alters the redox state of the host cell,
such as a host cell
which harbours a mutation in the thioredoxin reductase gene and/or a mutation
in the
glutathione reductase gene. In one embodiment of the method the polynucleotide
sequence is
a library of polynucleotide sequences, and in this case a library of
polynucleotide sequences
should be prepared prior to step a). Thus a library of polynucleotide
sequences is screened by
the method. Examples of libraries of polynucleotide sequences are given above,
such as a
sample of polynucleotide generated by mutating a parent polynucleotide
sequence encoding a
parent a ntimicrobial p olypeptide, o r a s ample o f p olynucleotide s
equences r epresenting t he
genome or polypeptides expressed by a specific organism or cell, or by two or
more organisms
or cells. Said library may be prepared by any method, e.g. any conventional
method known to
a person skilled in the art.
As part of introducing the polynucleotide sequence into a host cells, i.e. in
step a), said host
cells may be separated on the basis of e.g. if a polynucleotide sequence has
actually been
introduced into the host cell and/or if the host cell is viable and/or its
proliferation has not been
inhibited. This may typically be done by the use of a selection marker.
Examples of suitable
selection markers are given above. However, it is not essential to the
screening method to
separate non-viable host cells from viable host cells as the antimicrobial
polypeptide is
selected on the basis of its killing or cell proliferation inhibiting effect
on the indicator cell. If a
selection marker is used the host cells may typically be sub-cultured in the
absence of the
selection marker, as the selection marker may kill and/or inhibit
proliferation of the indicator
cell. Instead of sub-culturing the host cell a compound capable of
inactivating the selection
marker may be added to the culture. Methods for culturing and/or sub-culturing
host cells are
known to a person skilled in the art and include culturing on solid media,
e.g. a conventional
agar plate or culture in a liquid media, e.g. in a shake or a microtiter
plate.
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In step b) the host cells are cultivated in the absence of the inducer so that
said cells can
proliferate without being killed or its proliferation being inhibited by the
antimicrobial
polypeptide to which they are sensitive. The host cells may by cultured in the
presence of any
suitable nutrient medium using methods known in the art. For example, the cell
may be
cultivated by shake flask cultivation, on agar plates, in microtiter plates or
small-scale
fermentation. Typically a suitable nutrient medium comprises carbon and
nitrogen sources and
inorganic salts and may be available from commercial suppliers or be prepared
according to
published compositions (e.g., in catalogues of the American Type Culture
Collection). The host
cells may be cultivated in a suitable medium without an inducer f or as I ong
as necessary,
typically this may be for 10-24 hours, 10-36 hours, 10-48 hours, 10-72 hours,
such as 10
hours, 24 hours, 48 hours or 72 hours.
In step c) the host cells are cultivated in the presence of an inducer so as
to induce expression
of the antimicrobial polypeptide. Typically said cells are cultivated in the
same nutrient medium
as in step b) but with the exception that it now also contains the inducer. A
suitable nutrient
medium, method of cultivation and time of cultivation may be as described
above. The amount
of inducer depends on the type of inducer and type of inducible promoter and
is well known to
a person skilled in the art. For example if arabinose is used as inducer it
may typically be used
in the concentration range from 0.0001-20% arabinose, e.g. 0.001-10% or 0.01-
1% arabinose
and if IPTG is used as inducer it may typically be used in range of 0.001-1 mM
IPTG. The
presence of the inducer should induce expression of the antimicrobial
polypeptide by the host
cells and thereby kill or inhibit the proliferation of said host cells. The
dose of inducer may be
regulated to control for example whether the host cells are completely killed
or only their
proliferation is inhibited and/or it may be varied during the time period of
cultivation. If the dose
of inducer is varied during cultivation it may e.g. be lower in the beginning
to induce expression
of the antimicrobial polypeptide but only slightly affect the proliferation of
the host cells and
then increased to eventually kill the host cells.
In one embodiment of the invention the host cells may be lysed before step d),
i.e. after they
have had time to produce the AMP but before culturing them in the presence of
an indicator
cell and/or before culturing the indicator cells) in the presence of the AMP.
The AMP may by
itself lyse the cells or this may be performed by affecting the host cells by
external factors
capable of lysing of cells, such as addition of chloroform or lysozym,
infection with a lytic
phage or the host cells may be lysed by exposing them to sonication, high
temperature or an
acidic pH or by a combination of two or more factors. For example the host
cells may be lysed
by a xposing t hem to a t I east 6 0 d agrees C , s uch a s a t I east 7 0 d
agrees C , o r a t I east 8 0
degrees C, or at least 90 degrees C or at least 95 degrees C, e.g. between 65
and 95 degrees
C, or between 75 and 95 degrees C, or between 75 and 85 degrees C. The host
cells may also
be lysed by exposing them to an acidic pH, such as below pH 6, or below pH 5
or below pH 4
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or below pH 3, or below pH 2, e.g. between pH 2 and pH 5, or between pH 2 and
pH 4, or pH
between pH 2.5 and 3. In a particular embodiment of the present invention the
host cells are
lysed by a exposing them to a high temperature and an acidic pH, e.g. a
temperature between
65 and 95 degrees C and a pH between pH 2 and pH 6, particularly a temperature
between 75
and 85 degrees C and a pH between pH 2 and pH 4, more particularly a
temperature of 80
degrees C and a pH between pH 2.5 and 3.
In step d) the host cells are cultivated in the presence of an indicator cell,
so that the killing or
proliferation inhibiting effect of the antimicrobial polypeptide expressed by
the host cell can be
tested on the indicator cell. The host cells and indicator cells may be
cultivated in any suitable
nutrient media, by any suitable method and for any period of time, e.g. as
described above.
The host cells and the indicator cells may be in direct contact or in indirect
contact with each
other, e.g. they may be cultivated without direct contact but by a method
allowing diffusion of
the antimicrobial polypeptide expressed by the host cells to the indicator
cells. The viability
and/or proliferation of the indicator cells may be tested by any known method,
e.g. as
described below. In one embodiment of the invention the effect of the
antimicrobial polypeptide
expressed by the host cell may be tested on two or more differerit indicator
cells, e.g. the effect
of the AMP may be tested on a library of indicator cells. Typically this may
be performed by
cultivating the each of the different indicator cells separately in the
presence of the host cells
expressing the AMP(s) to distinguish the indicator cells from each other.
Host cells which express an antimicrobial polypeptide capable of killing or
reducing the
proliferation of the indicator cells are identified in step e). Different
criteria for selecting the host
cells) may be used. For example only host cells which greatly affect the
indicator cells may be
selected, such as host cells expressing an antimicrobial polypeptide capable
of completely
killing the indicator cell, or all host cells capable of affecting the
proliferation rate of the
indicator cell may be selected, e.g. all host cells are selected independent
of whether they kill
the indicator cell, inhibit the proliferation of the indicator cell slightly
or inhibit the proliferation of
the indicator cell greatly. If the effect of the antimicrobial polypeptide is
tested on two or more
different indicator cells one may e.g. select only host cells expressing an
antimicrobial
polypeptide which kills and/or inhibits the proliferation of two or more
different indicator cells or
one may select all host cells expressing an antimicrobial polypeptide capable
of killing or
inhibiting the proliferation of at least one type of indicator cell.
In step f) the polynucleotide sequence encoding an antimicrobial polypeptide
expressed by a
host cell selected in step e) is recovered from said host cell. Recovering of
said polynucleotide
may be performed by any known method. The polynucleotide sequences may be
amplified by
conventional methods, e.g. PCR amplification. The identified and amplified
polynucleotide
sequence may then be inserted into a host cell capable of producing said
antimicrobial
polypeptide. For example the antimicrobial polypeptide may be expressed
through fusion to
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another polypeptide which then may be exported or secreted by the host cell.
