A high proportion of the acetamido sugar residues in the peptidoglycan layer of B. anthracis vegetative cells are de-N-acetylated by the action of one or more specific peptidoglycan deacetylase(s). This modification has the effect of making the bacterial cell wall resistant to digestion by lysozyme, a ubiquitous enzyme in human secretions, blood and tissues. The feasibility of using the peptidoglycan deacetylase of B. anthracis as a new drug target is assessed in this R21 application. A specific inhibitor of the enzyme could render the bacteria unable to protect their cell walls from host lysozyme and they would then be rapidly killed. Twelve candidate peptidoglycan deacetylase genes have been tentatively identified in the genome of B. anthracis. The first specific aim is to screen expressed candidate deacetylases for enzyme activity. To facilitate development of suitable analytical methods and biochemical procedures for use with the B. anthracis deacetylase, the only confirmed peptidoglycan deacetylase gene, the pgdA gene from S. pneumoniae, will be cloned first and expressed. Important properties of the enzymes will be determined, including their pH optima, metal ion requirements, if any, and the effect of reducing agents on enzyme activity. In addition, amino acid residues critical for enzymatic activity will be identified by means of site-directed mutagenesis of highly conserved regions of the enzymes. The second specific aim is to mutationally inactivate candidate deacetylase genes in B. anthracis and then assess the effects of the mutations on peptidoglycan deacetylation and sensitivity to lysozyme. In the unlikely event that none of the candidate genes encode the active deacetylase, the gene will be identified using a transposon mutagenesis, procedure. The third specific aim is to determine if deacetylase-deficient mutants are no longer virulent in a mouse model of B. anthracis infection. Virulence of wild type and mutant (i.e., peptidoglycan deacetylase-deficient) spores of the Sterne strain of B. anthracis will be compared using A/J and BALB/c mice, which are differentially sensitive to inoculation of Sterne spores. Mice will be challenged by three different routes, subcutaneous, intranasal, and intratracheal inoculation of spores. Success of this project may lead to the development of another valuable means of treating infections caused by other Gram-positive bacterial pathogens, in addition to B. anthracis, by inhibiting the deacetylases which specifically render their cell wall peptidoglycans resistant to the action of lysozyme. ? ? ?
