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Title: Legionella Metaeffector Exploits Host Proteasome to Temporally Regulate Cognate Effector}
Article:
Abstract:
Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector, ” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.
Introduction:
Many bacterial pathogens encode a large array of “effector proteins, ” that manipulate host cellular processes during infection. Effector proteins are translocated from bacteria directly into the cytosol of host cells. This process is mediated by dedicated bacterial protein delivery systems, including the type III and the type IV secretion systems. In some cases, effector proteins delivered into host cells by a bacterium have opposing functions on a single host protein. For example, Legionella pneumophila DrrA( SidM) and LepB are effector proteins with opposing effects on the host Rab1 GTPase, with DrrA functioning as a guanine nucleotide exchange factor( GEF) and guanine nucleotide dissociation inhibitor-displacement factor( GDF), and LepB having GTPase-activating protein( GAP) activity[1],[2],[3],[4]. Similarly, the Salmonella enterica serovar typhimurium effectors SopE and SptP have GEF and GAP activities for the Rho family of GTPases[5],[6], respectively. Although the GEF activity of SopE is dominant in the host cell immediately after infection, degradation of SopE by the host proteasome alters the balance of these effectors, resulting in the GAP activity of SptP to be dominant later in infection[7]. Although differential regulation of gene transcription and post-translational modifications of effectors have also been shown to regulate their activities in host cells[8],[9], details on how these processes are controlled remain largely unknown; other effector-regulating mechanisms probably also exist. L. pneumophila is a gram-negative bacterium ubiquitously found in freshwater environments[10]. When phagocytosed by eukaryotic cells, L. pneumophila remodels the Legionella-containing phagosome to form a compartment that allows its intracellular replication[11],[12],[13]. As a result, L. pneumophila is able to replicate in a wide variety of phagocytic cells, from amoebae to macrophages; human infections can result in a severe pneumonia called Legionnaires' disease[14]. The Dot/Icm type IV secretion system is an essential virulence determinant that translocates L. pneumophila effector proteins into host cells during infection[15],[16]. These effector proteins control host cell functions to initiate trafficking of the L. pneumophila vacuole, promote host cell survival, modulate innate immune responses, and promote bacterial egress[17]. Although over 100 L. pneumophila effector proteins have been identified, the biochemical and cellular functions of most effector proteins remain unknown[17],[18]. Ubiquitin is a small, well-conserved peptide of 76 amino acids, present in all eukaryotes[19]. Ubiquitination of substrate proteins involves a cascade of reactions. At the last step of the cascade, an E3 ligase recognizes a substrate protein and transfers ubiquitins to the substrate from an E2 conjugating enzyme[20]. Ubiquitinated proteins are subjected to further cellular processes, most notably proteasomal degradation[21]. E3 ligases can be divided into several major families; HECT-type, RING-type, U-box-type and NEL-type[22],[23],[24]. The each family has distinct structural feature, while RING and U-box domains are closely related. Many but not all RING-type E3 ligases work as multi-subunit complexes called SCF complexes containing Skp1, Cullin and F-box proteins. U-box-type E3 ligases contain single U-box domain that serves as an E2-binding site, while L. pneumophila effector protein LubX carries two U-box domains, one of which functions as a substrate-binding site( Figure 1A, see below). NEL-type is the most recent addition to E3 ubiquitin ligase families[24]. NEL family is composed of IpaH/SspH family proteins from bacterial pathogens Shigella flexneri and Salmonella enterica[25],[26],[27] and more than 30 homologous proteins found in bacterial pathogens[24]. Most notably NEL-type E3 ligases seem to be prevalent among bacterial pathogens, whereas no homologous protein has found in eukaryotic cells. It is well documented that bacterial pathogens exploit the host ubiquitin-proteasome pathway by delivering effectors that function as E3 ubiquitin ligases or as deubiquitinating enzymes[24],[28],[29]. L. pneumophila encode one U-box protein( LubX)[30] and several F-box-containing proteins including AnkB/LegAU13/Lpg2144/Lpp2082[31],[32],[33],[34],[35]. We previously reported that LubX functions as a U-box-type E3 ubiquitin ligase in vitro and in host cells[30]. LubX mediates polyubiquitination of a host kinase Clk1, but its consequence remains unknown. Interestingly, the expression and translocation of LubX is induced upon infection and the levels of LubX within host cells come to maximum at later stages of infection, compared to other L. pneumophila effectors so far characterized. The gene encoding LubX is in close proximity to genes that encode several other type IV effectors, including VipD[36] and SidH[37]( Figure 1B). Surprisingly, analyses of these effector proteins led us to identify SidH as a target of LubX. LubX acts as a negative temporal regulator of SidH within host cells. This is the first example of the bacterial effector that targets and regulates a cognate effector within the host cells, and we propose the designation “metaeffector” for this class of bacterial effectors.