In particular the
antimicrobial polypeptide may be expressed through fusion to a polypeptide
which is larger, i.e.
for which the mass is larger, than the antimicrobial polypeptide. Said
polypeptide may have the
function of protecting the antimicrobial polypeptide of interest from
digestion within the cell and
thereby inactivation by the host cell enzymes and/or the polypeptide may have
the function of
lowering the effect of the antimicrobial polypeptide on the host cell so that
the host may
proliferate and continue expression of the antimicrobial polypeptide without
being significantly
affected by the expressed antimicrobial polypeptide, an effect which may occur
if the
antimicrobial polypeptide has not been incorporated into the polypeptide. The
identified and
amplified polynucleotide sequence encoding the antimicrobial polypeptide may
also be
mutated as described, vide supra, e.g., by random mutagenesis, by gene
shuffling, or by
synthesizing degenerate genes. These mutated nucleotide sequences may then be
screened
again according to steps a) to f) to identify nucleotide sequences encoding
new antimicrobial
polypeptides with an improved effect e.g. by lowering the concentration of
inducer in
subsequent screenings. In this context the term "improved effect" refers to
that the AMP(s)
identified by the second (or more) time the method is repeated has an effect,
such as
specificity towards indicator cells, activity or stability under given
conditions which is better as
compared to the same effect of the AMP(s) identified by the previous time the
method was
performed. A better specificity towards indicator cells(s) may for example be
that the number of
indicator cells which are sensitive to a given AMP may e.g. be higher or
lower. A better activity
may for example be that the concentration or amount of a given AMP which is
necessary to
inhibit proliferation of a given indicator cell may be lower. A better
stability may be that a
particular AMP is more stabile under e.g. a high temperature, an acidic or
alkaline pH or in the
presence of certain compounds such as detergents.
In one embodiment of the present invention the screening and/or testing method
of the present
invention is carried out by application of conventional plate assays so that
after introduction of
the polynucleotide sequence in the host cell in step a), said cells are
streaked out on a plate
comprising a nutrient medium without an inducer, but optionally with an
antibiotic or another
selectable m arker. A n a ntibiotic o r s electable m arker may b a a sed i f
the a xpression v ector
used for construction of the polynucleotide sequence encoding the
antimicrobial polypeptide
comprises a gene encoding resistance to said antibiotic or selectable marker.
The presence of
the selectable marker/antibiotic will make only those host cells survive into
which an
expression vector comprising a polynucleotide sequence has actually entered,
i.e. host cells
where the transformation, transfection or infection has been successful. In a
particular
embodiment a filter, such as a cellulose acetate filter is placed on top of
the plate with the
nutrient medium with or without a selection marker and the host cells are
streaked out on the
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filter. The plate is then incubated for a period of time to enable colony
formation of
transformed/transfected/infected host cells.
In a particular embodiment of the present invention the host cells are sub-
cultured, i.e. from the
plate one or more samples of host cells or if the host cells were cultured on
a filter said filter
comprising the host cells may be transferred to another plate. Said plate
comprises a nutrient
medium and it may comprise an inducer capable of inducing expression and
production of the
antimicrobial polypeptide comprised in the inserted expression vector. In a
particular
embodiment the colonies of host cells may be sub-cultured by transferring them
to another
plate by placing a filter on top of the plate and then stripping the colonies
of host cells of the
plate with the filter and transferring the filter to a new plate.
In a particular embodiment the host cells may, instead of being transferred
from a plate without
inducer to a plate with inducer (i.e. sub-cultured), be overlaid with another
layer of e.g. agarose
comprising the inducer, where the inducer may be arabinose. The plate is then
again
incubated for a period of time to allow expression of the antimicrobial
polypeptides.
The indicator cells may then be placed on top of the plate comprising the host
cells in a
suitable nutrient medium and incubated for a period of time allowing the
antimicrobial
polypeptide to exert its effect on the indicator cell. If the host cells have
been cultured on a
filter the filter may be removed before placing the indicator cells on top of
the plate comprising
the host cells. If the host cells have been culture in the presence of a
selection marker but they
have not been sub-cultured, i.e. the selection marker is still present; a
compound capable of
degrading/removing the selection marker may be added to the plate before
and/or together
with the indicator cell. For example if ampicillin has been used as selection
marker beta-
lactamase, which is capable of degrading ampicillin, may be added before
and/or together with
the indicator cells. If a transformed/transfected/infected host cell colony
inhibits proliferation of
the indicator cell in the area surrounding the host cell colony it may be
deduced that said host
cell express an antimicrobial polypeptide which inhibits the proliferation or
kills the indicator
cell. Inhibition of the proliferation of the indicator cell may be detected as
described below.
In another embodiment of the present invention the screening and/or testing
method may be
carried out in a liquid assay. Thus for example after introduction of the
polynucleotide
sequence into the host cells, said host cells may be seeded in a microtiter
plate or a shake
flask, at a concentration facilitating growth of individual colonies in each
well/flask, in a nutrient
medium without an inducer and cultured for a period of time necessary for
proliferation of the
host cells. Thereafter an inducer may be added to the nutrient medium and the
host cells may
be cultured for a period of time allowing expression of the AMP. In one
embodiment the
indicator strain may then be added to the microtiter plate/shake flask and
cultures are then
again cultured for a period of time allowing the AMP to act on the indicator
cells. Finally,
proliferation of the indicator cells during this period of time may be
measured by, e.g. optical
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density (OD). In another embodiment the culture media is used to test the
antimicrobial activity
on an indicator cell in another assay, such as a radial diffusion assay. The
host cells may be
lysed before using the culture media.
A combination of a solid plate assay and a liquid assay may also be used. For
example after
introducing the polynucleotide sequence encoding an antimicrobial activity
into a host cell, said
host cells may be cultured on a solid media, e.g. an agar plate to select for
viable cells
comprising the polynucleotide sequence and then the subsequent steps may be
performed in
a liquid media as e.g. described above.
In another embodiment of the present invention the screening and/or testing
method may be
performed by cloning the polynucleotide sequence encoding an AMP into a phage,
for
example lambda-ZAP (Stratagene, where expression is driven by the Lac
promoter, induced
by IPTG) and then infect the host cells (for example E.coli XL-1-Blue) with
the packaged
phages by e.g. mixing the phages and the host cells on a top agar solution and
plate them on
a LB plate. The plates are then incubated to prevent lysogenesis for a period
of time, e.g. at
42°C and for 3-4 hours. When tiny plaques are visible the infected host
cells are overlaid with
an indicator cell and an inducer, such as a strain of Bacillus and IPTG, to
induce the
expression of the polypeptide. The plates are then incubated for a period of
time at e.g. 37°C.
The host cells will lyse but they will still have time to produce the AMP
before dying. T he
synthesized polypeptide will be liberated and it may then inhibit
proliferation of the indicator
cells. After e.g. an overnight incubation inhibition of the proliferation of
the indicator cells may
be visible as clearing zones surrounding the host cells. For this embodiment
it is important that
the indicator strain can not be infected by the phage.
The present invention also relates to a method for screening a library of
polynucleotide
sequences encoding one or more antimicrobial polypeptide(s), comprising:
a) cultivating an indicator cell in the presence of the antimicrobial
polypeptide(s),
wherein the antimicrobial polypeptide(s) has/have been expressed by a host
which is sensitive to said antimicrobial polypeptide(s), and wherein
expression
of each of the polynucleotide sequences in the library has been under control
of an inducible promoter, and wherein the host cells comprising the library
polynucleotide sequence has been cultivated in the absence of an inducer and
subsequently in the presence of an inducer
b) selecting a host cell expressing an antimicrobial polypeptide which is
capable of
reducing the proliferation of the indicator cell
The present invention also relates to a method for testing the antimicrobial
activity of an
antimicrobial polypeptide comprising:
a) cultivating an indicator cell in the presence of the antimicrobial
polypeptide(s),
wherein the antimicrobial polypeptide(s) has/have been expressed by a host
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which is sensitive to said antimicrobial polypeptide(s), and wherein
expression
of each of the polynucleotide sequences in the library has been under control
of an inducible promoter, and wherein the host cells comprising the library
polynucleotide sequence has been cultivated in the absence of an inducer and
subsequently in the presence of an inducer
Both of said methods may further comprise the step of:
c) recovering the polynucleotide sequence encoding the antimicrobial
polypeptide from
the host cell.
