Title: Micro-organisms with
glycosylation modulating action of intestinal cell surface
United States Patent: 7,008,785
Issued: March 7, 2006
Inventors: Antoine; Jean-Michel (Maisons-Alfort,
FR); Freitas; Miguel (Paris, FR); Cayuela; Chantal (Paris, FR); Trugnan;
Germain (Montreuil, FR)
Assignee: Compagnie Gervais Danone (Levallois-Perret,
Appl. No.: 332243
Filed: July 4, 2001
PCT Filed: July 4, 2001
PCT NO: PCT/FR01/02147
371 Date: September 11, 2003
102(e) Date: September 11, 2003
PCT PUB.NO.: WO02/02800
PCT PUB. Date: January 10, 2002
Training Courses --Pharm/Biotech/etc.
The invention concerns micro-organism
strains, in particular of lactic acid bacteria, having a glycosylation
modulating effect of intestinal cell surface. The invention also concerns
a method for selecting micro-organism strains, in particular of lactic
acid bacteria, which consists in measuring the average fluorescence
intensity variation of HT29-MTX cells incubated in the presence of a
lectin coupled with a fluorochrome after being in contact with the
supernatant of the strain concerned. Said lactic acid bacteria strains can
be used, optionally in the form of their active fraction, for preparing
food compositions or medicines or food supplements, modulating
glycosylation of glycoproteins of intestinal epithelial cells.
Description of the Invention
FIELD OF THE
The invention concerns microorganisms
having a modulatory effect on surface glycosylation or on the composition
of sugars at the surface of intestinal cells, a method for selecting said
microorganisms, and their uses in the food and medical sectors.
BACKGROUND OF THE
The gastrointestinal mucosa consists of a
simple layer of epithelial cells which are at least partially covered on
the side of the intestinal lumen by a viscoelastic layer mainly consisting
of glycoconjugates. The epithelial cells synthesize the glycoconjugates
present at their surface, which are the intermediates in numerous
interactions, in particular with lectins or adhesins, with bacterial
toxins, or with antibodies, bacteria, viruses, parasites. These
glycoconjugates therefore constitute important intermediates in the
relationship between the host and the intestinal flora.
These glycoconjugates are, as their name indicates, glycosylated
compounds, that is to say on which are grafted sugar chains which are long
to a greater or lesser degree and which may be branched. In the healthy
human intestine, these sugars may be galactose (Gal), fucose (Fuc), N-acetylneuraminic
acid or sialic acid, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNac),
linked to each other by various bonds. Three of them may be in a terminal
position; they are galactose, fucose and sialic acid.
A host and its intestinal microflora function as a complex system in which
the microflora has a significant impact on the host. In the case of
nonpathogenic or probiotic microorganisms, a symbiotic or cooperative
relationship often exists between the host and the microorganisms, the
presence of the latter being necessary for good balance and good
functioning of the host's intestine. By contrast, the presence of
pathogenic microorganisms, which is more rare, may have negative
consequences by preventing or reducing the presence of probiotic
microorganisms, or even by having a parasitic action which is directly
harmful to the host.
The fragile balance between the host and the microflora is directly linked
to the intestinal environment and in particular to the quantitative and
qualitative presence of the surface glycoconjugates. Indeed, it is known
that some microorganisms, which may be probiotic or pathogenic, will be
sensitive to certain sugars at the terminal position. The intestinal
bacteria can modulate the pattern of glycosylation of the glycoconjugates
present at the surface of the intestinal cells, without their mode of
action being completely elucidated as will be seen hereinafter. The
bacteria can, on the one hand, induce the presence of such and such sugar
during glycosylation and, on the other hand, break down the sugars present
at the terminal position of a chain, which qualitatively and/or
quantitatively modifies the sugars present at the terminal position.
This modulation of the pattern of epithelial cell surface glycosylation
causes modification of the intestinal environment, it being possible for
the modified environment to promote the establishment of certain
microorganisms and/or to limit, or even avoid, the establishment of other
microorganisms. Modification of the environment, which may be generated by
the microflora, therefore has a direct impact on the balance between the
host and the microflora.
It would therefore be highly advantageous to have a model which makes it
possible to rapidly and easily select microorganisms according to their
action on the pattern of glycosylation. It would thus be possible to
identify microorganisms promoting the presence of one sugar or another,
and therefore promoting the establishment of beneficial microorganisms
such as, for example, probiotics and/or limiting the establishment of
Indeed, to the knowledge of the applicant, no microorganisms, in
particular lactic acid bacteria, have so far been identified which are
capable of finely acting accurately on the glycosylation pattern, and thus
of accurately modulating this glycosylation pattern and therefore the
implantation of microorganisms.
