Bacterial 2-Component Signal Transduction Systems: A Fluorescence Polarization Screen for Response Regulator-Protein Binding

Abstract

Two-component signal transduction systems are the primary means by which bacteria sense environmental change and integrate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the expression of virulence and antibiotic resistance. This makes bacterial TCST systems attractive targets for pharmacologic intervention. This paper describes a fluorescence polarization assay that quantifies the binding between bacterial DNA promoter segments and their cognate response regulator proteins. Using the Van RSTCST system from Enterococcus faecium, which encodes vancomycin resistance, the authors demonstrate inhibition of response regulator protein/promoter segment binding with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance measurements and gel shift measurements.

Keywords

fluorescence polarization response regulator bacterial signal transduction systems antibiotic resistance

References

Hoch JA , Silhavy TJ (eds): Two-Component Signal Transduction. Washington, DC: ASM Press, 1995.

Stock AM , Robinson VL , Goudreau PN : Two-component signal transduction. Ann Rev Biochem 2000;69:183-215.

West AH , Stock AM : Histidine kinases and response regulator proteins in two component signaling systems. Trends Biochem Sci 2001;26:369-376.

Giammarinaro P , Sicard M , Gasc AM : Genetic and physiological studies of the CiaH-CiaR two-component signal-transducing system involved in cefotaxime resistance and competence of Streptococcus pneumoniae. Microbiology 1999;145:1859-1869.

Throup JP , Koretke KK , Bryant AP , Ingraham KA , Chalker AF , Ge Y , et al: A genomic analysis of two-component signal transduction in Streptococcus pneumoniae. Mol Microbiol 2000;35:566-576.

Zahrt TC , Deretic V : Mycobacterium tuberculosis signal transduction system required for persistent infections. Proc Natl Acad Sci U S A 2001;98:12706-12711.

Kallipolitis BH , Ingmer H , Gahan CG , Hill C , Sogaard-Andersen L : Ces RK, a two-component signal transduction system in Listeria monocytogenes, responds to the presence of cell wall-acting antibiotics and affects beta-lactam resistance. Antimicrob Agents Chemother 2003;47:3421-3429.

Holman TR , Wu Z , Wanner BL , Walsh CT : Identification of the DNA-binding site for the phosphorylated VanR protein required for vancomycin resistance in Enterococcus faecium. Biochemistry 1994;33:4625-4631.

Hancock L , Perego M : Two-component signal transduction In Enterococcus faecalis. J Bacteriol 2002;184:5819-5825.

10.

Barrett JF , Hoch JA : Two-component signal transductionas a target for microbial anti-infective therapy. Antimicrob Agents Chemother 1998;42:1529-1536.

11.

Barrett JF , Goldschmidt RM , Lawrence LE , Foleno B , Chen R , Demers JP , et al: Antibacterial agents that inhibit two-component signal transduction systems. Proc Natl Acad Sci U S A 1998;95:5317-5322.

12.

Hilliard JJ , Goldschmidt RM , Licata L , Baum EZ , Bush K : Multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems. Antimicrob Agents Chemother 1999;43:1693-1699.

13.

Stephenson K , Hoch JA : Two-component and phosphorelay signal-transduction systems as therapeutic targets. Curr Opin Pharmacol 2002;2:507-512.

14.

Ulijasz AT , Kay BK , Weisblum B : Peptide analogues of the Van S catalytic center inhibit VanR binding to its cognate promoter. Biochemistry 2000;39:11417-11424.

15.

Smith EA , Erickson MG , Ulijasz AT , Weisblum B , Corn RM : Surface plasmon resonance imaging of transcription factor proteins: interactions of bacterial response regulators with DNA arrays on gold films. Langmuir 2003;19:1486-1492.

16.

LeTilly V , Royer CA : Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding. Biochemistry 1993;32: 7753-7758.

17.

Burke T , Bolger R , Checovich W , Lowery R : Measurement of peptide binding affinity using fluorescence polarization. In Kay B , Winter J McCafferty J (eds): Phage Display of Peptides and Proteins. San Diego, CA: Academic Press, 1996:305-325.

18.

Roychoudhury A , Zielinski NA , Ninfa AJ , Allen NE , Jungheim LN , Nica TI : Inhibitors of two-component signal transduction systems: inhibition of alginate gene activation in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 1993;90:965-969.

19.

Ulijasz AT , Weisblum B : Dissecting the Van RS signal transduction pathway with specific inhibitors. J Bacteriol 1999;181:627-631.

20.

Ulijasz AT , Grenader A , Weisblum B : A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme. J Bacteriol 1996;178:6305-6309.

21.

Mattison K , Kenney LJ . Phosphorylation alters the interaction of the response regulator OmpR with its sensor kinase EnvZ. J Biol Chem 2002;277:11143-11148.