U.S. patent application number 13/008440 was filed with the patent office on 2011-06-02 for inactivation of genes of the mep pathway.
This patent application is currently assigned to BIOAGENCY AG. Invention is credited to BORAN ALTINCICEK, MATTHIAS EBERL, HASSAN JOMAA.
Application Number | 20110129883 13/008440 |
Document ID | / |
Family ID | 7682435 |
Filed Date | 2011-06-02 |
United States Patent
Application |
20110129883 |
Kind Code |
A1 |
JOMAA; HASSAN ; et
al. |
June 2, 2011 |
INACTIVATION OF GENES OF THE MEP PATHWAY
Abstract
The invention relates to cells and organisms as well as to
methods for producing said cells and organisms, according to which
intermediates of the mevalonate-independent pathway for isoprenoid
biosynthesis (MEP pathway) are enriched by deleting or inactivating
genes. The derivatives can also be enriched by using enzyme
inhibitors. The enriched intermediates may be used as substrates in
enzyme activity tests. The inventive cells and organisms and the
enriched intermediates can further be used in the production of
medicaments.
Inventors: |
JOMAA; HASSAN; (GIESSEN,
DE) ; EBERL; MATTHIAS; (GIESSEN, DE) ;
ALTINCICEK; BORAN; (FERNWALD-ANNEROD, DE) |
Assignee: |
BIOAGENCY AG
HAMBURG
DE
|
Family ID: |
7682435 |
Appl. No.: |
13/008440 |
Filed: |
January 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10475510 |
Jan 29, 2007 |
7875279 |
|
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PCT/EP02/04134 |
Apr 13, 2002 |
|
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13008440 |
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Current U.S.
Class: |
435/131 ;
435/375 |
Current CPC
Class: |
A61P 19/10 20180101;
A61P 29/00 20180101; A61P 37/08 20180101; A61P 19/02 20180101; A61K
31/66 20130101; A61P 1/16 20180101; A61P 3/10 20180101; A61P 31/04
20180101; A61P 37/02 20180101; A61P 11/06 20180101; A61P 25/00
20180101; A61P 5/14 20180101; A61P 17/00 20180101; A61P 5/38
20180101; A61P 1/00 20180101; A61P 37/04 20180101; A61P 7/06
20180101; A61P 21/04 20180101; A61K 35/68 20130101; A61P 27/02
20180101 |
Class at
Publication: |
435/131 ;
435/375 |
International
Class: |
C12P 9/00 20060101
C12P009/00; C12N 5/00 20060101 C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2001 |
DE |
10119905.8 |
Claims
1. A process for preparing intermediates of the MEP pathway or
derivatives of these intermediates, comprising: modifying a gene
involved in the MEP pathway in selected cells or organisms to a
degree sufficient to delete, inactivate or change the gene, thereby
reducing or modifying the enzymatic activity of a gene product
derived from the modified gene with respect to the MEP pathway and
producing an accumulation of an intermediate of the MEP pathway or
a derivative of the intermediate within the selected cell or
organism.
2. A process according to claim 1, wherein the gene is the lytE
gene.
3. A process for preparing intermediates of the MEP pathway or
derivatives of these intermediates, comprising exposing selected
cells and organisms to an enzyme inhibitor.
4. A process according to claim 3, wherein the enzyme inhibitor is
capable of inhibiting the LytB enzyme.
5. A process according to claim 1, wherein the organisms are
selected from a group consisting of bacteria, algae, plants and
protozoa.
6. A process according to claim 1 further comprising concentrating
an intermediate of the MEP pathway.
7. A process according to claim 1 wherein the intermediate or
derivative of the intermediate is suitable for preparing substrates
for a GcpE enzyme or LytB enzyme.
8. A process according to claim 1 wherein the intermediate or
derivative of the intermediate is capable of activating gamma/delta
T cells.
9. A process according to claim 1 wherein the intermediate or
derivative of the intermediate obtained is
3-formyl-1-butypyrophosphate.
10. A method of activating gamma/delta T-cells comprising
contacting gamma/delta T-cells with bacterial cells in which the
lytB gene has been deleted or inactivated.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/475,510, filed Jan. 29, 2007, which is the U.S. national
stage application of International Patent Application No.
PCT/EP02/04134, filed Apr. 13, 2002.
BACKGROUND OF THE INVENTION
[0002] This invention refers to cells and organisms for which
intermediates of the mevalonate independent isoprenoid metabolism
pathway (MEP pathway) are enriched through deletion or inactivation
of genes. Furthermore, it refers to processes for producing
intermediates and products derived from the MEP pathway from
organisms, for which the genes according to the invention have been
deleted or inactivated and genetic engineering and convention
processes for producing these organisms. It also refers to the
application of enzyme inhibitors for enriching MEP pathway
intermediates. It also refers to the therapeutic application of
cells and organisms for which the genes or enzymes according to the
invention have been deleted or inhibited and the production of
medication from these cells and organisms.
[0003] The biosynthesis of isoprenoids using the classic
acetate/mevalonate pathway (Beytia E D, Porter J W, Annu Rev
Biochem, 1976; 45: 113-42) and an alternative, mevalonate
independent biosynthesis pathway, the 2-methyl-D-erythritol pathway
(MEP pathway, synonymous with DOXP pathway) is known (Rohmer M Nat
Prod Rep, 1999 October; 16(5): 565-74). Both pathways lead to
isopentenylpyrophosphate (IPP), the common precursor of all higher
isoprenoids. While the acetate/mevalonate pathway has been known
for some time and is fully understood, at present not all
biosynthetic steps in the reaction of the MEP pathway are
known.
[0004] In the past, various biotechnological processes have been
derived, based on the application of knowledge regarding the MEP
pathway:
[0005] 1. Inhibitors of various enzymes through the MEP pathway are
suitable as disinfectants and herbicides as the MEP pathway does
not occur in humans.
[0006] 2. Certain intermediates of the MEP pathway lead to a
massive stimulation of human gamma/delta T cells. These
intermediates are suitable as immunomodular medicines.
[0007] 3. Through the over-expression of certain genes of the MEP
pathway (e.g. DOXP synthase, LytB), the enriching of higher
isoprenes can be achieved as subsequent products of the MEP
pathway.
[0008] It was previously unknown that through the deletion of a
gene of the MEP pathway or through inactivation of the
corresponding enzyme, an intermediate of the MEP pathway can be
achieved.
[0009] It is known that human gamma/delta T cells are activated
through one or more intermediate of the MEP pathway. This means
that with the incubation of peripheral blood lymphocytes with
extracts from organisms which have an MEP pathway, there is a
selective proliferation and cytokine secretion of the gamma/delta T
cell population (Jomaa H, Feurle J, Luhs K, Kunzmann V, Tony H P,
Herderich M, Wilhelm M, FEMS Immunol Med Microbiol. 1999 September;
25(4): 371-8). The exact chemical composition of this activating
substance of substances is still unknown. Published data suggest
that 3-formyl-1-butylpyrophosphate plays a role as a hypothetical
intermediate of the MEP pathway in activating gamma/delta T cells
(Belmant C, Espinosa E, Poupot R, Peyrat M A, Guiraud M, Poquet Y,
Bonneville M, Fournte J J, J. Biol. Chem. 1999 Nov. 5; 274(45):
32079-84).
[0010] Consequently, it was shown that bacteria, where various
genes of the MEP pathway (e.g. DOXP reductoismerase, gepE) had been
deleted, were no longer able to activate gamma/delta T cells
(Altincicek B, Moll J, Campos N, Foerster G, Beck E, Hoeffler J F,
Grosdemange-Billiard C, Rodriguez-Concepcion M, Rohmer M, Boronat
A, Eberl M, Jomaa H, J. Immunol. 2001 Mar. 15; 166(6):3655-8). In
order to produce these deletion mutations it is necessary to
introduce genes of the mevalonate pathway using genetic engineering
into the bacteria. As a result, the bacteria can then survive in
the medium in the presence of mevalonate if the MEP pathway is no
longer functional (FIG. 1).
BRIEF SUMMARY OF THE INVENTION
[0011] The invention relates to cells and organisms as well as to
methods for producing said cells and organisms, according to which
intermediates of the mevalonate-independent pathway for isoprenoid
biosynthesis (MEP pathway) are enriched by deleting or inactivating
genes. The derivatives can also be enriched by using enzyme
inhibitors. The enriched intermediates may be used as substrates in
enzyme activity tests. The inventive cells and organisms and the
enriched intermediates can further be used in the production of
medicaments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The following explains the invention using the enclosed
figures:
[0013] FIG. 1 shows the principle of enriching the intermediates of
the MEP pathway through the deletion of genes of the MEP pathway.
Substantial steps of the MEP pathway occurring naturally in E. coli
are represented with the enzymes Dxs, Dxr, YgbP, YchB, YgbB, Gcpe,
LytB. In order to be able to delete genes of the MEP pathway, genes
of the mevalonate pathway (coding for Mvk, Pmk, Mpd) are introduced
through genetic engineering. Through the deletion of lytB, the
intermediates are enriched which activate the gamma/delta T
cells.
