Gut commensal bacterium and methods of using the same
United States Patent: 7,988,961
Issued: August 2, 2011
Inventors: Farrar; Mark
(Leeds, GB), Carding; Simon (Leeds, GB)
Assignee: Plant Bioscience
Appl. No.: 11/814,739
Filed: January 24, 2006
PCT Filed: January 24, 2006
PCT No.: PCT/GB2006/000222
371(c)(1),(2),(4) Date: July
PCT Pub. No.: WO2006/079790
PCT Pub. Date: August 03,
Executive MBA in Pharmaceutical Management, U. Colorado
The invention provides gut commensal
bacteria that have been modified to express one or more biologically
active polypeptides or protiens, the bacteria includes a promoter, such as
a xylanase promoter, which is induced in response to the presence of xylan
in the diet and which regulates the expression of the biologically active
polypeptide or protien.
Description of the
BRIEF SUMMARY OF THE DISCLOSURE
According to a first aspect of the invention there is provided a gut
commensal bacterium deposited under the provisions of the Budapest Treaty
at the National Collection of Industrial, Food and Marine Bacteria (NCIMB)
Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, Scotland AB21
9YA on Nov. 29, 2007 and assigned Accession No. 41521 (Bacteriodes ovatus
BO-KGF), 41522 (Bacteriodes ovatus BO-TGF) or 41523 (Bacteriodes ovatus
BO-MUIL2-S) modified to express one or more biologically active
polypeptides or proteins, the bacterium further comprising a promoter
which is induced in response to the presence of a dietary factor and which
regulates the expression of said biologically active polypeptide or
Throughout the description and claims of this specification, the words
"comprise" and "contain" and variations of the words, for example
"comprising" and "comprises", means "including but not limited to", and is
not intended to (and does not) exclude other moieties, additives,
components, integers or steps.
Throughout the description and claims of this specification, the singular
encompasses the plural unless the context otherwise requires. In
particular, where the indefinite article is used, the specification is to
be understood as contemplating plurality as well as singularity, unless
the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or
groups described in conjunction with a particular aspect, embodiment or
example of the invention are to be understood to be applicable to any
other aspect, embodiment or example described herein unless incompatible
An operon may be defined as a functional unit consisting of a promoter, an
operator and a number of structural genes. An example is the xylanase
operon. The structural genes commonly code for several functionally
related enzymes, and although they are transcribed as one (polycistronic)
mRNA, each has its separate translation initiation site. In the typical
operon, the operator region acts as a controlling element in switching on
or off the synthesis of mRNA. The xylanase operon is activated in the
presence of xylan.
Preferably, the promoter is constitutive and more preferably is the
xylanase promoter. Thus it will be appreciated that the expression of the
one or more biologically active polypeptides or protiens is controlled by
the presence of xylan in the diet. The bacteria can therefore be said to
comprise a xylan-inducible regulatory element.
Xylan is a water-soluble, gummy polysaccharide found in plant cell walls
and yielding xylose upon hydrolysis. It is therefore a common dietary
factor or component, accordingly the inclusion or exclusion of xylan in
the diet controls the expression of the biologically active polypeptide or
protien. The modified bacteria of the present invention therefore
advantageously provide an easily controllable expression system avoiding
repeated invasive dosing of an individual since the modified bacteria of
the present invention are also able to colonise the gut whilst
concomitantly minimising any adverse side-effects.
Preferably, the bacterium is obligate anaerobe and more preferably still
said bacterium is either Bacteroides ovatus or Prevotella.
Preferably, the bacterium in non-pathogenic to man.
"Biologically active" refers to the ability to perform a biological
function. The biologically active polypeptide or protein used in the
present invention can be either homologous to the bacterium or
heterologous thereto, derived from either eukaryotic or prokaryotic or
Specific examples of such polypeptides and proteins used in the present
invention preferably include insulin, growth hormone, prolactin,
calcitonin, luteinising hormone, parathyroid hormone, somatostatin,
thyroid stimulating hormone, vasoactive intestinal polypeptide, trefoil
factors, cell and tissue repair factors, transforming growth factor
.beta., keratinocyte growth factor, a structural group 1 cytokine adopting
an antiparallel 4.alpha. helical bundle structure such as IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF,
SCF, IFN-.gamma., EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFN.alpha./.beta.,
a structural group 2 cytokine which are often cell-surface associated,
form symmetric homotrimers and the subunits take up the conformation of
.beta.-jelly roll described for certain viral coat proteins such as the
TNF family of cytokines, eg TNF.alpha., TNF.beta., CD40, CD27 or FAS
ligands, the IL-1 family of cytokines, the fibroblast growth factor
family, the platelet derived growth factors, transforming growth factor p
and nerve growth factors, a structural group 3 cytokine comprising short
chain .alpha./.beta. molecules, which are produced as large transmembrane
pre-cursor molecules which each contain at least one EGF domain in the
extracellular region, eg the epidermal growth factor family of cytokines,
the chemokines characterised by their possession of amino acid sequences
grouped around conserved cysteine residues (the C-C or C-X-C chemokine
subgroups) or the insulin related cytokines, a structural group 4 cytokine
which exhibit mosaic structures such as the heregulins or neuregulins
composed of different domains, eg EGF, immunoglobulin-like and kringle
Alternatively, the biologically active polypeptide can be a receptor or
antagonist for biologically active polypeptides as defined above.
