Lantibiotic from Streptococcus mutans, method of production...

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai

Reexamination Certificate

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Reexamination Certificate

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06699839

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel antibiotic produced by
streptococcus mutans,
salts thereof, pharmaceutical compositions containing same, and to methods for the production and extraction thereof.
BACKGROUND OF THE INVENTION
Bacteriocins produced by Gram-positive bacteria can be defined as inhibitory substances having a bactericidal mode of action and an essential protein moiety (Jack et al., 1995, Microbiol. Rev. 59:171-200). Bacteriocins produced by
Streptococcus mutans
were termed mutacins by Hamada and Ooshima (Hamada et al, 1975, Arch. Oral Biol. 20:641-648). Although there are many reports which show that
S. mutans
produces inhibitory substances, only a few such inhibitors have been isolated and characterized as mutacins (Chikindas et al., 1995, Antimicrob. Agents Chemother. 39:2656-2660; Delisle, 1986, Microbios 46:21-28; Fukushima et al., 1985, Arch. Oral Biol. 30:229-234; Hamada et al., 1986, Zentralbl. Bakteriol. Hyg. A. 261:287-298; Hillman et al., 1984, Infect. Immun. 44:141-144; Kurita et al., 1988, J. Gen. Microbiol. 134:213-220; Loyola-Rodriguez et al., 1992, J. Gen. Microbiol. 138:269-274; Novak et al., 1994, J. Bacteriol. 176:4316-4320; Novák et al., 1996, Anal. Biochem. 236:358-360; Paul et al., 1975, Infect. Immun. 12:1375-1385; Takada et al., 1984, Infect. Immun. 44:370-378). The well studied mutacins include: RM-10 (Fukushima et al., 1985, Arch. Oral Biol. 30:229-234), C3603 (Takada et al., 1984, Infect. Immun. 44:370-378), JH 1000 (Hillman et al., 1984, Infect. Immun. 44:141-144), GS-5 (Paul et al., 1975, Infect. Immun. 12:1375-1385), MT 3791 and MT 6223 (Hamada et al., 1986, Zentralbl. Bakteriol. Hyg. A. 261:287-298;Loyola-Rodriguez et al., 1992, J. Gen. Microbiol. 138:269-274), mutacin-b (Delisle, 1986, Microbios 46:21-28), mutalipocins (Kurita et al., 1988, J. Gen. Microbiol. 134:213-220) and J-T8 (Chikindas et al., 1995, Antimicrob. Agents Chemother. 39:2656-2660; Novák et al., 1994, J. Bacteriol. 176:4316-4320;Novák et al., 1996, Anal. Biochem. 236:358-360).
A preliminary classification of mutacins has been published previously (Morency et al., 1995, Can. J. Microbiol. 41:826-831), but a definitive classification will have to await the complete chemical characterization of these substances. Among the known mutacins, only T8, which belongs to group J (Morency et al., 1995, Can. J. Microbiol. 41:826-831), was identified as a lantibiotic and its first 8 N-terminal amino acid residues were determined by Novák et al. (Novák et al., 1994, J. Bacteriol. 176:4316-4320). Lantibiotics are defined as bacterium-derived ribosomally synthesized lanthionine-containing peptides with antibiotic activity (Jack et al., 1995, Microbiol. Rev. 59:171-200; Bierbaum et al., 1993, Zentralbl. Bakteriol. 278:1-22;Jack et al., 1995, Trends Biotechnol. 13:269-278). They generally contain unsaturated amino acids like 2,3-didehydroalanine (dhA or U) (2)-2,3-didehydrobutyrine (dhB or O), and 2-aminobutyric acid (Abu). The lantibiotics are divided into two types (Jack et al., 1995, Microbiol. Rev. 59:171-200; Bierbaum et al., 1993, Zentralbl. Bakteriol. 278:1-22; Jack et al., 1995, Trends Biotechnol. 13:269-278; Jung, 1991, in: Nisin and Novel Lantibiotics., Jung et al., eds., pp. 1-34. E
SCOM
Science, Leiden). Type A comprises screw-shaped, amphipathic molecules with molecular masses between 2151 and 4635 Da and with 2 to 7 net positive charges. Type B consists of more globular molecules with molecular masses between 1825 and 2042 Da and with either no net charge or a net negative charge. They usually contain a higher proportion of modified amino acid residues than type A.
Only four streptococcal strains were shown to produce a lantibiotic: Streptococcus pyogenes producing streptococcin A-FF22 (Jack et al., 1994, Eur J Biochem 220:455-462),
Streptococcus salivarius
