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2.
Infect Immun ; 82(3): 1222-33, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24379287

ABSTRACT

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 cyclic di-GMP (c-di-GMP) has been shown to play key roles in the 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 the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the 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 actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the 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 of the host degradative pathways and establishment of a replicative vacuole.


Subject(s)
Bacterial Proteins/metabolism , Cyclic GMP/analogs & derivatives , Legionella pneumophila/metabolism , Legionnaires' Disease/metabolism , Cell Line, Tumor , Cyclic GMP/metabolism , Endocytosis/physiology , Endoplasmic Reticulum/metabolism , Escherichia coli Proteins/metabolism , Humans , Macrophages/metabolism , Phagocytes/metabolism , Phosphoric Diester Hydrolases/metabolism , Phosphorus-Oxygen Lyases/metabolism , Protein Transport/physiology , Signal Transduction/physiology , U937 Cells , Virulence/physiology
3.
J Biol Chem ; 286(36): 31136-44, 2011 Sep 09.
Article in English | MEDLINE | ID: mdl-21757706

ABSTRACT

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.


Subject(s)
Cyclic GMP/analogs & derivatives , Escherichia coli Proteins/metabolism , Legionella pneumophila/enzymology , Phosphoric Diester Hydrolases/metabolism , Phosphorus-Oxygen Lyases/metabolism , Protein Kinases/physiology , Bacterial Proteins , Cyclic GMP/biosynthesis , Genes, Bacterial , Phosphorylation
4.
Infect Immun ; 79(5): 1936-50, 2011 May.
Article in English | MEDLINE | ID: mdl-21321072

ABSTRACT

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.


Subject(s)
Bacterial Secretion Systems/genetics , Legionella pneumophila/genetics , Legionella pneumophila/pathogenicity , Protein Kinases/genetics , Amino Acid Sequence , Animals , Endoplasmic Reticulum/enzymology , Intracellular Space/enzymology , Legionella pneumophila/enzymology , Mice , Molecular Sequence Data , Protein Kinases/metabolism , Sequence Alignment , Virulence
5.
PLoS One ; 4(11): e7732, 2009 Nov 04.
Article in English | MEDLINE | ID: mdl-19888467

ABSTRACT

Pneumonia associated with Iegionnaires's 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.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/physiology , Drug Resistance, Multiple , Gene Expression Regulation, Bacterial , Legionella pneumophila/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/physiology , Acanthamoeba castellanii/microbiology , Alleles , Gene Deletion , Genetic Complementation Test , Humans , Legionella pneumophila/pathogenicity , Microbial Sensitivity Tests , Models, Genetic , Plasmids/metabolism , Reactive Oxygen Species , U937 Cells
6.
Proc Natl Acad Sci U S A ; 104(15): 6394-9, 2007 Apr 10.
Article in English | MEDLINE | ID: mdl-17405861

ABSTRACT

Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect. This feature guided the identification of Ivy orthologues in other Gram-negative bacteria. The structure of the evolutionary distant Pseudomonas aeruginosa Ivy orthologue was also determined in complex with hen egg white lysozyme, and its antilysozyme activity was confirmed. Ivy expression protects porous cell-wall E. coli mutants from the lytic effect of lysozyme, suggesting that it is a response against the permeabilizing effects of the innate vertebrate immune system. As such, Ivy acts as a virulence factor for a number of Gram-negative bacteria-infecting vertebrates.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Evolution, Molecular , Models, Molecular , Muramidase/antagonists & inhibitors , Phylogeny , Cluster Analysis , Crystallography , Protein Conformation , Species Specificity
7.
Microbiology (Reading) ; 151(Pt 10): 3337-3347, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16207916

