Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-05-23
2003-08-19
Housel, James (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023200, C536S023700, C530S350000, C435S069100, C435S320100, C435S471000, C435S069200, C435S472000, C435S005000, C424S185100, C424S190100, C424S234100, C424S244100, C424S009100
Reexamination Certificate
active
06608187
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the identification of C
1
bacteriophage genes that express protein involved in the lysis of bacterial cells during the phage life cycle, lysin and holin. The invention further relates to methods for lysing certain bacteria using lysin, which are useful for example in the treatment of an oral cavity bacterial infection.
BACKGROUND OF THE INVENTION
All double stranded DNA bacteriophages contain a lytic system consisting of a holin and at least one peptidoglycan hydrolase or “lysin”, which is capable of degrading the bacterial cell wall to allow phage release. In Gram positive bacteria, lysins can be endo-p-N-acetylglucosaminidases or N-acetylmuramidases (lysozymes), which act on the sugar moiety; an endopeptidase, which acts on the peptide cross bridge; or more commonly, an N-acetyhnuramoyl-L-alanine amidase, which hydrolyzes the amide bond connecting the sugar and peptide moieties (for review, see (Young et al., 2000)). Typically, the holin which is expressed in the late stages of infection forms a pore in the cell membrane allowing the lysin to gain access to the cell wall peptidoglycan and resulting in release of progeny phage. Exogenously added lysin can lyse the cell wall of healthy, uninfected cells, producing “lysis from without”.
The virulent C
1
bacteriophage specifically infects group C streptococci and produces a very powerful lysin (Evans, 1934; Maxted, 1957). Interestingly, the C
1
phage lysin has hydrolytic activity against cell walls of group A streptococci as well as group C streptococci (Cohen et al., 1975; Fischetti et al., 1971; Raina, 1981). This unique activity has been exploited as a tool in group A streptococcal studies to isolate surface molecules including M protein (Cohen et al., 1977; Fischetti et al., 1985), to extract DNA from lysed cells, and to make protoplasts when used in a hypertonic solution (Wheeler et al., 1980). The cell wall lysis activity of C
1
lysin is also potentially very useful in the treatment of oral cavity bacterial infections.
It is to be noted that the direct introduction of bacteriophages into an animal to prevent or fight diseases has certain drawbacks. Specifically, the bacteria must be in the right growth phase for the phage to attach. Both the bacteria and the phage have to be in the correct and synchronized growth cycles. Additionally, there must be the right number of phages to attach to the bacteria; if there are too many or too few phages, there will either be no attachment or no production of the lysing enzyme. The phage must also be active enough. The phages are also inhibited by many things including bacterial debris from the organism it is going to attack. Further complicating the direct use of bacteriophage to treat bacterial infections is the possibility of immunological reactions, rendering the phage non-functional.
Despite these drawbacks, attempts have been made to treat bacterial diseases with by the use of bacteriophages. U.S. Pat. No. 5,688,501 discloses a method for treating an infectious disease caused by bacteria in an animal with lytic or non-lytic bacteriophages that are specific for particular bacteria. U.S. Pat. No. 4,957,686 discloses a procedure of improved dental hygiene by introducing into the mouth bacteriophages parasitic to bacteria that readily adheres to the salivary pellicle.
C
1
lysin containing compositions are also useful in the topical treatment of wounds and burns to the skin. U.S. Pat. No. 6,056,954 discloses methods of the treatment of these injuries by the administration of partially purified C
1
lysin to a patient. U.S. Patent No. 6,056,955 discloses methods of treating dermatological infections caused by streptococcal bacteria by administering a semipurified C
1
lysin.
U.S. Pat. No. 5,985,271 discloses administration of therapeutic amounts of semipurified lysin to a patient to treat a streptococcal infection of the mouth, throat or nasal passages. U.S. Pat. No. 6,017,528 discloses semipurified C
1
lysin compositions for prophylactic and therapeutic treatment of streptococcal infection. However, these compositions comprised relatively impure preparations of the enzyme: the soluble fraction of C
1
bacteriophage infected bacterial cell lysates comprises a number of factors in addition to lysin. Thus, these methods suffer from a disadvantage of uncertainty of the amount of lysin enzyme administered, the presence and nature of any contaminants, and the resulting variability in dosages and safety profiles of these lysin preparations. Furthermore, these preparations have lower specific activity values (units of enzyme/gram of protein) due to the presence of other bacterial proteins. Substantially purified material is much more defined than the semipurified material disclosed in the prior art and is therefore much more desirable for ingestable therapeutic compositions. There is a need in the art for highly purified lysin so that it can be characterized, cloned, and used therapeutically.
SUMMARY OF THE INVENTION
The present invention is directed to a homogeneously purified C1 bacteriophage lysin protein (lysin) and a homogeneously purified holin protein (holin). Furthermore, the present invention is directed to both a specifically disclosed lysin and holin, as disclosed in SEQ ID NO:2 and SEQ ID NO:4, respectively. The present invention also includes proteins which are about 70% identical to the amino acid sequences as set forth in SEQ ID NO:2 and SEQ ID NO:4, as well as to pharmaceutical compositions comprising homogeneously purified lysin.
This invention also includes an isolated nucleic acid molecule encoding a Cl bacteriophage lysin protein and an isolated nucleic acid molecule encoding a C1 bacteriophage holin protein. Also included in the present invention are nucleic acids which encode amino acid sequences which are about 70% identical to the amino acid sequence set forth in SEQ ID NO:2 or the amino acid sequence set forth in SEQ ID NO:4. Specifically disclosed nucleic acid molecules whose nucleotide sequences are set forth in SEQ ID NO:1 and SEQ ID NO:2 are also included in this invention.
An expression vector comprising the C1 lysin gene and an expression vector comprising the C1 holin gene are included in the invention, as are host cells comprising one of these expression vectors.
A method of producing lysin protein or holin protein comprising culturing a host cell as mentioned above under conditions which allow the expression of lysin or holin are included in the invention. This method may further comprise purification of lysin or holin to homogeneity.
The present invention also includes a method of degrading streptococci susceptible to lysin. This method involves contacting the bacterial cell wall with lysin. These streptococci may include group A or C streptococci. A method of treating or preventing a streptococcal infection in a patient comprising administration of a pharmaceutical composition comprising lysin is also a part of the invention. The infection treated by this method may include oral cavity infections and may include administration of a lysin containing pharmaceutical composition directly to the oral cavity. Methods of coadministration of a second therapeutic agent in addition to the lysin containing pharmaceutical composition are also a part of this invention.
Anti-holin or an anti-lysin antibodies are included in the present invention.
The present invention also includes a method for detecting group C or group A streptococcal antigens. This method comprises detecting the group C or A streptococcal antigen in a clinical specimen suspected of containing group C or A streptococci contacted with an extraction reagent comprising a lytic amount of homogeneously purified lysin enzyme, thereby releasing group C or A streptococcal antigen into the extraction reagent using an immunological ligand receptor assay.
REFERENCES:
patent: 4957686 (1990-09-01), Norris
patent: 5604109 (1997-02-01), Fischetti et al.
patent: 5688501 (1997-11-01), Merril et al.
patent: 5985271 (1999-11-01), Fischetti et al.
patent: 6017528 (20
Fischetti Vincent A.
Nelson Daniel C.
Darby & Darby
Housel James
Li Bao Qun
The Rockefeller University
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