Screening and use of reagents which block or activate intein splicing utilizing natural or homologous exteins

Abstract

In accordance with the present invention, there are provided selection systems and methods for screening for agents that control splicing of inteins in their native host protein (extein) or in homologous exteins. Specifically, there are provided positive genetic selection systems for the screening of agents which inhibit or activate protein splicing which comprise: a host cell containing a chromosomal gene encoding either a drug-resistant form of a target enzyme or a wild-type target enzyme, and a plasmid-borne gene encoding either a drug-sensitive form of the target enzyme, which is dominantly cytotoxic upon interaction with the drug, or a dominantly cytotoxic form of the target enzyme. In these systems the plasmid-borne gene contains an intein, and the inhibition or activation of splicing of the dominant cytotoxic form of the target enzyme by a given reagent results in the survival or death of the host cell. More specifically, positive genetic selection systems that utilize the M. xenopi GyrA intein or M. tuberculosis DnaB helicase intein are provided. Similar reporter systems utilizing native or homologous exteins and systems utilizing controllable inteins are provided, as are methods of controlling in vivo expression of proteins by modulating protein splicing with inhibiting or activating agents, and methods of controlling the delivery of proteinaceous drugs in vivo by modulating protein splicing.

1 . A method of identifying an agent for antimicrobial activity against a microbial pathogen that naturally has an intein in an essential gene comprising screening for agents that block splicing of that intein in its native context or a homologous extein context.  
     
     
         2 . A method of identifying an agent for antimicrobial activity against a microbial pathogen that naturally has an intein in an essential gene comprising screening for agents that block splicing of that intein in a heterologous extein context that includes one or more native extein residues flanking one or both ends of the intein.  
     
     
         3 . A method of identifying agents with antimicrobial activity against  Mycobacterium tuberculosis  comprising screening for agents that inhibit splicing of the  Mycobacterium tuberculosis  DnaB intein.  
     
     
         4 . A method of identifying agents with antimicrobial activity against  Mycobacterium leprae  comprising screening for agents that inhibit splicing of the  Mycobacterium xenopi  or  Mycobacterium leprae  GyrA inteins.  
     
     
         5 . A method of identifying lead compounds with antimicrobial activity comprising identifying agents that inhibit splicing of an intein that is naturally present in that organism.