Method of screening anti-mycobacterial molecules

Details Method of screening anti-mycobacterial molecules Method of screening anti-mycobacterial molecules

1999-10-28

2003-06-03

Swartz, Rodney P (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C424S009200, C424S130100, C424S164100, C424S168100, C424S234100, C424S248100, C435S004000, C435S029000, C435S183000, C435S253100

Reexamination Certificate

active

06573064

ABSTRACT:

BACKGROUND OF THE INVENTION
Tuberculosis and leprosy, caused by the bacilli from the
Mycobacterium tuberculosis
complex and
M. leprae
, respectively, are the two major mycobacterial diseases. Other mycobacteriosis caused by a typical mycobacteria such as
M. avium, M. xenopi
, and
M. Kansasii
also represent major health problems worldwide.
M. avium
is a predominant strain isolated from T. B. patients with AIDS (Horburgh et al., 1991) and
M. xenopi
along with
M. kansasii
and
M. avium
, is the main agent of pulmonary infections due to opportunist mycobacteria in HIV seronegative patients. (M. Picardeau et al., 1995).
In addition, these atypical mycobacteriosis are often difficult to cure because of the lack of efficient drugs specifically directed against atypical mycobacteria. Pathogenic mycobacteria have the ability to survive within host phagocytic cells. The pathology of the tuberculosis infection derives from the interactions between the host and the bacteria, resulting from the damage the host immune response causes on tissues (Andersen & Brennan, 1994). In addition, the protection of the host against mycobacteria infection also depends on interactions between the host and mycobacteria.
Identification of the bacterial antigens involved in these interactions with the immune system is essential for the understanding of the pathogenic mechanisms of mycobacteria and the host immunological response in relation to the evolution of the disease. It is also of great importance for the improvement of the strategies for mycobacterial disease control through vaccination and immunodiagnosis.
Through the years, various strategies have been followed for identifying mycobacterial antigens. Biochemical tools for fractionating and analyzing bacterial proteins permitted the isolation of antigenic proteins selected on their capacity to elicit B- or T-cell responses (Romain et al., 1993; Sorensen et al., 1995). The recent development of molecular genetic methods for mycobacteria (Jacobs et al., 1991; Snapper et al., 1990; Hatful, 1993; Young et al., 1985) allowed the construction of DNA expression libraries of both
M. tuberculosis
and
M. leprae
in the &lgr;gt11 vector and their expression in
E. coli
. The screening of these recombinant libraries using murine polyclonal or monoclonal antibodies and patient sera led to the identification of numerous antigens (Braibant et al., 1994; Hermans et al., 1995; Thole & van der Zee, 1990). However, most of them turned out to belong to the group of highly conserved heat shock proteins (Thole & van der Zee, 1990; Young et al., 1990).
The observation in animal models that specific protection against tuberculosis was conferred only by administration of live BCG vaccine, suggested that mycobacterial secreted proteins might play a major role in inducing protective immunity. These proteins were shown to induce cell-mediated immune responses and protective immunity in a guinea pig or a mouse model of tuberculosis (Pal & Horwitz, 1992; Andersen, 1994; Haslov et al., 1995). Recently, a genetic methodology for the identification of exported proteins based on PhoA gene fusions was adapted to mycobacteria by (Lim et al., 1995). It permitted the isolation of
M. tuberculosis
DNA fragments encoding exported proteins, including the already known 19 kDa lipoprotein (Lee et al., 1992) and the ERP protein similar to the
M. leprae
28 kDa antigen (Berthet et al., 1995).
SUMMARY OF THE INVENTION
We have characterized a new
M. tuberculosis
exported protein name DES, identified by using the PhoA gene fusion methodology. The des gene, which seems conserved among mycobacterial species, encodes an antigenic protein highly recognized by human sera from both tuberculosis and leprosy patients but not by sera from tuberculous cattle. The results of allelic exchange experiments described in this application, indicate that the des gene is essential to the survival of mycobacteria.
The amino acid sequence of the DES protein contains two sets of motifs that are characteristic of the active sites of enzymes from the class II diiron-oxo protein family. Among this family, the DES protein presents significant homologies to soluble stearoyl-acyl carrier protein (ACP) desaturases. Three dimensional modeling demonstrates that the DES protein and the plant stearoyl-ACP desaturase share a conserved active site.
This invention also provides methods of identifying molecules capable of inhibiting the growth and/or survival of Mycobacteria species. In particular, the methods of this invention include screening molecules that can inhibit the activity of the DES protein. These methods comprise the steps of:
a) contacting the molecule with a strain of mycobacteria species containing an active DES protein or a DES like protein or a vector carrying an active DES protein gene or a vector containing a polynucleotide sequence encoding the active site of the DES protein;
b) measuring the inhibition of the growth of said mycobacteria strain; and
c) identifying the molecule that is reacting with the DES protein or with the active site of said protein carrying conserved residues.
To practice the methods of this invention, the purified DES protein may be a recombinant desaturase protein. The recombinant DES protein can be obtained from a recombinant mycobacterium host cell that was transformed with an expression vector containing a polynucleotide encoding the DES protein whose expression is controlled by regulatory sequences that function in mycobacteria. In one method of the invention, the recombinant expression vector is a plasmid derived from the pJAM2 plasmid (e.g. pJAM21). The invention also encompasses the pJAM2 and pJAM21 plasmids, as well as recombinant host cells transformed with the pJAM2 and pJAM21 plasmids. A recombinant host cell transformed with pJAM21 has been deposited at Collection Nationale de Cultures de Micro-organisms (CNCM) in Paris, France, on Jun. 23, 1998, under accession number I-2042.
Another aspect of this invention relates to molecules that have been screened according to the methods of this invention and identified as having antibiotic activity against mycobacteria.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with description, serve to explain the principles of the invention.


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