The TTSS-mediated translocation of bacterial effectors into host cells is an intricate mechanism that, although extensively studied, has not been completely unraveled [31
]. Here we have found that Y. pseudotuberculosis
engages the small GTPase Rho to control the delivery of effectors to the host cell. Activation of this signaling pathway is mediated by the YopB/YopD translocon in cooperation with the high affinity binding of invasin or YadA to β-1 integrins.
It has been put forward that pore formation and translocation of effector Yops into the host cells are not related processes [19
]. Pore formation has been recently implicated in mediating a caspase-1 dependent type of cell death in Salmonella
-infected macrophages [21
]. Shin and Cornelis [33
] have recently reported that insertion of translocation pores in macrophages infected with a multi-effector mutant of Y. enterocolitica
triggers activation of caspase-1. Here we ruled out that in our infection system, YopB/YopD-mediated pore formation induces caspase-1 dependent cell death. Thus, amounts of a specific caspase-1 inhibitor large enough to block IL-1 β production in macrophages, does not prevent LDH release in Hela cells. Also, glycine treatment that efficiently prevented cell lysis in Salmonella
infected macrophages failed to inhibit LDH release in Yersinia
-infected HeLa cells. Based on these findings, we sustain that in our experimental system pore formation-induced LDH release is related to the process of Yop translocation.
Both pore formation and translocation require activation of small Rho GTPases, as glucosylation of Rho, Rac and Cdc42 by C. difficile
toxin ToxB potently inhibits the two processes. We found that Rac activation is not likely to be involved in pore formation or translocation. Thus over-expression of a dominant negative form of Rac does not prevent uptake of membrane impermeable dyes in cells infected with the pore forming strain. In line with these results, a specific Rac inhibitor, NSC23766, that efficiently blocks Rac-mediated internalization, does not inhibit pore formation or translocation. On the other hand, we found that signaling downstream of Rho is essential for the control of Yops delivery. Treatment with C. botulinum
C3 toxin, that converts endogenous Rho A, B and C into dominant negative forms [3
], potently down-regulates pore formation and translocation without affecting bacterial adhesion or internalization considerably.
The type of host cell processes that Rho proteins regulate to promote translocation and pore formation most likely involves actin cytoskeleton rearrangements. Thus treatment with 2μg/ml actin polymerization inhibitor CD blocks pore formation [18
] and decreases the level of YopE translocation by more than 60%. In early studies aim at demonstrating that Yop translocation is mediated by extracellular bacteria, Sory et al studied the effect of 5μg/ml CD treatment on the delivery of Yop-cyclase fusion proteins by Y. enterocolitica
into murine macrophages [34
]. Compared to the dramatic effect on bacterial uptake (2000 fold inhibition), the authors suggest that Yop translocation was not sensitive to the action of CD. However, their results show that CD treatment decreased YopE-cyclase translocation by 32% and YopH-cyclase by 52%. Using 10 times less CD (0.5μg/ml for 30 min) and using a strain of Salmonella
ectopically expressing YopE, Rosqvist et al
reported that Yop translocation into HeLa cells was notably decreased [35
]. The authors also reported that the same was observed when YopE was delivered by Y. pseudotuberculosis
. Interestingly, our findings strongly suggest that actin polymerization required for pore formation and translocation is dependent on Rho, as inhibition of Rho A, B and/or C results in a decrease of the number of actin halos.
Adhesion of bacteria to host cell is crucial for the activation of the TTSS. In Y. pseudotuberculosis
two main adhesins, invasin and YadA, mediate tight binding to host cells by interaction with β1 integrin receptors. Here we show that in an inv/yadA
mutant, constitutive expression of the pH6 antigen confers good adhesion properties to host cells. In spite of that, we found that such mutants are defective in pore formation and Yop translocation, suggesting that interaction with β1 integrin receptors is essential for the two processes. Mota et al.
have shown that a minimal needle length is required for efficient functioning of the Yersinia
injectisome, and that this length correlates with the length of the YadA protein [36
]. We considered that the attachment imparted by pH6 antigen in the absence of invasin and YadA, might not provide that critical length. Our data suggest that this is not likely to be the case in our experimental system. First, a Y. pseudotuberculosis
strain expressing pH6 antigen is able to stimulate a YopB-dependent proinflammatory response, including activation of NFκB and ERK, and production of IL-8. Second, a single amino acid substitution in invasin (invD911E), that is not expected to change its length, failed to mediate efficient Yop translocation. This mutant promotes adhesion without inducing receptor clustering and subsequent β1 integrin-mediated signal transduction. Altogether, these results suggest efficient translocation requires high affinity binding of β1 integrin receptors and subsequent activation of signaling. It is still conceivable that, independent of integrin signaling, tight bacterial adhesion mediated by high affinity interaction with β1 receptors preconditions effective translocation. The fact that interfering with β1 integrin signaling by the action of a Src inhibitor impairs efficient translocation, would argue against that idea. Still, we cannot discard the possibility that Src activity might also be required for YopB/D-dependent Rho activation.
