Identification of antibacterial agents

Details Identification of antibacterial agents Identification of antibacterial agents

1999-05-06

2001-03-27

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S252100, C435S252800, C435S253400, C435S252500, C435S440000, C435S069100, C435S006120, C435S029000

Reexamination Certificate

active

06207407

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to methods of identifying genes and corresponding gene products which are required for survival.
SUMMARY OF THE INVENTION
The invention features a method for identifying a strain carrying a lethal conditional-sensitive mutation in a gene essential for survival. The method includes (a) growing organisms (e.g., cells) under first permissive conditions; (b) exposing organisms from step (a) to restrictive conditions for a period of time equivalent to at least two growth cycles (e.g., cell cycles); and (c) shifting the organisms from step (b) to second permissive conditions for a period of time equivalent to at least ten growth cycles (e.g., cell cycles). Following this treatment, mutant organisms which both (i) failed to grow when exposed to the restrictive conditions of step (b), and (ii) failed to resume growth when returned to the second permissive conditions of step (c) are selected (step (d)).
This selection process separates strains having lethal mutations from strains having nonlethal or static mutations. In general, a selected strain has a gene that is sensitive to the restrictive conditions and that is essential for growth. The transient shift to a restrictive condition results in loss of the gene product; this loss is lethal to the organism. For example, the gene product of the mutated gene is not functional under the restrictive conditions.
Another aspect of the invention features a method for identifying an essential gene in a strain. This method includes (a) growing organisms (e.g., cells) under first permissive conditions; (b) exposing organisms from step (a) to restrictive conditions for a period of time equivalent to at least two cell cycles; (c) shifting the organisms from step (b) to second permissive conditions for a period of time equivalent to at least ten cell cycles; (d) selecting a strain having a lethal mutation, wherein the strain (i) failed to grow when exposed to the restrictive conditions of step (b), and (ii) failed to grow when shifted to the second permissive conditions; (e) identifying a strain selected in step (d) which carries a recessive conditional lethal mutation; and (f) identifying from the strain of step (e) a gene corresponding to the gene encoding the recessive conditional lethal mutation. Preferably, failure to grow is defined by at least three logs of killing, i.e., if 1000 cells are grown under permissive conditions, shifted to restrictive conditions, and then shifted to second permissive conditions, statistically 999 cells are dead. This threshold is intended to identify the more sensitive conditional mutations. Failure to grow can also be defined as 1.5, 2, 3.5, 4, or 5 logs of killing. Another aspect of
Another aspect is a method for identifying a gene product target of a biocidal drug (e.g., antimicrobial, antiparasitic, or insecticidal), including the steps of: (a) growing strains (e.g., fungal, bacterial, parasitic, or insect) under first permissive conditions; (b) exposing the strains from step (a) to restrictive conditions for a period of time equivalent to at least two growth cycles; (c) shifting the strains from step (b) to second permissive conditions for a period of time equivalent to at least ten growth cycles; (d) selecting a strain having a gene carrying a conditional lethal mutation; and (e) identifying the gene product corresponding to the conditional lethal mutation, thereby identifying a gene product target of a biocidal drug.
Once a mutant organism (strain) is identified, routine techniques may be generally be used for transformation, amplification, isolation, purification, and sequencing the gene carrying the mutation. Essential survival genes are required for growth (e.g., metabolism, division, or reproduction). Such genes and gene products are useful in developing therapeutic agents such as antifungal, antibacterial, and antiparasitic agents; insecticidal agents; and preventive antimicrobial agents. Therapeutic agents can reduce or prevent growth, or decrease pathogenicity or virulence, and preferably, kill the organism. The genes and gene products identified by the invention can also be used to develop antimicrobial agents which are effective in preventing microbial infection, e.g., by inhibiting the establishment of a bacterial biofilm, in addition to agents which are useful in the treatment of an established infection.
Other features and advantages of the present invention will be apparent from the following detailed description of the invention, the examples, and also from the appending claims.
DETAILED DESCRIPTION OF THE INVENTION
The invention features a method of identifying mutant organisms having conditional-sensitive lethal mutations, and subsequently gene products thereof. The disclosed methods are useful for high-throughput (e.g., use of 96-well plates) screening of genomic or mutant libraries to rapidly identify genes, and corresponding gene products, which are essential for survival. By altering restrictive conditions, including incubation period, temperature, concentration of an antibiotic, a salt, pH, and so on, and by altering the threshold level of “lethal” (how many logs of killing), the strains can be prioritized.
The selected strains cannot survive under restrictive conditions. The effect of a lethal mutation cannot be reversed or overcome by shifting the organisms to permissive conditions. The selected genes and products thereof are therefore essential for survival of the organism under restrictive conditions. In contrast, strains having nonlethal or static mutations may grow very slowly or even appear inactive under restrictive conditions. The effect of a static mutation is reversible; organisms having static mutations resume metabolism and growth when shifted to permissive conditions. Selection of a conditional-sensitive lethal mutant organism allows identification of the gene carrying the lethal mutation and identification of the corresponding gene product, if any.
A conditional lethal mutation results in a gene or a protein which is not functional under restrictive conditions. A non-functional gene can have a defect in the promoter resulting in reduced or abnormal gene expression. A non-functional protein may have a conformational defect causing improper protein folding or abnormal protein degradation. Improper protein folding can result in partial or total failure to fold, to recognize a native substrate, and/or to bind and release the substrate.
Therapeutic agents can be developed from the identification of essential genes of organisms such as bacteria or fungi. Preferably, a gene product (e.g., a protein or an RNA molecule) identified by the methods disclosed herein is distinct from the gene products targeted by existing drugs such as antibiotic or antifungal agents. The disclosed gene selection methods establish that the gene product is essential for survival of the organism. Such an identified gene product therefore serves as a novel target for therapeutics based on a mechanism which is likely distinct from the mechanisms of existing drugs. Similarly, distinct from known compounds is a compound which inhibits the function of a gene product identified by methods disclosed herein, for example, by producing a phenotype or morphology similar to that found in the original mutant strain.
According to one aspect of the invention, a mutant collection is systematically screened to identify genes and preferably gene products which are targets for drugs. For example, an antimicrobial (e.g., antibacterial or antifungal) drug may act as a biocide by binding reversibly, or preferably irreversibly, to the identified gene or gene product target, and thereby impairing its function. Loss of the function (or the synthesis or the complete processing) of the gene product target will result in inhibition of microbial growth, and preferably will result in death of the microbe. This aspect includes a method for identifying biocidal agents, including the step of exposing a gene product corresponding to the wildtype sequence of a mutant sequence identified b

Affiliated with

Also associated with

Rating

Identification of antibacterial agents does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Identification of antibacterial agents, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Identification of antibacterial agents will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2439236