Discussion:
The temporal regulation of SidH mediated by L. pneumophila LubX E3 ligase and the host proteasome system illustrates a novel mechanism by which bacterial effectors are regulated. In the previously reported temporal regulation of the induced membrane ruffling by Salmonella effectors SopE and SptP, it was shown that these two effectors have distinct susceptibility towards ubiquitin-mediated proteasomal degradation[7]. Importantly, the determinants of the susceptibility are encoded in the effectors themselves. By contrast, the L. pneumophila SidH is an intrinsically stable protein within host cells; the effector protein LubX controls SidH instability in the host cytosol by directly targeting this protein for host ubiquitination. Thus LubX represents a bona fide metaeffector—a designation for an effector that regulates the function of another effector within the host cell. Discovery of an effector having a regulatory role on another effector provides unique insight into the sophisticated mechanisms that underlie the ability of L. pneumophila to coordinate the function of such a large array of effector proteins with diverse activities. As shown previously[30] and partly in Figure 4 as well, the intracellular level of LubX increases over time and reaches to the maximum at 10–12 hours post infection, which is far late stages compared to the critical time window in which SidH is polyubiquitinated and targeted to proteasomal degradation. It appears that relatively small amount of LubX detected within first couple of hours post infection is sufficient for the polyubiquitination and degradation of SidH( Figure 4). This raised the question on the role of LubX at late stages of infection. We previously reported that host kinase Clk1 is a substrate of LubX[30]. Together with the current findings, one of reasonable explanations would be that LubX has multiple targets within host cells including bacterial SidH and host Clk1. Accumulating lines of evidence suggest that effector proteins functioning as an E3 ubiquitin ligase are prevalent among many plant and animal bacterial pathogens. Some effector proteins which have been reported to possess E3 ligase activity do not show similarity to eukaryotic E3 ligases at sequence level. AvrPtoB of a plant pathogen Pseudomonas syringae shows structural similarity to U-box E3 ligase, whereas they do not share sequence level homology[44]. This suggests that there could be more bacterial E3 ligases than predicted by simple homology search. Structural analyses demonstrated that IpaH/SspH family proteins Shigella flexneri IpaH1. 4, IpaH3 and Salmonella enterica SspH2 show no similarity to eukaryotic E3 ligases and represents a new class of E3 ligase[25],[26],[27]. This class of E3 ligase, called NEL, seems to comprise a large family of bacterial E3 ligases, because more than 30 homologous proteins are found in a subset of bacterial pathogens[24]. Furthermore, there are more than 30 bacterial F-box proteins which are expected to be a key component of SCF E3 ligase complexes[28]. However, the targets of these bacterial E3 ligases remain largely unknown, and all previous studies have aimed to identify host target proteins. SidH is the first example of bacterial targets of the bacterial E3 ligase effector. Future studies without prejudice will reveal that some of bacterial effectors implicated in manipulation of the host ubiquitin system possess regulatory functions as metaeffector that coordinate the expression of effector functions spatiotemporally.
Many bacterial pathogens encode a large array of “effector proteins” that are essential for successful infection . By definition , effector proteins are synthesized in bacteria and transported from bacteria into host cells . Within host cells , effector proteins directly interact with host factors in order to modulate their functions . Effector expression , translocation or activity within host cells must be precisely regulated over infection stages . Here we demonstrate the first example of an effector protein which targets and regulates another effector within host cells: Legionella effector protein LubX targets another effector protein SidH to proteasome-mediated protein degradation in the host cells . Expression and delivery of these effector proteins are differentially regulated , which results in LubX-dependent SidH shutdown at late stages of infection . We propose the designation “metaeffector” for this class of bacterial effector protein: an effector that targets and regulates another effector within host cells . Future studies may reveal that metaeffectors which play critical roles in coordinating the functional expression of other effectors spatiotemporally are prevalent among bacterial pathogens .