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Query: UMLS:C0023241 (Legionella)
 6,990 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously, we had demonstrated that a Legionella pneumophila prepilin peptidase (pilD) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (pilE(L)) mutant. To determine whether these pilD-dependent defects were linked to type II secretion, we have constructed two new mutants of L. pneumophila strain 130b. Mutations were introduced into either lspDE, which encodes the type II outer membrane secretin and ATPase, or lspFGHIJK, which encodes the pseudopilins. Unlike the wild-type and pilE(L) strains, both lspDE and lspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of the pilD mutant, suggesting that their surfaces are distinct. The pilD, lspDE, and lspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, both lsp mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, the pilD mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel pilD-dependent mechanism for promoting L. pneumophila intracellular infection of human cells.
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PMID:Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila. 1125 62

The ability of Legionella pneumophila to grow and cause disease in the host is completely dependent on a type IV secretion system known as the Dot/Icm complex. This membrane-spanning apparatus translocates effector molecules into host cells in a process that is poorly understood but that is known to require the putative ATPase DotB. One possible role for DotB is suggested by its similarity to the PilT family of proteins, which mediate pilus retraction. To better understand the molecular behavior of DotB, we have purified the protein and shown that it forms stable homohexameric rings and hydrolyzes ATP with a specific activity of 6.4 nmol of ATP/min/mg of protein. ATPase activity is critical to the function of DotB, as alteration of the conserved Walker box lysine residue resulted in a mutant protein, DotB K162Q, which failed to bind or hydrolyze ATP and which could not complement a DeltadotB strain for intracellular growth in macrophages. Consistent with the ability of DotB to interact with itself, the dotBK162Q allele exhibited transdominance over wild-type dotB, providing the first example of such a mutation in L. pneumophila. Finally, the DotB K162Q mutant protein had a significantly enhanced membrane localization in L. pneumophila compared to wild-type DotB, suggesting a relationship between nucleotide binding and membrane association. These results are consistent with a model in which DotB cycles between the cytoplasm and the Dot/Icm complex at the membrane, where it hydrolyzes nucleotides to provide energy to the complex.
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PMID:The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth. 1499 96

The pulmonary pathogen Legionella pneumophila uses the Dot/Icm type IV secretion system (T4SS) to replicate inside host cells. This apparatus translocates proteins into macrophages to alter their endocytic pathway and enable bacterial growth. Although the secretion ATPase DotB is critical for T4SS function, its specific role in type IV secretion remains undefined. Due to similarity to the VirB11 and PilT ATPases, DotB has been proposed to play a role in assembly of the T4SS, retraction of pili and/or export of substrates. With the goal of understanding the protein's function(s), we isolated and characterized 30 dotB alleles using a variety of phenotypic and biochemical assays. Twenty-four of these alleles possess several dot/icm mutant phenotypes, including a complete lack of intracellular replication, plasmid mobilization and contact-dependent cytotoxicity. These 24 non-functional alleles fall into three classes: those with a known biochemical defect, those with a predicted enzymatic defect and those with an unknown defect. Six other alleles display partial activity in dot/icm phenotypic assays, thus constituting a fourth class. Two mutants in this class are unable to export a subset of T4SS substrates, providing the first evidence for a DotB function in substrate export and suggesting a possible role in substrate selection.
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PMID:Genetic analysis of the Legionella pneumophila DotB ATPase reveals a role in type IV secretion system protein export. 1594 50

The natural hosts of the bacterial pathogen Legionella pneumophila are amoebae and protozoa. In these hosts, as in human macrophages, the pathogen enters the cell through phagocytosis, then rapidly modifies the phagosome to create a compartment that supports its replication. We have examined L. pneumophila entry and behaviour during early stages of the infection of Dictyostelium discoideum amoebae. Bacteria were labelled with a red fluorescent marker, and selected proteins and organelles in the host were labelled with GFP, allowing the dynamics and interactions of L. pneumophila -containing phagosomes to be tracked in living cells. These studies demonstrated that entry of L. pneumophila is an actin-mediated process, that the actin-binding protein coronin surrounds the nascent phagosome but dissociates immediately after internalization, that ER membrane is not incorporated into a phagosome during uptake, that the newly internalized phagosome is rapidly transported about the cell on microtubules, that association of ER markers with the phagosome occurs in two steps that correlate with distinct changes in phagosome movement, and that the vacuolar H(+)-ATPase does not associate with mature replication vacuoles. These studies have clarified certain aspects of the infection process and provided new insights into the dynamic interactions between the pathogen and its host.
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PMID:Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. 1595 31

