Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1995-06-07
2001-08-07
Minnifield, Nita (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S320100, C435S173300, C435S173300, C435S069100, C435S242000, C435S091200, C530S300000, C530S350000, C530S328000, C530S388100, C530S387100, C530S389500
Reexamination Certificate
active
06271017
ABSTRACT:
Helicobacter pylori
(also described by the expression
H. pylori
) is a Gram negative bacterium found exclusively nowadays at the surface of the stomach mucosa in man, and more particularly around the lesions of the craters due to gastric and duodenal ulcers. This bacterium was initially called
Campylobacter pyloridis
(Warren et al. (1983) Lancet 1. 1273-1275).
Like most bacteria,
H. pylori
is sensitive to a medium of acidic pH but can however tolerate acidity in the presence of physiological levels of urea (Marshall et al. (1990). Gastroenterol. 99: 697-702). By hydrolysing urea to carbon dioxide and ammonia which are released into the microenvironment of the bacterium, the urease of
H. pylori
is assumed to permit the survival of the bacterium in the acidic environment of the stomach. Recently, studies conducted on animal models have provided elements suggesting that urease is an important factor in the colonization of the gastric mucosa.
Helicobacter pylori
(
H. pylori
) is presently recognized as the etiological agent of antral gastritis, and appears to be one of the cofactors required for the development of ulcers. Furthermore it seems that the development of gastric carcinomas may be linked to the presents of
H. pylori.
All of the strains isolated in the clinic from biopsies or gastric juice synthesize a very active urease, which is exposed at the surface of the bacterium and is one of the most immunogenic proteins of
H. pylori.
The urease is suspected of playing a role in the pathogenic process, a fact which has been confirmed by experiments performed on the pig which show that weakly producing urease strains obtained by chemical mutagenesis were incapable of colonizing the stomach of the pig. These results obtained after chemical mutagenesis do not make it possible to attribute with certainty the diminution or urease production to an inability to colonize the stomach, since other genes may be inactivated during generalized mutagenesis. Hence these are not controllable mutations and, consequently, this procedure has no real value in the creation of agents designed to diminish, and even prevent, the harmful effects of urease in the case of an infection by
H. pylori.
In addition to this role in the colonization of the stomach, it has been shown that urease as well as the ammonia released might have a direct cytotoxic effect on epithelial cells and an indirect effect by inducing an inflammatory response which might be responsible for the gastric lesions.
The urease hence is one of the most important determinants of pathogenecity and the construction of isogenic strains of
H. pylori
specifically inactivated in the genes responsible for the expression of urease, whether they be structural genes or accessory genes, are of primary importance for defining the role of urease in the colonization step, and for use in the construction of strains which can be used to protect individuals in a vaccination process, for example by the construction of attenuated strains.
Hitherto the urease genes had been localized on a 34 kb fragment of the
H. pylori
chromosome and had been associated with a 4.2 kb region present in this fragment. Four genes designated by the terms ureA, ureB, ureC and ureD had been associated with this region of 4.2 kb. This region led to the production of a urease-positive phenotype when the DNA of 4.2 kb was transferred by the intermediary of a shuttle vector to
Campylobacter jejuni.
However, the transformation of
E. coli
cells with the DNA of 4.2 kb previously described did not lead to the expression of urease activity in
E. coli.
The inventors have succeeded in defining the elements which, both genetically and from the point of view of growth conditions, are necessary for the expression in
E. coli
of a urease activity such as that obtained in
H. pylori
. In this regard, they have established that the expression of urease in
E. coli
was dependent on both the activation of the nitrogen regulatory system of
E. coli
and the presence of accessory genes to the urease structural genes. They have identified and isolated several genes which will sometimes be designated subsequently by the expression urease “accessory genes” which permit the functional expression or urease in
E. coli
and specify the maturation and regulation or urease in
H. pylori.
Hence the invention relates to a set of five novel determinant genes or which are at least likely to be implicated in the functional expression of urease in
H. pylori
and in
E. coli
, as well as each of these genes considered in isolation and independently of the other genes. It also relates to this set of genes, optionally modified, in combination with the urease structural genes designated by ureA, ureB, ureC and ureD and described in the publication (Labigne et al. (1991) J. Bacteriol. 173: 1920-1931)
Furthermore, the invention relates to novel agents for the in vitro detection of an infection due to
H. pylori
, as well as to compositions which can be used for protection against infection by
H. pylori.
Hence the object of the invention is a nucleotide sequence characterized in that it is constituted by or in that it comprises at lease one of the nucleic acid sequences corresponding to the genes called ureE, ureF, ureG, ureH, ureI and represented by the nucleotide sequences presented below:
or any part of at least one of these nucleic acid sequences.
A nucleotide sequence according to the invention is constituted either by DNA or by RNA.
The invention also relates to a nucleotide sequence modified with respect to the nucleotide sequence described above by deletion, addition, substitution or inversion of one or more nucleotides such that the functional properties of the polypeptides encoded in these genes are either conserved or attenuated, or even deleted, in comparison with the properties of the polypeptides UreE, UreF, UreG, UreH or UreI such as expressed by
H. pylori
, or such that this sequence does not express a polypeptide in
H. pylori.
According to a particular embodiment of the invention and in the context of the preceding definition, a nucleotide sequence is characterized in that it is constituted by or in that it comprises:
a) the set of nucleotide sequences corresponding to the genes called ureE, ureF, ureG, ureH, ureI and represented by the nucleotide sequences shown in
FIG. 4
or,
b) the set formed by the (variant) nucleotide sequences corresponding to these genes modified independently of each other such that the set of these variants codes for polypeptides having a functional homology with the polypeptides UreE, UReF, UreG, UreH or UreI such as expressed by
H. pylori
or, on the other hand, codes for modified peptides which attenuate or even suppress the functional properties of the polypeptides UreE, UreF, UreG, UreH or UreI such as expressed by
H. pylori.
Fragments (nucleotide sequences) of the above nucleotide sequences are of interest for different reasons and as examples it is possible to define:
fragments of the above-mentioned sequences which have conserved the capacity to code for polypeptides having a functional homology with the peptides such as obtained by expression of a gene selected from ureE, ureF, ureG, ureH, ureI in
H. pylori;
fragments coding for any part of the above polypeptides such as produced in
H. pylori
, and in particular coding for peptides or parts of polypeptides recognized by antibodies directed against
H. pylori
or capable of behaving as haptens or immunogens;
fragments of the above-mentioned sequences lacking the capacity to code for the polypeptides of
H. pylori
such as expressed by the genes ureE, ureF, ureG, ureH, and ureI;
fragments coding for polypeptides or peptides having properties attenuated or even deleted in comparison with the properties of the polypeptides encoded in the genes ureE, ureF, ureG, ureH, ureI of
H. pylori.
Such fragments have advantageously at least 15 nucleotides, and preferably at least 20 nucleotides.
The genes ureE, ureF, ureG, ureH, ureI are present on a
H. pylori
chromosome; these genes are s
Cussac Valerie
Ferrero Richard
Labigne Agnes
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Minnifield Nita
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