Legionella pneumophila regulates the activity of UBE2N by deamidase-mediated deubiquitination
Ontology highlight
ABSTRACT:
SUBMITTER: Dr. Ninghai Gan
PROVIDER: S-SCDT-EMBOJ-2019-102806 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The Legionella pneumophila effector MavC induces ubiquitination of the E2 ubiquitin-conjugating enzyme UBE2N by transglutamination, thereby abolishing its function in the synthesis of K63 -type polyubiquitin chains. The inhibition of UBE2N activity creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular L. pneumophila replication. Here, we show that prolonged inhibition of UBE2N activity by MavC restricts intracellular bacterial replication and that the activity of UBE2N is restored by MvcA, an ortholog of MavC (50% identity) with ubiquitin deamidase activity. MvcA functions to deubiquitinate UBE2N-Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA-UBE2N-Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our study establishes a deubiquitination mechanism catalyzed by a deamidase, which, together with MavC, imposes temporal regulation of the activity of UBE2N during L. pneumophila infection.
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Project description:Legionella pneumophila, the etiological agent of Legionnaires' disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.
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Project description:Effectors delivered into host cells by the Legionella pneumophila Dot/Icm type IV transporter are essential for the biogenesis of the specialized vacuole that permits its intracellular growth. The biochemical function of most of these effectors is unknown, making it difficult to assign their roles in the establishment of successful infection. We found that several yeast genes involved in membrane trafficking, including the small GTPase Ypt1, strongly suppress the cytotoxicity of Lpg0695(AnkX), a protein known to interfere severely with host vesicle trafficking when overexpressed. Mass spectrometry analysis of Rab1 purified from a yeast strain inducibly expressing AnkX revealed that this small GTPase is modified posttranslationally at Ser(76) by a phosphorylcholine moiety. Using cytidine diphosphate-choline as the donor for phosphorylcholine, AnkX catalyzes the transfer of phosphorylcholine to Rab1 in a filamentation-induced by cAMP(Fic) domain-dependent manner. Further, we found that the activity of AnkX is regulated by the Dot/Icm substrate Lpg0696(Lem3), which functions as a dephosphorylcholinase to reverse AnkX-mediated modification on Rab1. Phosphorylcholination interfered with Rab1 activity by making it less accessible to the bacterial GTPase activation protein LepB; this interference can be alleviated fully by Lem3. Our results reveal reversible phosphorylcholination as a mechanism for balanced modulation of host cellular processes by a bacterial pathogen.
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Project description:The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.
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Project description:Legionella pneumophila is an important opportunistic pathogen for which environmental reservoirs are crucial for the infection of humans. In the environment, free-living amoebae represent key hosts providing nutrients and shelter for highly efficient intracellular proliferation of L. pneumophila, which eventually leads to lysis of the protist. However, the significance of other bacterial players for L. pneumophila ecology is poorly understood. In this study, we used a ubiquitous amoeba and bacterial endosymbiont to investigate the impact of this common association on L. pneumophila infection. We demonstrate that L. pneumophila proliferation was severely suppressed in Acanthamoeba castellanii harboring the chlamydial symbiont Protochlamydia amoebophila The amoebae survived the infection and were able to resume growth. Different environmental amoeba isolates containing the symbiont were equally well protected as different L. pneumophila isolates were diminished, suggesting ecological relevance of this symbiont-mediated defense. Furthermore, protection was not mediated by impaired L. pneumophila uptake. Instead, we observed reduced virulence of L. pneumophila released from symbiont-containing amoebae. Pronounced gene expression changes in the presence of the symbiont indicate that interference with the transition to the transmissive phase impedes the L. pneumophila infection. Finally, our data show that the defensive response of amoebae harboring P. amoebophila leaves the amoebae with superior fitness reminiscent of immunological memory. Given that mutualistic associations between bacteria and amoebae are widely distributed, P. amoebophila and potentially other amoeba endosymbionts could be key in shaping environmental survival, abundance, and virulence of this important pathogen, thereby affecting the frequency of human infection.IMPORTANCE Bacterial pathogens are generally investigated in the context of disease. To prevent outbreaks, it is essential to understand their lifestyle and interactions with other microbes in their natural environment. Legionella pneumophila is an important human respiratory pathogen that survives and multiplies in biofilms or intracellularly within protists, such as amoebae. Importantly, transmission to humans occurs from these environmental sources. Legionella infection generally leads to rapid host cell lysis. It was therefore surprising to observe that amoebae, including fresh environmental isolates, were well protected during Legionella infection when the bacterial symbiont Protochlamydia amoebophila was also present. Legionella was not prevented from invading amoebae but was impeded in its ability to develop fully virulent progeny and were ultimately cleared in the presence of the symbiont. This study highlights how ecology and virulence of an important human pathogen is affected by a defensive amoeba symbiont, with possibly major consequences for public health.
