Many Gram-negative pathogenic bacteria of both animals and plants have evolved type III and/or type IV secretion systems (T3/4SSs) as an essential virulence determinant. These secretion systems are large protein complexes spanning the bacterial envelope that are dedicated to the transfer of protein or DNA substrates into target cells to subvert host defense and other signaling cascades for the benefit of the invading pathogen. We refer the reader to some excellent reviews on these topics [15–17]. In the last decade, an enormous body of work has established the role of T3/4SS effector proteins in bacterial virulence. Similarity in structure or function to eukaryotic proteins allows them to interfere with many different cellular processes, including cytoskeleton rearrangement and intracellular trafficking. The biochemical functions they fulfill within the host cell remain undetermined for the plethora of bacterial T3/4SS effectors identified to date. This is a major challenge for understanding the molecular basis of pathogenicity. Recent advances in this field include the discovery of the different T3/4SS effectors of mammalian bacterial pathogens that have the capacity to interfere with the host's Rho GTPase activity, to reorganize the actin cytoskeleton, and to allow or prevent bacterial internalization ([18] and references therein). Another example of the intriguing co-evolution between a pathogen and its host is the type III secretion system (T3SS) effector-mediated suppression of localized programmed cell death, which is triggered in plants when a specific resistance protein recognizes a specific avirulence protein of the pathogen ([19] and references therein).

Two other Salmonella SPI-1 T3SS effectors, SopA and SopB, are functionally regulated by host ubiquitination. SopB is a phosphoinositide phosphatase that modulates vesicle trafficking by altering the phosphoinositide metabolism. It was shown to be monoubiquitinated and degraded, although probably not via the proteasome [25]. SopA, a protein required for the elicitation of intestinal inflammation, has been shown to be ubiquitinated within the host cell by the membrane-anchored RING-type E3 Ub ligase HsRMA1, and degraded by the proteasome in an HsMRA1-dependent manner [26]. The authors suggest that HsRMA1-dependent ubiquitination of SopA is involved in the escape of bacteria from Salmonella-containing vacuoles into the cytosol of epithelial cells. Recently, the same research group identified SopA as a HECT-type E3 Ub ligase. They identified the catalytic cysteine residue of SopA, and showed the formation of a transient E3-ubiquitin intermediate. These new data are in line with the mono-Ub state of SopA previously detected in the absence of HsRMA1 [26]. Interestingly, this SopA HECT ligase activity is not required for the escape of bacteria from Salmonella-containing vacuoles into the cytoplasm, but seems to be involved in Salmonella-induced transepithelial migration of polymorphonuclear neutrophils (PMNs) [27]. The recruitment of these inflammatory cells depends on several factors, including interleukin (IL)-8 secretion, and is considered an important factor for the development of Salmonella-induced enteritis. The bacterial or host target proteins for the HECT Ub ligase activity of SopA may well be involved in PMN migration, but are unknown at this stage. Together, the data suggest that SopA displays multiple functions during Salmonella infection.

Yersinia sp. and Pseudomonas aeruginosa may also modulate the activity of their effectors by a similar strategy. Yersinia, the causal agent of plague (Y. pestis) and gastrointestinal disorders (Y. pseudotuberculosis and Y. enterocolitica), is an extracellular pathogen that injects several effectors through its T3SS to provoke disease [28]. Several of these effectors have been shown to interfere with actin cytoskeleton dynamics that are involved in blocking phagocytosis and subsequent bacterial killing. Y. pseudotuberculosis YopE contributes to virulence by inducing depolymerization of actin filaments in the host cells early after contact with Y. pseudotuberculosis via the inhibition of Rho GTPases, which control rearrangements of the actin skeleton. This activity also prevents the formation of pores in the host membranes and subsequent host cell death, and thus enables a prolonged colonization of the host [29,30]. In Y. enterocolitica–infected cells, YopE is polyubiquitinated on lysine K75 and targeted for proteasome degradation [31]. At this time, it is not clear whether the host-mediated YopE destruction is beneficial to the host's defense or to Y. enterocolitica infection. On the one hand, the degradation products of YopE could be a bacterial antigen source for the host to fend off later infections [31,32]. On the other hand, removing YopE could pave the way for YopT (a cysteine protease [33]) and YopO (a kinase [34]), two other T3SS effectors also targeting actin rearrangements in the host cell. Intriguingly, YopE and S. typhimurium SptP both have GTPase-activating protein activity that indirectly inhibits the pathogen-induced actin polymerization, but it is interesting to note that YopE is actively degraded by the host UPS, whereas SptP has a much longer half-life. These two effector proteins, translocated by different pathogens, seem to have evolved a similar strategy for blocking actin polymerization, yet subtle differences in interaction with the host UPS seem to reflect differences in infection strategy.

