Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach.
Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type III secretion mechanism. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis and Yersinia enterocolitica translocate cytotoxin YopE across the host cell plasma membrane. Several lines of evidence suggest that tyrosine phosphatase YopH follows 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-dependent adenylate cyclase. The YopE-cyclase and YopH-cyclase hybrids were readily secreted by Y. enterocolitica. The N-terminal domain required for secretion was not longer than 15 residues of YopE and 17 residues of YopH. Internalization into eukaryotic cells, revealed by cAMP production, only required the N-terminal 50 amino acid residues of YopE and the N-terminal 71 amino acid residues of YopH. YopE and YopH are thus modular proteins composed of a secretion domain, a translocation domain, and an effector domain. Translocation of YopE and YopH across host cell's membranes was also dependent on the secretion of YopB and YopD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bacterial pathogens.
Sory
M P
MP
Microbial Pathogenesis Unit, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Boland
A
A
Lambermont
I
I
Cornelis
G R
GR
D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013045:Species Specificity; D015008:Yersinia enterocolitica / Q000276:immunology* / Q000472:pathogenicity; D015011:Yersinia pseudotuberculosis / Q000276:immunology* / Q000472:pathogenicity
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C063239:yopE protein, Yersinia; D000242:Cyclic AMP; D017027:Protein Tyrosine Phosphatases; C067571:yopH protein, Yersinia; D000262:Adenylyl Cyclases