Disruption of target cell adhesion structures by the Yersinia effector YopH requires interaction with the substrate domain of p130Cas.

The docking protein p130Cas has, together with FAK, been found as a target of the Yersinia virulence effector YopH. YopH is a protein tyrosine phosphatase that is delivered into host cells via the bacterial type III secretion machinery, and the outcome of its activity is inhibition of host cell phagocytosis. In the present study using p130Cas-/- cells, and p130Cas-/- cells expressing variants of GFPp130Cas, we show that this docking protein, via its substrate domain, is responsible for subcellular targeting of YopH in eukaryotic cells. Since YopH inhibits phagocytosis, p130Cas was expected to be critical for signalling mediating bacterial internalization. However, p130Cas-/- cells did not exhibit reduced capacity to internalize Yersinia. On the other hand, when a dominant negative variant of p130Cas was expressed in these cells, the phagocytic capacity was severely impaired. Moreover, the p130Cas-/- cells displayed a marked reduced sensitivity towards YopH-mediated detachment compared to wild-type cells. Transfecting these cells with full-length p130Cas rendered cells hypersensitive to both mechanical and Yersinia-mediated detachment. This hypersensitivity was not seen upon transfection with the dominant negative substrate domain-deleted variant of p130Cas. This implicates p130Cas as a prominent regulator of cell adhesion, where its substrate-binding domain has a significant function.

Interaction between the Yersinia tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein, Fyb.

Pathogenic Yersinia species can evade phagocytosis by injecting virulence effectors that interfere with the phagocytic machinery of host cells. One of these virulence effectors is the protein tyrosine phosphatase YopH. Through its enzymatic activity, YopH interferes with the initial phagocytic process by affecting signalling for cytoskeletal rearrangements. Fyb (Fyn-binding protein), which is an immune cell-specific adaptor protein, has been identified as a substrate of YopH in macrophages. In this study, the interaction between YopH and Fyb is studied. We show that YopH binds to Fyb via different regions in both phosphotyrosine-dependent and phosphotyrosine-independent ways. The phosphotyrosine substrate binding N-terminal part (1-130) of YopH as well as the C-terminal catalytic region binds to Fyb in a phosphotyrosine-dependent manner. We also show that a central part of YopH (130-260) interacts with the Fyb C-terminus (548-783) in a phosphotyrosine-independent manner. Further, we demonstrate that the N-terminal binding region of YopH is important for YopH-mediated functions on macrophages such as dephosphorylation of Fyb, blockage of phagocytosis, and cytotoxic effects.

Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion.

The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). We have previously shown that the Drosophila Alk RTK is crucial for visceral mesoderm development during early embryogenesis. Notably, observed Alk visceral mesoderm defects are highly reminiscent of the phenotype reported for the secreted molecule Jelly belly (Jeb). Here we show that Drosophila Alk is the receptor for Jeb in the developing visceral mesoderm, and that Jeb binding stimulates an Alk-driven, extracellular signal-regulated kinase-mediated signalling pathway, which results in the expression of the downstream gene duf (also known as kirre)--needed for muscle fusion. This new signal transduction pathway drives specification of the muscle founder cells, and the regulation of Duf expression by the Drosophila Alk RTK explains the visceral-mesoderm-specific muscle fusion defects observed in both Alk and jeb mutant animals.

Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism.

The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.

Resistance to phagocytosis by Yersinia.

Enteropathogenic species of the genus Yersinia penetrate the intestinal epithelium and then spread to the lymphatic system, where they proliferate extracellularly. At this location, most other bacteria are effectively ingested and destroyed by the resident phagocytes. Yersinia, on the other hand binds to receptors on the external surface of phagocytes, and from this location it blocks the capacity of these cells to exert their phagocytic function via different receptors. The mechanism behind the resistance to phagocytosis involves the essential virulence factor YopH, a protein tyrosine phosphatase that is translocated into interacting target cells via a type III secretion machinery. YopH disrupts peripheral focal complexes of host cells, seen as a rounding up of infected cells. The focal complex proteins that are dephosphorylated by YopH are focal adhesion kinase and Crk-associated substrate, the latter of which is a common substrate in both professional and non-professional phagocytes. In macrophages additional substrates have been found, the Fyn-binding/SLP-76-associated protein and SKAP-HOM. Phagocytosis is a rapid process that is activated when the bacterium interacts with the phagocyte. Consequently, the effect exerted by a microbe to block this process has to be rapid and precise. This review deals with the mechanisms involved in impeding uptake as well as with the role of the YopH substrates and focal complex structures in normal cell function.