Y. pseudotuberculosis and Y. pestis YopJ, and their homologue YopP in Y. enterocolitica, (phrased YopJ/P when we refer to Y. pseudotuberculosis YopJ and Y. enterocolitica YopP proteins at the same time) were shown to interfere with the host inflammatory response via both the MAPK and the anti-apoptotic NF-κB signaling pathways (Figure 3), which prevents the production of pro-inflammatory cytokines such as TNFα and IL-8, and triggers apoptosis. YopJ/P homologues are found in S. typhimurium (protein AvrA) and in some plant pathogenic bacteria [46]. These proteins were assigned as C55 cysteine proteases [47], and by sequence comparison were originally hypothesized to be deSUMOylating enzymes, even though no specific substrate could be identified [48,49]. Recently, different research groups have identified YopJ/P as deubiquitinating enzymes directly involved in the inhibition of the inflammatory response [43–45,50]. Zhou and collaborators showed that Y. pseudotuberculosis YopJ can deubiquitinate the K63-poly-Ub chain of TRAF6 (and TRAF2) inducing the inhibition of both the MAPK and NF-κB pathways. Y. pseudotuberculosis YopJ was also shown to deubiquitinate the K48-poly-Ub chain from IκBα, preventing proteasome-mediated degradation of IκBα and the resulting NF-κB nuclear translocation [50]. In addition, Y. enterocolitica YopP can cleave TRAF6 and NEMO K63-poly-Ub chains, but this activity could only be proven in vitro [44]. Recently, Thiefes and colleagues showed that Y. pestis YopP was not able to deubiquitinate TRAF6, but rather that it cleaved the K63-Ub chains of TAK1 and TAB1 [43]. The versatility of Y. pseudotuberculosis YopJ was further emphasized by the finding that it could remove the single Ub linked to IKKβ [45]. Tampering with Ub in the inflammatory pathway is achieved so efficiently by YopJ of Y. pseudotuberculosis and Yop P of Y. enterocolitica that Y. pestis has evolved a less invasive strategy. Indeed, Y. pestis YopJ is believed to be injected by the T3SS less efficiently than YopP, and, as a consequence, does not result in fast apoptosis of macrophages, but rather weakens the inflammatory response, enabling the pathogen to be transported within the macrophages to target organs [51]. These reports emphasize the crucial role of bacterial effectors in the deubiquitination of key components in the MAPK and NF-κB pathways, on both K48- and K63-poly-Ub chains (Figure 3). Interestingly, the eukaryotic deubiquitinating enzymes cylindromatosis tumor suppressor protein and A20, which are required for cell homeostasis [52–54], can also deubiquitinate the K63-polyubiquitin chains of signaling intermediates of the NF-κB pathway, but are not active on the K48 chains [50,55]. This illustrates that pathogens clearly use mechanisms that are familiar to the host. Even though YopJ/P seem to have an extended deubiquitinating activity (both of K48- and K63-poly-Ub chains), these proteins don't seem to be able to cleave SUMOylated proteins. Indeed, YopJ/P are deubiquitinating enzymes in vitro, with no activity on SUMOylated proteins [50]. The cysteine residue (C172) that was originally defined for the deSUMOylation activity [49] is also required for the deubiquitinating activity [44,50]. We would like to add that recent work shows that Y. pseudotuberculosis YopJ blocks signaling by binding and acetylating residues in the activation loop of MEKK6 (a mitogen-activated protein kinase kinase upstream of c-Jun NH₂-terminal kinase and p38) [56]. The authors hypothesize that a similar activity could prevent phosphorylation of IKKβ, thus preventing further activation of the NF-κB pathway [56].

Evidently more work is necessary to unravel the mode of action of the YopJ/P family of T3SS effectors. It would be interesting to re-evaluate the function of YopJ/P homologues widely present in plant pathogenic bacteria and currently identified as deSUMOylating enzymes [46,57–59].

The facultative intracellular pathogen Shigella flexneri, causal agent of shigellosis in humans, has among its repertoire of T3SS effectors a protein called OspG that is structurally related to a kinase. This protein was shown to be injected into epithelial cells, where it weakens the host innate immune response [60]. OspG negatively regulates the NF-κB inflammatory response by interfering with the proteasome-dependent degradation of IκBα. OspG binds and inhibits ubiquitinated E2 Ub-conjugating enzymes: it interacts with UbcH5, which is necessary for the Ub supply to the E3 Ub ligase SCF^βTrCP, the specific SCF complex that controls IκBα degradation (Figure 3). OspG displays a kinase activity inducing its autophosphorylation. This activity is necessary for the function of OspG, but doesn't seem to be involved in the phosphorylation of IκBα prior to its K48-polyubiquitination and proteasome-mediated degradation. The mechanism by which OspG inhibits the SCF^βTrCP-mediated IκBα degradation still has to be elucidated. This T3SS effector is injected into host cells during the early stages of infection [61]. An attractive hypothesis is that the inhibition of the NF-κB inflammatory response in these early stages facilitates cellular colonization by a limited number of luminal bacteria [60]. Considering that OspG can interact with different E2 Ub-conjugating enzymes, this effector might interfere with other UPS targets in the host cells as well.