The embodiments described above concerning preparatory steps which may be
performed
before step a), preparation of a library of polynucleotide sequences encoding
one or m ore
antimicrobial polypeptides, preparation of a host cell comprising a
polynucleotide sequence
encoding an antimicrobial polypeptide, cultivating a host cell comprising said
polynucleotide
sequence in the absence of an inducer, cultivating a host cell comprising said
polynucleotide
sequence in the presence of an inducer, and additional steps such as lysing
the host cell may
also be used in the two above mentioned methods.
The indicator cell may cultivated in the presence of the antimicrobial
polypeptide as described
above for the previous method by cultivating it directly or indirectly in the
presence of the host
cell expressing the antimicrobial polypeptide(s). If the indicator cell is
cultured in a liquid media
the antimicrobial polypeptide(s) may be added to the culture of the indicator
cell, e.g. the
culture broth into which the host cells have expressed the AMP may be added to
the indicator
cell. The AMP may be purified before adding it to the indicator cell.
In another embodiment the host cell expressing the AMP(s) has been cultured on
solid media,
e.g. on a filter placed on agar, and the antimicrobial polypeptide(s) has/have
diffused into said
solid media; the host cells may subsequently be removed and the indicator cell
may be added
to the solid media and thereby be cultivated in the presence of the
antimicrobial polypeptide.
In a particular embodiment of the above two methods a library of indicator
cells is cultivated in
the presence of the AMP(s). This may in particular be performed by cultivating
each of the
different indicator cells separately in the presence of the AMP(s). For
example if the indicator
cells are cultivated in a liquid media, such as in a microtiter plate or a
shake flask, each of the
different indicator cells may be cultivated as separate culture, e.g. in
separate wells of a
microtiter plate or separate shake flasks. If the indicator cells are cultured
on solid media, they
may be cultured as separate colonies or on separate agar plates.
The recovered polynucleotide sequence of step c) may also be further
manipulated as
described above and the methods) may be repeated one or more times as
described above to
identify an AMP with an improved effect.
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Methods for testing viability and proliferation rates
Methods for testing the viability and/or proliferation rate of cells are well
known to a person
skilled in the art. For example if the cells are cultured on a plate, e.g. an
agar plate which may
be in the presence of a selection marker such as an antibiotic, the presence
of a colony and/or
the size of a colony can be used to evaluate the viability and/or the
proliferation rate of the
cells. Typically cells are spread over an agar plate comprising a selection
marker and then the
plates of are cultured for a period of time, such as overnight. The presence
of visible colonies
indicates cells which cells are viable.
The viability and/or proliferation rate of cells cultured in suspension may be
measured by
measuring the optical density (OD) of the cultures.
Other methods include staining with MTT (3-(3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) which stains viable cells blue, Alamar Blue or
2,3,5-
triphenyltetrazolium chloride. For example cells may plated on e.g. an agar
plate, a microtiter
plate or they may grow in suspension and then the dye is added so that it is
possible to e.g.
visually identify cells which are alive. The amount of dye after a certain
culture time may also
be used to evaluate the proliferation rate of the cells. For example if the
same number of cells
have been seeded in e.g. suspension or on a microtiter plate and then allowed
to proliferate for
a certain period of time, such as 24 hours, the cells may then be stained
subsequently and the
amount of dye present used to evaluate the proliferation rate of the cells.
A further method for testing viability and proliferation rates is to measure
expression of a
reporter gene present in the indicator strain, for example the Green
Fluorescent Protein (GFP),
Luciferase (LUC), beta-Glucoronidase (GUS) or beta-galactosidase (GAL) by the
use for
example of a Victor Wallac 1420 multilabel counter (Wallac Danmark A/S). The
growth of the
indicator cells may be correlated with the measurement of the reporter protein
present in the
culture.
The antimicrobial activity may also be tested in a radial diffusion assays as
e.g. described in
WO 03/044049A1 based on the protocol published in (Lehrer et al., (1991 )
Ultrasensitive
assays for endogenous antimicrobial polypeptides J Immunol Methods 137: 167-
173) with
some modifications. Briefly, indicator cells are added to an underlay of
agarose into which
several holes are made, e.g. by solidifying the agarose on a Nunc omnitray
plate containing a
nunc-ImmunoTSP PS rack inside to obtain 96 holes in the agarose media. The
samples are
subsequently added to the holes and incubated for a period of time, e.g. at 37
degrees C for 3
hours t o a Ilow f or the a ntimicrobial a ctivity t o a xert i is a ction o n
t he i ndicator c ell. N ext a n
overlay (LB media with agar) is poured on top of the plate and the plate is
incubated again to
allow for the antimicrobial activity to exert its action on the indicator
cell, e.g. it may be
incubated for 10-24 or 10-48 hours to allow the growth of t he indicator
cells. Antimicrobial
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activity is detected as bacterial clearing zones around the wells. Any method
of staining may
be used to distinguish living cells from dead cells as described above.
Use of antimicrobial polypeptides
Antimicrobial polypeptides identified by the method of the present invention
may be used in a
variety of applications, for example it may be used within areas such medical
care, cosmetic
care, animal feed, cleaning systems or for various industrial applications.
Medical aaplications
The invention also relates to the use of an antimicrobial polypeptide found by
the present
invention as a medicament. Further, an antimicrobial polypeptide or
composition comprising
said antimicrobial polypeptide found by the present invention may also be used
for the
manufacture of a medicament for controlling or combating microorganisms, such
as fungal
organisms or bacteria.
The antimicrobial polypeptide found by the present invention may also be used
as an
antimicrobial veterinarian or human therapeutic or prophylactic agent. Thus,
the antimicrobial
polypeptide found by the present invention may be used in the preparation of
veterinarian or
human therapeutic agents or prophylactic agents for the treatment of a
microbial, such as
fungal infection, bacterial infection or viral infection; it may also be used
for multi-resistant
infections. Examples of infections or diseases were an AMP of the present
invention may be
used include cystic fibrosis (CF), ventilator-associated pneumonia (VAP),
candidiasis, HIV and
nasal carriage of S, aureus.
The antimicrobial polypeptide found by the present invention may also be used
in wound
healing composition or products such as bandages, medical devices such as,
e.g., catheters.
Thus, the antimicrobial polypeptides found by the present invention may be
useful as a
disinfectant, for example it may be used for topical application, e.g. for the
treatment of acne,
infections in the skin, wounds, chronic wounds, bruises and the like. Other
examples of
infections for which an AMP of the present invention may be used include
treatment of
infections in the eye or the mouth, and diabetic foot ulcers.
Cosmetic applications
An AMP of the present invention may for example be used in a variety of
different products for
personal care such as lotions, creams, gels, ointments, soaps, shampoos,
conditioners or oral
care p roducts s uch a s mouth w ash, i n a ntiperspirants o r d eodorants; i
n foot b ath s alts; for
cleaning and disinfection of contact lenses, teeth (oral care), or for anti-
dandruff hair products,
such as shampoos.
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Feed applications
Another application for an antimicrobial polypeptide identified by the present
invention is the
employment of said antimicrobial polypeptide in animal feed products. The term
animal
includes a II a nimals, i ncluding h uman b eings. E xamples o f a nimals a re
n on-ruminants, a nd
ruminants, such as cows, sheep and horses. In a particular embodiment, the
animal is a non-
ruminant animal. Non-ruminant animals include mono-gastric animals, e.g. pigs
or swine
(including, b ut n of I invited t o, p iglets, growing p igs, a nd s ows); p
oultry s uch a s t urkeys a nd
chicken (including but not limited to broiler chicks, layers); young calves;
and fish (including but
not limited to salmon).
The t erm feed or f eed p roduct m eans any compound, preparation, m fixture,
or composition
suitable for, or intended for intake by an animal.
It may also be used as an antimicrobial in food products and would be
especially useful as a
surface antimicrobial in cheeses, fruits and vegetables and food on salad
bars.
Other uses include preservation of foods, beverages or food ingredients.
Cleanings systems
Further, it is contemplated that the antimicrobial polypeptides found by the
present invention
can advantageously be used in a cleaning-in-place (C.LP.) system for cleaning
of process
equipment of any kind.
The a ntimicrobial p olypeptides found b y t he p resent i nvention m ay a
dditionally b a a sed f or
cleaning surfaces and cooking utensils in food processing plants and in any
area in which food
is prepared or served such as at hospitals, nursing homes, restaurants,
especially fast food
restaurants, delicatessens and the like.
In general it is contemplated that the antimicrobial polypeptides found by the
present invention
may be useful for cleaning, disinfecting or inhibiting microbial growth on any
hard surface.
Examples of surfaces, which may advantageously be contacted with the
antimicrobial
polypeptides of the invention are surfaces of process equipment used e.g.
dairies, chemical or
pharmaceutical process plants, water sanitation systems, oil processing
plants, paper pulp
processing plants, water treatment plants, and cooling towers. The
antimicrobial polypeptides
found by the present invention should be used in an amount, which is effective
for cleaning,
disinfecting or inhibiting microbial growth on the surface in question.
Industrial applications
Typically, antimicrobial polypeptides are useful at any locus subject to
contamination by
bacteria, fungi, yeast or algae, including places such as aqueous systems,
e.g. water cooling
systems, laundry rinse water, oil systems such as cutting oils, lubricants,
oil fields and the like,
where microorganisms need to be killed or where their growth needs to be
controlled.
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However, the antimicrobial polypeptides found by the present invention may
also be used in all
applications for which known antimicrobial compositions are useful, such as
protection of
wood, latex, adhesive, glue, paper, cardboard, textile, leather, plastics,
caulking, and feed.
It may also be used as a preservation agent or a disinfection agent in water
based paints.
The antimicrobial polypeptides found by the present invention may also be
useful for microbial
control of water lines, and for disinfection of water, in particular for
disinfection of industrial
water.
Another example is use of an AMP of the present invention for preservation of
enzyme
formulations.
EXAMPLES
Materials
Host cells
E. coli origami: E.coli origami cells (Novagen) are a K-12 derivative strain
harbouring mutations
in both the thioredoxin reductase (trx8) and gluthathione reductase (gor)
genes allowing
disulfide bond formation in the cytoplasm.
E.coli TOP10: E.coli cells in which disulfide bond formation is not possible
from Invitrogen
Antimicrobial peptides
Plectasin is an antimicrobial peptide derived from Pseudoplectania nigrella
(SEQ ID N0:2 in
PA 2001 01732).
AFP is an antifungal protein derived from Aspergillus giganteus
Novispirin G10 is an alpha-helical antimicrobial polypeptide which does not
contain any
disulfide bonds. Novispirin G10 (WO 02/00839 SEQ ID No. 17) is obtained by
rational design
based on homology to SMAP-29, an ovine cathelicidin peptide.
AMP/alasmid constructs
pDR18-Plectasin: The AMP Plectasin was cloned into a version of the pBAD/glll
A plasmid
made in-house which does not comprise the glll secretion signal, whereby
Plectasin is
expressed in the cytoplasm of the host cell.
The promoter of both of the above pBAD/glll A plasmids is regulated by
arabinose, i.e.
expression of a polynucleotide sequence inserted into the plasmid is induced
by the presence
of arabinose.
pHHA: Is the same plasmid as pDR18-Plectasin but without the nucleotide
sequence encoding
Plectasin (i.e. it is the control vector for expression of Plectasin).
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pHHA-Cm: Is the same plasmid as pHHA but where the nucleotide sequence
encoding
ampicillin resistance has been exchanged with a nucleotide sequence encoding
chloramphenicol resistance.
pDR18-Cm-Plectasin: The pHHA-Cm plasmid into which the nucleotide sequence
encoding
Plectasin has been cloned.
pDR54, pDR55, pDR56, pDR57 and pDR58: Different variants of Plectasin created
by site-
directed m utagenesis a nd c toned i nto a version o f t he p BAD/gl I I A p
lasmid m ade i n-house
which does not comprise the glll secretion signal, whereby the variants are
expressed in the
cytoplasm of the host cell.
pHH: The pBad/glll plasmid comprising the gill secretion signal which directs
expression of
polypeptides into the periplasm.
pDRSS-Novispirin G10: The pHH plasmid into which the nucleotide sequence
encoding
Novispirin G10 was cloned.
pHHA900-AFP: Construct containing the AFP encoding sequence in the pBAD/glll
plasmid
where the promoter is regulated by arabinose and the expression of AFP is
directed to the
cytoplasm.
Indicator cells
Bacillus subtilis
Bacillus subtilis 1315-1: a Bacillus subfilis strain which is resistant to
chloramphenicol
Fusarium longypes
Staphylococcus carnosus
E.coli TOP10 cell (see above for further description).
Culture media
The composition of the RM media is described in the protocol from Invitrogen
relating to the
use of the pBAD/glll A, B and C plasmids (V450-01).
Methods
Radial diffusion assay
The protocol describing the radial diffusion assay method used in below
examples is described
in the patent WO 03/044049A1 based on the protocol published in (Lehrer et
al., (1991 )
Ultrasensitive assays for endogenous antimicrobial polypeptides J Immunol
Methods 137: 167-
173) with some modifications. Briefly, Target bacteria (5x106 colony forming
units (CFU) were
added to 50 ml of underlay agarose (1 % low electro-endosmosis agarose, 0.03%
Trypticase
soy broth, 10 mM sodium phosphate, pH 7.4, 37 degrees C). Suspension was
solidified on a
Nunc omnitray plate containing a nunc-ImmunoTSP PS rack inside to obtain 96
holes in the
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agarose media. Samples were added to the holes and incubated at 37 degrees C
for 3 hours.
A 25 ml overlay of LB media with agar was poured on top and the plate was
incubated
overnight (LB media, 7.5% Agar). Antimicrobial activity was detected as
bacterial clearing
zones around the wells. Living cells were counterstained by adding 6 ml, 6 mM
MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Thiazolyl blue). All
standard protocols
have been described elsewhere (Sambrook, Fritsch, and Maniatis, 1989).
Lysis of cells with hot acid
Cells which were cultured in 96-well microtiter plates were lysed by hot acid
by adding
100microlitre of sodium phosphate buffer 1 M pH 2.3 to the wells. The final pH
was
approximately 3. The plates were incubated overnight at 80 degrees C in a low
condensation
incubator with shaking. Next day, 75 microlitre of sodium phosphate buffer 0.5
M pH 13 was
added to the wells. The final pH was approximately 7.