Such microorganisms modulating the glycosylation pattern could find a use
in particular in pharmaceutical or food compositions or food supplements.
Indeed, they can make it possible to optimize the functioning of the
intestinal cells and the good balance of the microflora.
The document WO 99/29833 describes a novel bacterial strain having certain
properties, for example an antimicrobial activity. However, the modalities
of its action are not detailed, and in particular the question of the
possible action of the bacterium on sugars is never addressed.
Bry L. et al., in "A model of host-microbial interactions in an open
mammalian ecosystem", Science, Vol. 273, pp. 1380-1383, Sep. 6, 1996,
studied in vivo (in mice) the influence of Bacteroides thetaiotaomicron
on fucosylation. Bacteroides thetaiotaomicron is part of the
intestinal flora in humans and in mice. The model used is nevertheless
limited to the study of fucosylation, with the exclusion of other types of
glycosylation. It is moreover an in vivo model, and therefore longer and
more complex to use than an in vitro model.
However, this model made it possible to emit the hypothesis of the
existence of a soluble factor secreted by B. thetaiotaomicron, and
which is thought to play the role of a signal bringing about the
modification of surface glycosylation. This unidentified soluble factor is
therefore thought to act without there being direct contact between the
bacteria and the target cells.
To the knowledge of the applicant company, no method has been developed to
date which makes it possible to select in vitro, easily and rapidly,
various microorganisms, in particular various strains of lactic acid
bacteria, according to their precise action on the pattern of
glycosylation of the intestinal epithelial cells, and more precisely on
the modulation or variation of the composition of each of the sugars.
In the context of the present application, the term "lactic acid bacteria"
denotes bacteria capable of producing lactic acid, and in particular the
nonpathogenic bacteria chosen from the group comprising Streptococcus,
Lactobacillus, Lactococcus, Bifidobacterium and Leuconostoc, in
particular B. breve, B. longum, L. lactis, S. thermophilus, L. casei,
L. helveticus and L. bulgaricus.
DESCRIPTION OF THE INVENTION
A subject of the present invention is
therefore such a method of selection in vitro, which comprises the
- bringing the culture supernatant for
the strain of microorganisms, in particular of lactic acid bacteria,
into contact with cells of a cell line model representing the intestinal
- extracting the cells and incubating
with various lectins coupled to a fluorochrome,
- measuring the mean fluorescence
intensity (MFI) of each lectin, comparing with the measurements
performed on a control sample in order to evaluate the variation in MFI
induced by the bacteria, selecting the strain of microorganisms inducing
a variation of at least 20% for at least one sugar.
The latter step may in particular be carried out on plates, the
measurement of MFI being performed by a probe, according to techniques
known to persons skilled in the art, or by flow cytometry. In the case of
flow cytometry, it is also possible to compare the graph obtained,
representing the variation of fluorescence as a function of the number of
cells, with that obtained with a control sample, and to select the strain
of microorganisms inducing a variation in the shape of the graph.
Any cell line model representing intestinal epithelial cells may be used
in this method of selection. Persons skilled in the art will know how to
choose an appropriate cell line model among the known models, for example
the lines Caco-2 or HT29-MTX, which are cancerous intestinal epithelial
cells in culture. These lines are considered as reproducing all or some of
the mechanisms of regulation of the systems in vivo and in particular the
production of glycoconjugates. Any line exhibiting this characteristic may
be used in the method according to the invention. As regards the HT29-MTX
line, reference may be made to Lesuffleur et al., "Growth adaptation to
methotrexate of HT-29 human colon carcinoma cells is associated with their
ability to differentiate into columnar absorptive and mucus-secreting
cells", 1990, Cancer Res., 50:6334-6343.
Centrifugation and filtration of the culture medium in which each strain
of microorganisms to be tested has been incubated are carried out in order
to extract the ("trial") supernatant therefrom. Without wishing to be
bound by any theory, the applicant considers that the supernatant contains
the soluble factor constituting the signal for modulating glycosylation.
The supernatant therefore constitutes an active fraction of a strain of
microorganisms. Brought into contact with the intestinal epithelial cells,
it will cause or not one or another modification of glycosylation
according to the strain of microorganisms tested.
Thus, in Bacteroides, the soluble factor was identified as being a
low molecular weight molecule (less than 8 kD) and that it is
heat-sensitive. It can be assumed that the soluble factor from another
strain will exhibit similar, if not identical, characteristics.
