Yersinia pseudotuberculosis Spatially Controls Activation and Misregulation of Host Cell Rac1

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Abstract
Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell. Many disease-causing bacteria transfer proteins into host cells, interfering with defense against infections. Bacteria often do this by manipulating host proteins that send signals. This study analyzes how one such bacterial pathogen manipulates the host signaling protein Rac1. The proteins YopT and YopE, which are made by several pathogens, including the agent of bubonic plague, had been presumed to inactivate Rac1. The authors show here that this model is too simple, and that pathogens are able to both inactivate and maintain activation of a host protein in a single cell. In this work, the pathogen divides up the Rac1 population into two pools, each with different potentials to send signals. One pool is found in the host cell cytoplasm and is unable to function properly. The other pool of Rac1 is sent into the nucleus, where it still sends an appropriate signal. Therefore, a bacterial pathogen is shown to allow signaling from one site in the host cell, while preventing it from occurring at a different site. Such locale-dependent events within single cells were not previously thought to play a role in microbial pathogenesis.