[0014] FIG. 2 shows the activation of gamma/delta T cells from the
blood of healthy donors, measured as an expression of CD25, through
extracts from various bacteria sources (wild type, wtDeltagcpE,
wtDeltalytB) in various dilution stages. IPP serves as a control
with an end concentration of 10 uM (IPP activates gamma/delta T
cells substantially weaker than the intermediate according to the
invention, but is suitable as a control for the test system). The
wtDeltagcpE mutants were produced analogously to the wtDeltagcpB
mutants.
DETAILED DISCLOSURE OF THE INVENTION
[0015] Surprisingly, it was found that bacteria, whose lytB gene
had been deleted (example 1) activated gamma/delta T cells
significantly more strongly than typical bacteria (example 2, FIG.
2). An essential participation of the lytB gene in the MEP pathway
was displayed (example 3). A blockage of the MEP pathway at the
level of the lytB enzyme thus leads to the intermediate, which is
responsible for the gamma/delta T cells, being enriched.
[0016] Therefore, a process is available, through which the
enriching of intermediates of the MEP pathway is achieved through
deletion, mutation or functional inactivation of the corresponding
genes. The enriching of the intermediates of the MEP pathway can
also be achieved through inhibiting the enzymatic function of the
corresponding polypeptide.
[0017] DNA sequences, which code for a polypeptide with the amino
acids represented in SEQ ID NO: 2 or for an analogue or derivative
of the polypeptide according to SEQ ID NO: 2, are particularly
suitable for carrying out the process according to the process,
where one or more amino acids are deleted, added or substituted by
other amino acids without the enzymatic effect of the polypeptide
being substantially reduced.
[0018] Furthermore, the invention is defined by claims 1-17.
Further images of the invention are defined in the subordinate
claims.
[0019] The genes and their genetic products (polypeptide) are
listed in the sequence protocol with their primary structures and
are allocated as follows:
[0020] SEQ ID NO: 1 lytB-gene
[0021] SEQ ID NO:2 lytB-protein.
[0022] The sequences come from escherichia coli.
[0023] Apart from the DNA sequences named in the sequence protocol,
others are suitable which have another DNA code as a result of the
degeneration of the genetic code but which code for the same
polypeptide or for an analogue or derivative of the polypeptide.
This also includes sequences which come from organisms other than
E. coli, specifically, other bacteria, algae, plants and protozoa,
and which are recognized, based on sequence comparisons or function
analyses, as homologous to the sequences named in the sequence
protocol.
[0024] The invention refers to cells and organisms and the
production of cells and organisms, for which the genes according to
the invention are functionally inactivated principally as a result
of known methods. The genes do not have to be fully inactivated but
instead can have their function reduced or modified. This can be
achieved by the following: [0025] Complete or partial deletion of
the genes [0026] Substitution of the gene through an artificial DNA
sequence or a gene for a selection marker [0027] Insertion of a
gene for a selection marker [0028] Deletion, insertion and
substitution of one or several base pair [0029] Mutations in the 5'
and 3' area of the coding sequences (influence of promoter,
enhancer, terminator sequences, ribosome conjugates) [0030]
Introduction of DNA constructs which code for antisense DNA [0031]
Application of mutagene agents, ionising radiation, UV radiation
[0032] Screening for spontaneous mutants.
[0033] The inactivation or modification of the sequences according
to the invention can occur in bacteria, algae, plants and protozoa.
In order to maintain stable mutants, it can be necessary to
introduce the genes of the acetate/mevalonate pathway partially or
in full and, if necessary, to add mevalonate or another
intermediate of the MEP pathway to the medium. Alternatively or
additionally, intermediates of the MEP pathway or derivatives or
analogues to these intermediates (e.g. 3-methyl-3-buten-1-ol,
3-methyl-2-buten-1-ol) can be added to the medium.
[0034] Cells and organisms can also be used, which do not naturally
have the MEP pathway, if genes of the MEP pathway have been
introduced through genetic engineering and bio-engineering methods.
It is also possible to achieve enriching of intermediates of the
MEP pathway of their derivatives by only incompletely introducing
the genes of the MEP pathway. Mammal and insect cells, lower and
higher fungi, slime mold and various protozoa, among others, are
suitable for this.
[0035] Apart from through genetic methods, inactivation or
reduction of the enzymatic activity of the polypeptides according
to the invention can also be achieved through enzyme inhibitors
which are added to the culture medium of the organisms or cell
extracts from the organisms. The enzyme inhibitors can have
synthetic or natural conjugates which reversibly or irreversibly
inhibit the function of the polypeptide through competitive or
allosteric interactions.
