Lantibiotic

Details Lantibiotic Lantibiotic

2001-12-21

2004-08-10

Meller, Michael (Department: 1654)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C424S093440

Reexamination Certificate

active

06773912

ABSTRACT:

This invention relates to lantibiotics, organisms producing such lantibiotics, and to the uses of both the organisms and lantibiotics produced therefrom.
BACKGROUND
Bacterial infection in humans is a problem of both considerable personal concern, and economic importance in the heath field.
Streptococcal infections are particularly prevalent causing ailments ranging from dental caries and minor throat infections to serious diseases such as scarlet fever, rheumatic fever and acute glomerulonephritis.
In order to reduce the incidence of streptococcal infections, it is desirable to control or prevent the growth of the harmful causative bacteria. One approach towards this is to provide bacteriocins and like substances (including lantibiotics) active against streptococci and organisms capable of producing such substances, which are suitable for use in controlling or preventing the growth of harmful streptococci bacteria.
A number of bacteriocins are known. Examples of bacteriocins derived from gram positive bacteria are given in Tagg et al, (1976),
Bacteriol Rev
. Vol. 40, pp 722-756. Further examples of such bacteriocins are lacticin 481 from
Lactobacillus lactis
(Piard et al, (1992),
Applied and Environmental Microbiology
, Vol. 58, pp 279-284), variacin from
Micrococcus varians
(U.S. Pat. No. 5,981,261) and the bacteriocins from
Streptococcus thermophilus
described in EP 0643136.
Streptococcus salivarius
has also long been known to have a high incidence of lantibiotic production (Dempster RP et al (1982)
Arch Oral Biol
27:151). One lantibiotic which has been characterised from
S. salivarius
is salivaricin A (Ross et al (1993)
Appi Envir Microbiol
59:2014). However, while demonstrating inhibitory activity against a number of streptococcal species, the activity was bacteriostatic rather than bacteriocidal. Salivaricin A and microorganisms which produce it therefore do not provide a complete answer to controlling streptococcal infections.
The applicants have now identified a further antibacterial protein from
S. salivarius
This protein, which the applicants have found to have bacteriocidal efficacy rather than being simply bacteriostatic, is the primary focus of the present invention.
SUMMARY OF THE INVENTION
Accordingly, in one aspect, the present invention may broadly be said to consist in an antibacterial protein which can be isolated from
S. salivarius strains K
12 and K30, which has a molecular mass of approximately 2733 Da as determined by ion-spray mass spectrometry, and the N-terminal amino acid sequence Gly-Gly-Gly-Val-Ile-Gln, or an antibacterial fragment or variant thereof which has greater than 80% amino acid sequence homology with the protein.
The protein of the invention has been termed by the applicants “salivaricin B”, with the full nucleotide and amino acid sequences being given in
FIG. 2
, together with the sequences of a portion of the leader peptide.
Conveniently, salivaricin B is obtained by expression of a DNA sequence coding therefor in a host cell or by cult g producer strains
S. salivarius K
12 or K30.
In a further aspect, the invention provides an antibacterial composition which includes a protein as defined above or an organism which can express a protein as defined above.
In still a further aspect, the invention provides a therapeutic formulation comprising salivaricin B as defined above or an antibacterial fragment or variant thereof in combination with a diluent, carrier and/or excipient.
In yet a further aspect, the invention provides a therapeutic formulation comprising an organism capable of expressing salivaricin B as defined above, or an antibacterial fragment or variant thereof, in combination with a diluent, carrier and/or excipient.
Preferably, said organism is capable of expressing salivaricin B alone or in combination with a secondary antibacterial agent.
More preferably, said secondary antibacterial agent is a BLIS; most preferably salivaricin A2. The full nucleotide and amino acid sequences for Salivaricin A2 are given in
FIG. 3
, together with the sequences of the leader peptide.
Conveniently, said organism is selected from
S. salivarius
strains K12 and K30.
In a particularly preferred embodiment, the therapeutic formulations are in the form of foods or drinks, most preferably in the form of dairy product-based foods or drinks.
Alternative forms are medicaments and confectioneries.
In still a further aspect, the invention provides an organism which expresses salivaricin B.
Preferably, said organism is selected from
S. salivarius
strains K12 and K30.
In still further aspects of the invention, there are provided methods of treating an individual to at least inhibit growth of harmful streptococcal bacteria in the upper respiratory tract comprising the step of administering an effective amount of salivaricin B orally to said individual.
Preferably, said salivaricin B is administered as part of a therapeutic composition.
Conveniently, m said method said inhibitory effect is caused by colonising at least part of the upper respiratory tract of an individual with a viable non-pathogenic organism which expresses salivaricin B.
Preferably, said organism is administered as part of a food or drink.
More preferably, said organism is a
S. salivarius
strain selected from strains K12 and K30.
In yet a further embodiment, said method includes a preliminary step of pre-treating said individual to at least reduce the bacterial population present in the upper respiratory tract.
Preferably, said pre-treatment comprises the step of administering an antibiotic, preferably erythromycin, orally to said individual.
In yet a further embodiment, the invention provides a method of treatment of a patient against infections of the upper respiratory tract caused by Streptococcal organisms which comprises the steps of:
(i) orally administering to said patient an amount of an antibiotic effective to reduce the numbers of streptococci present; and
(ii) administering, to the resulting bacterially depopulated environment, BLIS producing
S. salivarius
organism(s) to repopulate said environment
Although the invention is broadly as described above, it will be appreciated by those persons skilled in the art that the invention is not limited thereto but also includes embodiments of which the following description gives examples, together with the aspects fully defined in the appended claim set.


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patent: 5872001 (1999-02-01), Caufield et al.
patent: 2268744 (1998-04-01), None
Caufield et al., Computer Sequence Search Display, WO 9817685, 1998.*
Piard et al., “Structure, Organization, and Expression of thelctGene for Lacticin 481, a Novel Lantibiotic Produced byLactococcus lactis”; J. Biol. Chem. 1993 Aug. 5; 268(22):16361-68.
John Tagg, “Prevention-Still Better Than a Cure”, Microbiology Australia, Jul. 1996, pp. 18-20.
Kalmokoff, et al., “Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal AnaerobeButyrivibrio fibrisolvensOR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A”, Applied and Environmental Microbiology, vol. 65, No. 5, May 1999, pp. 2128-2135.
Ross, et al., “Isolation and Characterization of the Lantibiotic Salivaricin A and Its Structural GenesalAfromStreptococcus salivarius20P3”, Applied and Environmental Microbiology, vol. 59, No. 7, Jul. 1993, pp. 2014-2021.

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