In this work we demonstrated that the Rho GTPase RhoG is targeted by three Yersinia pseudotuberculosis virulence factors. In the absence of Yop expression, RhoG was recruited to the site of bacterial attachment as a result of a high-affinity invasin-β1 integrin interaction (Fig. ), and RhoG inactivation resulted in reduced bacterial uptake efficiency (Fig. ). A novel FRET biosensor (Fig. ) was used to observe accumulation of activated RhoG at sites of bacterial attachment (Fig. ). The translocated toxins YopE (a Rho GAP) and YopT (a prenylcysteine endoprotease) both targeted RhoG: YopE inactivated RhoG and YopT cleaved and mislocalized RhoG (Fig. , , and ). The various methods of RhoG manipulation by Y. pseudotuberculosis are depicted in Fig. .
FIG. 9. Model of RhoG manipulation by Yersinia pseudotuberculosis. (A) RhoG is activated in response to invasin-mediated signaling. When there is no expression of antiphagocytic factors, invasin binding to β1 integrin activates RhoG. Activated (GTP-bound) (more ...)
In evaluating the effect of RhoG inactivation upon invasin-mediated signaling, we used a dominant-negative RhoG allele (encoding the T17N mutation) as well as RNAi-mediated depletion. In our system, both inactivating approaches yielded similar results, causing a defect in invasin-mediated uptake (Fig. ). In other systems dominant-negative mutants have been shown to generate potentially artifactual findings (21
). Patel and Galan (37
) have shown that dominant-negative Cdc42 interferes with Salmonella enterica
-induced ruffle formation, whereas Cdc42 RNAi does not. Similarly, Hakeda-Suzuki et al. (21
) found that inactivating mutations in Drosophila melanogaster
Rac1 do not affect the establishment of cell polarity, whereas dominant-negative mutants do. The system used here—invasin-β1 integrin-mediated signaling through RhoG and Rac1—seems insensitive to such confounding results.
Differences between Rac1 and RhoG recruitment to nascent phagosomes are notable. Patel et al. (38
) observed that Rac1, in the absence of activating signals, is recruited to nascent Fc receptor phagosomes, concluding that Rac1 activation occurs after recruitment to phagosomes. In contrast, our observations indicate that inactive RhoG is not recruited to nascent phagosomes (Fig. ), suggesting that the mechanisms for recruitment and activation may differ between the two highly similar GTPases. It is possible that different factors mediate RhoGDI dissociation for each GTPase, especially since each interacts with a different RhoGDI isoform. Rac1 shows specificity for RhoGDI-1, while RhoG is sequestered by RhoGDI-3 (5
). Alternatively, the difference in recruitment may be due to variations in each of the PBRs. That is, both GTPases are liberated from their respective RhoGDIs by a similar mechanism but localize differently in the absence of activation due to differences in the number of basic residues in the PBR, since the number of C-terminal basic residues has been shown to influence subcellular localization (59
The fate of YopT-cleaved RhoG is unclear. In the case of Rac1, cleavage exposes a nuclear localization signal, leading to the accumulation of activated Rac1 in the nucleus (35
), but no such signal exists in RhoG. Differences between YopT-mediated RhoG and Rac1 mislocalization are depicted in Fig. . Triton X-114 fractionation of cells coexpressing RhoG and YopT (Fig. ) showed an increase in the aqueous form of RhoG, which most likely localizes to the cytosol. This aberrantly localized pool of RhoG, which most likely lacks C-terminal prenylation and thus cannot be sequestered by RhoGDI, may be a new signaling niche for RhoG. Furthermore, the cleaved pool of RhoG is most likely not inactivated by YopE, since evidence exists for cytosolic Rac1 not being susceptible to YopE inactivation (57
). Exposure of the cytosolic compartment to active RhoG may lead to a number of atypical outcomes, ranging from altered immune signaling to altered cytoskeletal rearrangement events. Triton X-114 fractionation shows a marked accumulation of YopT-cleaved RhoG in the aqueous fraction, which is absent in cleaved Rac1 (Fig. ; compare aqueous fractions in RhoG and Rac1 panels). This difference is most likely due to the fact that Triton X-114 does not disrupt the nucleus, which contains a significant fraction of YopT-cleaved Rac1 (Fig. ).
