Characterization of YopT Effects on Rho GTPases in Yersinia enterocolitica-infected Cells

Abstract

Pathogenic yersiniae employ a type III secretion system for translocating up to six effector proteins (Yersinia outer proteins (Yops)) into eukaryotic target cells. YopT is a cysteine protease that was shown to remove the C-terminal isoprenoid group of RhoA, Rac, and CDC42Hs. Here we characterized the cell biological and biochemical activities of YopT in cells infected with pathogenic Yersinia enterocolitica. Bacterially injected YopT located to cell membranes from which it released RhoA but not Rac or CDC42Hs. In the infected cells RhoA was dissociated from guanine nucleotide dissociation inhibitor-1 (GDI-1) and accumulated as a monomeric protein in the cytosol, whereas Rac and CDC42Hs remained GDI-bound. Direct transfer of isoprenylated RhoA to YopT and RhoA modification could be reconstituted in vitro by guanosine 5-3-O-(thio)triphosphate loading of a recombinant RhoA⅐GDI-1 complex. Finally, in macrophages infected with a Yersinia strain selectively translocating YopT podosomal adhesion structures required for chemotaxis as well as phagocytic cups mediating uptake of yersiniae were disrupted. These findings indicate that bacterially translocated YopT acts on membrane-bound and GDI-complexed RhoA but not Rac or CDC42, and this is sufficient for disruption of macrophage immune functions.

Targeting Rac1 by the Yersinia Effector Protein YopE Inhibits Caspase-1-mediated Maturation and Release of Interleukin-1β

“…It should be mentioned that, although the effect of YopE and YopT on caspase-1 activation was comparable upon overexpression in HEK293T cells and mediated by their effect on Rho GTPases (as shown by the use of specific YopE and YopT mutants), the effect of YopE was much more pronounced in the case of Yersinia infected macrophages. Although the underlying mechanism of Rho GTPase inhibition by YopE and YopT is different, they both have been shown to inhibit Rac1, RhoA, and Cdc42 in vitro or upon overexpression (4,5,28,37). However, in Yersinia infected macrophages, YopE and YopT have been shown to specifically inhibit Rac1 and RhoA, respectively (28,37).…”

“…Although the underlying mechanism of Rho GTPase inhibition by YopE and YopT is different, they both have been shown to inhibit Rac1, RhoA, and Cdc42 in vitro or upon overexpression (4,5,28,37). However, in Yersinia infected macrophages, YopE and YopT have been shown to specifically inhibit Rac1 and RhoA, respectively (28,37). The latter observation together with our own data showing that IL-1␤ release from YopPE Ϫ -infected macrophages is much higher compared with the release from YopPT Ϫ -infected cells point to a major role for Rac1 in caspase-1 activation and the proteolytic maturation of proIL-1␤ in Yersinia infected macrophages.…”

“…Most probably, this can be explained by a difference in the Yop concentration in cells upon overexpression or infection, respectively. In this context, it should be mentioned that whereas YopE and YopT inactivate RhoA, Rac1, and Cdc42 in vitro or upon overexpression (5, 28), when they are injected into cells by Yersinia, they are remarkably specific, inactivating selectively Rac1 and RhoA, respectively (28,37). Therefore, the more potent effect of YopE deletion on proIL-1␤ maturation in infected cells suggests a major role for Rac1.…”

Bacterial Toxins Induce Sustained mRNA Expression of the Silencing Transcription Factor klf2 via Inactivation of RhoA and Rhophilin 1

“…YopT, a cysteine protease, inactivates RhoA by cleavage adjacent to a prenylated cysteine located near the carboxy terminus, resulting in membrane release and cytoplasmic redistribution of RhoA (60). However, some studies also showed some effect of YopT on Rac and Cdc42 in biochemical assays performed in vitro (61), but after infection of living cells, YopT seems to act mainly on RhoA (2).…”

Immunomodulatory Yersinia outer proteins (Yops)–useful tools for bacteria and humans alike

“…94 In vivo, the preferred target of YopT is RhoA. 95 YopT is the prototype of a new protein fold subfamily of proteases, which among others encompasses toxin B from E. coli or the avirulent protease AvrPphB of the plant pathogen Pseudomonas syringae. 96 This classification was based on predictions and homology analysis, although the actual 3D-structure of YopT has not been resolved yet.…”