Abstract
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd ) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.
Highlights
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells
YscU Binds to the Disordered Segment of YscP—Previous genetic analysis showed that yscP deletion abolishes Yersinia outer protein (Yop) secretion and that the wild-type phenotype can be partially rescued by the A268F, Y287G, and V292T suppressor mutations within YscU [10]
A direct interaction between YscP and YscUC homologues in P. aeruginosa has been observed previously by nuclear magnetic resonance (NMR) [22]; it was shown that a conserved N-terminal segment of PscP was responsible for its interaction with PscUC
Summary
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. Yersinia spp. share a common virulence plasmid that encodes a type III secretion system (T3SS) supporting the secretion of the Yersinia outer protein (Yop) effectors from the bacterium into eukaryotic host cells [1]. Activation of the T3SS impairs the secretion of the early substrates but triggers the secretion of the Yop effectors (late substrates) [10, 11] This modification of the secretion pattern was first described by Macnab and co-workers [12,13,14] in the flagellum and is called the substrate specificity switch. A minimal needle length is required to support Yop secretion [17] Together, these results suggest that the needle length is tightly regulated and that YscP functions as a molecular ruler [16]. A systematic deletion analysis of YscP in Yersinia enterocolitica led to the identification of domains with specific functions [11, 19], including two distinct
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