The bacterium expresses the biologically active polypeptide or protein and
the antigen from nucleic acid contained within it. The nucleic acid may
comprise one or more nucleic acid constructs in which nucleic acid
encoding the biologically active polypeptide and nucleic acid encoding the
antigen are under control of appropriate regulatory sequences for
expression in the bacterium.
The bacterium may also express the biologically active polypeptide or
protein as a vaccine.
Preferably, the bacterium of the present invention may be modified to
express a plurality of biologically active polypeptides or proteins.
According to a further aspect of the invention there is provided a
pharmaceutical comprising a gut commensal bacterium modified to express
one or more biologically active polypeptides or protiens, the bacterium
further comprising a promoter which is induced in response to the presence
of a dietry factor and which regulates the expression of said biologically
active polypeptide or protien.
Preferably, the pharmaceutical is provided as a composition in a
physiologically acceptable carrier, diluent or excipient.
Preferably, the pharmaceutical comprises any one or more of the features
According to a further aspect of the invention there is provided use of a
gut commensal bacterium modified to express one or more biologically
active polypeptides or protiens, the bacterium further comprising a
promoter which is induced in response to the presence of a dietry factor
and which regulates the expression of said biologically active polypeptide
or protien, in the manufacture of a medicament for the treatment of
chronic infammatory bowel disease.
Preferably, the use further comprises any one or more of the features
According to a further aspect of the invention there is provided a method
of delivering one or more biologically active polypeptides or proteins or
antigens or enzymes or vaccine which comprises administering to a subject
a gut commensal bacterium which expresses one or more of said biologically
active agents expression of which is under control of a promoter which is
activated in the presence of a dietry factor.
Preferably, bacterium expresses more than one biologically active
polypeptide or protein or antigen or enzyme or vaccine or a combination
Preferably, the method comprises the administration of a mixture of
bacteria expressing a variety of biologically active polypeptides or
proteins or antigens or enzymes or vaccines or a combination thereof.
Thus it will be appreciated that in this embodiment of the invention there
is provided, for example and without limitation, bacteria capable of
expressing IL2 and bacteria capable of expressing IL12 and/or IL9 and
optionally bacteria capable of expressing a cell and tissue repair factor.
Preferably, the method includes any one or more of the features herein
Bacteroides ovatus, is a major commensal colonic Gram-negative bacterium
in humans and rodents for which cloning systems are available that allow
the introduction of foreign DNA into the organism and integration into the
genome (Tancula et al. 1992). This organism is also one of only a few that
are able to degrade the polysaccharide xylan. We provide evidence for the
successful engineering of B. ovatus to produce murine IL2 (MuIL2)
intracellularly under the control of the xylanase promotor which is active
in the presence of xylan. Our results demonstrate that B. ovatus can be
induced to produce biologically active MuIL2 in response to xylan. We have
also engineered a second strain to secrete MuIL2 by adding the B. fragilis
enterotoxin secretion signal sequence to the protein. The recombinant
strains produced MuIL2 only in the presence of xylan as determined by
enzyme-linked immunosorbent assay of cell lysates and culture
supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate
that MuIL2 produced by both B. ovatus strains was biologically active.
Moreover, this activity could be blocked by an anti-IL2 neutralising
According to a further aspect of the invention there is provided a method
of treating chronic inflammation of the gut comprising administering to an
individual suffering from such a condition a pharmaceutically effective
amount of a gut commensal bacterium modified to express one or more
biologically active polypeptides or protiens, the bacterium further
comprising a promoter which is induced in response to the presence of a
dietry factor and which regulates the expression of said biologically
active polypeptide or protien.
The use of bacteria of the invention as drug delivery vehicles offers a
means of delivering immunomodulatory factors, such as cytokines, and other
biologically active molecules directly to the site action to treat chronic
inflammation of the gut.