We predict that upon integrin clustering, RhoA could be recruited and generate a signal that polymerizes actin. It is well documented that invasin engagement of β1 integrin receptors triggers Rac1-mediated signals that induce bacterial internalization into epithelial cells [15
]. This Rac1-mediated mechanism involves Arp2/3, PIP 4,5 and capping-proteins [30
]. Results from our GTP-Rho pull down assays suggest that bacteria producing invasin and YadA (YP29) can also mediate Rho activation in a YopB-independent manner. There are further evidences in the literature that engagement of β1 integrin receptors can stimulate RhoA activation. Wong and Isberg have shown that RhoA is recruited at the nascent phagosome in Cos1 cells infected with a yopE yopT
mutant of Y pseudotuberculosis
]. Werner et al have reported that interaction of invasin-coated beads with α5β1 integrin in synovial fibroblast results in beads uptake by a process that is RhoA-dependent [37
]. Also, activation of RhoA by engagement of α5β1 integrins by Ipa invasins has been implicated in the internalization of Shigella
to HeLa cells [38
]. Alternatively, β1 integrin may indirectly facilitate Rho activation by a focal adhesion kinase (FAK) -dependent pathway. Such a mechanism of Rho activation has been described for the regulation of microtubules stabilization at the leading edge of mouse fibroblasts [40
], and involves targeting of Rho to GM1-rich domains in the plasma membrane, where it can interact with downstream effectors.
We envision a model in which high affinity binding to β1 integrin receptors, in addition to stimulating Rac activation, triggers Rho activation (). Subsequently, YopB/D insertion into the plasma membrane stimulates increased Rho activation, and the cooperative activation of Rho stimulates Yop translocation. A central question is how Rho activation regulates Yop translocation. We hypothesize that Rho signaling induces changes in the host cell, such as actin polymerization, that are required for an efficient translocation process. One possibility is that, cell molecules present in specialized membrane microdomains, such as lipid rafts, are required for efficient translocation. These membrane microdomains would be recruited at the site of bacteria-host cell contact, as a result of Rho GTPases activation and actin polymerization. More injectisomes could then interact with lipid rafts at the site of bacteria, and more effector Yops would be translocated. Once proper amounts of Yops are delivered into the host cell, the process would be shut down to avoid further cell damage caused by excessive signaling. We based our hypothesis, in part, on the fact that Salmonella
-YopB homologues bind to cholesterol [41
], and that lipid raft are required for translocation in Salmonella
and EPEC [41
]. Interestingly, actin polymerization and Rho GTPases activation have been shown to be involved in lipid raft clustering in B cells [42
], T cells [43
] and NK cells [44
Model for the Requirement of Rho Activation and Actin Polymerization for Pore Formation and Efficient Translocation
Why is Rho-dependent, but not Rac-dependent, actin polymerization required for translocation? Rho GTPases transmit signals that control the formation of distinct cytoskeletal structures through the interaction with different nucleating machineries. Cdc42 and Rac mediate nucleation of branched actin filaments through the Arp2/3 protein complex, leading to lamellipodia formation. On the other hand, Rho proteins stimulate unbranched actin filaments formation, such as those in stress fibers, via interaction with formins. It could be speculated that only F-actin structures generated by formins are important for translocation. The effect of the expression of dominant negative mutants of the formin mDia1 on translocation will be investigated in future studies.
Findings from two studies that investigate translocation of TTSS effector proteins by Salmonella
in real time [45
] indicate that effector translocation occurs right after host cell contact, with a half maximal rate of about 4 min. In our experimental model we detect the strongest Rho activation after 10 to 15 min infection with a YopEHJ
bacteria. This is probably due to accumulation of GTP-Rho in the absence of the Rho inhibitors YopE and YopT. The decrease in the levels of GTP-Rho after 15 min is presumably due by the action of endogenous GAPs. We envision that during infection with wild type bacteria, the kinetics of Rho activation would be much faster. Translocation of Salmonella
SipA and SopE, and Shigella
IpaC were found to follow a linear kinetic [45
]. Interestingly, however, slopes of IpaB secretion kinetics curves seemed to vary at different time points, suggesting that the speed of injection changes during the course of the translocation process resembling a slow-fast-slow type of mechanism. This type of translocation kinetic is what we would expect in our model.
How does our model fit with the mechanism of Yop translocation in Y. pestis
? Although closely related to Y. pseudotuberculosis
, Y. pestis
lacks invasin and YadA. Unless Y. pestis
has yet-unidentified adhesins that interact with β1 integrin receptors, we envision that the bacteria would activate Rho only by the stimulus elicited by YopB/D. In this situation, Rho activation would be limited, and therefore, one should expect that Y. pestis
would be less effective for Yop translocation. A recent report suggests that, in macrophages, Y. pestis
translocate less YopJ than a Y. enterocolitica
strain expressing invasin and YadA [47
]. However, in this report the authors suggest that this is most likely due to a difference between the YopJ protein from the two Yersinia
species. We have preliminary results suggesting that Y. pestis
deliver much less YopE in HeLa cells than Y. pseudotuberculosis
It has been proposed that, because bacterial effectors are directly injected within cell cytosol, the TTSS does not need to trigger signals through cell surface receptor [48
]. Our data suggest that, although not essential, signal stimulated by engagement of β1 integrin receptors greatly enhances Yop translocation.