Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species

Deep sequencing defines the transcriptional map of L. pneumophila and identifies growth phase-dependent regulated ncRNAs implicated in virulence.

International audienceThe bacterium Legionella pneumophila is found ubiquitously in aquatic environments and can cause a severe pneumonia in humans called Legionnaires' disease. How this bacterium switches from intracellular to extracellular life and adapts to different hosts and environmental conditions is only partly understood. Here we used RNA deep sequencing from exponentially (replicative) and post exponentially (virulent) grown L. pneumophila to analyze the transcriptional landscape of its entire genome. We established the complete operon map and defined 2561 primary transcriptional start sites (TSS). Interestingly, 187 of the 1805 TSS of protein-coding genes contained tandem promoters of which 93 show alternative usage dependent on the growth phase. Similarly, over 60% of 713 here identified ncRNAs are phase dependently regulated. Analysis of their conservation among the seven L. pneumophila genomes sequenced revealed many strain specific differences suggesting that L. pneumophila contains a highly dynamic pool of ncRNAs. Analysis of six ncRNAs exhibiting the same expression pattern as virulence genes showed that two, Lppnc0584 and Lppnc0405 are indeed involved in intracellular growth of L. pneumophila in A. castellanii. Furthermore, L. pneumophila encodes a small RNA named RsmX that functions together with RsmY and RsmZ in the LetA-CsrA regulatory pathway, crucial for the switch to the virulent phenotype. Together our data provide new insight into the transcriptional organization of the L. pneumophila genome, identified many new ncRNAs and will provide a framework for the understanding of virulence and adaptation properties of L. pneumophila

Conjugation of Oligo-His Peptides to Magnetic γ-Fe 2 O 3 @SiO 2 Core–Shell Nanoparticles Promotes Their Access to the Cytosol

International audienceThe endosomal entrapment of functional nanoparticles is a severe limitation to their use for biomedical applications. In the case of magnetic nanoparticles (MNPs), this entrapment leads to poor heating efficiency for magnetic hyperthermia and suppresses the possibility to manipulate them in the cytosol. Current strategies to limit their entrapment are based on their functionalization with cell-penetrating peptides in order to promote their translocation directly across the cell membrane or their endosomal escape. However, these strategies suffer from potential release of free peptides in cell and to the best of our knowledge there is currently a lack of effective methods for the cytosolic delivery of MNPs after incubation with cells. Herein, we report the conjugation of fluorescently labelled cationic peptides to γ-Fe2O3@SiO2 core-shell nanoparticles by click chemistry to improve MNP access to the cytosol. We compare the effect of Arg9 and His4 peptides. On one hand, Arg9 is a classical cell-penetrating peptide, able to enter cells by direct translocation and on the other hand, it has been demonstrated that sequences rich in histidine residues promote endosomal escape, most probably by the proton sponge effect. The methodology developed allows to have a high co-localization of the peptides and core-shell nanoparticles in cells and to attest that the grafting onto nanoparticles of peptides rich in histidine promotes NP access to the cytosol. The endosomal escape was confirmed by a calcein leakage assay and by ultrastructural analysis in transmission electron microscopy. No toxicity of the nanoparticles functionalized with peptides was found. We show that our conjugation strategy is compatible with the addition of multiple substrates and can thus be used for the delivery of cytoplasm-targeted therapeutics

Interaction of the Anti-Proliferative GPER Inverse Agonist ERα17p with the Breast Cancer Cell Plasma Membrane: From Biophysics to Biology

International audienceThe peptide ERα17p, which corresponds to the 295-311 fragment of the hinge/AF2 domains of the human estrogen receptor α (ERα), exerts apoptosis in breast cancer cells through a mechanism involving the G protein-coupled estrogen-dependent receptor GPER. Besides this receptor-mediated mechanism, we have detected a direct interaction (Kd value in the micromolar range) of this peptide with lipid vesicles mimicking the plasma membrane of eukaryotes. The reversible and not reversible pools of interacting peptide may correspond to soluble and aggregated membrane-interacting peptide populations, respectively. By using circular dichroism (CD) spectroscopy, we have shown that the interaction of the peptide with this membrane model was associated with its folding into β sheet. A slight leakage of the 5(6)-fluorescein was also observed, indicating lipid bilayer permeability. When the peptide was incubated with living breast cancer cells at the active concentration of 10 μM, aggregates were detected at the plasma membrane under the form of spheres. This insoluble pool of peptide, which seems to result from a fibrillation process, is internalized in micrometric vacuoles under the form of fibrils, without evidence of cytotoxicity, at least at the microscopic level. This study provides new information on the interaction of ERα17p with breast cancer cell membranes as well as on its mechanism of action, with respect to direct membrane effects

Conjugation of Oligo-His Peptides to Magnetic γ-Fe 2 O 3 @SiO 2 Core–Shell Nanoparticles Promotes Their Access to the Cytosol

International audienc

Conjugation of oligo-His peptides to magnetic γ-Fe2O3@SiO2 core-shell nanoparticles promotes their access to the cytosol

The endosomal entrapment of functional nanoparticles is a severe limitation to their use for biomedical applications. In the case of magnetic nanoparticles (MNPs), this entrapment leads to poor heating efficiency for magnetic hyperthermia and suppresses the possibility to manipulate them in the cytosol. Current strategies to limit their entrapment are based on their functionalization with cell-penetrating peptides in order to promote their translocation directly across the cell membrane or their endosomal escape. However, these strategies suffer from potential release of free peptides in cell and to the best of our knowledge there is currently a lack of effective methods for the cytosolic delivery of MNPs after incubation with cells. Herein, we report the conjugation of fluorescently labelled cationic peptides to γ-Fe2O3@SiO2 core-shell nanoparticles by click chemistry to improve MNP access to the cytosol. We compare the effect of Arg9 and His4 peptides. On one hand, Arg9 is a classical cell-penetrating peptide, able to enter cells by direct translocation and on the other hand, it has been demonstrated that sequences rich in histidine residues promote endosomal escape, most probably by the proton sponge effect. The methodology developed allows to have a high co-localization of the peptides and core-shell nanoparticles in cells and to attest that the grafting onto nanoparticles of peptides rich in histidine promotes NP access to the cytosol. The endosomal escape was confirmed by a calcein leakage assay and by ultrastructural analysis in transmission electron microscopy. No toxicity of the nanoparticles functionalized with peptides was found. We show that our conjugation strategy is compatible with the addition of multiple substrates and can thus be used for the delivery of cytoplasm-targeted therapeutics

The <em>Legionella pneumophila</em> F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication.

International audienceThe 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

Structural bases for the involvement of phosphatidylinositol-4,5-bisphosphate in the internalization of the cell-penetrating peptide Penetratin

International audienceCell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2, and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photocrosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photocrosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization

Characterization of an acetyltransferase that detoxifies aromatic chemicals in Legionella pneumophila

International audienceLegionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires' disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these environments. The molecular mechanisms by which this bacterium detoxifies these chemicals remain poorly understood. In particular, the expression and functions of XMEs (xenobiotic-metabolizing enzymes) that could contribute to chemical detoxification in L. pneumophila have been poorly documented at the molecular and functional levels. In the present paper we report the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens and Philadelphia). Strain-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to detoxify aromatic amine chemicals and grow in their presence. The present study provides a new enzymatic mechanism by which the opportunistic pathogen L. pneumophila biotransforms and detoxifies toxic aromatic chemicals. These data also emphasize the role of XMEs in the environmental adaptation of certain prokaryotes

A cationic motif upstream Engrailed2 homeodomain controls cell internalization through selective interaction with heparan sulfates

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells