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Find video protocols related to scientific articles indexed in Pubmed.
Defective lysosome maturation and Legionella pneumophila replication in Dictyostelium ArfGAP ACAP-A mutant cells.
J. Cell. Sci.
PUBLISHED: 09-04-2014
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Dictyostelium discoideum ACAP-A is an Arf-GTPase-activating protein involved in cytokinesis, cell migration and actin cytoskeleton dynamics. In mammalian cells, ACAP family members regulate endocytic protein trafficking. Here we explored the function of ACAP-A in the endocytic pathway of D. discoideum. In the absence of ACAP-A, reduced fusion efficacy of post-lysosomes with the plasma membrane resulted in the accumulation of post-lysosomes. Moreover, internalized fluid-phase showed extended intracellular transit time and transfer kinetics of phagocyted particles from lysosomes to post-lysosomes was reduced. Neutralization of lysosomal pH, one essential step in lysosome maturation, was also delayed. Whereas expression of ACAP-A-GFP in acapA(-) cells restored normal particle transport kinetics, a mutant ACAP-A protein with no GAP activity towards the small GTPase ArfA failed to complement this defect. Together these data support a role for ACAP-A in maturation of lysosomes into post-lysosomes through an ArfA-dependent mechanism. In addition, we reveal that ACAP-A is required for efficient intracellular growth of Legionella pneumophila, a pathogen known to subvert the endocytic host cell machinery for replication. This further emphasizes the role of ACAP-A in the endocytic pathway.
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Three antagonistic c-di-GMP-catabolizing enzymes promote differential Dot/Icm effectors delivery and intracellular survival at the early steps of Legionella pneumophila infection.
Infect. Immun.
PUBLISHED: 12-30-2013
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Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages causing an acute pneumonia in humans. The second messenger c-di-GMP has been shown to play key roles in regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118) and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities which are all required for survival and intracellular replication of L. pneumophila. Mutants of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for escape of Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated action of several Type 4 Secretion System Dot/Icm effectors proteins, which exhibit a differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged others appeared over- and under-translocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion to the host degradative pathways and establishment of a replicative vacuole.
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Molecular mimicry and original biochemical strategies for the biogenesis of a Legionella pneumophila replicative niche in phagocytic cells.
Microbes Infect.
PUBLISHED: 03-08-2013
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Legionella pneumophila is a paradigm of highly adapted intravacuolar pathogens that acquired the rare ability to replicate within a phagocytic cell. Here, we review recent progress about the role of Type 4 secretion system effectors involved in the biogenesis of the replicative niche, the Legionella containing vacuole.
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The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3-5)-cyclic dimeric GMP synthesis.
J. Biol. Chem.
PUBLISHED: 07-13-2011
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A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3-5)-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.
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Protein kinase LegK2 is a type IV secretion system effector involved in endoplasmic reticulum recruitment and intracellular replication of Legionella pneumophila.
Infect. Immun.
PUBLISHED: 02-14-2011
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Legionella pneumophila is the etiological agent of Legionnaires disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting the Legionella phagosome. Many Legionella effectors display distinctive eukaryotic domains, among which are protein kinase domains. In silico analysis and in vitro phosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemic L. pneumophila Lens strain. Except for LegK5, the Legionella protein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant of legK2 exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed by L. pneumophila to establish a replicative niche and evade host cell defenses.
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The TolC protein of Legionella pneumophila plays a major role in multi-drug resistance and the early steps of host invasion.
PLoS ONE
PUBLISHED: 07-20-2009
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Pneumonia associated with Iegionnairess disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.
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Micriamoeba tesseris nov. gen. nov. sp.: a new taxon of free-living small-sized Amoebae non-permissive to virulent Legionellae.
Protist
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Investigation of soil amoebae in 11 cooling towers allowed us to isolate a major unknown small-sized amoeba population (SZA). However, SZA did not appear to be specific to cooling tower ecosystems since they are also a major amoeba population found in muds isolated from different points of a water treatment plant. The SSU-rDNA sequences from SZA strains did not match any known database sequences, suggesting that SZA constitutes a new amoeba taxon. We isolated and further described one of the SZA that we named Micriamoeba tesseris. The phylogenetic analyses showed that Micriamoeba tesseris belongs to the Amebozoa and branched together with genus Echinamoeba+Vermamoeba vermiformis. Phylogenetic analyses within the Micriamoeba group distinguished different subgroups of Micriamoeba strains according to their origin, i.e. cooling tower or mud. Although Micriamoeba are able to feed on viable E. coli cells, they do not uptake virulent Legionella pneumophila strains, thus enabling them to avoid infection by Legionella. Consequently, Micriamoeba is not directly involved in L. pneumophila multiplication. However, an indirect role of Micriamoeba in Legionella risk is discussed.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.