[0036] The cells and organisms according to the invention,
including complete plants and parts of plants, can be reproduced
and cultivated through known processes. A co-culture with other
cells or organisms, including those which do not have an MEP
pathway, is also possible. The enriched intermediates of the MEP
pathway or their derivatives can be obtained through breaking down
the cells or from the culture. Various known methods are suitable
for purifying the intermediates, including chromatography,
electrophoreses and precipitation (e.g. as barium salts).
[0037] The enriched intermediates of the MEP pathway are suitable
for various applications. It has been found that the intermediates
contain the product of the GcpE enzyme and the substrate of the
LytB enzyme. Thus, the intermediates can be used as substrates in
the enzyme activity test for LytB and GcpE. In the activity test
for GcpE, the reverse reaction is observed. This type of enzyme
activity test is suitable for finding GcpE and LytB inhibitors.
[0038] The enriched intermediates of the MEP pathway are also
suitable for producing medicines. The effectiveness of the
substances is based on the activation of gamma/delta T cells.
Depending on the area of application, the immunity can be
strengthened (e.g. against tumors) or immunological tolerance
against auto-antigens and allergens can be induced. Areas of
application are the treatment of immune, auto-immune diseases and
allergies.
[0039] For example: allergies, multiple sclerosis, rheumatoid
arthritis, Hashimoto's thyroiditis, myasthenia gravis, lupus
erythematosus, diabetes mellitus, primary biliary cirrhosis, active
chronic hepatitis, adrenalitis/Addison's disease, polymyositis,
dermatomyositis, auto-immune haemolytic anaemia, myocardial and
cardiac infections, scleroderma, uveitis (phacouveitis, sympathetic
ophthalmia), pemphigus vulgaris, pemphigoid, pernicious anaemia,
auto-immune atrophic gastritis, inflammatory disease of the
intestines such as Crohn's disease and colitis ulcerosa,
inflammatory disease of the lungs such as asthmatic diseases and
bronchitis.
[0040] The application is preferred for morbus Crohn, colitis
ulcerosa, multiple sclerosis, asthma, chronic bronchitis,
allergies.
[0041] Other applications are infections of the bone, especially
osteoporosis.
[0042] The intermediates of the MEP pathway can be isolated from
the organisms according to the invention or used as raw extracts
for medical application. The complete organisms can also be used
living or dead. The intermediates and organisms according to the
invention can be used alone or in combination with other
medications. Application as adjuvant for strengthening or for
modulation of an immune response is also included. Preferred
methods of application are oral, inhalative and rectal application,
as well as application on the skin or mucous membranes.
[0043] The following are suitable as pharmaceutical compositions:
tablets, drops, capsules, pills, granules, suppositories,
solutions, suspensions and emulsions, pastes, ointments, gels,
creams, lotions, powders and sprays.
[0044] Tablets, drops, capsules, pills and granules can contain the
active ingredients alongside the usual carriers such as (a) fillers
and mixers, e.g. starch, lactose, cane sugar, glucose, mannitol and
silicic acid, (b) binders, e.g. carboxymethylcelulose, alginate,
gelatine, polyvinylpyrrolidone, (c) moisturizers, e.g. glycerine,
(d) explosive, e.g. agar-agar, calcium carbonate and sodium
carbonate, (e) emulsifier, e.g. paraffin (f) re-absorption
accelerator, e.g. quanternary ammonium conjugates, (g) nets, e.g.
cetylalcohol, glycerine monostearate, (h) absorbers, e.g. kaolin
and bentonite and (i) lubricant, e.g. talcum, calcium and magnesium
stearate and solid polyethylglycol or mixtures of the substances
listed in (a) to (i). Moreover, the conjugates according to the
invention can be included in other carriers such as plastics
(plastic chains for local treatment), collages or bone cement.
[0045] Tablets, drops, capsules, pills and granules can be produced
with the usual, if necessary opaque, casings and cases and also
combined such that the active ingredients are only released, or
preferably with a delay, in a specific section of the intestinal
tract where embedders can be used, e.g. polymer substances and
waxes.
[0046] The active ingredients can also exist in micro-capsule form
in one or more of the above carriers.
[0047] Suppositories can contain the usual water soluble or
insoluble carriers along with the active ingredients, e.g.
polyethylglycol, fats, e.g. cocoa fat and higher ester (e.g. C14
alcohol with C16 fatty acid) or mixtures of same.