producing salivaricin A (Ross et al., 1993, Appl. Environ. Microbiol. 59:2014-2021),
Streptococcus mutans
T8 producing mutacin J-T8 (Parrot et al., 1990, Can. J. Microbiol. 36:123-130; Parrot et al., 1992, Congrès de I'AMQ, p. 22; Novak et al., 1993, ASM meeting Abstr. No. A-44, p. 8; Novak et al., 1994, J. Bacteriol 196:4316-4320).
Streptococcus mutans
strain B-Ny266 has been previously shown to inhibit 98% of the
S. mutans
strains tested (Morency et al., 1995, Can. J. Microbiol. 41:826-831) and all tested Gram-positive bacteria including
Listeria monocyto genes, Clostridium sporogenes, Mycobacterium phleï, Staphylococcus aureus, Enterococcus faecalis
and
Bacillus subtilis
(Parrot et al., 1990, Can. J. Microbiol. 36:123-130). A preliminary characterization of the antibacterial effect of this strain permitted to hypothesize that at least one mutacin was responsible therefor. However, the mutacin(s) of B-Ny266 has yet to be purified. Furthermore, it remains to be determined whether the wide antibacterial activity associated with strain B-Ny266 is attributable to a single proteinaceous substance or shared by two or more proteinaceous substances therein (Morency et al., 1995, Can. J. Microbiol. 41:826-831; and references therein). It follows, that the determination of the amino sequence of the antibacterial substance(s) present in B-Ny266 had yet to be carried out.
There thus remains a need to purify to homogeneity the bacterial substance(s) carrying the demonstrated antibacterial activity present in
S. mutans
strain B-Ny266. There further remains a need to identify the amino acid sequence of this substance.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference.
SUMMARY OF THE INVENTION
The invention concerns the purification to homogeneity of the proteinaceous substance having bactericidal activity produced by
Streptococcus Mutans
B-Ny266. More particularly, the present invention concerns the purification to homogeneity of a lantibiotic type A produced by
Streptococcus mutans
B-Ny266, hereinafter termed mutacin B-Ny266.
The invention also relates to the amino acid sequence of mutacin B-Ny266. More particularly, the invention relates to the primary amino acids sequence of pro-B-Ny266 the precursor protein of mutacin B-Ny266: F-K-S-W-S-F-C-T-P-G-C-A-K-T-G-S-F-N-S-Y-C-C (SEQ ID NO:1). Further, the present invention relates to pro-B-Ny266 sequence being posttranslationnally modified to yield the proposed sequence: F-K-
A
-W-U-F-
A
-
Abu
-P-G-
A
-A-K-O-G-
A
-F-N-
U
-Y-
A
(SEQ ID NO:4; underlined residues participate in lanthionine or b-methyllanthionine bridges).
The present invention further relates to methods of purifying to homogeneity Mutacin B-Ny266 from
S. mutants
strain B-Ny266. Further, the invention relates to methods of purifying to homogeneity type A mutacins from
S. mutants
being close relatives to mutacin B-Ny266. More particularly, the invention relates to methods for the preparation of mutacin B-Ny266 and pharmaceutically acceptable salts thereof comprising cultivating microorganisms capable of expressing mutacin B-Ny266, preferably
S. mutants
B-Ny266, under condition whereby mutacin B-Ny266 is expressed, isolated, secreted and extracted from cell pellets of the strain expressing B-Ny266 in the culture medium, and if desired, converting the isolated mutacin B-Ny266 to a pharmaceutically acceptable salt thereof,
S. Mutans
B-Ny266 has been deposited at the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Md.) on Aug. 22, 1997 and granted the accession number ATCC 202022.
The invention in addition relates to pharmaceutical compositions comprising mutacin B-Ny266 and pharmaceutically acceptable carriers or excipients. Further, the invention relates to pharmaceutical compositions comprising as active ingredient, mutacin B-Ny266 or a pharmaceutically acceptable salt thereof, and associated with a pharmaceutically acceptable carrier or excipient.
The present invention therefore further relates to a purified preparation of mutacin B-Ny266. More particularly, the invention relates mutacin B-Ny266 purified to more

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