The advantages of this unique form of therapeutic delivery is that it is a
convenient and simple means of delivering biologically active proteins
directly to their site of action, avoiding the inconvenience and systemic
exposure associated with parenteral therapy
Bacterial Strains, Plasmids and General DNA Manipulations
E coli DH5.alpha. and J53/R751 were grown in LB medium. Cultures of E. coi
J53/R751 were supplemented with 200 .mu.g trimethoprim ml.sup.-1. B.
ovatus V975 was grown anaerobically at 37.degree. C. in brain heart
infusion (BHI) broth supplemented with 10 .mu.g haemin ml.sup.-1 or in
routine growth medium (RGM) prepared as described by Hespell et al. (1987)
and supplemented with 0.1% (w/v) glucose. Where xylan was required, a hot
water-soluble fraction of oatspelt xylan was prepared by the method of
Hespell and O'Bryan (1992) and added to media at a concentration of 0.2%
(w/v). Transfer of plasmids to B. ovatus from E. coli J53/R751 was carried
out by conjugation as described by Valentine et al. (1992). pBT2 (Tancula
et al. 1992) was selected in E. coli using 50 .mu.g kanamycin ml.sup.-1.
B. ovatus transconjugants were selected on BHI-haemin agar containing 200
.mu.g gentamicin ml.sup.-1 and 5 .mu.g tetracycline ml.sup.-1.
Transconjugants were subsequently grown in medium containing 1 .mu.g
tetracycline ml.sup.-1. E. coli was transformed by the method of Hanahan
(1983). General DNA manipulations were carried out as described by
Sambrook et al. (1990).
Construction of MuIL2-producing and control B. ovatus strains
MuIL2-producing strain BOMuIL2. The MuIL2 gene was PCR-amplified from cDNA
cloned in pUC13 using primers MuIL2F1 (GCGCATATGGCACCCACTTC MGCTCCAC;SEQ
ID NO:1 Ndel site in bold) and MuIL2R1 (GCGGGATCCTT
ATTGAGGGCTTGTTGAGATGATG; SEQ ID NO:2 BamH1 site in bold). A portion of the
B. ovatus xylanase operon encompassing the 3' half of the orf gene and
region between this gene and the xyl gene was amplified from plasmid pOX1
(Whitehead and Hespell 1990) using primers ORFF1 (GCGGGATCCATGGAGCA
TGAATGCGTCA; SEQ ID NO:3 BamHI site in bold) and ORFR1 (CATATGTTA
TATTTTTGAGTMTAAACATTCTAC; SEQ ID NO:4 Ndel site in bold). The MuIL2 and
ORF PCR products were cloned into pGEM-T (Promega) to create plasmids
pGEM-MuIL2 and pGEM-ORF respectively. MuIL2 was removed from pGEM-MuIL2
with Ndel and ligated into Ndel-digested PGEM-ORF to create pORF-MuIL2.
The insert was sequenced to verify the construct. The ORF-MuIL2 construct
was removed from pORF-MuIL2 by BamHI digestion and cloned into the BamHI
site of pBT2 to create pBOMuIL2. This plasmid was transferred into B.
ovatus by conjugation and integration of the plasmid into the genome of
transconjugants was confirmed by PCR. MUIL2-secreting strain BOMuIL2-S. B.
ovatus strain BOMuIL2-S was constructed in the same way as strain BOMuIL2
except that the MuIL2 gene was PCR-amplified using primers BFTSIGIL2F (GACATATGMGAATGTAAAGTTACTTTTAA
TGCTAGGAACCGCGGCATTATTAGCTGCAGCACCCACTTCAAGCTCCAC; SEQ ID NO:5 signal
sequence coding region is underlined, Ndel site in bold) and MuIL2R1. This
led to the creation of plasmids pGEM-MuIL2-S, pORF-MuIL2-S and pBOMulL2-S.
Control strain BT2. The control strain containing pBT2 without the MuIL2
gene was constructed as follows. The same portion of the off gene as used
above was PCR amplified with primers ORFF1 and ORFR2 (GGATCCTTATATTTTTGAGTAAT
AAACATTCTAC; SEQ ID NO:6 BamHI site in bold) and cloned into pGEM-T to
create pGEM-ORFB. The insert was removed with BamHI and cloned into the
BamHI site of pBT2 to create pBT-ORF. This plasmid was transferred into B.
ovatus as described above.
Preparation of Samples of B. ovatus producing MuIL2.
B. ovatus strains V975, BT2, BOMuIL2 and BOMuIL2-S were grown in 10 ml RGM
with or without xylan for 24 h. Strains BOMuIL2 and BOMuIL2-S were also
grown for 16 h without xylan and then with xylan for a further 8 h.
Following incubation, cells were harvested (5000 g, 30 min, 4.degree. C.).
Supernatants were removed and frozen. Cells were washed once in 10 ml RGM
and resuspended in 5 ml distilled water. Cells were disrupted by
sonication on ice for 4.times.20 sec at 12 .mu.m (Soniprep 150, MSE).
