Abstract
Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.
Highlights
In 1952, Hills and Spurr showed that virulent strains of Yersinia pestis (Pasturella pestis) were unable to grow and divide when incubated at 37uC; instead, they required incubation at 27uC [1]
This was based on the observation that 2.5 mM calcium in the growth medium blocked Yersinia outer proteins (Yops) secretion, while depletion of calcium induced massive Yop secretion that results in stop of bacteria proliferation [3,4,5]
The yscU mutant P264A was severely suppressed in autoproteolysis leading to an almost complete inhibition of Yop secretion [16]
Summary
In 1952, Hills and Spurr showed that virulent strains of Yersinia pestis (Pasturella pestis) were unable to grow and divide when incubated at 37uC; instead, they required incubation at 27uC [1]. Kupferberg and Smith demonstrated that addition of 2.5 mM calcium to the growth medium supported growth of Y. pestis at 37uC [2] This unusual requirement for calcium was later shown to be correlated to the massive synthesis and secretion of a number of proteins, called Yersinia outer proteins (Yops). Yersinia employs the T3SS to secrete Yops into the external environment and to translocate Yops into the cytoplasm of eukaryotic target cells [11]. Several seminal and general discoveries have been made based on the calcium effect, including T3SS mediated secretion, translocation, and target cell induced expression of effector proteins [12,13,14]
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