Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-09-30
2001-10-30
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007320, C435S069100, C435S320100, C536S023100, C536S023400
Reexamination Certificate
active
06309817
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of identifying substances that combat infections and diseases caused by prokaryotes, and more particularly to methods of identifying substances that exhibit anti-bacterial/anti-microbial effects.
BACKGROUND OF THE INVENTION
As a consequence of the widespread use and perhaps even misuse of antibacterial drugs, strains of drug-resistant pathogens have emerged. Antibiotic-resistant bacterial strains have been associated with a variety of infections, including tuberculosis, gonorrhea, staphylococcal and pneumococcal infections, and the bacteria most commonly associated with pneumonia, ear infections and meningitis. More importantly, infectious disease remains the largest cause of mortality in the world.
The typical response to an ineffective antibiotic has simply been change antibiotics. Unfortunately, this alternative no longer offers a guarantee of success. For example, certain strains of enterococci are resistant to vancomycin—a drug formerly considered to be the ultimate weapon against many different types of bacteria. The World Health Organization has expressed concern that the development of new drugs is not keeping pace with the numbers of antibiotics which become ineffective. World Health Report 1996: Fighting Disease, Fostering Development, Executive Summary (World Health Organization 1996). Despite ongoing research, there remains a pressing need to develop new antibiotics. There is also a need for antibacterials that are effective in treating disease while not stimulating the emergence of resistant strains.
Bacteria respond to nutritional stress by the coordinated expression of different genes. This facilitates their survival in different environments. Among these differentially regulated genes are the genes responsible for the expression of virulence determinants. The expression of these genes in a sensitive or susceptible host allows for the establishment and maintenance of infection or disease. Virulence genes encode toxins, colonization factors and genes required for siderophores production or other factors that promote this process.
Virulence genes in bacteria express a variety of factors that allow the organism to invade, colonize and initiate an infection in humans and/or animals. These genes are not necessarily expressed constantly (constitutively), however. That is, the bacterium is not always “infectious”. In many circumstances, the expression of virulence genes is controlled by regulatory proteins known as repressors in conjunction with a corresponding operon(s) or operator(s). In prokaryotes, one class of repressors is activated upon binding to or forming a complex with a transition metal ion such as iron or zinc. When the repressor is activated, it binds the operator thereby preventing production of virulence determinants.
Virulence determinants are most often expressed when the bacterial pathogen is exposed to nutritional stress. An iron-poor environment is an example of such a condition. In this environment, insufficient iron is present to maintain the repressor in its active state. In the inactive form, the repressor cannot bind to target operators. As a result, virulence genes are de-repressed and the bacterium is able to initiate, establish, promote or maintain infection.
The expression of these virulence determinants is in many bacterial species is co-regulated by metal ions. In many instances the metal co-factor that is involved in vivo is iron. In the presence of iron, the repressor is activated and virulence gene expression is halted.
This pattern of gene regulation is illustrated by the following example. The bacterium that causes diphtheria produces one of the most potent toxins known to man. The toxin is only produced under conditions of iron deprivation. In the presence of iron, the bacterial repressor (which in this species is known as diphtheria toxin repressor protein, abbreviated “DtxR”) binds iron and undergoes conformational changes that activate it and allow it to bind a specific DNA sequence called the tox operator. The tox operator is a specific consensus DNA sequence found upstream of the gene that produces the diphtheria toxin. Binding of DtxR to this site thereby prevents toxin expression. Typically, during infection of a human or animal host the diphtheria bacillus (or other pathogenic/opportunistic bacteria) grows in an environment that rapidly becomes restricted in several key nutrients. Paramount among these essential nutrients is iron, and when iron becomes limiting the diphtheria bacillus begins to produce the toxin. Moreover, the constellation of virulence genes that DtxR controls becomes de-repressed and the diphtheria bacillus becomes better adapted to cause an infection. In the case of diphtheria, the toxin kills host cells thereby releasing required nutrients including iron.
SUMMARY OF THE INVENTION
The present invention is directed to a simple and accurate method for identifying substances that repress or prevent or attenuate virulence gene expression in an infectious microorganism and phenotypically convert it to a non-pathogen. The method may be practiced to identify substances effective against any pathogenic (infectious) prokaryote whose pattern of virulence determinant expression (or a portion thereof) is under the regulatory control of a metal ion-dependent repressor. Thus, Applicants' invention can be employed to identify substances that provide a therapeutic or medicinal benefit to humans and animals.
A first aspect of the present invention is directed to a method for screening test substances to identify non-metal ion activators of a metal ion-dependent repressor of virulence determinants expression in a virulent or opportunistic prokaryotic pathogen. Substances that are identified as activators of the repressor may be developed as antibiotics or anti-bacterial substances. Accordingly, this method involves:
(a) providing recombinant cells comprising a first recombinant DNA segment containing a first promoter operably linked to a first regulatory gene encoding a first repressor native to or functional in a given prokaryote, a second DNA segment containing a second promoter operably linked to a first operator that binds said first repressor and a second regulatory gene encoding a second repressor, and a third recombinant DNA segment comprising a third promoter operably linked to a second operator that binds the second repressor, and a reporter gene;
(b) culturing said recombinant cells in medium substantially free of metal ion activators of said first repressor and which contains a selection agent that directly or indirectly causes a detectable response upon expression or lack of expression of the reporter gene;
(c) adding a non-metal ion test substance to said medium; and
(d) determining whether the response occurs as an indication of whether said test substance activates said first repressor.
In preferred embodiments, the first regulatory gene encodes a diphtheria tox repressor (DtxR) protein that is the native DtxR protein, or a fragment, variant or homologue of DtxR, and the first operator binds the DtxR protein and is native tox operator (toxo) or a toxo fragment, or a variant of a DtxR consensus binding sequence. In more preferred embodiments the second regulatory gene encodes the tetracycline repressor (TetR), the second operator comprises the tetracycline operator (tetO), the reporter gene encodes chloramphenicol acetyltransferase and the selection agent is chloramphenicol. The medium comprises a chelating agent that binds metal ion activators of the first repressor.
In other preferred embodiments, the first and second recombinant DNA segments are contained in a first vector, preferably a plasmid, and the third recombinant DNA segment is contained in a second vector such a lysogenic phage.
Another embodiment of this aspect of the invention entails providing a solution containing (a) purified repressor native to or functional in a given prokaryote; (b) a DNA construct comprising in operable association, a promoter, an operator and a report
Murphy John R.
Sun Li
Boston Medical Center Corporation
Lerner David Littenberg Krumholz & Mentlik LLP
Stucker Jeffrey
Winkler Ulrike
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