Unbroken cells and cell debris were removed by centrifugation (13,000 g,
20 min, 4.degree. C.). Lysates and supernatants were lyophilized and
resuspended in 0.5 ml distilled water.
Assays for Detection of MuIL2
An ELISA incorporating native rat anti-mouse IL2 (clone JES6-1A12) and
biotinylated rat anti-mouse IL2 (clone JES65H4) as capture and detection
antibodies respectively, was used to quantify levels of MuIL2 produced by
recombinant strains of B. ovatus and was carried out according to
manufacturer's instructions (BD Pharmingen). Recombinant MuIL2 (rMulL2;
Sigma) was used as a control to obtain a standard curve. An IL2 bioassay
using the indicator cell line CTLL-2 (Gillis et al. 1978) was used to
detect the presence of biologically active MuIL2 in samples (Wadhwa et al.
2000). Briefly, cells were incubated with dilutions of test samples or
control rMuIL2 in 96-well plates in duplicate for 18 h. Cells were then
pulsed with 0.5 .mu.Ci [.sup.3H]thymidine, harvested after 4 h and the
radioactivity incorporated into DNA estimated by scintillation counting.
The assay was also performed in the presence of an IL2 neutralising
antibody (clone JES6-1A12). This was added to samples at a concentration
of 5 .mu.g ml.sup.-1 1 h before addition of cells.
Detection of MuIL2 transcription by RT-PCR
B. ovatus V975, BT2, BOMuIL2 and BOMuIL2-S were grown in RGM without xylan
for 16 h. A preinduction sample was taken from cultures of BOMuIL2 and
BOMuIL2-S before xylan was added to induce transcription of the xylanase
operon. Samples were taken from all four cultures after 1 h. Total RNA was
extracted from cell samples using the RNeasy kit (Qiagen) followed by
treatment with TURBO DNA-free.TM. (Ambion) to remove any residual
contaminating DNA. RT-PCR was performed using the AccessQuick.TM. RT-PCR
System (Promega) and primers for the orf-Muil2 fusion (CCGATGGTACCTGCCATTAAA
(SEQ ID NO:7) and CTGTGCTTCCGCTGAGG) SEQ ID NO:8 or the gyrA gene (CTCCATGTCGG
TCATCGTTTC (SEQ ID NO:9) and CAAAGGATMCGCATTGCCCA (SEQ ID NO:10)) as a
positive control. As a negative control the reaction was performed without
the addition of reverse transcriptase.
Construction of B. ovatus strains
In order to construct a strain of B. ovatus capable of expressing MuIL2 in
a xylan-inducible manner, the MuIL2 gene (minus native signal sequence)
and 3' portion of the orf gene of the xylanase operon were PCR-amplified
and ligated in pGEM-T to give plasmid pORF-MuIL2. An ATG start codon was
positioned before the sequence encoding the mature MuIL2 as part of an
Ndel site. This ensured translation of the protein. The use of this Ndel
site for cloning resulted in a single base change (G to A) in the
non-coding region between off and the MuIL2 gene compared to the wild-type
region between off and xyl. However, this was not expected to affect MuIL2
expression. The construction of plasmid pBOMuIL2 in FIG. 1 (see Original Patent)
comprises the 3' portion of the B. ovatus off gene and entire MuIL2 gene
amplified by PCR, ligated together in pBluescript then subcloned into pBT2
to create pBOMuIL2. Only restriction sites used for cloning are shown in
the Figure. tet, tetracycline resistance for selection in B. ovatus; kan,
kanamycin resistance for selection in E. coli; oriV, origin of
replication; repA, repB, repC encode replication functions and mob is
required for mobilization from E. coli to B. ovatus. The pBOMuIL2 plasmid
(FIG. 1) was then successfully transferred to B. ovatus V975. The
MuIL2-secreting strain, B. ovatus BOMuIL2-S, was constructed in the same
way except that the forward primer used to PCR-amplify the MuIL2 gene,
contained the sequence coding for the B. fragilis enterotoxin secretion
signal sequence. A control strain, B. ovatus BT2 was also constructed by
cloning only the off gene into pBT2. Successful construction of the MuIL2
and MulL2-S expression strains, and BT2 control strain was confirmed by
PCR and nucleotide sequencing (data not shown).
Claim 1 of 12 Claims
1. A recombinant Bacteroides ovatus gut
commensal bacterium deposited at the National Collection of Industrial,
Food and Marine Bacteria (NCIMB) Accession No. 41521, 41522 or 41523
wherein the bacterium comprises a xylanase promoter that regulates
expression of a polypeptide or protein and wherein the promoter is induced
in the presence of dietary xylan.
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