Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
1997-05-14
2001-04-24
Hobbs, Lisa J. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S006120, C435S320100, C435S252300, C435S254110, C435S325000, C536S023200, C536S024300
Reexamination Certificate
active
06221640
ABSTRACT:
BACKGROUND
The genus Enterococcus has been established as a separate genus from Streptococcus, based on nucleic acid hybridization studies (Schleifer, K. H. and R. Kilpper-Bälz,
Int. J. Syst. Bacteriol.
344:31-34 (1984)). The enterococci include the species
E. faecalis, E. faecium, E. avium, E. casseliflavus, E. durans, E. gallinarum, E. malodoratus, E. raffinosus, B. pseudoavium, E. soliltarius, E. mundtii
, and
E. hirae
(Murray, B. E., “Enterococci,” pp. 1415-1420 In Gorbach, S. L., et al., eds.,
Infectious Diseases
, W. B. Saunders Co., Harcourt Brace Jovanovich, Inc., Philadelphia, 1992).
Enterococcus faecalis
and
Enterococcus faecium
are the two most clinically important strains of the genus Enterococcus, accounting for over 95% of all enterococcal infections. As part of the normal flora of the human bowel and genital tract, the enterococci had not been thought to cause serious infection. In recent years, however, the enterococci have emerged as clinically important pathogens responsible for 5-15% of bacterial endocarditis, 15% of intra-abdominal pelvic and wound infections, 5-10% of spontaneous peritonitis, 5-10% of nosocomial bacteremia and 15% of nosocomial urinary tract infections (ibid). Enterococcal isolates are increasingly responsible for nosocomial infections and are a common cause of morbidity and mortality. Recently they have been cited as the second most common pathogen isolated from hospitalized patients (Schaberg, D. R. et al.,
Am. J. Med.
91:(suppl. 3B) 72S-75S (1991)).
The increase in enterococcal disease is most likely due to an increase in the use of invasive devices, an increase in the number of seriously ill patients and an increase in the use of antimicrobial agents to which enterococci have developed resistance (Nicoletti, G. and Stefani,
Eur. J. Clin. Microbiol. Infect. Dis.
14: (suppl. 1) 33S-37S (1995)).
Enterococci are intrinsically resistant to a large number of antimicrobial agents including beta lactams, polymyxins and lincosamides. In addition, many species have developed resistance to a number of other antimicrobial agents including ampicillin, aminoglycosides, chloramphenicol, erythromycin and vancomycin. Many strains of enterococci now exhibit multiple drug resistance. Some nosocomial isolates of enterococci have displayed resistance to essentially every useful antimicrobial agent, exemplifying the increased difficulty in treating and controlling enterococcal infections (Jones, R. N. et al.,
Diagn. Microbiol. Infect. Dis.
21:95-100 (1995); Jones, R. N. et al.,
Diagn. Microbiol. Infect. Dis.
21:85-93 (1995)).
The incidence of resistance to antimicrobial agents among enterococci is continuing to rise at an alarming rate. The ability of this genus to develop and acquire new resistance has lead, in some cases, to ineffective treatments with agents currently available. The development of a new generation or class of antimicrobial agent is clearly needed to solve the growing threat which enterococcal infections present.
The design of effective antibiotics should exploit the biological differences between the pathogen and host. Designing new antibiotics requires the identification of potential targets in enterococci such as
Enterococcus faecalis
. The search for exploitable differences in the enzymatic pathways of
E. faecalis
and humans is hindered by the limited understanding of the biology of enterococci.
SUMMARY OF THE INVENTION
The invention relates to isolated and/or recombinant nucleic acids which encode Enterococcus (or enterococcal) aminoacyl-tRNA synthetases, including those isolated from naturally occurring enterococci. The invention also relates to recombinant nucleic acid constructs and vectors comprising nucleic acid having a sequence which encodes an enterococcal aminoacyl-tRNA synthetase, or portions of such enzyme. These nucleic acids and DNA constructs can be used in host cells to produce recombinant enterococcal aminoacyl-tRNA synthetases.
A further embodiment of the invention is antisense nucleic acid which can hybridize to the nucleic acid which encodes an aminoacyl-tRNA synthetase of enterococci. In cells, antisense nucleic acid can inhibit the function of a nucleic acid which encodes an aminoacyl-tRNA synthetase of enterococci.
The invention also relates to proteins or polypeptides, referred to herein as isolated and/or recombinant enterococcal aminoacyl-tRNA synthetases, and more specifically, phenylalanyl-, tryptophanyl-, isoleucyl-, leucyl-, tyrosyl-, and seryl-tRNA synthetases. These enzymes are useful in the biochemical separation of the amino acid which they specifically recognize and in quantitations of the amino acid and ATP. Antibodies which bind to these enzymes can be made and can be used in the purification and study of the enzymes.
The above recombinant enterococcal aminoacyl-tRNA synthetases can be produced in host cells described herein. Tester strains, which are cells engineered to rely on the function of the tRNA synthetase encoded by an introduced cloned gene, are also an embodiment of the invention. Tester strains can be used to test the effectiveness of drug candidates in the inhibition of the essential tRNA synthetase enzyme encoded by the introduced cloned gene. The isolated and/or recombinant enterococcal phenylalanyl-, tryptophanyl-, isoleucyl-, leucyl-, tyrosyl-, and seryl-tRNA synthetases can be used in methods for detecting and identifying inhibitors of their activities. In these ways, potential inhibitors of the enzyme can be screened for antimicrobial or antibiotic effects, without requiring the culture of pathogenic strains of Enterococcus, such as
Enterococcus faecalis.
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Weygand-Dura{haeck over (s)}ević, I., et al., “Yeast Seryl-tRNA Synthetase Expressed inEscherichia coliRecognizes Bacterial Serine-Specific tRNAs in vivo,”Eur. J. Biochem., 214:869-877 (1993).
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Avruch Anthony S.
Gallant Paul L.
Nair Shamila
Sassanfar Mandana
Shen Xiaoyu
Cubist Pharmaceuticals, Inc.
Hamilton Brook Smith & Reynolds P.C.
Hobbs Lisa J.
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