The Legionella pneumophila F-box protein Lpp 2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication. Cell Microbiol

Institut Pasteur, Biologie des Bactéries Intracellulaires, Departement Genomes et Génétique, F-75015 Paris, France.
Cellular Microbiology (Impact Factor: 4.92). 03/2010; 12(9):1272-91. DOI: 10.1111/j.1462-5822.2010.01467.x
Source: PubMed


The environmental pathogen Legionella pneumophila encodes three proteins containing F-box domains and additional protein-protein interaction domains, reminiscent of eukaryotic SCF ubiquitin-protein ligases. Here we show that the F-box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella-containing vacuole. Single, double and triple mutants of the F-box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP-1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo, and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two-hybrid screen and co-immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein-protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.

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Available from: Carmen Buchrieser, May 18, 2014
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    • "The Icm/Dot substrate AnkB subverts amino acid metabolism and protein degradation by hijacking the host cell ubiquitination machinery and the proteasome to create nutrients for bacterial growth (Al-Khodor et al., 2010). AnkB harbors several eukaryotic domains: an F-box domain that allows interaction with the host SCF1 ubiquitin ligase complex, two ANK domains, which mediate protein-protein interactions in eukaryotes and a CaaX motif that is modified by farnesylation (Price et al., 2009, 2010a,b; Ensminger and Isberg, 2010; Ivanov et al., 2010; Lomma et al., 2010). Farnesylation of AnkB leads to localization of the effector to the LCV membrane, and intracellular replication of L. pneumophila fails when farnesylation is blocked. "
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    ABSTRACT: Legionella pneumophila is a ubiquitous environmental bacterium that thrives in fresh water habitats, either as planktonic form or as part of biofilms. The bacteria also grow intracellularly in free-living protozoa as well as in mammalian alveolar macrophages, thus triggering a potentially fatal pneumonia called "Legionnaires' disease." To establish its intracellular niche termed the "Legionella-containing vacuole" (LCV), L. pneumophila employs a type IV secretion system and translocates ~300 different "effector" proteins into host cells. The pathogen switches between two distinct forms to grow in its extra- or intracellular niches: transmissive bacteria are virulent for phagocytes, and replicative bacteria multiply within their hosts. The switch between these forms is regulated by different metabolic cues that signal conditions favorable for replication or transmission, respectively, causing a tight link between metabolism and virulence of the bacteria. Amino acids represent the prime carbon and energy source of extra- or intracellularly growing L. pneumophila. Yet, the genome sequences of several Legionella spp. as well as transcriptome and proteome data and metabolism studies indicate that the bacteria possess broad catabolic capacities and also utilize carbohydrates such as glucose. Accordingly, L. pneumophila mutant strains lacking catabolic genes show intracellular growth defects, and thus, intracellular metabolism and virulence of the pathogen are intimately connected. In this review we will summarize recent findings on the extra- and intracellular metabolism of L. pneumophila using genetic, biochemical and cellular microbial approaches. Recent progress in this field sheds light on the complex interplay between metabolism, differentiation and virulence of the pathogen.
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    • "L. pneumophila require AnkB to replicate in various hosts, including U937 macrophages, A. castellanii, A. polyphaga, as well as for pulmonary infection in a mouse model (Al-Khodor et al., 2008; Lomma et al., 2010; Price et al., 2010). The AnkB effector itself is posttranslationally modified by farnesylation, which mediates its localization on the membrane of the L. pneumophila replication vacuole (Price et al., 2010). "
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    ABSTRACT: The Gram-negative bacterium Legionella pneumophila is ubiquitous in freshwater environments as a free-swimming organism, resident of biofilms, or parasite of protozoa. If the bacterium is aerosolized and inhaled by a susceptible human host, it can infect alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. A sophisticated cell differentiation program equips L. pneumophila to persist in both extracellular and intracellular niches. During its life cycle, L. pneumophila alternates between at least two distinct forms: a transmissive form equipped to infect host cells and evade lysosomal degradation, and a replicative form that multiplies within a phagosomal compartment that it has retooled to its advantage. The efficient changeover between transmissive and replicative states is fundamental to L. pneumophila's fitness as an intracellular pathogen. The transmission and replication programs of L. pneumophila are governed by a number of metabolic cues that signal whether conditions are favorable for replication or instead trigger escape from a spent host. Several lines of experimental evidence gathered over the past decade establish strong links between metabolism, cellular differentiation, and virulence of L. pneumophila. Herein, we focus on current knowledge of the metabolic components employed by intracellular L. pneumophila for cell differentiation, nutrient salvaging and utilization of host factors. Specifically, we highlight the metabolic cues that are coupled to bacterial differentiation, nutrient acquisition systems, and the strategies utilized by L. pneumophila to exploit host metabolites for intracellular replication.
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    • "Here, AnkB likely recruits host proteins through its ANK domains that are then polyubiquitinated through interaction of the F-box domain with the SCF1 ubiquitin ligase (Fig. 2) (Price et al., 2009; 2010a; Lomma et al., 2010). This generates an LCV that is densely decorated with polyubiquitinated proteins (Dorer et al., 2006; Price et al., 2009; Lomma et al., 2010), which can be detected immediately upon invasion of L. pneumophila into amoebae or human cells (Fig. 2). Polyubiquitination of proteins is a conserved eukaryotic post-translational modification involved in a plethora of cellular processes including signalling, protein localization and protein turnover (Sadowski et al., 2012). "
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    ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, invades and proliferates within a diverse range of free-living amoeba in the environment but upon transmission to humans the bacteria hijack alveolar macrophages. Intracellular proliferation of L. pneumophila in two evolutionarily distant hosts is facilitated by bacterial exploitation of conserved host processes that are targeted by bacterial protein effectors injected into the host cell. A key aspect of microbe-host interaction is microbial extraction of nutrients from the host but understanding of this is still limited. AnkB functions as a nutritional virulence factor and promotes host proteasomal degradation of polyubiquitinated proteins generating gratuitous levels of limiting host cellular amino acids. L. pneumophila is auxotrophic for several amino acids including cysteine, which is a metabolically preferred source of carbon and energy during intracellular proliferation, but is limiting in both amoebae and humans. We propose that synchronization of bacterial amino acids auxotrophy with the host is a driving force in pathogenic evolution and nutritional adaptation of L. pneumophila and other intracellular bacteria to life within the host cell. Understanding microbial strategies of nutrient generation and acquisition in the host will provide novel antimicrobial strategies to disrupt pathogen access to essential sources of carbon and energy.
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