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

Abstract

SummaryNeisseria 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 twohybrid 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 bacteriahost interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opainteracting 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 twohybrid 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 Opaþ 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.

OIP5 is a highly expressed potential therapeutic target for colorectal and gastric cancers

“…No. NM_007280) was originally identified as an Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein (1). OIP5 is homologous to OIP4, which is expressed in tumors and some normal tissues (2).…”

Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli

“…In order to localize the domains of Tir involved in its various functions, we constructed a series of Tir deletion mutants. Previously, a yeast twohybrid system has been used successfully in the identi®ca-tion of eukaryotic proteins interacting with a prokaryotic bait (Williams et al, 1998); therefore, we were interested in investigating whether the interaction between Tir and intimin could occur when both of these proteins were expressed in the yeast cytosol. In conjunction with a geloverlay technique, the yeast two-hybrid system was found to be a valuable tool for studying interactions under non-denaturing conditions between intimin and truncated forms of Tir.…”

LAP2α-binding protein LINT-25 is a novel chromatin-associated protein involved in cell cycle exit

“…OIP5 was previously found by yeast two-hybrid analysis to interact with Neisseria gonorrhoeae opacityassociated (Opa) proteins (Williams et al, 1998). Opa proteins are found in the outer membrane of N. gonorrhoeae and are involved in gonococcal adhesion to and invasion of human epithelial cells.…”