We have used RhoG-ELMO FRET to show RhoG inactivation in the presence of YopE, but this does not directly demonstrate that YopE is a RhoG GAP. Roppenser et al., however, have shown that purified Y. enterocolitica
YopE acts as a GAP for RhoG (42
), so it is likely that the Y. pseudotuberculosis
YopE used in this study is also a RhoG GAP, especially since Y. enterocolitica
YopE and Y. pseudotuberculosis
YopE are 94% identical at the primary protein sequence level.
We have observed that Rac1 and RhoG are able to signal redundantly during invasin-mediated uptake of bacteria, but RhoG and Rac1 functions do not overlap. Inactivation of RhoG by YopE or mislocalization by YopT may be a way of expanding the targeted Rho GTPase repertoire by Y. pseudotuberculosis
. Inactivating Rac1 is crucial for antiphagocytosis, but it may be insufficient to cripple the antibacterial activity of immune cells such as neutrophils. By targeting RhoG, a factor that is required for the generation of ROS by neutrophils, Y. pseudotuberculosis
drastically interferes with an important arm of the host immune defense. Infection of RhoG-deficient mice with Y. pseudotuberculosis
may shed light on the importance of RhoG in the context of a fully intact immune system, especially since RhoG−/−
mice show no apparent defects in immune system development (53
). In addition, RhoG is uniquely localized compared with other Rho GTPases. The active form of RhoG has been shown to localize to the plasma membrane as well as to perinuclear structures that may be of Golgi or endoplasmic reticulum nature (5
). This is in contrast to active Rac1, which localizes almost exclusively to the plasma membrane. By inhibiting both Rho GTPases, Y. pseudotuberculosis
also increases the range of compartments it is able to target. YopE displays a perinuclear localization pattern when expressed in CHO cells (28
), so targeting GTPases that reside in that vicinity is entirely possible. The significance of such differential compartment targeting is not clear but may contribute to successful establishment and progression of disease.
We investigated the interplay between RhoG and Rac1 signaling during phagocytic uptake (Fig. ) and found evidence for both linear and parallel signaling depending on the size of the ingested particle. Both GTPases are activated in response to bacterial binding, but inactivation of only one does not affect uptake efficiency in the presence of a constitutively active form of either GTPase. In contrast, large-particle uptake efficiency is significantly decreased when either GTPase is inactivated, but Rac1 inactivation cannot be overcome by expression of constitutively active RhoG, indicating that large-particle uptake requires signaling from RhoG to Rac1. Active Rac1, on the other hand, can bypass the loss of RhoG function in this case. Observed differences between results described here and previous work may be attributed to assays used. Most previous conclusions have been based on observed changes in cell morphology (17
) and assays directly looking at GTPase activation (27
) or effector/GEF interaction (55
), while we investigated the issue using a functional phagocytosis assay. This assay demonstrates that the requirement for a particular GTPase is partly determined by the nature of the signaling event being assayed.
Roppenser et al. recently reported that RhoG is targeted by the related organism Yersinia enterocolitica
via invasin and YopE (42
). We demonstrate here that Y. pseudotuberculosis
YopE and invasin behave similarly. In addition, we demonstrate that Y. pseudotuberculosis
YopT effectively cleaves RhoG (Fig. ). Roppenser et al. conclude that, in response to bacterial attachment, Rac1 signaling depends on RhoG. Although we believe this to be correct, we find that if the surface of the phagocytic particle is sufficiently small, activated RhoG can bypass a defect in Rac1 signaling. This may be a consequence of the activation of a small pool of Rac1 or of the direct replacement of Rac1 by RhoG and activation of downstream effectors that may normally associate with Rac1 signaling. Clearly, as the surface area of the phagosome is increased, such bypass cannot occur.
Pathogenic Yersinia species encode sophisticated systems to dampen the host response and proliferate successfully. Misregulation of RhoG appears to be yet another clever adaptation by these organisms that has not been uncovered previously, and precise ramifications of this misregulation remain to be investigated. Our study has shed light on RhoG's unique nature among GTPases that are misregulated by Y. pseudotuberculosis. Despite a high degree of primary sequence similarity to such GTPases as Rac1, RhoG's distinctive subcellular localization and crucial role in proper neutrophil function make it stand out as a novel target of pathogenic Yersinia.