[0048] Ointments, pastes, creams and gels can contain the usual
carriers along with the active ingredients, e.g. animal and
vegetable fats, waxes, paraffin, starch, tragant, cellulose
derivatives, polyethylglycol, silicone, bentonite, silicic acid,
talcum and zinc oxide or mixtures of same.
[0049] Powders and sprays can contain the usual carriers along with
the active ingredients, e.g. lactose, talcum, silicic acid,
aluminium hydroxide, calcium silicate and polyamide powder or
mixtures of same. In addition, sprays can contain propellants such
as CFCs.
[0050] Solutions and emulsions can contain the usual carriers along
with the active ingredients, such as solvents, solubilisers and
emulsifiers, e.g. water, ethylalcohol, isopropylalcohol,
ethylcarbonate, ethylacetate, benzylalcohol, benzylbenzoate,
propylenglycol, 1,3-butylenglycol, dimethylformamide, oils,
especially cotton seed oil, peanut oil, maize oil, olive oil,
ricinus oil and sesame oil, glycerine, glycerine formal,
tetrahydrofurylalcohol, polyethylglycols and fatty acid ester of
sorbitol or mixtures of same.
[0051] Particularly beneficial is the selection of a medical
application which also contains a substance which can be recognized
by the immune system as a foreign object or auto-antigen.
Example 1
Construction of lytB deletion mutant
[0052] Construction of the Gene Exchange Plasmid
pKO3-.DELTA.lytB
[0053] In order to produce a lytB deletion mutant from E. coli, the
pKO3 vector was used (Link, A. J.; Philips, D.; Church, G. M.; J.
Bacteriol 179, 6228-6237). In order to produce the deletion design,
two sequences were amplified downstream and upstream of the lytB
gene in two asymmetrical PCR stages. The primers were used in a
1:10 molar ratio (50 nM and 500 nM). Both PCR products were fused
in a second PCR amplification to form one product. The product was
cloned using the pCR-TA-TOPO cloning kit (Invitrogen) and recloned
using the restriction interfaces Bam HI and Sal I in the pKO3
vector. The following primers were used:
TABLE-US-00001 lytB-N-out, (SEQ ID NO: 3)
5'-TAGGATCCccggcctagatgactgcg-3'; ltyB-N-in, (SEQ ID NO: 4)
5'-CCCATCCACTAAACTTAAACAcaacaggatctgcatgttacg-3'; ltyB-C-in, (SEQ
ID NO: 5) 5'-TGTTTAAGTTTAGTGGATGGGcgtgaagtcgattagtcat-3';
ltyB-C-out, (SEQ ID NO: 6) 5'-TAGTCGACagaaccacccatgatcacc-3'.
[0054] The restriction interfaces are underlined. Overlapping
sequences, which define a 21 bp-"in frame" insertion, are printed
in bold.
Construction of the Synthetic Mevalonate Operon pSC-MVA
[0055] In order to be able to produce mutants whose individual
genes of the MEP pathway are deleted, first of all a genetically
altered E. coli source was produced which was able to use
mevalonate from the culture medium for the synthesis of IPP. To do
this, a synthetic operon was constructed which contained the gene
for the following enzyme of the mevalonate pathway from
saccharomyces cerevisiae (yeast): mevalonate kinase (ERG12),
phosphomevalonate kinase (ERG8) and
diphosphomevalonate-decarboxylase (ERG19). The three genes were
amplified in three asymmetrical PCR stages with genome yeast DNA as
a matrix, with the primers being used in a 1:10 molar ratio (50 nM
and 500 nM). Ribosome binders were included with the primers. The
three PCR products were mixed so that they could hybridize with the
overlapping areas and were amplified using the external primer as a
fragment. The product was cloned in the pBAD vector using
pBAD-TA-TOPO cloning kits (Invitrogen) and verified using
restriction and sequence analysis. The following primer set was
used:
TABLE-US-00002 Mev-kin-Sc-for: (SEQ ID NO: 7)
5'-TAGGAGGAATTAACCATGTCATTACCGTTCTTAACT-3' Mev-kin-Sc-rev: (SEQ ID
NO: 8) 5'-TTGATCTG ATGAAGTCCATGGTAAATT-3' Pmev-kin-Sc-for: (SEQ ID
NO: 9) 5'-ACTTCAT CAGATCAAATGTCAGAGTTGAGAGCCTTC-3' Pmev-kin-Sc-rev:
(SEQ ID NO: 10) 5'-GAGTATTAT ATTTATCAAGATAAGTTTC-3'; Decarb-Sc-for:
(SEQ ID NO: 11) 5'-GATAAAT TAATACTCATGACCCGTTACACAGCATCC-3'
Decarb-Sc-rev: (SEQ ID NO: 12) 5'-TTATTCCTTTGGTAGACCAGT-3'.
