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Molecular microbiology

Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth.


PMID 9466265

Abstract

Neisseria gonorrhoeae opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast two-hybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two-hybrid system in identifying bacteria-host interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opa-interacting proteins (OIPs). We identified five different OIPs, designated OIP1-OIP5, two of which are homologous to human proteins--thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa-PK interactions were confirmed by in vitro and in vivo assays independent of the yeast two-hybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa-negative E. coli in in vitro binding assays. Using anti-PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co-localizes with intracellular Opa+ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two-hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.