ABSTRACT

The tol-pal genes are necessary for maintaining the outer-membrane integrity of Gram-negative bacteria. These genes were first described in Escherichia coli, and more recently in several other species. They are involved in the pathogenesis of E. coli, Haemophilus ducreyi, Vibrio cholerae and Salmonella enterica. The role of the tol-pal genes in bacterial pathogenesis was investigated in the phytopathogenic enterobacterium Erwinia chrysanthemi, assuming that this organism might be a good model for such a study. The whole Er. chrysanthemi tol-pal region was characterized. Tol-Pal proteins, except TolA, showed high identity scores with their E. coli homologues. Er. chrysanthemi mutants were constructed by introducing a uidA-kan cassette in the ybgC, tolQ, tolA, tolB, pal and ybgF genes. All the mutants were hypersensitive to bile salts. Mutations in tolQ, tolA, tolB and pal were deleterious for the bacteria, which required high concentrations of sugars or osmoprotectants for their viability. Consistent with this observation, they were greatly impaired in their cell morphology and division, which was evidenced by observations of cell filaments, spherical forms, membrane blebbing and mislocalized bacterial septa. Moreover, tol-pal mutants showed a reduced virulence in a potato tuber model and on chicory leaves. This could be explained by a combination of impaired phenotypes in the tol-pal mutants, such as reduced growth and motility and a decreased production of pectate lyases, the major virulence factor of Er. chrysanthemi.


Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Dickeya chrysanthemi/pathogenicity , Dickeya chrysanthemi/ultrastructure , Membrane Proteins/metabolism , Bacterial Proteins/genetics , Cichorium intybus/microbiology , Cloning, Molecular , Dickeya chrysanthemi/genetics , Dickeya chrysanthemi/physiology , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Membrane Proteins/genetics , Molecular Sequence Data , Movement , Mutation , Plant Diseases/microbiology , Plant Leaves/microbiology , Plant Roots/microbiology , Sequence Analysis, DNA , Solanum tuberosum/microbiology , Virulence
8.
Microbiology (Reading) ; 151(Pt 7): 2487-2497, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16000739

ABSTRACT

Curli are necessary for the adherence of Escherichia coli to surfaces, and to each other, during biofilm formation, and the csgBA and csgDEFG operons are both required for their synthesis. A recent survey of gene expression in Pseudomonas aeruginosa biofilms has identified tolA as a gene activated in biofilms. The tol genes play a fundamental role in maintaining the outer-membrane integrity of Gram-negative bacteria. RcsC, the sensor of the RcsBCD phosphorelay, is involved, together with RcsA, in colanic acid capsule synthesis, and also modulates the expression of tolQRA and csgDEFG. In addition, the RcsBCD phosphorelay is activated in tol mutants or when Tol proteins are overexpressed. These results led the authors to investigate the role of the tol genes in biofilm formation in laboratory and clinical isolates of E. coli. It was shown that the adherence of cells was lowered in the tol mutants. This could be the result of a drastic decrease in the expression of the csgBA operon, even though the expression of csgDEFG was slightly increased under such conditions. It was also shown that the Rcs system negatively controls the expression of the two csg operons in an RcsA-dependent manner. In the tol mutants, activation of csgDEFG occurred via OmpR and was dominant upon repression by RcsB and RcsA, while these two regulatory proteins repressed csgBA through a dominant effect on the activator protein CsgD, thus affecting curli synthesis. The results demonstrate that the Rcs system, previously known to control the synthesis of the capsule and the flagella, is an additional component involved in the regulation of curli. Furthermore, it is shown that the defect in cell motility observed in the tol mutants depends on RcsB and RcsA.


Subject(s)
Bacterial Proteins/biosynthesis , Biofilms/growth & development , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Bacterial Adhesion , Escherichia coli/genetics , Escherichia coli/physiology , Gene Expression Regulation, Bacterial , Multienzyme Complexes/metabolism , Phosphoprotein Phosphatases/metabolism , Protein Kinases/metabolism , Transcription Factors/metabolism
9.
J Bacteriol ; 187(6): 2038-49, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15743952

