Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
2001-08-21
2004-08-10
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S190100, C424S192100, C424S193100, C424S234100, C424S243100, C435S183000, C435S191000, C435S212000, C435S220000, C530S350000
Reexamination Certificate
active
06773706
ABSTRACT:
BACKGROUND OF THE INVENTION
General Background and State of the Art: This invention is directed to an enzyme from Gram-positive bacteria, designated sortase-transamidase, nucleic acid segments encoding the enzyme, and methods of use of the enzyme.
Human infections caused by Gram-positive bacteria present a medical challenge due to the dramatic increase in multiple antibiotic resistance strains in recent years. Gram-positive bacteria that can cause serious or fatal infections in humans include Staphylococcus, Streptococcus, Enterococcus, Pneumococcus, Bacillus, Actinomyces, Mycobacterium, and Listeria, as well as others. Infections caused by these pathogens are particularly severe and difficult to treat in immunologically compromised patients. These include patients suffering from infection with the Human Immunodeficiency Virus (HIV), the virus that causes AIDS, as well as patients given immune-suppressive agents for treatment of cancer or autoimmune diseases. In particular, infections caused by various Mycobacterium species, including
M. tuberculosis, M. bovis, M. avium
, and
M. intracellulare
, are frequently the cause of disease in patients with AIDS.
Therefore, it is apparent that new target sites for bacterial chemotherapy are needed if such pathogenic organisms are to be controlled.
A unique characteristic of these pathogens and many Gram-positive bacteria is their surface display of proteins anchored to the cell wall. In fact, many of these molecules are known to be involved in essential cellular functions, including pathogenesis in a susceptible host. Thus, a possible disruption in this anchoring process may prove to be an effective treatment against these disease-causing elements.
The anchoring of surface molecules to the cell wall in Gram-positive bacteria has been demonstrated to involve a conserved pathway, culminating in recognition of a conserved cleavage/anchoring site by some previously uncharacterized cellular machinery. Molecules whose ultimate location is the cell wall must invariably be translocated across the single cellular membrane of these organisms. This is mediated for all cell wall anchored proteins by the well studied secretory pathway, involving cleavage of an amino-terminal signal peptide by a type I signal peptidase. Upon translocation of the molecule out of the cytoplasm, a mechanism must be present that extracellularly recognizes this protein as a substrate for anchoring. This process has been previously shown to involve the carboxyl-terminally located cell wall sorting signal, consisting of a highly conserved motif such as LPXTG (SEQ ID NO:1), in which X can represent any of the twenty naturally occurring L-amino acids, followed by a series of hydrophobic residues and ultimately a sequence of positively-charged residues. Thus, once amino-terminally modified and successfully secreted, a polypeptide with this carboxyl-terminal sequence can present itself as a substrate to be processed by the anchoring machinery. At this time, cleavage of the sorting signal after the threonine residue is coupled with covalent linkage of the remainder of the polypeptide to the free amino group of the pentaglycine crossbridge in the cell wall.
It is this transpeptidation reaction that anchors mature surface proteins to the peptidoglycan layer, from which point the molecules can serve their biological functions. Therefore, there is a need to isolate and purify the enzymes that catalyze this reaction. There is also a need to identify the genes encoding such enzymes in order that the enzymes can be produced by genetic engineering techniques.
Additionally, there is also a need to develop new methods for displaying proteins or peptides on the surfaces of bacteria. For many purposes, it is desirable to display proteins or peptides on the surfaces of bacteria so that the proteins or peptides are accessible to the surrounding solution, and can, for example, be bound by a ligand that is bound specifically by the protein or peptide. In particular, the display of proteins on the surface of bacteria is desirable for the preparation of vaccines, the linkage of molecules such as antibiotic molecules or diagnostic reagents to cells, for screening reagents such as monoclonal antibodies, and for the selection of cloned proteins by displaying the cloned proteins, then observing their reaction with specific reagents such as antibodies. One way of doing this has been with phage display (G. P. Smith, “Filamentous Fusion Phage: Novel Expression Vectors that Display Cloned Antigens on the Virion Surface,”
Science
228:1315-1316 (1985)). However, phage display is limited in its practicality, because it requires that the protein being displayed to be inserted into a coat protein of filamentous phage and retain its activity while not distorting the conformation of the coat protein, allowing functional virions to be formed. In general, this technique is therefore limited only to small peptide and proteins.
