Lysosomal evasion of legionella pneumophilia the effector.

Legionella pneumophila is a Gram-negative facultative intracellular bacterium found in freshwater environments that has co-evolved to survive and proliferate in various amoeba and protozoan species, which serve as the natural host for the bacterium. Humans are an accidental host of L. pneumophila, where infection occurs upon inhalation of aerosolized water droplets that contain the bacteria. Intracellular proliferation of L. pneumophila in alveolar macrophages is essential for manifestation of pneumonia, designated as Legionnaires’ Disease. Biogenesis of the legionella containing vacuole (LCV) occurs via interception of ER-Golgi vesicle trafficking and avoids the default endosomal/lysosomal degradation pathway. Intracellular proliferation of L. pneumophila within protozoa and macrophages is dependent on the Dot/Icm type IV secretion system (T4SS) apparatus, which is comprised of 27 proteins and is responsible for translocating over 350 different effector proteins into the host cell. Many of these effector proteins contain eukaryotic-like domains and motifs, which have been acquired through interkingdom horizontal gene transfer from various aquatic eukaryotic hosts. While L. pneumophila contains the largest repertoire of effector proteins, known for an intracellular pathogen, most of which are not required for survival and proliferation in mammalian macrophages. It is more likely that the large repertoire of effector proteins constitutes a toolbox utilized by L. pneumophila to survive and replicate within various protozoan species. The diversion of the L. pneumophila-containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for disease manifestation. Many of the ~350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes; but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophilia in human monocyte-derived macrophages (hMDMs), amoebae and for intrapulmonary proliferation in mice. This is shown by both single cell analysis during confocal microscopy and by quantifying colony forming units (CFUs). We have shown the mavE null mutant fails to remodel the LCV with ER-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. Importantly, during infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE (39-172) was resolved to 1.8 Å, revealing a eukaryotic NPxY motif that binds with phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. We show that point mutations within the NPxY motif results in attenuation of L. pneumophila in both hMDMs and amoeba, and the substitution defects of P78 and D64 results in fusion of the LCV to the lysosomes, with no remodeling by the ER, leading to bacterial degradation. Following ectopic expression of MavE, a proximity-dependent biotin identification (BioID) strategy was used to screen for MavE-interacting proteins in mammalian cells. These data show that MavE interacts with a host protein, acyl-CoA binding domain containing 3 (ACBD3), which co-localizes with the LCV. ACBD3 plays an essential role in the sorting and modification of proteins exported from the endoplasmic reticulum through its interaction with the integral membrane protein giantin. We have shown the mavE null mutant-containing LCV fails to colocalize with ACBD3, similar to the Dot/Icm translocation-defective mutant. There are areas of homology of ACBD3 with proteins found in Amoebozoa, indicative of a possible conserved binding motif. We conclude that the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion by interacting with the host protein ACBD3, which is involved in ER-Golgi vesicle trafficking and is likely conserved throughout evolution

Maturation of heterogeneity in afferent synapse ultrastructure in the mouse cochlea

Auditory nerve fibers (ANFs) innervating the same inner hair cell (IHC) may have identical frequency tuning but different sound response properties. In cat and guinea pig, ANF response properties correlate with afferent synapse morphology and position on the IHC, suggesting a causal structure-function relationship. In mice, this relationship has not been fully characterized. Here we measured the emergence of synaptic morphological heterogeneities during maturation of the C57BL/6J mouse cochlea by comparing postnatal day 17 (p17, ∼3 days after hearing onset) with p34, when the mouse cochlea is mature. Using serial block face scanning electron microscopy and three-dimensional reconstruction we measured the size, shape, vesicle content, and position of 70 ribbon synapses from the mid-cochlea. Several features matured over late postnatal development. From p17 to p34, presynaptic densities (PDs) and post-synaptic densities (PSDs) became smaller on average (PDs: 0.75 to 0.33; PSDs: 0.58 to 0.31 μ

An Indispensable Role for the MavE Effector of in Lysosomal Evasion

ABSTRACT Diversion of the -containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires’ pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in have been shown to interfere with various host pathways and processes, but no effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C-terminal transmembrane helix, three copies of tyrosine-based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of in both hMDMs and amoeba. The substitution defects of P and D are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of is indispensable for phagosome biogenesis and lysosomal evasion. Intracellular proliferation of within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires’ pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the -containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway. The pathogen has evolved to inject ∼350 protein effectors into the host cell where they modulate various host processes, but no effector has ever been identified to be indispensable for lysosomal evasion. We show that the MavE effector localizes to the poles of the LCV membrane and is essential for lysosomal evasion and intracellular growth of and for intrapulmonary proliferation in mice. The crystal structure of MavE shows an NPxY eukaryotic motif essential for ER-mediated remodeling and lysosomal evasion by the LCV. Therefore, the MavE effector of is indispensable for phagosome biogenesis and lysosomal evasion

Feasibility pilot trial for the Trajectories of Recovery after Intravenous propofol versus inhaled VolatilE anesthesia (THRIVE) pragmatic randomised controlled trial

Introduction Millions of patients receive general anaesthesia for surgery annually. Crucial gaps in evidence exist regarding which technique, propofol total intravenous anaesthesia (TIVA) or inhaled volatile anaesthesia (INVA), yields superior patient experience, safety and outcomes. The aim of this pilot study is to assess the feasibility of conducting a large comparative effectiveness trial assessing patient experiences and outcomes after receiving propofol TIVA or INVA.Methods and analysis This protocol was cocreated by a diverse team, including patient partners with personal experience of TIVA or INVA. The design is a 300-patient, two-centre, randomised, feasibility pilot trial. Patients 18 years of age or older, undergoing elective non-cardiac surgery requiring general anaesthesia with a tracheal tube or laryngeal mask airway will be eligible. Patients will be randomised 1:1 to propofol TIVA or INVA, stratified by centre and procedural complexity. The feasibility endpoints include: (1) proportion of patients approached who agree to participate; (2) proportion of patients who receive their assigned randomised treatment; (3) completeness of outcomes data collection and (4) feasibility of data management procedures. Proportions and 95% CIs will be calculated to assess whether prespecified thresholds are met for the feasibility parameters. If the lower bounds of the 95% CI are above the thresholds of 10% for the proportion of patients agreeing to participate among those approached and 80% for compliance with treatment allocation for each randomised treatment group, this will suggest that our planned pragmatic 12 500-patient comparative effectiveness trial can likely be conducted successfully. Other feasibility outcomes and adverse events will be described.Ethics and dissemination This study is approved by the ethics board at Washington University (IRB# 202205053), serving as the single Institutional Review Board for both participating sites. Recruitment began in September 2022. Dissemination plans include presentations at scientific conferences, scientific publications, internet-based educational materials and mass media.Trial registration number NCT05346588