| S-EPMC6520448 | biostudies-literature
Project description:Legionella pneumophila are important opportunistic pathogens for which environmental reservoirs such as protists are crucial for the infection of humans. Free-living amoebae are considered key hosts providing nutrients and shelter for highly efficient intracellular proliferation of L. pneumophila, which eventually leads to lysis of the amoeba host cell. Yet, the significance of other bacterial players for L. pneumophila ecology is poorly understood. In this study we used a ubiquitous amoeba and their bacterial endosymbiont to investigate the impact of this common association on L. pneumophila infection. We demonstrate that Acanthamoeba castellanii harboring the chlamydial symbiont Protochlamydia amoebophila were able to erase L. pneumophila and, in contrast to symbiont-free amoebae, survived the infection and were able to resume growth. Environmental amoeba isolates harboring P. amoebophila were equally well-protected, and fresh environmental isolates of L. pneumophila were equally well-erased, suggesting ecological relevance of this symbiont-mediated protection. We further show that protection was not mediated by impaired L. pneumophila uptake. Instead, we observed reduced virulence of L. pneumophila released from symbiont-containing amoebae that is strongly supported by transcriptome data. Interference with transition to the transmissive phase is thus likely the basis for this protection. Finally, our data indicate that the defensive response of amoebae harboring P. amoebophila leaves the amoebae with superior fitness reminiscent of immunological memory. Given that mutualistic associations between bacteria and amoebae are widely distributed, P. amoebophila and potentially other amoeba endosymbionts could be key elements in shaping environmental survival, abundance and virulence of this important pathogen thereby affecting frequency of human infection.
2019-04-14 | GSE125876 | GEO
Project description:Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication.
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Project description:L. pneumophila, an important facultative intracellular bacterium, infects the human lung and environmental protozoa. At least fifteen phospholipases A (PLA) are encoded in its genome. Three of which, namely PlaA, PlaC, and PlaD, belong to the GDSL lipase family abundant in bacteria and higher plants. PlaA is a lysophospholipase A (LPLA) that destabilizes the phagosomal membrane in absence of a protective factor. PlaC shows PLA and glycerophospholipid: cholesterol acyltransferase (GCAT) activities which are activated by zinc metalloproteinase ProA via cleavage of a disulphide loop. In this work, we compared GDSL enzyme activities, their secretion, and activation of PlaA. We found that PlaA majorly contributed to LPLA, PlaC to PLA, and both substrate-dependently to GCAT activity. Western blotting revealed that PlaA and PlaC are type II-secreted and both processed by ProA. Interestingly, ProA steeply increased LPLA but diminished GCAT activity of PlaA. Deletion of 20 amino acids within a predicted disulfide loop of PlaA had the same effect. In summary, we propose a model by which ProA processes PlaA via disulfide loop cleavage leading to a steep increase in LPLA activity. Our results help to further characterize the L. pneumophila GDSL hydrolases, particularly PlaA, an enzyme acting in the Legionella-containing phagosome.
| S-EPMC5701174 | biostudies-literature
Project description:FIH-mediated post-translational modification through asparaginyl hydroxylation of eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing vacuole (LCV) membrane and its modification by FIH, our data show that FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function.
| S-EPMC5337513 | biostudies-literature
Project description:L. pneumophila propagates in eukaryotic cells within a specialized niche, the Legionella-containing vacuole (LCV). The infection process is controlled by over 330 effector proteins delivered through the type IV secretion system. In this study, we report that the Legionella MavH effector localizes to endosomes and remodels host actin cytoskeleton in a phosphatidylinositol 3-phosphate (PI(3)P) dependent manner when ectopically expressed. We show that MavH recruits host actin capping protein (CP) and actin to the endosome via its CP-interacting (CPI) motif and WH2-like actin-binding domain, respectively. In vitro assays revealed that MavH stimulates actin assembly on PI(3)P-containing liposomes causing their tubulation. In addition, the recruitment of CP by MavH negatively regulates F-actin density at the membrane. We further show that, in L. pneumophila-infected cells, MavH appears around the LCV at the very early stage of infection and facilitates bacterium entry into the host. Together, our results reveal a novel mechanism of membrane tubulation induced by membrane-dependent actin polymerization catalyzed by MavH that contributes to the early stage of L. pneumophila infection by regulating host actin dynamics.
| S-EPMC10381072 | biostudies-literature