Example 1
Growth inhibition of Bacillus subtilis by the antimicrobial peptide Plectasin
E. coli origami cells were transformed with pDR18-Plectasin and cultured
overnight at 37°C on
LB-agar plates containing tetracycline (tet), kanamycin (kan) and ampicillin
(amp) to select for
viable origami cells comprising the pDR18-Plectasin construct. Three viable
origami colonies
were selected and transferred to a cellulose acetate filter (Schleider &
Schull) placed on top of
an agar plate without any antibiotics. The filter-agar plates were incubated
overnight at 37°C
before transferring the filters to a new agar plate containing 0.1 % arabinose
to induce
expression of Plectasin. The filter-agar, 0.1 % arabinose plates were
incubated overnight at 37
degrees C. One of the plates were treated with vapours of chloroform, which
lyses cells, by
pouring chloroform on a filter and then placing the agar-filter plate on top
of this upside-down
for 10 minutes. Afterwards the filter-agar plate is remove and left open for
15 minutes to
evaporate the remaining chloroform before transferring the filter to a new
agar-filter, 0.1
arabinose plate. After this a top-layer of agar comprising approximately 4x106
CFU Bacillus
subtilis were poured on top of both the chloroform treated filter-agar plate
and the non-treated
plate. The plates with top-layer were then incubated overnight at 37 degrees
C. The next day
clearing zones in the top-layer (Bacillus subtilis cells) were detected by
staining with a 0.2 mM
MTT-solution, w hich s tains v fable c ells b lue. T hus t he p resence o f c
tearing z ones ( no b lue
cells) surrounding the origami colonies indicates that the proliferation of
the Baeillus subtilis
cells near the origami cells is inhibited and thereby that the polynucleotide
expressed by said
host cells has an antimicrobial activity towards said indicator cells.
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The results are shown in table 1 as whether or not a clearing zone is present
in the top-layer
over each of 6 colonies of origami cells transformed with the pDRl8-Plectasin
construct. A
clearing zone is defined as the absence of or presence of only a few blue
cells in the top-layer
over each of the origami colonies, which indicates that the Bacillus subtilis
cells, which are
more or less uniformly distributed throughout t he top-layer, above the
origami colonies are
dead.
Table 1:
AMP/plasmid construct Plate treated with Plate not treated with
chloroform
chloroform
Clearing zone
+/-
pDR18-Plectasin no.1 + +
pDR18-Plectasin no.2 + +
pDR18-Plectasin no.3 + +
The above results show that expression of Plectasin by the origami cells is
able to act on other
cells .(in this case Bacillus subtilis) than the host cells it is expressed by
and kill those cells.
Furthermore, it also shows that this effect is independent on whether or the
host cells are lysed
with chloroform.
Example 2
The effect of Plectasin on the host cells (E coli origami)
E. coli origami cells were transformed with pDR18-Plectasin and cultured
overnight at 37
degrees C o n L B-agar plates c ontaining tet, kan a nd a mp t o s elect for v
fable o rigami c ells
comprising the pDR18-Plectasin construct. Four viable origami colonies were
selected and two
were streaked out on agar plates comprising tet, kan and amp while the other
two were
streaked out on agar plates comprising tet, kan, amp and 0.1 % arabinose to
induce expression
of Plectasin from the pDR18-Plectasin construct. The plates were incubated
overnight at 37
degrees C.
Results:
On the agar plates without arabinose a line of origami cells was present
clearly indicating that
the origami cells had grown overnight.
On the agar plates comprising arabinose only very few origami cells were
present indicating
that the presence of arabinose had inhibited the proliferation of said cells.
Thus that the
proliferation of the origami cells was almost completely inhibited by the
presence of the AMP
Plectasin as arabinose induce expression of Plectasin.
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Example 3
The effect of Plectasin on host cells not capable of forming disulfide bridges
TOP10 cells were transformed with pDR18-Plectasin and cultured overnight at 37
degrees C
on LB-agar plates containing amp to select for viable TOP10 cells comprising
the pDR18-
Plectasin construct. Four viable TOP10 colonies were selected and two were
streaked out on '
agar plates comprising amp while the other two were streaked out on agar
plates comprising
amp and 0.1 % arabinose to induce expression of Plectasin from the pDR18-
Plectasin
construct. The plates were incubated overnight at 37 degrees C.
Results:
A line of TOP10 cells was present at both the plates comprising arabinose and
those without
arabinose. Thus induction of Plectasin by arabinose did not inhibit
proliferation of the TOP10
cells indicating that formation of disulfide bonds) is important for the
antimicrobial activity of
Plectasin as expression of Plectasin in origami cells, in which disulfide bond
formation is
possible, inhibited proliferation of these cells (see example 2).
Example 4
Growth inhibition of Bacillus subtilis by Plectasin diffused into solid media
The method used in this example is similar to the method described in example
1 with the
exception that in the present example the acetate filter containing the
origami cells is removed
from the arabinose plate before it is overlaid with the indicator strain.
E. coli origami cells were transformed independently with pHHA or pDR18-
Plectasin plasmid
and cultured overnight at 37 degrees C on LB-agar plates containing
tetracycline (tet),
kanamycin (kan) and ampicillin (amp) to select for viable origami cells
comprising the plasmid.
Six viable origami colonies were selected containing each of the constructs
and transferred to
a cellulose acetate filter placed on top of an agar plate without any
antibiotics. The filter-agar
plates were incubated two nights at 37 degrees C. Next, the filters were
transferred to a new
agar plate containing 0.1 % arabinose to induce expression of the peptide. The
filter-agar, 0.1
arabinose plates were incubated overnight at 37 degrees C. Next day, the
acetate filter was
removed from the plates and a top-layer of agar comprising approximately 3x105
CFU Bacillus
subtilis were poured on top of the plate. The plates with top-layer were then
incubated
overnight at 37 degrees C. The next day clearing zones in the top-layer
(Bacillus subtilis cells)
were detected by staining with a 0.2 mM MTT-solution, which stains viable
cells blue.
The results are shown in table 2 as the presence ("+") or absence ('=") of a
clearing zone in the
top-layer over the place where each of the 6 colonies origami cells
transformed with the
pHHA/pDR18-Plectasin construct had been growing before removing the acetate
filter. A
clearing zone is defined as the absence of or presence of only a few blue
cells in the top-layer,
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which indicates that the Bacillus subtilis cells, which are more or less
uniformly distributed
throughout the top-layer, are dead.
Table 2:
AMP/plasmid construct Clearing zone
(-/+)
pHHA no. 1 -
pHHA no. 2 -
pHHA no. 3 -
pDR18-Plectasin no.1 +
pDR18-Plectasin no.2 +
pDR18-Plectasin no.3 +
Compared to the results of example 1 this results indicate that the Plectasin
is capable of
diffusing through the acetate filter into the agar after it is produced by the
E.coli origami cells.
Example 5
Growth inhibition of Bacillus subtilis by Plectasin diffused into solid media
without sub-culturing
the host cells expressing Plectasin
The method used in this example is similar to the method described in example
4 with the
exception that only ampicillin was used as a selection marker after
transformation of the E.coli
origami cells and beta-lactamase was added to the top-layer of agar comprising
Bacillus
subtilis. Beta-lactamase is capable of degrading ampicillin and thereby
allowing the Bacillus
subtilis to grow.
In brief, E.coli origami cells were transformed independently with pHHA or
pDR18-Plectasin
plasmid and cultured for 2 nights at 37 degrees C on a LB-agar plate
comprising ampicillin
(amp) to select for viable E.coli origami cells comprising the plasmid.
Thereafter, an acetate
filter was placed on top the agar plate comprising the transformed cells and
the acetate filter
was then stripped off and transferred to a another agar plate comprising 0.1 %
arabinose and
ampicillin. The plate comprising the acetate filter was incubated overnight at
37 degrees C.
Next day, the acetate filter was removed from the plate and a top-layer of
agar comprising 0,25
Units of beta-lactamase and approximately 3x105 CFU Bacillus subtilis was
poured on top of
the plate. The plate with top-layer was incubated overnight at 37 degrees C.
The next day,
clearing zones in the top-layer (Bacillus subtilis cells) were detected by
staining with a 0.2 mM
MTT-solution, which stains viable cells blue.
