Mycobacterium tuberculosis superoxide dismutase

Details Mycobacterium tuberculosis superoxide dismutase Mycobacterium tuberculosis superoxide dismutase

1999-11-12

2003-02-11

Swartz, Rodney P (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S141100, C424S146100, C424S150100, C424S164100, C424S168100, C424S234100, C435S252100, C435S253100, C435S863000, C435S864000, C435S865000, C435S866000, C514S924000, C530S300000, C530S350000

Reexamination Certificate

active

06517845

ABSTRACT:

BACKGROUND OF THE INVENTION
Superoxide dismutase catalyzes the conversion of superoxide radicals (O
2
−
) into molecular oxygen (O
2
) and hydrogen peroxide (H
2
O
2
). The conversion of superoxide radicals is generally beneficial to a cell, since such molecules can react with the cell's genomic DNA to induce mutations.
Superoxide dismutases (SOD) have been classified based on the inorganic atoms they require for activity. Three SOD families have been identified: those requiring manganese (MnSOD), those requiring iron (FeSOD), and those requiring copper and zinc (Cu, ZnSOD).
MnSODs have been found in mitochondria and prokaryotes, whereas FeSODs have been found in prokaryotes, primitive eukaryotes, and some plants. Cu, ZnSODs were originally found in eukaryotes and later found in several bacterial.
Macrophages are an important arm of a vertebrate's immune system. Such cells can kill pathogens such as bacteria by engulfing the pathogen and bombarding it with superoxide radicals. Therefore, a secreted Cu, ZnSOD may play a role in the survival of bacterial pathogens, especially those known to survive and grow in macrophages.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a secreted Cu, ZnSOD in
Mycobacterium tuberculosis
. It has been found that antibodies which specifically bind this
M. tuberculosis
SOD are useful in detecting the presence of the bacterium. It has also been discovered that tuberculosis patients develop antibodies against the
M. tuberculosis
Cu,ZnSOD. Thus, a patient producing antibodies against
M. tuberculosis
Cu,ZnSOD is diagnostic for tuberculosis in that patient.
Accordingly, the invention features an antibody, such as a monoclonal antibody, which specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, which is the amino acid sequence of the
M. tuberculosis
Cu,ZnSOD. Specific binding of an antibody to the polypeptide means that it does not substantially bind to other components within a sample. A Cu,ZnSOD or copper/zinc superoxide dismutase is a polypeptide that facilitates conversion of superoxide radicals to molecular oxygen and hydrogen peroxide, and whose superoxide dismutase activity is dependent on the presence of copper and zinc atoms or ions.
The invention also includes a method of detecting
M. tuberculosis
infection in a mammal by (1) providing a polypeptide comprising the amino acid sequence of SEQ ID NO:2; (2) contacting the polypeptide with a biological sample (e.g., a human serum sample) collected from the mammal, the contacting performed under conditions sufficient to allow an antibody to bind to the polypeptide; and (3) determining the presence of antibody bound to the polypeptide, wherein the presence of the antibody indicates
M. tuberculosis
infection in the mammal. This method optionally includes the step of removing antibodies which do not bind to the polypeptide.
The invention also features a method of testing whether a compound inhibits superoxide dismutase activity of a polypeptide by (1) contacting a polypeptide with the compound, the polypeptide being a Cu,ZnSOD (also called a copper/zinc superoxide dismutase) and having an amino acid sequence which is at least 50% (e.g., at least 60, 70, 80, 90 or 100%) identical to SEQ ID NO:2; (2) measuring the level of superoxide dismutase activity; and (3) comparing the level of superoxide dismutase activity in the presence of the compound with the level of superoxide dismutase activity in the absence of the compound. The compound is said to inhibit the superoxide dismutase activity of the polypeptide when the level of superoxide dismutase activity in the presence of the compound is lower than the level of superoxide dismutase activity in the absence of the compound. The polypeptide can be within a cell such as a bacterium, e.g., in the periplasm of the bacterium.
To facilitate the detection or testing methods of the invention, the polypeptide can be bound to a solid support (e.g., a plastic support such as a microtiter plate). In addition, the polypeptide can be covalently bound to a solid support bead such as Sepharose. The covalent linkage between the polypeptide and a support can be achieved by methods well known in the art. For example, the polypeptide can be covalently linked to a support by reacting it with chemically activated forms of the support (e.g., CNBr-activated Sepharose 4B or EAH Sepharose 4B, available from Pharmacia). In addition, equal amounts of the polypeptide can be deposited in each well of a microtiter plate, thereby creating an array on which multiple compounds can be tested in parallel or multiple samples can be assayed for the presence of
M. tuberculosis.
DETAILED DESCRIPTION
The invention relates to an antibody useful for detecting
M. tuberculosis
in a sample, methods of detecting
M. tuberculosis
infection in a mammal, and methods of testing a compound for its ability to inhibit SOD activity. These aspects of the invention arise from the discovery of a novel Cu,ZnSOD produced by
M. tuberculosis.
I. Polypeptides
The Cu,ZnSOD polypeptides useful in the methods of the invention include the
M. tuberculosis
Cu,ZnSOD polypeptide described below. The Cu,ZnSOD useful in the methods of the invention are not limited to the naturally occurring sequence. Cu,ZnSOD containing substitutions, deletions, or additions can also be used, provided that those polypeptides retain at least one activity associated with the naturally occurring polypeptide and are at least 50% identical to the naturally occurring sequence.
To determine the percent identity of two polypeptide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100).
The determination of percent homology and identity between two sequences can be accomplished using a mathematical algorithm. An example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al., Proc Natl Acad Sci USA 87:2264-2268 (1990), modified as in Karlin et al., Proc Natl Acad Sci USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J Mol Biol 215:403-410 (1990). BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules useful in the methods of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers et al., CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences is determined using any of the above-described techniques with allowances for gaps. In calculating percent identity, only exact matches are counted.
An example of a Cu,ZnSOD that is not naturally occurring, though useful in the methods of the invention, is a Cu,ZnSOD-glutathione-S-transferase fusion prote

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