IDENTIFICATION OF THE YOPE AND YOPH DOMAINS REQUIRED FOR SECRETION AND INTERNALIZATION INTO THE CYTOSOL OF MACROPHAGES, USING THE CYAA GENEFUSION APPROACH

Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type IU secretion mechanism. Upon infection of cultured epithelia l tells, extracellular Yersinia pseudotuberculosis and Yersinia entero colitica translocate cytotoxin YopE across the host cell plasma membra ne. Several lines of evidence suggest that tyrosine phosphatase YopH f ollows the same pathway. We analyzed internalization of YopE and YopH into murine PU5-1.8 macrophages by using recombinant Y. enterocolitica producing truncated YopE and YopH proteins fused to a calmodulin-depe ndent adenylate cyclase, The YopE-cyclase and YopH-cyclase hybrids wer e readily secreted by Y. enterocolitica, The N-terminal domain require d for secretion was not longer than 15 residues of YopE and 17 residue s of YopH. Internalization into eukaryotic cells, revealed by cAMP pro duction, only required the N-terminal 50 amino acid residues of YopE a nd the N-terminal 71 amino acid residues of YopH. YopE and YopH are th us modular proteins composed of a secretion domain, a translocation do main, and an effector domain, Translocation of YopE and YopH across ho st cell's membranes was also dependent on the secretion of YopB and Yo pD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bac terial pathogens.