Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells.
Pathogenic bacteria of the genus Yersinia release in vitro a set of antihost proteins called Yops. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis transfers YopE across the host cell plasma membrane. To facilitate the study of this translocation process, we constructed a recombinant Yersinia enterocolitica strain producing YopE fused to a reporter enzyme. As a reporter, we selected the calmodulin-dependent adenylate cyclase of Bordetella pertussis and we monitored the accumulation of cyclic AMP (cAMP). Since bacteria do not produce calmodulin, cyclase activity marks the presence of hybrid enzyme in the cytoplasmic compartment of the eukaryotic cell. Infection of a monolayer of HeLa cells by the recombinant Y. enterocolitica strain led to a significant increase of cAMP. This phenomenon was dependent not only on the integrity of the Yop secretion pathway but also on the presence of YopB and/or YopD. It also required the presence of the adhesin YadA at the bacterial surface. In contrast, the phenomenon was not affected by cytochalasin D, indicating that internalization of the bacteria themselves was not required for the translocation process. Our results demonstrate that Y. enterocolitica is able to transfer hybrid proteins into eukaryotic cells. This system can be used not only to study the mechanism of YopE translocation but also the fate of the other Yops or even of proteins secreted by other bacterial pathogens.
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Microbial Pathogenesis Unit, Université Catholique de Louvain, Brussels, Belgium.
Cornelis
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GR
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active; D001886:Bordetella pertussis / Q000201:enzymology / Q000235:genetics; D000242:Cyclic AMP / Q000096:biosynthesis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012449:Safety; D014774:Virulence; D015008:Yersinia enterocolitica / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity
D037361:Adenylate Cyclase Toxin; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C078909:YopB protein, Yersinia; C078910:YopD protein, Yersinia; C063239:yopE protein, Yersinia; D000242:Cyclic AMP; D000262:Adenylyl Cyclases