[0056] Overlapping sequences are printed in bold and sequences
which define ribosome conjugates are in italics.
[0057] In order to check the functionality of the operon, the
sensitivity to fosmidomycin from bacteria which have been
transformed with the synthetic operon was tested in the presence of
mevalonate. As expected, bacteria grew, which contained
fosmidomycin at a reduced rate as long as the medium contained
mevalonate. Without mevalonate, the bacteria died under
fosmidomycin.
Construction of the Deletion Mutant wt.DELTA.lytB
[0058] The plasmid pKO3-DeltalytB was transformed in the E. coli
K-12 source DSM No. 498 (ATCC 23716), which had previously been
transformed with pSC-MVA. The medium was supplemented with 100 uM
mevalonate. After 1 hour incubation at 30.degree. C., bacteria with
integrated plasmid were selected through a temperature shift to
43.degree. C. As a result of the subsequent test for sucrose
resistance and chloramphenicol sensitivity, the bacteria, which had
lost the vector sequences, were selected and then analyzed through
PCR for the desired gene type.
Example 2
Activation of Gamma/Delta T Cells Through Enriched Intermediates of
the MEP Pathway
[0059] The enriching of intermediates of the MEP pathway was
detected from the ability of these intermediates to activate
gamma/delta T cells. Various bacteria sources (wild type,
wtDeltagcpE, wtDeltalytB) were cultivated in liquid cultures up an
optical thickness of approximately 0.8. Obtaining low molecular
extracts (low molecular weight, LMW) with an exclusion limit of 3
kDa occurs as described (Jomaa H, Feurle J, Luhs K, Kunzmann V,
Tony H P, Herderich M and Wilhelm M, FEMS Immunol Med Microbiol,
25:371). Lymphocytes are obtained from the peripheral blood of
three healthy donors through the ficoll-density gradient
centrifugation. For each test, 2 lots of 10.sup.5 of the cells
obtained are shown in a volume of 0.2 ml RPMI-1640-Medium (Life
Technologies), which was enriched with 25 mM HEPES, 2 mM
L-glutamine, 0.025 mg/ml gentamycin, 100 U/ml human interleukin-2
(IL-2) (all from Life Technologies), and 10% human AB serum
(Bavarian Red Cross). LMW preparations were added to various
solutions, IPP (sigma) was used in a final concentration of 10 uM
as a positive control. The incubation was carried out at 37.degree.
C. and 5% CO.sub.2 in the incubator. After 72 hours, the cells were
harvested and analyzed in a throughflow cytometer. The expression
of the activation marker CD25 was measured on the surface of V
gamma 9.sup.+ T cells using the monoclonal antibodies CD25-PE
(B1.49.9), V gamma 9-FITC (Immu360) and CD3-PC5 (UCHT1) from the
Beckman-Coulter Company. Extracts from the wild type bacteria
source activated the gamma/delta T cells at a concentration of
1:500 (corresponds to approx. 2.times.10.sup.7 bacteria/ml).
Extracts from the DeltagcpE-deletion mutants led to a significantly
reduced activation. By contrast, the activation by extracts from
the DeltalytB-deletion mutants was considerably stronger than
through extracts from the wild type source. A significant
gamma/delta T cell activation was also measured at a concentration
of 1:12500 (corresponds to approx. 8.times.10.sup.5 bacteria/ml)
(FIG. 2).
Example 3
Participation of lytB in the MEP Pathway
[0060] All lytB deletion mutants obtained grew strictly mevalonate
dependent. In order to investigate this observation more closely,
the deletion mutants wtDeltalytB were complemented by a wild type
lytB gene on a plasmid. The lytB gene was amplified with the primer
eclytbfor (5'-GGATCCATGCAGATCCTGTTGGCCAAC-3', SEQ ID NO: 13) and
ecltybrev (5'-AAGCTTTTAATCGACTTCACGAATATCG-3', SEQ ID NO: 14) from
genomic E. coli DNA and cloned in the pCR2.1-TOPO vector. The
insert was recloned through the restriction interfaces BamHI and
HindIII and in the expression vector pQE30. Bacteria from the
wtDeltalytB source, which were transformed with this construct,
were able to grow without mevalonate. This result confirms that
lytB is an essential participant in the MEP metabolism pathway. The
enriched intermediates therefore come from the MEP pathway.