ABSTRACT

Curli fibers could be described as a virulence factor able to confer adherence properties to both abiotic and eukaryotic surfaces. The ability to adapt rapidly to changing environmental conditions through signal transduction pathways is crucial for the growth and pathogenicity of bacteria. OmpR was shown to activate csgD expression, resulting in curli production. The CpxR regulator was shown to negatively affect curli gene expression when binding to its recognition site that overlaps the csgD OmpR-binding site. This study was undertaken to clarify how the interplay between the two regulatory proteins, OmpR and CpxR, can affect the transcription of the curli gene in response to variation of the medium osmolarity. Band-shift assays with purified CpxR proteins indicate that CpxR binds to the csgD promoter region at multiple sites that are ideally positioned to explain the csg repression activity of CpxR. To understand the physiological meaning of this in vitro molecular phenomenon, we analyzed the effects of an osmolarity shift on the two-component pathway CpxA/CpxR. We establish here that the Cpx pathway is activated at both transcriptional and posttranscriptional levels in response to a high osmolarity medium and that CpxR represses csgD expression in high-salt-content medium, resulting in low curli production. However, csgD repression in response to high sucrose content is not mediated by CpxR but by the global regulatory protein H-NS. Therefore, multiple systems (EnvZ/OmpR, Cpx, Rcs, and H-NS) appear to be involved in sensing environmental osmolarity, leading to sophisticated regulation of the curli genes.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Trans-Activators/metabolism , Water-Electrolyte Balance/physiology , Base Sequence , Escherichia coli/metabolism , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Promoter Regions, Genetic/physiology , Repressor Proteins/metabolism , Saline Solution, Hypertonic , Trans-Activators/genetics
10.
J Infect Dis ; 191(6): 939-48, 2005 Mar 15.
Article in English | MEDLINE | ID: mdl-15717270

ABSTRACT

Sepsis is initiated by interactions between microbial products and host inflammatory cells. Toll-like receptors (TLRs) are central innate immune mediators of sepsis that recognize different components of microorganisms. Peptidoglycan-associated lipoprotein (PAL) is a ubiquitous gram-negative bacterial outer-membrane protein that is shed by bacteria into the circulation of septic animals. We explored the inflammatory effects of purified PAL and of a naturally occurring form of PAL that is shed into serum. PAL is released into human serum by Escherichia coli bacteria in a form that induces cytokine production by macrophages and is tightly associated with lipopolysaccharide (LPS). PAL activates inflammation through TLR2. PAL and LPS synergistically activate macrophages. These data suggest that PAL may play an important role in the pathogenesis of sepsis and imply that physiologically relevant PAL and LPS are shed into serum and act in concert to initiate inflammation in sepsis.


Subject(s)
Bacterial Outer Membrane Proteins/immunology , Escherichia coli/immunology , Lipopolysaccharides/immunology , Lipoproteins/immunology , Membrane Glycoproteins/agonists , Peptidoglycan/immunology , Receptors, Cell Surface/agonists , Animals , Bacterial Outer Membrane Proteins/blood , Cell Line , Drug Synergism , Escherichia coli K12/immunology , Escherichia coli Proteins , Humans , Lipoproteins/blood , Macrophage Activation , Macrophages, Peritoneal/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Peptidoglycan/blood , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Sepsis/physiopathology , Toll-Like Receptor 2 , Toll-Like Receptors
11.
Biochimie ; 84(5-6): 391-7, 2002.
Article in English | MEDLINE | ID: mdl-12423782

ABSTRACT

The Tol proteins are involved in outer membrane stability of Gram-negative bacteria. The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane. These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA. The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope. Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented.


Subject(s)
Colicins/metabolism , Escherichia coli Proteins/metabolism , Membrane Proteins/metabolism , Escherichia coli/metabolism , Protein Transport/physiology
12.
J Bacteriol ; 184(16): 4620-5, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12142433

ABSTRACT

The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Periplasmic Proteins , Amino Acid Sequence , DNA Mutational Analysis , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed/physiology , Protein Binding/genetics , Protein Structure, Tertiary
13.
J Bacteriol ; 184(3): 754-9, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11790745

ABSTRACT

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.


Subject(s)
Cell Membrane/physiology , Cell Wall/physiology , Escherichia coli/physiology , Lipoproteins/physiology , Proteoglycans , Bacterial Outer Membrane Proteins/metabolism , Carrier Proteins/metabolism , Dimerization , Escherichia coli Proteins/metabolism , Fungal Proteins/metabolism , Gene Dosage , Peptidoglycan , Protein Binding , Recombinant Proteins
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