The results were similar to the results of example 4, i.e. there were no
clearing zones present
in the top-layer comprising Bacillus subtilis in the areas corresponding to
where the E.coli
origami cells expressing the control plasmid pHHA had been growing, while
clearing zones
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were present in the areas corresponding to where the E.coli origami cells
expressing the
Plectasin-expressing plasmid pDR-Plectasin had been growing.
The present example provides a method where it is not necessary to sub-culture
the host cells.
Example 6
Growth inhibition of Bacillus subtilis by Plectasin variants diffused into
solid media without sub-
culturina the host cells expressing the variants
The method used in this example is similar to the one described in example 5.
In brief, E.coli origami cells were transformed independently with pHHA (empty
control vector),
pDR18-Plectasin (wild type Plectasin) and five constructs encoding for
different Plectasin
variants (pDR54, pDR55, pDR56, pDR57 and pDR58) and plated on LB-agar plates
with
ampicillin and incubated overnight at 37 degrees C to select for viable E.coli
origami cells
comprising the construct.
Four viable E.coli origami colonies from each construct were picked and
transferred to a
cellulose acetate filter placed on top of an agar plate with ampicillin. The
filter-agar plate was
incubated two nights at 37 degrees C before transferring the filter to a new
agar plate
comprising ampicillin and 0.1 % arabinose to induce expression of
Plectasin/Plectasin variants.
The plate was incubated overnight at 37 degrees C. Next day, the acetate
filter was removed
from the plate and a top-layer of agar comprising 0.25 Units of beta-lactamase
and
approximately 3x105 CFU Bacillus subtilis were poured on top of the plate. The
plate with top-
layer was then incubated overnight at 37 degrees C before staining with 0.2 mM
MTT-solution
to detect clearing zones in the top-layer.
Results:
Clearing zones were present in the top-layer comprising Bacillus subtilis
corresponding to the
areas where E.coli origami cells comprising pDR-Plectasin (wild-type
Plectasin) or pDR54,
pDR55, pDR56, pDR57 and pDR58 (Plectasin variants) had been growing before
removing the
acetate filter, while no clearing zone was present in the area corresponding
to where the E.coli
origami cells comprising pHHA (empty control vector) had been growing.
Furthermore, the
clearing zones were larger and more defined at the areas where the E.coli
origami cells
expressing the Plectasin variants had been growing than were the E.coli
origami cells
expressing wild-type Plectasin had been g rowing. T his indicates that the
Plectasin variants
have a higher antimicrobial activity towards Bacillus subtilis than wild-type
Plectasin has.
Example 7
Growth inhibition of a chloramphenicol-resistant strain of Bacillus subtilis
by Plectasin diffused
into solid media
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The method used in this example is similar to the method used in example 4
with the exception
that chloramphenicol was used as a selection marker and a chloramphenicol-
resistant strain of
Bacillus subtilis (1315-1 ) was used as indicator strain.
In brief, E, coli origami cells were transformed independently with pHHA-Cm
and pDR18-Cm
Plectasin constructs and cultured overnight at 37 degrees C on LB-agar plates
comprising
chloramphenicol to select for viable E.coli origami cells comprising the
construct. Six viable
origami colonies from each construct were picked and transferred to a
cellulose acetate filter
placed on top of an agar plate with chloramphenicol. The filter-agar plate was
incubated for
two nights at 37 degrees C before transferring the filters to an agar plate
comprising
chloramphenicol and 0.1 % arabinose to induce expression of Plectasin. The
plates were
incubated overnight at 37 degrees C. Next day, the acetate filter was removed
from the plate
and a top-layer of agar comprising approximately 3x105 CFU Bacillus subtilis
(1315-1 ) resistant
to chloramphenicol were poured on top of the plate. The plates with top-layer
were then
incubated overnight at 37 degrees C before staining with 0.2 mM MTT-solution
to detect
clearing zones in the top-layer comprising the Bacillus subtilis cells (1315-1
).
Results:
The results are shown in table 3 as the presence "+" or absence "-" of a
clearing zone in the
top-layer corresponding to the area where E.coli origami cells comprising
either the pHHA-Cm
or pDRl8-Cm-Plectasin constructs had been growing. A clearing zone is defined
as the
absence of or presence of only a few blue cells in the top-layer, which
indicates that the
Bacillus subtilis cells (1315-1 ), which are more or less uniformly
distributed throughout the top-
layer, are dead.
Table 3:
AMP/plasmid construct Clearing zone (-/+)
pHHA-Cm no. 1 -
pHHA-Cm no. 2 -
pHHA-Cm no. 3 -
pHHA-Cm no. 4 -
pHHA-Cm no. 5 -
pHHA-Cm no. 6 -
pDR18-Cm-Plectasin no.1 +
pDR18-Cm-Plectasin no.2 +
pDR18-Cm-Plectasin no.3 +
pDR18-Cm-Plectasin no.4 +
pDR18-Cm-Plectasin no.5 +
pDR18-Cm-Plectasin no.6 +
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Compared to the previous examples these results indicate that different
selection markers may
be used depending on whether or not the indicator strain is resistant or not
to the selection
marker.
Example 8
Growth inhibition of Bacillus subtilis by Plectasin diffused into solid media
without using
cellulose acetate filters
Briefly, E. coli origami cells were transformed independently with pHHA
(control empty vector)
and pDR18-Plectasin (wild type Plectasin) and the transformed cells were
plated on LB-agar
plates with ampicillin and incubated overnight at 37 degrees C to select for
viable E.coli
origami cells comprising the plasmid constructs.
For each plasmid construct three viable E.coli origami colonies were picked
and transferred to
an LB-agar plate and incubated for three days at 37 degrees C. Next, the plate
was overlaid
with a top layer of agar comprising 0.2% arabinose and incubated overnight at
37 degrees C to
induce expression of Plectasin. Next day, the plate was overlaid with a top-
layer of agar
comprising approximately 3x105 CFU Bacillus subtilis. The plate was then
incubated overnight
at 37 degrees C before staining with 0.2 mM MTT-solution to detect clearing
zones in the top-
layer comprising the Bacillus subtilis cells
Results:
The results are shown in table 4 as the presence "+" or absence "=' of a
clearing zone in the
top-layer corresponding to the area where E.coli origami cells comprising
either the pHHA or
pDR18-Plectasin plasmid constructs had been growing. A clearing zone is
defined as the
absence of or presence of only a few blue cells in the top-layer, which
indicates that the
Bacillus subtilis cells, which are more or less uniformly distributed
throughout the top-layer, are
dead.
Table 4:
AMP/plasmid construct Clearing zone (-/+)
pHHA no. 1 -
pHHA no. 2 -
pHHA no. 3
pDR18-Plectasin no.1 +
pDR18-Plectasin no.2 +
pDR18-Plectasin no.3 +
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In comparison to the previous examples the present results show that the same
results are
obtained as previous (Plectasin expressed by E.coli origami cells is able to
inhibit the growth of
Bacillus subtilis) even when an acetate filter is not used.
Example 9
High-through-put screeninct of Plectasin variants for Growth inhibition of
Staphylococcus
carnosus in a liauid microtiter format.
The nucleotide sequence encoding Plectasin was randomly mutagenized by Error-
Prone PCR
to create a library of nucleotide sequences encoding Plectasin variants. The
library was cloned
into the pHHA vector and transformed into E.coli origami cells. Transformed
colonies were
grown overnight at 37 degrees C on LB-agar plates containing ampicillin and
0.2% glucose to
select for viable origami cells comprising the plasmid. Glucose was added to
reduce the basal
expression level of the pBAp promoter. Colonies were picked using a colony
picker and
inoculated in microtitter plates containing 200 microlitre of liquid media,
either LB or TB, and
ampicillin. Cultures were grown overnight at 37 degrees C in a low
condensation incubator
(Kuhner) with shaking. Next day, 15 microlitre of the overnight cultures were
transferred to new
microtitter plates containing fresh media, either LB or TB, with ampicillin.
