The applicant proposes a program of research and study to prepare him for a career in the study of bacterial pathogenesis. The research will be conducted in the laboratory of Dr. John Mekalanos at Harvard Medical School. The emergence and spread of bacterial resistance to antibiotics poses a serious challenge to our ability to treat infections. New methods of finding antimicrobials will be needed to develop antibiotics that avoid current mechanisms of resistance. This proposal describes a method for finding novel proteins that interfere with bacterial growth or the expression of virulence factors needed by bacteria to cause disease. These proteins will be identified from a large library of """"""""aptamers"""""""", or random peptides expressed as fusions to carrier proteins. The proteins will be used to identify bacterial target molecules which are essential for bacterial growth or virulence factor expression. Evenually, they may also be used as """"""""lead compounds"""""""" for the development of small molecules which bind to the same target. The library of aptamers has been constructed, and aptamers with interesting activities have been identified through selections and screening. The proposal describes the analysis of these aptamers, including some which inhibit an enzyme required for nucleotide metabolism, thymidylate synthase, and others which interfere with bacterial growth. Genetic methods will be used to determine which molecules' functions are inhibited by these growth-inhibiting aptamers. This proposal also describes the use of additional genetic selections or screens which will identify random peptides with several activities. The PhoBR two-component regulator controls the expression of genes in response to environmental phosphate concentration. Selections are described which will be used to identify aptamers that inhibit various components of the PhoBR system. The molecular targets of these inhibitors will be determined. Additional selections will be used to identify aptamers that have streptomycin-like activities. Selections are described which will identify aptamers that inhibit the expression of toxin-coregulated pili in Vibrio cholerae. These pili are required for establishment of human infection by V. cholerae. Methods for improving the affinity of aptamers for their targets and biochemical approaches for studying the targets of aptamers are described. Ultimately, study of the structural interaction between aptamers and their targets may provide information useful for the development of new types of antibiotics.
