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.
Highlights
After ingestion by a host, Yersinia pseudotuberculosis gains a foothold in intestinal lymph nodes by moving across M cells into Peyer’s patches [1]
In cells transfected with CFP-tagged Rac1, binding by the GTP-loaded form of Rac1 to YFP-tagged p21-binding domain (PBD) was measured by intermolecular fluorescent resonance energy transfer from the CFP moiety to the YFP moiety after 440 nm excitation (Figure 1A)
Engagement of integrin receptors by the Y. pseudotuberculosis invasin protein results in activation of Rac1, but activation was localized around phagosomal cups that harbored Y. pseudotu
Summary
After ingestion by a host, Yersinia pseudotuberculosis gains a foothold in intestinal lymph nodes by moving across M cells into Peyer’s patches [1]. As is true for the closely related Y. enterocolitica, entry into regional lymph nodes in the first few hours after infection requires the bacterial outer membrane protein invasin [2,3], which binds multiple b1 integrin receptors [1,4]. Invasin-mediated uptake by cultured cells involves internalization of bacteria into membrane-bound compartments [9]. This adhesion event initiates actin rearrangements [4] controlled by the small guanosine triphosphatase (GTPase) Rac1 [10,11]. The details that link integrin engagement to Rac activation are unclear, it has been argued that during integrin adhesion events, GTP loading produces a signal that releases cytosolic Rac from the protein Rho GDP-dissociation inhibitor (RhoGDI) [13]. Overproduction and localization studies argue that actin dynamics promoted by Arp2/3 and the lipid phosphoinositol-4,5-phosphate, as well as the small GTPase Arf, are players downstream from Rac during uptake [14]
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