The emergence of multidrug resistant infections coupled with the continuous threat of biological warfare has created the need for the discovery of new antimicrobial targets. This project will utilize a novel genetic strategy to functionally interrogate microbial genomes for gene products required for cell viability. Making the connection between gene sequence and protein essentiality is ever so crucial since 30 to 50 percent of microbial ORFs have no assigned biological function. E. coli will serve as a paradigm for enteric infections, since strains have been engineered with very high transformation efficiencies.The main objective of this proposal is to detect essential bacterial genes, while simultaneously obtaining stable, cyclic peptides that inhibit their function(s).
The specific aims of this project are: (1) Identification of the cellular targets for individual toxic peptides, (2) Selection of potential antimicrobial targets and analysis of the structural and chemical determinants required for their inhibition, and (3) Genome-wide detection of metabolic functions required for cell viability. The results of our efforts will provide new targets and lead compounds for antibiotic design.The research plan will require the production and selection of vast numbers of cyclic peptide sequences in E. coli. These libraries will be obtained using the Split Intein Circular Ligation Of Peptides and ProteinS SICLOPPS) technology. The experimental method will involve screening for bactericidal/bacteriostatic peptides using replica plating and/or flow cytometry. Each identified toxic peptide will be co-transformed with every ORF found in the host genome. The obstructed metabolic function(s) will be restored by over-expression of the targeted protein(s). The genomic constructs encoding essential, peptide-inhibited functions will be recovered by PCR and their identity revealed by sequencing or by hybridizing onto DNA arrays. Essential genes conserved across bacterial species and not represented in humans will be selected for further studies. For each potential target selected, the intracellular peptide library will be re-screened to detect all inhibitors of the isolated biological function. The family of retrieved peptides will be aligned to identify conserved amino acids required for inhibition.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
7R01AI053212-05
Application #
7327573
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Korpela, Jukka K
Project Start
2003-01-01
Project End
2007-12-31
Budget Start
2006-09-01
Budget End
2006-12-31
Support Year
5
Fiscal Year
2006
Total Cost
$133,200
Indirect Cost
Name
University of Nevada Las Vegas
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
098377336
City
Las Vegas
State
NV
Country
United States
Zip Code
89154
Nilsson, Lisa O; Louassini, Mostafa; Abel-Santos, Ernesto (2005) Using siclopps for the discovery of novel antimicrobial peptides and their targets. Protein Pept Lett 12:795-9