Timothy Kaminsky

BACTERIAL mRNA AS A TARGET FOR ANTIMICROBIALS. Timothy Kaminsky¹, Norbert Hill², Yasmin Razia³, Jay-Ming Wang¹, Marvin Coughenour², and Jeffrey S. McKinney^2,3. Biology Department, Washington University, St. Louis, MO¹; Departments of Molecular Microbiology² and Pediatrics³, Washington University School of Medicine, St. Louis, MO^2,3.

Our laboratory has developed a novel system of gene regulation in bacteria that is based on specific mRNA cleavage. This system uses oligonucleotide molecules called External Guide Sequences (EGSs). EGSs hybridize to complementary target mRNA, forming a substrate that is cleaved by intracellular RNase P, disrupting the normal flow of genetic information from DNA to mRNA to protein. As proof of principle, my lab has used this system to create conditionally lethal or avirulent bacterial phenotypes.

We are now further dissecting the mechanistic details of EGS effects in the intracellular milieu of bacteria. One such molecular detail involves the relationship, if any, between EGS efficacy and the relative supply of the protein encoded by a given EGS’s target mRNA. Intuitively, other parameters being equal, I hypothesize that the phenotypic effect of an EGS is significantly dependent upon the relative intracellular supply of the protein encoded by the specific mRNA targeted by that EGS.

My research employs two model systems in Salmonella that manifest distinct phenotypes that we can reliably and sensitively measure. The first model incorporates green fluorescent protein (GFP) variants. In particular, I am working with GFP variants with different protein half-lives in Salmonella. This system should allow me to directly assess the relevance of protein half-life in the context of EGS down regulation of a given mRNA target.

Toward this end, I have measured GFP expression in several independent ways: single cell microscopy, fluorescent emission from clonal CFUs on plates, and fluorescent phenotype kinetics in liquid cultures as assessed by FACS analysis. Given the broad dynamic range of the GFP mediated flourescence in the Salmonella I study, I am also exploring FACS sorting, as a means to conduct high throughput screening of large libraries of candidate EGSs. This screening is motivated by our desire to derive more generalizable rules for designing effective EGSs. This could enhance their utility as a broadly applicable tool for artificial down-regulation of bacterial gene expression.

So-called essential genes in bacteria encode one set of bacterial mRNAs we are particularly interested in down-regulating, given the implications of using mRNAs as novel molecular targets for new antimicrobial maneuvers. Thus, we are studying a second model system in which we use EGSs designed to down-regulate dapB encoded diaminopimelate (DAP) biosynthesis. The DAP biosynthetic pathway is essential to bacterial, but not human, lysine and peptidoglycan (and thus cell wall) biosynthesis. We have produced a dapB deficient Salmonella strain and demonstrated this mutant exhibits lethal cell wall morphology defects, consistent with DAP’s essential role in peptidoglycan synthesis. Accordingly, we are investigating dapB mRNA as a potential target for EGSs intended as a test case for future antibacterial agents.