P. syringae causes bacterial speck disease on susceptible plants. HopM1 is a P. syringae T3SS effector required for virulence and is known to suppress the plant host cell wall–associated defense [72]. Recently, Nomura and colleagues showed that during bacterial infection of Arabidopsis plants, HopM1 mediates the proteasome-dependent elimination of AtMIN7, a plant protein involved in cell wall–associated host defense [78]. Interestingly, HopM1 has no classical E3 Ub ligase features, leading to the hypothesis that HopM1 may act as an adaptor protein mediating the recognition of AtMIN7 by the plant UPS. AtMIN7 is a GEF of the adenosine diphosphate ribosylation factor (ARF) subfamily. ARF-GEFs are important for vesicle trafficking by activation of Ras-like small GTPases [82]. Several lines of evidence show that vesicle trafficking plays an important role in plant immunity [72,83–85]. When challenged with a ΔCEL P. syringae mutant strain (lacking the hopM1 gene), AtMIN7 knock-out plants accumulate less callose deposits and are more susceptible to infection than wild-type plants. Altogether, the data suggest that the role of HopM1 in virulence is to inhibit vesicle trafficking associated with cell wall–associated host defense by targeting AtMIN7 for degradation by the host UPS [78].

Agrobacterium tumefaciens causes crown gall disease on a broad range of plants. The bacterium uses a type IV secretion system (T4SS) not only to translocate effectors into eukaryotic cells, but also to mediate the transfer of a single-stranded DNA molecule (transferred [T]-DNA), resulting in genetic colonization of the host [93–95]. VirF is a T4SS effector that determines host range and is necessary for full virulence on certain host plants [96,97]. VirF was the first prokaryotic protein shown to contain a conserved F-box domain [98]. F-box proteins (FBPs) are key components of the SCF type E3 Ub ligase complex, because they recruit the target protein for destruction by the 26S proteasome. Through the F-box domain, FBPs interact with the SKP1 component of the E3 Ub ligase complex [99]. The Arabidopsis homologues of the yeast SKP1 protein, ASK1 and ASK2, were isolated as interactors of VirF [98]. The F-box of VirF was shown to be essential not only for this interaction in vitro, but also for virulence. To determine the precise role of VirF in the infection process, recent studies are aimed at identifying the target proteins destined for ubiquitination and possibly degradation by the proteasome. Recently, it was shown that VirF interacts with the plant protein VirE2-interacting protein 1 (VIP1), which leads to degradation of VIP1 and, indirectly, of the effector protein VirE2 [100]. VirE2 is a single-stranded DNA-binding protein that is transported independently from the T-DNA by the T4SS into the host cell [101,102], where it cooperatively binds the T-DNA to facilitate nuclear uptake [103,104] and protect it from degradation [105]. VirE2 contains functional nuclear localization signals [104,106], but these signals overlap with the DNA-binding domain [106,107], which make it difficult to show an in vivo function of the nuclear localization signal region in nuclear uptake of the T-complex. The Arabidopsis protein VIP1 was identified as an interactor of VirE2 [108]. This protein was shown to interact with karyopherin-α, a member of the importin family involved in nuclear import of proteins via recognition of their nuclear localization signals. Citovsky and colleagues suggested a role for VIP1 in A. tumefaciens infection as a molecular adaptor between VirE2 and karyopherin-α that results in nuclear uptake of the T-complex [109]. Recently, Tzfira and colleagues proposed that VirF, which binds to VIP1 but not VirE2, is involved in the nuclear proteasome-dependent degradation of VIP1 and indirectly in that of VirE2, and may thus play a role in uncoating the T-complex from VirE2 molecules prior to integration of the T-DNA in the host genome [100]. A host-dependent role in virulence for VirF by indirectly targeting another effector protein for degradation is intriguing; yet, it remains to be determined whether VirF is able to destabilize VIP1 and VirE2 when complexed with T-DNA and, more importantly, during infection. In preliminary experiments, using the C-terminal part of VirF (lacking the F-box domain) as bait in a yeast two hybrid screen, several Arabidopsis proteins have been identified as putative targets of VirF (E. Jurado-Jácome, P. Hooykaas, and A. Vergunst, unpublished data). Among these are proteins that have been shown to be involved in host defense–related processes. Although the interaction of some of these proteins has been confirmed in vitro, the relevance of these interactions during infection and their VirF-mediated degradation remains to be confirmed. As described above, a function in disarming host proteins involved in defense against bacterial attack and suppression of the immune response seems to be a general mode of action for effectors of both mammalian and plant pathogens.

Our screen also revealed conserved F-box–encoding genes among several sequenced plant pathogens, including Xanthomonas sp., and several P. syringae pathovars. In summary, our in silico analysis indicates a wide range of bacteria, from the class of Chlamydiae to α-, β- and γ-proteobacteria, predicted to contain putative FBPs. It will be interesting to find out whether the bacteria with putative FBPs can indeed inject these proteins as substrates of the T3/4SS into host cells, where they could interfere with UPS-controlled mechanisms to benefit in the survival of the pathogen.