Method for identifying antibiotic targets
Inventors
Williams, David Hugh • Turner, Arthur Keith
Assignees
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Publication Number
US-11359195-B2
Publication Date
2022-06-14
Expiration Date
Abstract
Disclosed are methods related to identifying an essential gene which serves as an antibiotic target in a bacterium.
Core Innovation
The invention describes a method for identifying an essential gene which serves as an antibiotic target in a bacterium. The method generates a pool of mutant bacteria by transposon mutagenesis using two or more different activating transposons (TnAs), each TnA including a promoter. Insertion of a transposon into bacterial DNA disrupts gene function or increases transcription of a gene at or near the insertion site in a position-dependent manner, yielding an insertion rate of at least one transposon per 10 base pairs of bacterial DNA.
The method grows bacteria from the mutant pool in the presence of antibiotic at concentrations of about 0.5, about 1, and about 2× minimum inhibitory concentration (MIC) to produce at least three test cultures. The invention compares the distribution of TnA insertions between test cultures. This comparison identifies TnA insertion sites that disrupt essential gene function and insertion sites positioned to enhance essential gene transcription such that the essential gene product overexpresses to a level that functions as a sink for the antibiotic and alters the effect of the antibiotic on the bacterium.
Using these changes in insertion-site distribution, the method identifies a putative essential gene necessary for viability under all conditions of growth used in the antibiotic exposure. The method further identifies such an essential gene as a target of the antibiotic in the bacterium. The core concept is that position-dependent activation from activating transposons enables detection of essential genes whose overexpressed products act as an antibiotic sink.
Claims Coverage
The document includes one independent claim (clm-00001). It contains a method with three principal inventive components: generating a pool using two or more activating transposons with promoter-driven, position-dependent transcription effects; testing under antibiotic at multiple MIC-based concentrations; and comparing insertion-site distributions to identify essential genes whose insertion either disrupts essential function or creates activation-driven sink behavior.
Activating transposon mutant pool for position-dependent essential gene activation
Generating a pool of mutant bacteria by transposon mutagenesis with two or more different activating transposons (TnAs), wherein each TnA comprises a promoter such that transposon insertion into bacterial DNA disrupts the function or increases the transcription of a gene at or near the insertion site in a position-dependent manner, and wherein the transposon mutagenesis yields an insertion rate of at least one transposon per 10 base pairs of bacterial DNA.
Antibiotic growth at multiple MIC concentrations to create test cultures
Growing bacteria from the mutant pool in the presence of antibiotic at a concentration of about 0.5, about 1 and about 2× minimum inhibitory concentration (MIC) to produce at least three test cultures.
Insertion distribution comparison to identify essential genes and antibiotic-sink targets
Comparing the distribution of TnA insertions between test cultures to identify: (i) TnA insertion sites which disrupt essential gene function; and (ii) TnA insertion sites which are positioned such that essential gene transcription is enhanced such that the essential gene product is overexpressed to a level where it functions as a sink for said antibiotic and so alters the effect of the antibiotic on said bacterium, thereby identifying a putative essential gene which is necessary for viability under all conditions of growth used in step (b) and which serves as a target of said antibiotic in said bacterium.
Across the independent claim, essential antibiotic target genes are identified by combining activating transposon mutagenesis with promoter-driven, position-dependent transcription effects, antibiotic growth at multiple MIC concentrations, and comparison of TnA insertion distributions to distinguish essential gene disruption from activation-driven antibiotic sink behavior.
Stated Advantages
Documented Applications
Identifying essential bacterial genes that serve as antibiotic targets by using activating transposons that create insertion-site effects where essential gene transcription is enhanced to produce an antibiotic sink (including antibiotic target process identification in multiple bacterial taxa, and an example of ciprofloxacin resistance profiling in Salmonella Typhi).
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