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1.
PLoS Pathog ; 2(5): e46, 2006 May.
Article in English | MEDLINE | ID: mdl-16710455

ABSTRACT

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the intracellular multiplication (Icm)/defective organelle trafficking (Dot) type IV secretion system (T4SS) to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs) do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI) metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3) kinases (PI3Ks) are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot-secreted proteins which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P) in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4)P was found to be present on LCVs using as a probe either an antibody against PI(4)P or the PH domain of the PI(4)P-binding protein FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1). Moreover, the presence of PI(4)P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4)P to anchor secreted effector proteins to the LCV.


Subject(s)
Bacterial Proteins/metabolism , Legionella pneumophila/physiology , Phosphatidylinositol Phosphates/physiology , Vacuoles/metabolism , Animals , Bacterial Proteins/genetics , Biomarkers/metabolism , Cell Line , Dictyostelium/growth & development , Humans , Legionella pneumophila/growth & development , Mice , Molecular Sequence Data , Mutation , Organelles/metabolism , Phagocytosis/physiology , Phosphatidylinositol 3-Kinases/deficiency , Phosphatidylinositol 3-Kinases/physiology , Phosphatidylinositol Phosphates/metabolism , Protein Transport , Tissue Distribution , Vacuoles/microbiology
2.
J Mol Evol ; 61(5): 706-15, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16211423

ABSTRACT

We and others recently identified an almost-intact human endogenous retrovirus (HERV), termed HERV-K(HML-2.HOM), that is usually organized as a tandem provirus. Studies on HERV proviral loci commonly rely on the analysis of single alleles being taken as representative for a locus. We investigated the frequency of HERV-K(HML-2.HOM) single and tandem alleles in various human populations. Our analysis revealed that another HERV-K(HML-2) locus, the so-called HERV-K(II) provirus, is also present as a tandem provirus allele in the human population. Proviral tandem formations were identified in various nonhuman primate species. We furthermore examined single nucleotide polymorphisms (SNPs) within the HERV-K(HML-2.HOM) proviral gag, prt, and pol genes, which all result in nonsense mutations. We identified four proviral haplotypes displaying different combinations of gag, prt, and pol SNPs. Haplotypes harboring completely intact proviral genes were not found. For the left provirus of the tandem arrangement a haplotype displaying intact gag and prt genes and a mutated pol was found in about two-thirds of individuals from different ethnogeographic origins. The same haplotype was always found in the right provirus. The various haplotypes point toward multiple recombination events between HERV-K(HML-2.HOM) proviruses. Based on these findings we derive a model for the evolution of the proviral locus since germ line integration.


Subject(s)
Endogenous Retroviruses/genetics , Evolution, Molecular , Haplotypes/genetics , Alleles , Base Sequence , Humans , Molecular Sequence Data , Polymorphism, Single Nucleotide/genetics , Proviruses/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid
3.
Microbiology (Reading) ; 151(Pt 1): 167-182, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15632436

ABSTRACT

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.


Subject(s)
Acanthamoeba castellanii/growth & development , Acanthamoeba castellanii/microbiology , Bacterial Proteins/genetics , Legionella pneumophila/growth & development , N-Acetylglucosaminyltransferases/genetics , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Humans , Legionella pneumophila/genetics , Legionella pneumophila/metabolism , Macrophages/microbiology , Molecular Sequence Data , Mutation , N-Acetylglucosaminyltransferases/chemistry , N-Acetylglucosaminyltransferases/metabolism , Operon , Sequence Alignment , Sequence Homology, Amino Acid
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