Plates were shaken
for 4 hours at 37 degrees C in a low condensation incubator before 15
microlitre of 1
arabinose was added to the cultures to induce expression of the Plectasin-
variants. Plates
were incubated overnight at 37 degrees C in a low condensation incubator with
shaking. Next
day, a hot acid hydrolysis of the cultures was performed and 25microlitre of
the hydrolyzed
cultures was analyzed on a radial diffusion assay to test for antimicrobial
activity against
Staphylococcus carnosus.
Results are shown in table 5 (see below) as whether or not a clearing zone is
present in the
agar media ("=' or "+") or if the clearing zones are particular large ("++"),
Table 5:
1 2 3 4 5 6 7 8 9 10 11 12
A - + + ++ ++ _ _ _ _ _ _ _
g _ _ _ _ _ ++ _ _ _ + _ _
C _ _ _ _ _ _ _ _ _ _ _ +
p _ + _ _ _ _ _ _ _ + _ _
E _ + _ _ _ _ ++ _ + _ _ _
_ _ _ _ _ _ _ + _ _ + _
G ++ _ + _ _ _ _ _ _ _ +- _
_ _ _ _ _ _ _ _ ++ - -
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Results presented in table 5 showed that very week clearing zones were
detected on the radial
diffusion p lates from t he I yzed c ultures a xpressing P lectasin w ild type
( A2 a nd A 3) o r from
some of the Plectasin variants (B10, C12, D2, D10, E2, E9, F8, F11, G3, G11).
No clearing
zones were observed from the control plasmid (A1 ). In contrast, larger
clearing zones were
detected on some of the lyzed cultures expressing different Plectasin variants
(A4, A5, B6, E7,
G1 and H9). These results indicate that this assay can discriminate between
E.coli cells
expressing peptide variants with different antimicrobial activity and it can
be used to find
peptides with improved antimicrobial activity.
Example 10
Test of the antimicrobial activity of different Plectasin variants on
Staphylococcus carnosus in a
liquid assay using chloroform to Iyse the cells.
E.coli origami cells were transformed with different constructs (plasmids):
pHHA (control empty
vector), pDR18-Plectasin (wild type Plectasin) and pDR54, pDR55, pDR56, pDR57
and pDR58
(Plectasin variants). Transformed cells were plated on LB-agar plates with
ampicillin and
incubated overnight at 37 degrees C to select for viable origami cells
containing the plasmids.
For each construct two viable origami colonies were picked and inoculated into
5ml LB with
ampicillin. The tubes were incubated overnight at 37 degrees C with shaking.
Next day, 500
microlitre of the inoculated cultures were transferred to tubes containing 5
ml fresh LB with
ampicillin. Two tubes were prepared from each overnight culture. The cultures
were then
grown at 37 degrees C with shaking for approximately 4 hours until the ODsoo
was
approximately 1.5. For each construct 50 microlitre of 10% arabinose was then
added to one
of the tubes to induce polypeptide synthesis. The tubes were incubated
overnight at 37
degrees C with shaking. Next day, lysis of the cultures was perfiormed using
chloroform in the
following way: 150microlitre of the overnight cultures were transferred to 1.5
ml tubes and
50microlitre of chloroform was added to each tube. The tubes were inverted
several times to
mix the samples and incubated for 30 minutes at room temperature. Next,
100microlitre of the
upper phase of each tube was transferred to a new tube. Finally, an aliquot of
15microlitre of
the I yzed c ultures w as analyzed o n a r adial d iffusion a ssay t o t est
for antimicrobial a ctivity
against Staphylococcus carnosus.
Results are shown in table 6 (see below) as whether or not a clearing zone is
present in the
agar media ("-" or "+") or if the clearing zones are larger ("++"). Columns 1,
3, 5, 7, 9 and 11
correspond to samples not treated with arabinose. Columns 2, 4, 6, 8, 10 and
12 correspond to
samples treated with arabinose. Samples A1-A4 correspond to control vector
pHHA, A5-8
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correspond to pDR18-plectasin, A9-12 correspond to pDR54, B1-4 correspond to
pDR55, B5-8
correspond to pDR56, and B9-12 correspond to pDR57 and C1-2 correspond to
pDR58.
Table 6:
1 2 3 4 5 6 7 8 9 10 11 12
A - - - - - + - + - + - +
B - ++ - ++ - ++ - ++ - ++ - ++
C - ++
Results in table 6 show that cells treated with arabinose expressing wild type
Plectasin and the
five variants gave clearing zones in the radial assay. No clearing zones were
observed from
the cells comprising the control plasmid or from the cells not induced with
arabinose.
Additionally, four Plectasin variants, pDR55, pDR56, pDR57 and pDR58 showed
bigger
clearing zones than wild type, indicating that the antimicrobial activity of
these variants was
higher than the wild type. However, the size of the clearing zone from the
variant pDR54 was
similar to the one from cells expressing the wild type Plectasin, suggesting
that the
antimicrobial activity of this peptide was similar to the wild-type Plectasin.
These results
indicate that this assay can, discriminate between E.coli cells expressing
peptide variants with
different antimicrobial activity.
Example 11
Test of the antimicrobial activity of different Plectasin variants on
Staphylococcus carnosus in a
liguid assay using hot acid to Iyse the cells.
This example was performed similar to example 9, with the exception that
instead of testing
the antimicrobial activity of a library of Plectasin variants on
Staphylococcus carnosus the
antimicrobial activity of wild-type Plectasin was compared with that of the
Plectasin variants
encoded by pDR54, pDR55, pDR56, pDR57 and pDR58 was tested.
In brief, E.coli origami cells were transformed with pHHA (control empty
vector), pDR18
Plectasin (wild type plectasin) and pDR54, pDR55, pDR56, pDR57 and pDR58
(Plectasin
variants). Three viable transformed origami colonies from each construct were
picked and
cultured first in the absence of arabinose (polypeptide inducer) and
subsequently in the
presence of arabinose (as described in example 9). The cells were then lysed
by hot acid and
25 microlitre of each cell-culture was analyses on a radial diffusion assay to
test for
antimicrobial activity against Staphylococcus carnosus.
Results are shown in table 7 (see below) as whether or not a clearing zone is
present in the
agar media ("-" or "+") or if the clearing zones are larger ("++"). Samples A1-
A3 correspond to
control vector pHHA, A4-6 correspond to pDR18-plectasin, A7-9 correspond to
pDR54, A10-12
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correspond to pDR55, B1-3 correspond to pDR56, and B4-6 correspond to pDR57,
B7-9
correspond to pDR58, and B10-12 correspond to Blank.
Table 7:
1 2 3 4 5 6 7 8 9 10 11 12
A - - - - _ - + + + + + +
B + + + ++ ++ ++ ++ ++ ++ - _ -
Results in table 7 showed that no clearing zones were obtained from the hot
acid hydrolyzed
cultures corresponding to the control pHHA plasmid, or wild type Plectasin
(pDR18) or from
liquid media without E.coli cells (Blank). In contrast, the hot acid
hydrolyzed cultures
corresponding to the five Plectasin variants, pDR54, pDR55, pDR56, pDR57 and
pDR58,
showed clearing zones, indicating that the antimicrobial activity of these
variants was higher
than the wild type. Additionally, the clearing zones obtained f rom pDR57 and
pDR58 were
bigger than the ones obtained from the other three variants, suggesting that
their antimicrobial
activity was probably higher than for the other variants.
Example 12
Growth inhibition of E.coli TOP10 by Novispirin G10 in a liguid assay using
hot acid to Iyse the
cells
This example was performed similar to examples 9 and 11 with the following
exceptions:
In the present example the antimicrobial activity of Novispirin G10 was tested
towards E.coli
TOP10 cells.
