Immunolocalization of the Mip protein of intracellularly and extracellularly grown Legionella pneumophila

The macrophage infectivity potentiator (Mip) protein is an important factor in the optimal intracellular survival of Legionella pneumophila in protozoa and human cell lines. In this study we have localized the Mip protein in Legionella grown on buffered charcoal yeast extract (BCYE) agar as well as in Legionella which were ingested by Acanthamoeba castellanii. Immunogold techniques have shown that Mip is exposed on the cell surface of extracellularly grown bacteria. In A. castellanii infected with Legionella the Mip protein was also detected on host membranes which exhibited a multilamellar structure. The morphology of these structures is similar to that of respirable vesicles of amoebas by which live legionellas may be transmitted to humans. It can be assumed that the accumulation of Mip protein in the multilamellar host membranes increases the infection potential.     

Crystal structure of Mip, a prolylisomerase from Legionella pneumophila

The human pathogen Legionella pneumophila, the etiological agent of the severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits a peptidyl prolyl cis-trans isomerase (PPIase) activity, which appears to be important for infection. Here we report the 2.4 A crystal structure of the Mip protein from L. pneumophila Philadelphia 1 and the 3.2 A crystal structure of its complex with the drug FK506. Each monomer of the homodimeric protein consists of an N-terminal dimerization module, a long (65 A) connecting alpha-helix and a C-terminal PPIase domain exhibiting similarity to human FK506-binding protein. In view of the recent significant increase in the number of reported cases of Legionnaires' disease and other intracellular infections, these structural results are of prime interest for the design of new drugs directed against Mip proteins of intracellular pathogens.     

Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPIase) activity

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macrophage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506-binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K-12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP-type PPIases of eukaryotes. However, the N-terminal extension of Mip and/or the substitutions of the variable amino acids in the C-terminal FKBP core leads to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc-Ala-Xaa-Pro-Phe-4-nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein-folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.     

The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells

We analysed eight monoclonal antibodies (mAbs) directed against the Mip (macrophage infectivity potentiator) protein, a virulence factor of the intracellular pathogen Legionella pneumophila. Mip belongs to the FK506-binding proteins (FKBPs) and exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Five of the mAbs recognised epitopes in the C-terminal, FKBP-homologous domain of Mip, which is highly conserved among all Legionella species. Upon immunological binding to Mip, all but one of these mAbs caused inhibition of the PPIase activity in vitro. mAb binding to the N-terminal domain of Mip did not influence its enzymatic activity. All but one of the PPIase inhibiting mAbs were able to significantly inhibit the early establishment and initiation of an intracellular infection of the bacteria in Acanthamoeba castellanii, the natural host, and in the human phagocytic cell line U937. These data demonstrate for the first time that for the virulence-enhancing property of the L. pneumophila Mip protein, an intact active site of the enzyme is an essential requirement.     

Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration

The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), binds specifically to collagen IV. We identified a surface-exposed Mip-binding sequence in the NC1 domain of human collagen IV α1. The corresponding collagen IV-derived peptide (P290) co-precipitated with Mip and competitively inhibited the Mip-collagen IV binding. Transmigration of Legionella pneumophila across a barrier of NCI-H292 lung epithelial cells and extracellular matrix was efficiently inhibited by P290. This significantly reduced transmigration was comparable to the inefficient transmigration of PPIase-negative Mip mutant or rapamycin-treated L. pneumophila. Based on NMR data and docking studies a model for the mode of interaction of P290 and Mip was developed. The amino acids of the hydrophobic cavity of Mip, D142 and to a lesser extent Y185 were identified to be part of the interaction surface. In the complex structure of Mip(77-213) and P290, both amino acid residues form hydrogen bonds to P290. Utilizing modelling, molecular dynamics (MD) simulations and structural data of human PPIase FKBP12, the most related human orthologue of Mip, we were able to propose optimized P290 variants with increased binding specificity and selectivity for the putative bacterial drug target Mip.     

Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix

Guinea pigs are highly susceptible to Legionella pneumophila infection and therefore have been the preferred animal model for studies of legionellosis. In this study guinea pig infections revealed that the Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to the bacterial dissemination within the lung tissue and the spread of Legionella to the spleen. Histopathology of infected animals, binding assays with components of the extracellular matrix (ECM), bacterial transmigration experiments across an artificial lung epithelium barrier, inhibitor studies and ECM degradation assays were used to elucidate the underlying mechanism of the in vivo observation. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), was shown to bind to the ECM protein collagen (type I, II, III, IV, V, VI). Transwell assays with L. pneumophila and recombinant Escherichia coli HB101 strains revealed that Mip enables these bacteria to transmigrate across a barrier of NCI-H292 lung epithelial cells and ECM (NCI-H292/ECM barrier). Mip-specific monoclonal antibodies and the immunosuppressants rapamycin and FK506, which inhibit the peptidyl prolyl cis/trans isomerase (PPIase) activity of Mip, were able to inhibit this transmigration. By using protease inhibitors we found that the penetration of the NCI-H292/ECM barrier additionally requires a serine protease activity. Degradation assays with (35)S-labelled ECM proteins supported the finding of a concerted action of Mip and a serine protease. The described synergism between the activity of the collagen binding Mip protein and the serine protease activity represents an entirely new mechanism for bacterial penetration of the lung epithelial barrier and has implications for other prokaryotic and eukaryotic pathogens.     

