Abstract
Numerous Gram-negative bacterial pathogens employ type III secretion systems (T3SSs) to inject effector proteins into eukaryotic cells. The activation of the type III secretion (T3S) process is tightly controlled in all T3SSs. In Yersinia pestis, the secretion of effector proteins, termed Yersinia outer proteins (Yops), is regulated by the activity of the YopN/SycN/YscB/TyeA complex. YopN is a secreted protein that interacts with the SycN/YscB chaperone via an N-terminal chaperone-binding domain (CBD) and with TyeA via a C-terminal TyeA-binding domain (TBD). Efficient YopN secretion is dependent upon its N-terminal secretion signal (SS), CBD, and the SycN/YscB chaperone. In this study, we investigate the role of the YopN CBD in the regulation of Yop secretion. Analysis of YopE/YopN hybrid proteins in which the YopN SS or SS and CBD were replaced with the analogous regions of YopE indicated that the YopN CBD or SycN/YscB chaperone play a role in the regulation of Yop secretion that is independent of their established roles in YopN secretion. To further analyze the role of the YopN CBD in the regulation of Yop secretion a series of tetra-alanine substitution mutants were generated throughout the YopN CBD. A number of these mutants exhibited a defect in the regulation of Yop secretion but showed no defect in YopN secretion or in the interaction of YopN with the SycN/YscB chaperone. Finally, conditions were established that enabled YopN and TyeA to regulate Yop secretion in the absence of the SycN/YscB chaperone. Importantly, a number of the YopN CBD mutants maintained their defect in the regulation of Yop secretion even under the established SycN/YscB chaperone-independent conditions. These studies establish a role for the CBD region of YopN in the regulation of Yop secretion that is independent from its role in YopN secretion or in the binding of the SycN/YscB chaperone.
Highlights
Numerous Gram-negative bacterial pathogens use type III secretion systems (T3SSs) to inject effector proteins into eukaryotic cells (Ghosh, 2004)
We examined the role of the YopN secretion signal (SS), chaperone-binding domain (CBD), and SycN/YscB chaperone in the calcium-dependent regulation of Yersinia outer proteins (Yops) secretion
The YopN SS could be functionally replaced by the analogous region of YopE, indicating that the YopN SS has no unique role in the regulation of Yop secretion
Summary
Numerous Gram-negative bacterial pathogens use type III secretion systems (T3SSs) to inject effector proteins into eukaryotic cells (Ghosh, 2004). 70 kb plasmid encoding a T3SS that is required for virulence (Ben-Gurion and Shafferman, 1981; Ferber and Brubaker, 1981; Cornelis et al, 1998). These T3SSs function to inject at least five effector proteins, termed Yersinia outer proteins (Yops) into targeted eukaryotic cells (Trosky et al, 2008). The injected effector proteins act to block bacterial phagocytosis or suppress the production of proinflammatory cytokines. These activities allow the yersiniae to survive and multiply in the extracellular milieu of their hosts
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