Novispirin G10 was expressed in E.coli TOP10 cells which do not carry the trxB
and gor
mutations as E.coli origami cells, since the activity of the antimicrobial
polypeptide does not
require the formation of disulfide bonds in the molecule.
The expression of Novispirin G10 was directed to the periplasm, instead of the
cytoplasm,
which was the case of the other antimicrobial polypeptides tested in the other
examples.
Last, in this experiment, E.coli TOP10 was used as indicator cells.
Briefly, E. coli Top10 cells were t ransformed w ith p HH ( control a mpty
vector), a nd p DRSS-
Novispirin G10. Three viable transformed E. coli TOP10 colonies from each
construct were
picked and cultured f first in the absence of arabinose and subsequently in
the presence of
arabinose (as described in example 9). The cells were then lysed with hot acid
and 25
microlitre of each cell-culture was analysed on a radial diffusion assay to
test for antimicrobial
activity of Novispirin G10 against E. coli TOP10.
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Results are shown in table 8 as whether or not a clearing zone is present in
the agar media ("-"
or "+"). Samples A1-3 corresponds to the control vector pHH and samples A4-A6
corresponds
to the pDRSS-Novispirin G10.
Table 8:
1 2 3 4 5 6
A - - - + + +
Results in table 8 showed that no clearing zones were obtained from the
cultures transformed
with the control vector pHH. In contrast, the cultures transformed with
Novispirin G10, showed
clearing zones. These results indicate that E,coli TOP10 cells are sensitive
to the antimicrobial
polypeptide Novispirin G10.
Example 13
Growth inhibition of E. coli TOP10 upon expression of Novispirin G10
In order to evaluate whether E. coli TOP10 as host cells are sensitive to
Novispirin G10, the
following experiment was conducted in liquid media as disclosed in example 1
of patent
application WO 00/73433 (under "Growth inhibition of E.coli upon expression of
various
AMP's") with some modifications. Briefly, fresh overnight cultures of cells
containing either the
pHH (control) or pDRS5-Novispirin G10 plasmid were diluted 300-fold into 150
micro liter of
RM media or RM media containing 0.1 % arabinose in a microtiter plate and
incubated at 37
degrees C with vigorous shaking. The growth curve was monitored by measuring
OD450 at
regular intervals using an ELISA reader. The percentage of growth inhibition
was calculated by
taking the end point OD measurement of each sample divided by the end point OD
measurement obtained from cells containing the control vector and multiplied
by 100. The
formula is as follows: (1-(sample OD-empty well OD/control vector OD-empty
well OD)), where
the empty well value correspond to a well where no cells had been growing
(Blank).
Results are presented in table 9 and show that Novispirin G10 inhibited 90%
cell growth when
expression of Novispirin G10 was directed to the periplasm, in contrast to the
control vector
that only inhibited 18% (see below).
Table 9:
Plasmid % inhibition
pHH 18%
pDRSS-Novispirin G10 gp%
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These results indicate that the host cells, E. coli TOP10 are sensitive to
Novispirin G10 when
they are expressing it in the periplasm.
Example 14
Growth inhibition of Fusarium lonaypes by AFP
E. coli origami cells were transformed with pHHA900-AFP and pHHA, respectively
and
cultured overnight at 37 degrees C on LB-agar plates containing tetracycline
(tet), kanamycin
(kan) and ampicillin (amp) to select for viable origami cells comprising said
constructs. Two
viable origami colonies containing each of the constructs were selected and
used to inoculate
3 o ther L B-agar p lates w ith t et, kan a nd a mp before c ulturing s aid p
lates for 3 d ays a t 3 7
degrees C. A cellulose acetate filter (Schleider & Schull) was placed on top
of each of the
plates and the colonies were striped off with the filters. The filters were
then place on new LB-
agar plates containing 0.1 % arabinose with the colonies facing up to induce
expression of the
AFP. The filter-agar, 0.1 % arabinose plates were incubated overnight at 37
degrees C before
treating them with chloroform as described in example 1. After this a top-
layer of 6 ml agar
comprising approximately 103 spores/ml Fusarium longypes were poured on top of
the
chloroform treated filter-agar plate. The plates with top-layer were then
incubated for 3 days at
room temperature. The pink color of the Fusarium longypes mycelium facilitated
the detection
of the clearing zones in the top layer.
The results are shown in table 10 as whether or not a clearing zone is present
in the top-layer
over each of 2 colonies of origami cells transformed with the pHHA900-AFP and
pHHA
construct, respectively. A clearing zone is defined as the absence of or
presence of only a bit
of mycelium in the top-layer over each of the origami colonies, which
indicates that the
Fusarium longypes mycelium, which is more or less uniformly distributed
throughout the top-
layer, above the origami colonies has not grown.
Table 10:
AMP/plasmid construct Plate treated with chloroform
Clearing zone +/-
pHHA900-APF no. 1 +
pHHA900-AFP no. 2 +
pHHA no. 1 -
pHHA no. 2 -
The results shown in table 10 shows that the indicator cells Fusarium longypes
present on top
of the origami cells transformed with the construct comprising AFP are dead,
while origami
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cells which were transformed with the control vector had no effect on the
Fusarium longypes
indicator cells. Thus this indicates that expression of AFP by the origami
cells is capable of
killing or inhibiting the Fusarium longypes cells.
Example 15
Growth inhibition of Fusarium longypes by AFP diffused into solid media
The method used in this example is similar to the method described in example
14 with the
difference that in this example the acetate filter containing the E.coli
origami cells is removed
from the arabinose plate before it is overlaid with the fungus Fusarium
longypes which is used
as an indicator strain.
Additionally, this example is very similar to example 4, with the exception
that in the present
example AFP is the antimicrobial polypeptide (instead of Plectasin in example
4) and Fusarium
longypes is the indicator cells (instead of Bacillus subtilis in example 4).
As described in example 14, E. coli origami cells were transformed with
pHHA900-AFP and
pHHA, respectively and cultured overnight at 37 degrees C on LB-agar plates
containing
tetracycline (tet), kanamycin (kan) and ampicillin (amp) to select for viable
E.coli origami cells
comprising said constructs. Two viable E.coli origami colonies containing each
of the
constructs were selected and used to inoculate a LB-agar plate with tet, kan
and amp before
culturing said plate for 3 days at 37 degrees C. A cellulose acetate filter
was placed on top of
the plate and the colonies were striped off with the filter. The filter was
then placed on a new
LB-agar plate containing 0.1 % arabinose with the colonies facing up to induce
expression of
the AFP. The filter-agar, 0.1% arabinose plate was incubated overnight at 37
degrees C. Next
day, i n t his a xample, h owever, i nstead o f t reating t he filter w ith c
hloroform a s d escribed i n
example 14, the acetate filter was removed from the plate as described in
example 4. After
this, a top-layer of 6 ml agar comprising approximately 103 spores/ml Fusarium
longypes was
poured on top of the plate. The plate with top-layer was then incubated for 3
days at room
temperature. The pink colour of the Fusarium longypes mycelium facilitated
detection of
clearing zones in the top layer.
The results are shown in table 11 as whether or not a clearing zone is present
in the top-layer
over the place where each of the 2 E.coli origami colonies transformed with
either pHHA900
AFP or pHHA construct had been growing before removing the acetate filter. A
clearing zone is
defined as the absence of or presence of only a bit of mycelium in the top-
layer, which
indicates that t he Fusarium longypes mycelium, which is more or less a
niformly distributed
throughout the top-layer, has not grown.
Table 11:
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AMPlplasmid construct Clearing zone +/-
pHHA900-APF no. 1 +
pHHA900-APF no. 2 +
pHHA no.1 -
pHHA no.2 -
Compared to the results of example 14, the results in table 11 indicate that
AFP is capable of
diffusing through the acetate filter into the agar media after it is produced
by the E. coli origami
cells.
48
