Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Statutory Invention Registration
1999-08-19
2002-05-07
Jordan, Charles T. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S071200
Statutory Invention Registration
active
H0002021
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of a gene, pbp-nv2, encoding a novel high molecular weight penicillin binding protein (PBP), PBP-Nv2, from
Streptococcus pneumoniae
and the use of the gene and its encoded protein in a screen for new inhibitors of bacterial cell wall biosynthesis.
BACKGROUND
The emergence of antibiotic resistance in common pathogenic bacterial species has justifiably alarmed the medical and research communities. The emergence and rapid spread of beta-lactam resistance in
Streptococcus pneumoniae
has been particularly problematic. This organism is responsible for many respiratory tract infections, and resistance to beta-lactam drugs has been attributed to a modification of one or more of the penicillin-binding proteins (PBPs). Furthermore, penicillin-resistant
Streptococcus pneumoniae
are frequently resistant to other commonly used antibiotics, such as erythromycin. These multi-drug resistant (MDR) organisms are a real threat to humans, particularly children and the elderly. Increasingly, the only drug that can be used to treat infections with MDR organisms is vancomycin, and there is considerable concern that the bacteria could also develop resistance to vancomycin.
The PBPs are involved in bacterial cell wall synthesis. The cell wall comprises a peptidoglycan layer which provides mechanical rigidity for the bacterium. The peptidoglycan layer is composed of a sugar backbone (alternating residues of N-acetylglucosamine and N-acetylmuramic acid) attached to a pentapeptide (also referred to as “stem peptide”) containing D and L amino acid residues. In the formation of the mature peptidoglycan, a lipid-linked disaccharide-pentapeptide is translocated across the cytoplasmic membrane, exposing the pentapeptide sidechains to the cell surface. Transglycosylation of the sugar residues then leads to polymerization of the backbone sugar residues. Further stabilization of the nascent peptidoglycan occurs by a transpeptidation enzymatic reaction that crosslinks adjacent pentapeptide moieties. The high molecular weight PBPs catalyze these final steps in peptidoglycan synthesis. Without the crosslinking step the peptidoglycan structure is severely weakened and susceptible to degradation. Indeed, the crosslinking step constitutes the target of action for antibiotic compounds such as penicillin and other beta-lactam drugs.
When effective as antibiotic agents, beta-lactam drugs interact with PBPs to form an acyl-enzyme intermediate. This intermediate is resistant to hydrolysis. Mechanistically, beta-lactam drugs act as irreversible inhibitors. Resistance to beta-lactam drugs in
Streptococcus pneumoniae
arises through mutation events such that one or more low-affinity “mosaic” PBPs replace a wild-type PBP. The molecular basis of resistance has in a few cases been correlated with specific mutations within a PBP gene. The discovery of new antibacterial compounds against the transpeptidase domain of PBP-Nv2 or to an unexploited target (e.g. the transglycosylase domain) would be particularly useful against
Streptococcus pneumoniae
infections.
SUMMARY OF THE INVENTION
The present invention is designed to meet the aforementioned need and provides, inter alia, isolated nucleic acid molecules that encode a novel PBP from
Streptococcus pneumoniae
. The invention also provides protein products encoded by the gene, in substantially purified form.
Having the cloned pbp-nv2 gene of
Streptococcus pneumoniae
enables the production of recombinant PBP-Nv2 protein and derivatives thereof for the implementation of assays and screens to identify new inhibitory compounds targeted at the peptidoglycan biosynthetic pathway.
In one embodiment the present invention relates to the isolated gene pbp-nv2 that encodes a novel
Streptococcus pneumoniae
PBP, PBP-Nv2,. The gene comprising the nucleotide sequence is identified as SEQ ID NO. 1.
In another embodiment the present invention relates to a novel protein molecule, PBP-Nv2, wherein said protein molecule comprises the sequence identified as SEQ ID NO. 2.
In another embodiment, the present invention relates to a soluble form of PBP-Nv2 (designated PBP-Nv2
S
) wherein PBP-Nv2
S
comprises amino acid residues 85 through 821, inclusive, of SEQ ID NO.2.
In a further embodiment the present invention relates to a ribonucleic acid molecule encoding PBP-Nv2 protein, said ribonucleic acid molecule comprising the sequence identified as SEQ ID NO. 3:
In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates the
Streptococcus pneumoniae
pbp-nv2 gene in operable linkage to gene expression sequences enabling said PBP gene to be transcribed and translated in a host cell.
In still another embodiment the present invention relates to homologous or heterologous host cells that have been transformed or transfected with a vector carrying the cloned pbp-nv2 gene from
Streptococcus pneumoniae
such that said gene is expressed in the host cell.
In a still further embodiment, the present invention relates to a method for identifying compounds that bind the
Streptococcus pneumoniae
PBP-Nv2 protein or fragment thereof.
REFERENCES:
Hakenbeck et al (J. Bacteriology, vol. 180, No. 7, pp. 1831-1840, 1998.*
Ellerbrok et al (European Journal of Biochemistry, vol. 144, pp. 637-641, 1984.*
Lehninger (Principles of Biochemistry, Worth Publishers, Inc., New York, NY, Sally Anderson, June Fox, Eds, 1982.*
Potgieter et al (Current Microbiology, vol. 24, pp. 289-294, 1992.*
El Kharroubi et al (Biochemical Journal vol. 280, pp. 63-469, 1991.*
Wu et al (Antimicrobial Agents and Chemotherapy vol. 36(3) pp. 533-539, 1992.*
Reichmann P, Konig A., Linares J., Alcaide F., Tenover FC., McDougal L., Swidsinski S., Hakenbeck R., “A global gene pool for high-level cephalosporin resistance in commensal Streptococcus species andstreptococcus pneumoniae”, Journal of Infectious Diseases, 176(4): 1001-12, (Oct. 1997).
Stingele F., Mollet B., “Disruption of the gene encoding penicillin-binding protein 2b (pbpb) causes altered cell morphology and cease in exopolysaccharide production inStretococcus thermophilusSfi6”,Molecular Microbiology, 22(2): 357-66, (Oct. 1996).
Coffey TJ., Dowson CG., Daniels M., Spratt BG., Spratt BG., “Horizontal spread of an altered penicillin-binding protein 2B gene betweenStreptococcus pneumoniaeandStreptococcus oralis”, FEMS Microbiology Letters, 110(3): 335-9, (Jul. 1, 1993).
Martin C., Briese T., Hakenbeck R., “Nucleotide sequences of genes encoding penicillin-binding proteins fromStreptococcus pneumoniaeandStreptococcus oraliswith high homology toEscherichia colipenicillin binding proteins 1a and 1b”, Journal of Bacteriology, 174(13): 4517-23, (Jul. 1992).
Hoskins JoAnn
Jaskunas, Jr. Stanley Richard
Rockey Pamela Kay
Zhao Genshi
Cohen Charles E.
Eli Lilly and Company
Jordan Charles T.
Parker III Raymond S.
Thomson M.
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