The supernatant obtained from the culture medium which has not been in
contact with the bacteria is used as a "control".
An appropriate quantity of the "trial" and "control" supernatants are
incorporated into the culture medium in which the cells of the cell line
model, for example HT29-MTX, are placed for an appropriate period. This
period may be, for example, from 1 to 15 days.
In a particular embodiment, it is possible to add bacteria in place of the
supernatant. In this case, the bacteria may be added in the form of a
suspension in the presence of penicillin G and of a strong buffer making
it possible to inhibit bacterial development without preventing the
viability of the bacteria, and acidification of the medium.
After this period of incubation, the cells of the cell line model are
detached and resuspended, and the same number of "trial" and "control"
cells is incubated separately with a lectin of which the specificity
toward sugars (nature of the sugar, nature of the linkage) is known.
Each lectin, which is coupled to a fluorochrome, will bind to the sugar
for which it is specific.
The "trial" and "control" cell suspensions are each washed several times,
and the reactivity of each lectin is quantified by fluorescence
measurements called MFI (mean fluorescence intensity).
This measurement may be carried out by any appropriate means, in
particular by means of flow cytometry (FACScan, Bencton-Dickinson) or by
direct fluorimetric measurements on plates.
When flow cytometry is used, a graph representing the variation of
fluorescence as function of the number of cells is obtained for each
lectin. For each flow cytometry graph, the mean fluorescence intensity
corresponds to the median value.
Two comparisons are possible. On the one hand, regardless of the mean of
the measurement of fluorescence intensity, it is possible to compare
quantitatively the "trial" MFI with the "control" MFI. On the one hand, in
the case of flow cytometry, it is also possible to compare qualitatively
the "trial" result with the "control" result, and more precisely to
compare the graph obtained with the "trial" suspension with that obtained
with the "control" suspension, that is to say to compare the shape of the
It is estimated that a decrease or an increase in this MFI of at least
about 20%, preferably at least about 28%, still more preferably at least
about 35%, reveals a significant change in the reactivity of each lectin,
and therefore of the sugar composition of the glycoproteins present at the
surface of the epithelial cells.
This method therefore makes it possible to observe and to quantify in a
simple and rapid manner the presence of each lectin on the sugar chains,
and therefore to quantify the sugar whose lectin is specific. It is then
possible to compare the values obtained without and with microorganisms,
and thus to evaluate the glycosylation modulating effects of each strain
of microorganisms, in order to select the strains having the desired
effect: increase in such a sugar, decrease in another.
The subject of the invention is also the strains of microorganisms, in
particular of lactic acid bacteria, capable of modulating the pattern of
intestinal epithelial cell surface glycosylation, and more particularly
the strains of microorganisms, in particular of lactic acid bacteria,
capable of being selected by the method described above.
Such microorganisms therefore make it possible to modulate glycosylation,
and therefore to reestablish or modify the intestinal environment in order
to promote the establishment of probiotic microorganisms and to avoid or
limit the establishment of pathogenic microorganisms.
It is known, for example, that the receptor allowing adhesion of
Escherichia coli contains galactose and sialic acid; for
Streptococcus pyogenes and Listeria monocytogenes, it is
galactose; for Helicobacter pylori, it is fucose; and for
Entamoeba histolytica, it is galactose and N-acetylgalactosamine.
The strains which are the subject of the invention may be in particular
the following strains: CNCM collection No. I-2492, CNCM collection No.
I-2493, CNCM collection No. I-2494 (CNCM=Collection Nationale de Cultures
de Micro-organismes-Institut Pasteur-28, rue du Dr. Roux-75724 Paris Cedex
15-France), deposited on 20 Jun. 2000.
It is possible to use such microorganisms, in particular such lactic acid
bacteria, or their active fraction, that is to say the fraction comprising
the soluble factor, for the preparation of food compositions or
medicaments or food supplements modulating intestinal epithelial cell
surface glycosylation. These compositions may comprise a single strain, or
several strains, of microorganisms modulating glycosylation.
Thus, compositions are available which make it possible to maintain or
reestablish a proper host-intestinal flora balance, or which make it
possible to promote the establishment of one or another strain of
microorganisms. They therefore make it possible to optimize the
functioning of the intestinal cells and good balance of the microflora. It
is possible in particular to use lactic acid bacteria, and to incorporate
them into dairy products.
Claim 1 of 1 Claim
1. A biologically pure
Bifidobacterium animalis strain of lactic acid bacteria deposited under
the number CNCM I-2494.
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