Antimicrobial agents, diagnostic reagents, and vaccines based on unique apicomplexan parasite components

Abstract

This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphofructokinase and not generally operative in animals or encoded by the plastid DNA, to develop compositions that interfere with Apicomplexan growth and survival. Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences to which antibodies, antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors.

1 . A pharmaceutical composition having a parasite with a chorismate synthase gene that is knocked out.  
     
     
         2 . An immunogenic composition comprising an attenuated parasite of  claim 1 .  
     
     
         3 . The parasite of  claim 1  is  T. gondii.    
     
     
         4  An immunogenic composition comprising a cDNA molecule encoding chorismate synthase, said molecule complementary to an mRNA from  T. gondii.    
     
     
         5 . An assay for a candidate inhibitor of  T. gondii,  said assay comprising: 
 (a) adding a chorismate synthase—green fluorescent reporter protein DNA construct to a parasite of the  T. gondii  species;    (b) contacting the parasite with the candidate inhibitor;    (c) comparing the amounts of green fluorescent reporter protein in the parasite in the presence and absence of the candidate inhibitor; and    (d) inferring that the candidate inhibitor is an inhibitor of the parasite if there is significantly less reporter protein when the candidate inhibitor is present.    
     
     
         6 . A method for detecting a life cycle stage in a sample tested for  T. gondii  said method comprising: 
 (a) determining an amount of chorismate synthase present in the sample; and    (b) comparing the amount to amounts of standards determined from known life cycle stages.    
     
     
         7 . The method of  claim 6 , wherein the sample is derived from a cat.  
     
     
         8 . A recombinant expression system capable of producing chorismate synthase.  
     
     
         9 . A polyclonal antibody specific for chorismate synthase from  T. gondii.    
     
     
         10 . A high throughput assay for chorismate synthase, said assay comprising: 
 (a) detecting the product of a phosphate release assay; and    (b) inferring from (a) the quantity of chorismate synthase.    
     
     
         11 . A cDNA having an amino acid sequence shown in  FIG. 19 .  
     
     
         12 . A cDNA molecule having a nucleotide sequence shown in  FIG. 18 .  
     
     
         13 . A genomic DNA molecule having a nucleotide sequence shown in  FIG. 21 .  
     
     
         14 . A method for detecting the life stage of  T. gondii  in a biological sample, said method comprising: 
 (a) detecting isocitrate lyase with an amino acid sequence capable of being encoded by a cDNA molecule of  T. gondii.      
     
     
         15 . A DNA molecule having a sequence in accord with a sequence belonging to the group of sequences in GeneBank Assn. Number ASF 157612, 157613, 157614, 157615.  
     
     
         16 . A method to inhibit growth of  T. gondii,  said method comprising: 
 (a) selecting an inhibitor of plant plastic acetyl—CoA carboxylase; and    (b) contacting  T. gondii  with the inhibitor.