Therefore, there is a need for a more general method of peptide and protein display.
INVENTION SUMMARY
The present invention is directed to sortase-transamidase enzymes from Gram-positive bacteria, particularly the products of the surface protein sorting genes (srtA and srtB) of
Staphylococcus aureus
, and methods for their use, particularly in the areas of drug screening and peptide and protein display and as targets for bacteriocidal compounds or antibiotics.
One aspect of the present invention is a substantially purified sortase-transamidase enzyme from a Gram-positive bacterium, the enzyme catalyzing a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, the sorting signal having a motif of LPX
3
X
4
G therein, wherein sorting occurs by cleavage between the fourth and fifth residues of the LPX
3
X
4
G motif. Typically, the Gram-positive bacterium is a species selected from the group consisting of but not limited to
Staphylococcus aureus, S. sobrinus, Enterococcus faecalis, Streptococcus pyogenes
, and
Listeria monocytogenes.
Preferably, the Gram-positive bacterium is
S. aureus
, and more preferably, the enzyme is the product of the srtA gene or the srtB gene of
S. aureus.
Preferably, the enzyme has a molecular weight of about 23,539 or about 29, 076 daltons and the sorting signal further includes: (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif; and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif, wherein X
3
is any of the twenty naturally-occurring L-amino acids and X
4
is selected from the group consisting of alanine, serine, and threonine.
The enzyme includes an amino acid sequence of: (1)
M-K-K-W-T-N-R-L-M-T-I-
A-G-V-V-L-I-L-V-A-A-Y-L-F-A-K-P-H-I-D-N-Y-L-H-D-K-D-K-D-E-K-I-E-Q-Y-D-K-N-V-K-
E-Q-A-S-K-D-K-K-Q-Q-A-K-P-Q-I-P-K-D-K-S-K-V-A-G-Y-I-E-I-P-D-A-D-I-K-E-P-V-Y-P-
G-P-A-T-P-E-Q-L-N-R-G-V-S-F-A-E-E-N-E-S-L-D-D-Q-N-I-S-I-A-G-H-T-F-I-D-R-P-N-Y-
Q-F-T-N-L-K-A-A-K-K-G-S-M-V-Y-F-K-V-G-N-E-T-R-K-Y-K-M-T-S-I-R-D-V-K-P-T-D-V-
G-V-L-D-E-Q-K-G-K-D-K-Q-L-T-L-I-T-C-D-D-Y-N-E-K-T-G-V-W-E-K-R-K-I-F-V-A-T-E-V-
K (SEQ ID NO:3)
and (2) sequences incorporating one or more conservative amino acid substitutions in SEQ ID NO:3, wherein the conservative amino acid substitutions are any of the following: (1) any of isoleucine, leucine, and valine for any other of these amino acids; (2) aspartic acid for glutamic acid and vice versa; (3) glutamine for asparagine and vice versa; and (4) serine for threonine and vice versa.
Alternatively, the enzyme can include an amino acid sequence of: (1)
M-R-M-K-
R-F-L-T-I-V-Q-I-L-L-V-V-I-I-I-I-F-G-Y-K-I-V-Q-T-Y-I-E-D-K-Q-E-R-A-N-Y-E-K-L-Q-Q-K-
F-Q-M-L-M-S-K-H-Q-A-H-V-R-P-Q-F-E-S-L-E-K-I-N-K-D-I-V-G-W-I-K-L-S-G-T-S-L-N-Y-
P-V-L-Q-G-K-T-N-H-D-Y-L-N-L-D-F-E-R-E-H-R-R-K-G-S-I-F-M-D-F-R-N-E-
Liu Gwen
Mazmanian Sarkis
Schneewind Olaf
Ton-That Hung
Dreger Ginger R.
Heller Ehrman White & McAuliffe LLP
Navarro Mark
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