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.
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PMID:Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection. 1644 84

As part of its pathogenesis, Legionella pneumophila persists within human alveolar macrophages in non-acidified organelles that do not mature into phagolysosomes. Two L. pneumophila genes, lpg0971 and lpg1905, are predicted to encode ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) that share sequence similarity with human CD39/NTPDase1. The predicted products possess five apyrase conserved domains that are typical of eukaryotic ecto-NTPDases. In this study, we found that an lpg1905 mutant was recovered in lower numbers from macrophages, alveolar epithelial cells and the amoeba, Hartmannella vermiformis compared with wild-type L. pneumophila and an lpg0971 mutant. Similar to human CD39, recombinant purified Lpg1905 exhibited ATPase and ADPase activity and possessed the ability to inhibit platelet aggregation. Mutation of a conserved Glu159 residue that is essential for CD39 activity inhibited ATPase and ADPase activity of Lpg1905. In addition, enzyme activity was inhibited in the presence of the specific ecto-NTPDase inhibitor, ARL67156. The entry and replication defect of the lpg1905 mutant was reversed upon transcomplementation with lpg1905 but not lpg1905E159A encoding an enzymatically inactive form of the protein. Although several protozoan parasites exhibit ecto-NTPDase activity, including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi, this is the first time a bacterial ecto-NTPDase has been implicated in virulence.
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PMID:A bacterial ecto-triphosphate diphosphohydrolase similar to human CD39 is essential for intracellular multiplication of Legionella pneumophila. 1738 84

Proteins containing FIC (filamentation induced by cyclic adenosine monophosphate) domains are found in both prokaryotic and eukaryotic organisms, but their function has remained elusive. Recent studies indicate that bacterial FIC domain-containing proteins disrupt host cell processes after being delivered into eukaryotic host cells: The Vibrio parahaemolyticus VopS protein interferes with Rho guanine triphosphatase (GTPase) function, and the Legionella pneumophila AnkX protein disrupts the microtubule-dependent transport of vesicles. Analysis of the VopS protein revealed that the FIC domain covalently modifies Rac by transferring adenosine 5'-monophosphate (AMP) to a threonine residue in the switch 1 region of the protein. Thus, FIC domain-mediated AMPylation is involved in the posttranslational regulation of protein function, and this activity has been subverted by microbial pathogens to modulate cellular functions during infection.
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PMID:Bacterial FIC Proteins AMP Up Infection. 1929 28

Legionella pneumophila is an intracellular pathogen causing pneumonia-like disease in humans. A 43-kb putative heavy metal efflux gene island was found on the L. pneumophila genome. Large Legionella deletion strains of the metal efflux genes were tested in human THP-1-derived macrophages and amoebal Acanthamoeba castellanii cells and were able to survive and replicate similar to the wild type, suggesting that they do not play a significant role within the intracellular environment. Examination of the sequence of this genomic island revealed that some genes were not accurately annotated and there were no known metal-responsive regulators encoded in this region. Therefore, functional roles of these metal resistance genes were tested by conducting metal resistance assays. Individual genes were cloned in an expression vector and expressed in an appropriate metal-sensitive Escherichia coli background with varying concentrations of the tested metal. Of the 11 efflux systems, a role was determined only for one. A Cu(I)-translocating P(IB)-type ATPase was shown to be encoded by lpg1024. This gene, termed copA, complemented a copper-sensitive (Delta copA) E. coli strain in trans and was able to confer copper resistance.
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PMID:The metal efflux island of Legionella pneumophila is not required for survival in macrophages and amoebas. 1989 45

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae.
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PMID:Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. 2033 53

In the course of Legionnaires' disease, the bacterium Legionella pneumophila affects the intracellular vesicular trafficking of infected eukaryotic cells by recruiting the small guanosine triphosphatase (GTPase) Rab1 to the cytosolic face of the Legionella-containing vacuole. In order to accomplish this, the Legionella protein DrrA contains a specific guanine nucleotide exchange activity for Rab1 activation that exchanges guanosine triphosphate (GTP) for guanosine diphosphate on Rab1. We found that the amino-terminal domain of DrrA possesses adenosine monophosphorylation (AMPylation) activity toward the switch II region of Rab1b, leading to posttranslational covalent modification of tyrosine 77. AMPylation of switch II by DrrA restricts the access of GTPase activating proteins, thereby rendering Rab1b constitutively active.
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PMID:The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b. 2065 Nov 20


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