Sequence CWU 1
1
141951DNAEscherichia coliCDS(1)..(951) 1atg cag atc ctg ttg gcc aac
cca cgt ggt ttt tgt gcc ggg gta gac 48Met Gln Ile Leu Leu Ala Asn
Pro Arg Gly Phe Cys Ala Gly Val Asp1 5 10 15cgc gct atc agc att gtt
gaa aac gcg ctt gcc att tac ggc gca ccg 96Arg Ala Ile Ser Ile Val
Glu Asn Ala Leu Ala Ile Tyr Gly Ala Pro 20 25 30ata tat gtc cgt cac
gaa gtg gtg cat aac cgc tac gtg gtc gat agc 144Ile Tyr Val Arg His
Glu Val Val His Asn Arg Tyr Val Val Asp Ser 35 40 45ctg cgc gag cgt
gga gct atc ttt att gag cag atc agc gaa gtg ccg 192Leu Arg Glu Arg
Gly Ala Ile Phe Ile Glu Gln Ile Ser Glu Val Pro 50 55 60gac ggc gcg
atc ctg atc ttc tcc gca cat ggt gtt tct cag gcg gta 240Asp Gly Ala
Ile Leu Ile Phe Ser Ala His Gly Val Ser Gln Ala Val65 70 75 80cgt
aac gaa gcg aaa agc cgt gat ttg acg gta ttc gac gcc acc tgt 288Arg
Asn Glu Ala Lys Ser Arg Asp Leu Thr Val Phe Asp Ala Thr Cys 85 90
95ccg ctg gtg acc aaa gtg cat atg gaa gtc gcc cgc gcc agc cgt cgt
336Pro Leu Val Thr Lys Val His Met Glu Val Ala Arg Ala Ser Arg Arg
100 105 110ggc gaa gag tct att ctc atc ggt cac gcc ggg cac ccg gaa
gtg gaa 384Gly Glu Glu Ser Ile Leu Ile Gly His Ala Gly His Pro Glu
Val Glu 115 120 125ggg acg atg ggg cag tac agc aac cct gaa ggg gga
atg tat ctg gtc 432Gly Thr Met Gly Gln Tyr Ser Asn Pro Glu Gly Gly
Met Tyr Leu Val 130 135 140gaa tcg cct gac gat gtg tgg aaa ctg acg
gtc aaa aac gaa gag aag 480Glu Ser Pro Asp Asp Val Trp Lys Leu Thr
Val Lys Asn Glu Glu Lys145 150 155 160ctc tcc ttt atg acc caa acc
acg ctg tcg gta gat gac acg tct gat 528Leu Ser Phe Met Thr Gln Thr
Thr Leu Ser Val Asp Asp Thr Ser Asp 165 170 175gtg atc gac gcg ctg
cgt aaa cgc ttc ccg aaa att gtc ggt ccg cgc 576Val Ile Asp Ala Leu
Arg Lys Arg Phe Pro Lys Ile Val Gly Pro Arg 180 185 190aaa gat gac
atc tgc tac gcc acg act aac cgt cag gaa gcg gta cgc 624Lys Asp Asp
Ile Cys Tyr Ala Thr Thr Asn Arg Gln Glu Ala Val Arg 195 200 205gcc
ctg gca gaa cag gcg gaa gtt gtg ttg gtg gtc ggt tcg aaa aac 672Ala
Leu Ala Glu Gln Ala Glu Val Val Leu Val Val Gly Ser Lys Asn 210 215
220tcc tcc aac tcc aac cgt ctg gcg gag ctg gcc cag cgt atg ggc aaa
720Ser Ser Asn Ser Asn Arg Leu Ala Glu Leu Ala Gln Arg Met Gly
Lys225 230 235 240cgc gcg ttt ttg att gac gat gcg aaa gat atc cag
gaa gag tgg gtg 768Arg Ala Phe Leu Ile Asp Asp Ala Lys Asp Ile Gln
Glu Glu Trp Val 245 250 255aaa gag gtt aaa tgc gtc ggc gtg act gcg
ggc gca tcg gct ccg gat 816Lys Glu Val Lys Cys Val Gly Val Thr Ala
Gly Ala Ser Ala Pro Asp 260 265 270att ctg gtg cag aat gtg gtg gca
cgt ttg cag cag ctg ggt ggt ggt 864Ile Leu Val Gln Asn Val Val Ala
Arg Leu Gln Gln Leu Gly Gly Gly 275 280 285gaa gcc att ccg ctg gaa
ggc cgt gaa gaa aat att gtt ttc gaa gtg 912Glu Ala Ile Pro Leu Glu
Gly Arg Glu Glu Asn Ile Val Phe Glu Val 290 295 300ccg aaa gag ctg
cgt gtc gat att cgt gaa gtc gat taa 951Pro Lys Glu Leu Arg Val Asp
Ile Arg Glu Val Asp305 310 3152316PRTEscherichia coli 2Met Gln Ile
Leu Leu Ala Asn Pro Arg Gly Phe Cys Ala Gly Val Asp1 5 10 15Arg Ala
Ile Ser Ile Val Glu Asn Ala Leu Ala Ile Tyr Gly Ala Pro 20 25 30Ile
Tyr Val Arg His Glu Val Val His Asn Arg Tyr Val Val Asp Ser 35 40
45Leu Arg Glu Arg Gly Ala Ile Phe Ile Glu Gln Ile Ser Glu Val Pro
50 55 60Asp Gly Ala Ile Leu Ile Phe Ser Ala His Gly Val Ser Gln Ala
Val65 70 75 80Arg Asn Glu Ala Lys Ser Arg Asp Leu Thr Val Phe Asp
Ala Thr Cys 85 90 95Pro Leu Val Thr Lys Val His Met Glu Val Ala Arg
Ala Ser Arg Arg 100 105 110Gly Glu Glu Ser Ile Leu Ile Gly His Ala
Gly His Pro Glu Val Glu 115 120 125Gly Thr Met Gly Gln Tyr Ser Asn
Pro Glu Gly Gly Met Tyr Leu Val 130 135 140Glu Ser Pro Asp Asp Val
Trp Lys Leu Thr Val Lys Asn Glu Glu Lys145 150 155 160Leu Ser Phe
Met Thr Gln Thr Thr Leu Ser Val Asp Asp Thr Ser Asp 165 170 175Val
Ile Asp Ala Leu Arg Lys Arg Phe Pro Lys Ile Val Gly Pro Arg 180 185
190Lys Asp Asp Ile Cys Tyr Ala Thr Thr Asn Arg Gln Glu Ala Val Arg
195 200 205Ala Leu Ala Glu Gln Ala Glu Val Val Leu Val Val Gly Ser
Lys Asn 210 215 220Ser Ser Asn Ser Asn Arg Leu Ala Glu Leu Ala Gln
Arg Met Gly Lys225 230 235 240Arg Ala Phe Leu Ile Asp Asp Ala Lys
Asp Ile Gln Glu Glu Trp Val 245 250 255Lys Glu Val Lys Cys Val Gly
Val Thr Ala Gly Ala Ser Ala Pro Asp 260 265 270Ile Leu Val Gln Asn
Val Val Ala Arg Leu Gln Gln Leu Gly Gly Gly 275 280 285Glu Ala Ile
Pro Leu Glu Gly Arg Glu Glu Asn Ile Val Phe Glu Val 290 295 300Pro
Lys Glu Leu Arg Val Asp Ile Arg Glu Val Asp305 310
315326DNAArtificial sequencelytB-N-out primer 3taggatcccc
ggcctagatg actgcg 26442DNAArtificial sequenceltyB-N-in primer
4cccatccact aaacttaaac acaacaggat ctgcatgtta cg 42540DNAArtificial
sequenceltyB-C-in primer 5tgtttaagtt tagtggatgg gcgtgaagtc
gattagtcat 40627DNAArtificial sequenceltyB-C-out primer 6tagtcgacag
aaccacccat gatcacc 27736DNAArtificial sequenceMev-kin-Sc-forward
primer 7taggaggaat taaccatgtc attaccgttc ttaact 36833DNAArtificial
sequenceMev-kin-Sc-reverse primer 8ttgatctgcc tcctatgaag tccatggtaa
att 33942DNAArtificial sequencePmev-kin-Sc-forward primer
9acttcatagg aggcagatca aatgtcagag ttgagagcct tc 421034DNAArtificial
sequencePmev-kin-Sc-reverse primer 10gagtattatc ctcctattta
tcaagataag tttc 341142DNAArtificial sequenceDecarb-Sc-forward
primer 11gataaatagg aggtaatact catgacccgt tacacagcat cc
421221DNAArtificial sequenceDecarb-Sc-reverse primer 12ttattccttt
ggtagaccag t 211327DNAArtificial sequenceeclytbfor primer
13ggatccatgc agatcctgtt ggccaac 271428DNAArtificial
sequenceecltybrev primer 14aagcttttaa tcgacttcac gaatatcg 28
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