Translocation of a hybrid YopE‐adenylate cyclase from Yersinia enterocolitica into HeLa cells

Abstract

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.

YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane

“…At least 11 Yop proteins are secreted by the yscencoded type III secretion system of Yersinia. Of these, YopE, YopH, YopM, and YpkA are translocated into the cytosol of the eukaryotic cells (Rosqvist et al, 1994;Sory and Cornelis, 1994;Persson et al, 1995;Sory et al, 1995;Boland et al, 1996;Hå kansson et al, 1996a), and YopB and YopD are essential for the targeting of the Yop effectors into the cells (Rosqvist et al, 1991;Sory and Cornelis, 1994;Persson et al, 1995;Sory et al, 1995;Hå kansson et al, 1996b). YopK is expressed at a much lower level compared to most other Yops, with the exception of YpkA (Holmströ m et al, 1995).…”

“…Frithz-Lindsten, unpublished). This principle was first demonstrated for the Yop ( Yersinia outer protein) virulence determinants of Yersinia (Rosqvist et al, 1994;Sory and Cornelis, 1994) and infection of cells with this pathogen is also the most extensively studied model system for this mechanism. The secretion of Yops across the bacterial membranes is mediated by a type III secretion system (Salmond et al, 1993) encoded by the ysc genes (for a review, see Cornelis, 1992;Straley et al, 1993;Forsberg et al, 1994b).…”

“…In Y. pseudotuberculosis and Yersinia enterocolitica, YpkA, YopE, YopH, and YopM have been shown to be translocated (Rosqvist et al, 1991;Sory and Cornelis, 1994;Persson et al, 1995;Sory et al, 1995;Boland et al, 1996;Hå kansson et al, 1996a). YpkA exhibits Ser/Thr protein kinase activity and displays homology with eukaryotic protein kinases (Galyov et al, 1993).…”

YscM1 and YscM2, two Yersinia enterocolitica proteins causing downregulation of yop transcription

“…Translocation of YopE by the yscM1, yscM2 mutant was assayed using the YopE 130 -Cya reporter described by Sory and Cornelis (1994).…”

“…The first group includes YopE, YopH, YopM and YopO/YpkA, which are intracellular effector proteins that are injected into eukaryotic cells by extracellular Yersinia adhering at the cell surface. The second group (YopB, YopD and possibly others) forms an apparatus that is deployed at the surface of the bacterial cell in order to translocate the effectors across the eukaryotic cell membrane Sory and Cornelis, 1994;Sory et al, 1995;Hå kansson et al, 1996;Boland et al, 1996; for review, Cornelis and Wolf-Watz, 1997). YopN is thought to act as a stop valve blocking the deployment of the translocation apparatus and the release of the effectors.…”