Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-09-10
2003-02-18
Nashed, Nashaat T. (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C435S253400, C530S324000, C536S023700
Reexamination Certificate
active
06521596
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to polypeptide antibiotics and to the identification of genetic loci associated with expression of the antibiotics. The invention particularly describes a purified lanthionine-containing antimicrobial agent, DNA encoding the protein, and methods and compositions for treatments employing the antibiotic.
2. Description of Related Art
The phenotypic ally similar group of bacteria collectively known as the mutans streptococci are considered the major etiologic agents responsible for dental caries and have been implicated as major causative agents in other infectious and transmissible diseases, such as endocarditis. The species most commonly associated with dental caries is
Streptococcus mutans
. Attempts to better understand the genetic determinants that contribute to the cariogenic potential of this organism, as well as its natural history as an agent of an infectious disease, have only recently been explored. For example, a molecular approach has been employed to delineate the structure, function, and regulation of a number of different enzymes including the glucosyltransferases that are involved with sugar metabolism, an important virulence factor responsible for the metabolic conversion of sucrose to extracellular polymers to form critical mass and provide for fermentable substrates (Hamada, et al., 1986).
The other two major factors thought to be involved with the pathogenicity of
S. mutans
are its acidogenic/aciduric properties (Caufield, et al., 1990A) and its ability to elaborate poorly characterized bacteriocin-like substances, generally known as mutacins, which may provide a selective force necessary for sustained colonization in a milieu of densely packed competing organisms found in plaque (Buchman, et al., 1988). Collectively called “mutacins” these agents kill other bacteria of the same or closely related species. The mutacins are only similar in name and host producer, as their properties differ widely. Mutacins associated with plasmid-containing strains of
S. mutans
have been designated as either Group I or II (mutacin I and mutacin II. Therefore, the production of mutacins is one characteristic of
S. mutans
that appears to contribute to its ability to colonize and be sustained, particularly in the oral cavity. In this regard, mutacins may be considered virulence factors.
The mutans streptococci produce several different bacteriocin-like inhibitory substances, collectively called mutacins. To date, most remain only partially characterized (Hamada, et al., 1986; Loyola, et al., 1992). Reasons for limited success in characterization of these substances are likely to be that: 1) they are made in small quantities; 2) production occurs only under special cultivation conditions; 3) a lack of production in liquid media; and 4) difficulty in isolating these ‘mutacins’ from the media.
Bacteriocins are traditionally defined as proteinaceous substances capable of exerting lethal or bactericidal effects on other bacteria within the same species or against closely related species (Tagg, et al., 1976). Among the gram-positive bacteria, however, this definition becomes somewhat less precise because inhibitory substances exhibit a wider spectrum of activity. Moreover, bacteriocins are not always proteins and do not fit the traditional definition originally intended for the better studied gram-negative bacteriocins, e.g., colicin. Recent studies involving the molecular characterization and sequencing of several gram-positive bacterial inhibitory polypeptides, including nisin, epidermin, pep5 and subtilin, among others, reveal ribosomally translated, but post-translationally processed peptides having modified amino acids and thioether linkages. Collectively, these polypeptide antibiotics are termed lantibiotics or lanthionine-containing antibiotics (Allgaier, et al., 1986; Buchman, et al., 1988). It is now clear that mutacin II group belongs to the lanthionine-containing family of antibiotics.
A few characteristics of certain mutacins have been determined. “Mutacin” activity appears to be associated with the presence of a 5.6 kb plasmid in
S. mutans
; however mutacin production in plasmid-containing strains of mutans streptococci is not plasmid-encoded (Caufield, et al., 1990A). Among strains of
S. mutans
harboring a 5.6 kb plasmid, at least two distinct, but closely related mutacin producing/immunity groups exist. Efforts have consequently focused on locating and characterizing the chromosomal locus responsible for mutacin expression. Initial attempts to identify the gene(s) responsible for mutacin expression employed transpositional mutagenesis using transposon Tn916 as the mutagen (Caufield, et al., 1990B). Five different chromosomal loci associated with mutacin expression have been identified.
Unfortunately, very little is known about the many described extracts with “mutacin” activity, such as their composition and under what conditions they are made. Because the compositions investigated were made in small quantities and only under certain poorly defined conditions, attempts to partially isolate and biochemically characterize “mutacins” have yielded various results. Past studies, primarily using crude cell-free extracts, led to the description of mutacins with different molecular weights and differential sensitivity to pH and digestive enzymes (Caufield, et al. (1985, 1990A, 1990B), Loyola-Rodriguez, et al. (1992), Delisle (1986), Pinto Alves, et al. (1992)). The relevance and usefulness of these results are particularly confounded by the lack of purity of the compositions.
A recognized need is the development of antimicrobials effective against both a broad and a defined, but limited range of microorganisms. The present state of knowledge concerning mutacin-type antibiotics suggests that these compounds have potential as a class of antibacterials. However, owing to the lack of purity of the different mutacins and the inability to obtain such compounds in sufficient quantities, specific mutacins have yet to be clearly identified or developed for further use.
SUMMARY OF THE INVENTION
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing purified mutacin compositions and methods for the treatment of microbial infections in animals, including humans. The mutacin antimicrobials are isolated from
Streptococcus mutans
and show activity against several species of gram-positive bacteria. The invention also provides for the molecular cloning of the mutacin gene.
As used herein, “mutacin” is used to designate an antimicrobial agent isolatable from
S. mutans
and characterized by the properties disclosed herein. These properties include an apparent molecular weight of about 2,500 Da as determined by HPLC-size exclusion chromatography, thermostability up to 100° C., resistance to a pH range of about 4-10, an isoelectric point of >8.4 and insolubility in water-immiscible organic solvents such as CHCl
3
. The term “isolatable from” as used herein, is intended to describe the various sources from which a mutacin in accordance with the present invention may be obtained. Appropriate. sources are considered to be virtually all
S. mutans species, with T
8 and UA96 being particularly preferred, and also recombinant host cells engineered to express mutacin.
It is recognized that the literature describes impure compositions containing several mutacins elaborated by
S. mutans
and related species. However these “mutacin” containing compositions are different from the mutacin described herein in several important respects, including physical and biological properties, in addition to the significant factor of their purification.
The first area of disparity is in the predicted molecular weight characteristics of “mutacins”. Parrot et al. (1990) describe four “mutacin extracts” with M
r
's ranging in the about 3500 Da to <12,000 Da as measured by dialysis, while Delisle (1986) describes a mutacin with an M
r
of between 3500 and 6000 Da also by dialysi
Caufield Page W.
Novak Jan
Fronda Christian L.
Gifford, Krass, Groh Sprinkle, Anderson & Citkowski, P.C.
Nashed Nashaat T.
The UAB Research Foundation
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