Extracellular metalloproteinase from Legionella pneumophila

Using ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on DNP-hexamethylenediamine-Sepharose and gramicidin S-Sepharose and gel filtration, a metalloproteinase was isolated from the cultural fluid of L. pneumophila (strain Philadelphia-1) grown for 20 hours. The enzyme was purified 1606-fold with a 31% yield. The enzyme has a Mr of 38,000, pI approximately 4.0 and optimum of proteolytic activity at pH 6.0-7.0, 55 degrees C. The proteinase is the most stable within the pH range of 6.0-9.0. The enzyme contains one atom of zinc per molecule. The amino acid composition of metalloproteinase is close to that of thermolysin and is characterized by a high methionine content--17 residues out of 348. In the B-chain of oxidized bovine insulin the enzyme hydrolyzes the bonds precedent to the amino groups of leucine, phenylalanine and tyrosine. The enzyme is inhibited by chelating agents--Na2-EDTA and o-phenanthroline as well as by diethylpyrocarbonate. The serine and thiol proteinase inhibitors do not influence the enzyme activity. Under the given conditions of cultivation metalloproteinase is the major endopeptidase produced by L. pneumophila. Thus, the proteolytic system of Legionelles is characterized by the combination of metalloproteinase and the earlier described phenylalanine aminopeptidase.     

Interspecies sequence differences in the Mip protein from the genus Legionella: implications for function and evolutionary relatedness

The nucleotide sequence of the mip genes and their inferred amino acid sequences were determined from 35 Legionella species and compared with the published sequences for L . pneumophila , L . micdadei and L . longbeachae . The sequences were 69–97% conserved at the nucleotide level and 82–99% at the amino acid level, with total conservation of amino acids determined to be associated with sites known to be involved in peptidyl prolyl cis–trans isomerase activity. No apparent difference could be determined in the arrangement of amino acids that would predict a functional difference in Mip from species associated with disease and Mip from species isolated only from the environment. Additionally, a phylogenetic comparison of the sequences with published 16S RNA sequences, using both genetic distance and maximum parsimony methods, was performed. Few relationships were apparent that were well supported by both data sets, the most robust being a clade comprising {[( cincinnatiensis , longbeachae , sainthelensi , santicrucis ) gratiana ] ( moravica , quateirensis , shakespearei , worsleiensis ) anisa , bozemanii , cherrii , dumoffii , gormanii , jordanis , parisiensis , pneumophila , steigerwaltii , tucsonensis , and wadsworthii  }.     

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a V_max of 12 µmol/min/mg protein and a K_m of 45 μM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5–7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.     

Functional analysis of the multi-copper oxidase from Legionella pneumophila

Multicopper oxidases have been described to have functions in copper tolerance, manganese oxidation, and iron oxidation in a range of bacteria. The putative cytoplasmic membrane multicopper oxidase from Legionella pneumophila was investigated. The mcoL gene was found to be critical for aerobic extracellular growth under either iron-limiting conditions or in the presence of ferrous Fe(II) iron, as a sole source of this essential metal. The mcoL mutants showed minor growth defects when grown in the presence of Fe(III) as the iron source. In contrast, intracellular growth and survival was not affected by the absence of the mcoL gene regardless of available iron concentration. The evidence presented here could indicate a possible role for mcoL in prevention of the toxic effects of ferrous iron during aerobic conditions. However, a function in high-affinity acquisition of iron could also be possible given the inability of the McoL mutants to grow aerobically under iron-limiting conditions.     

Legionella pneumophila secretes a mitochondrial carrier protein during infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.     

Chemical composition of a lipopolysaccharide from Legionella pneumophila

Lipopolysaccharide isolated from Legionella pneumophila (Phil. 1) was examined for chemical composition. The polysaccharide split off by mild acid hydrolysis contained rhamnose, mannose, glucose, quinovosamine, glucosamine and 2-keto-3-deoxyoctonate, in molar proportions 1.6:1.8:1.0:1.5:4.1:2.7. Heptoses were absent and glucose was probably mainly phosphorylated. The carbohydrate backbone of the lipid A part consisted of glucosamine, quinovosamine and glycerol, in the molar ratios 3.9:1.0:3.4, with glycerol as a phosphorylated moiety. A complex fatty acid substitution pattern comprising eight O-ester-linked, exclusively nonhydroxylated acids, and nineteen amide-linked, exclusively 3-hydroxylated acids was revealed. Both straight- and branched (iso and anteiso) carbon chains occurred. The major hydroxy fatty acid was 3-hydroxy-12-methyltridecanoic acid and six others were of a chain-length above 20 carbon atoms, with 3-hydroxy-20-methyldocosanoic acid as the longest. Two dihydroxy fatty acids, 2,3-dihydroxy-12-methyltridecanoic and 2,3-dihydroxytetradecanoic acids, were also detected. These results suggest that L. pneumophila contains a rather complex and unusual lipopolysaccharide structure of considerable biological and chemotaxonomic interest.Abbreviations LPS lipopolysaccharide - PS polysaccharide - KDO 2-keto-3-deoxy-octonate - GC gas chromatography - GC-MS gas chromatograph-mass spectrometer combined instrument - CI chemical ionization - EI electron impact - HF hydrofluoric acid - TFA trifluoroacetyl - TMS trimethylsilyl     

Legionella pneumophila: homeward bound away from the phagosome

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嗜肺军团菌效应蛋白质介导的新型泛素化过程

泛素化是真核细胞特有的蛋白质翻译后修饰方式,调节真核细胞内多种重要生理过程,例如蛋白质稳态、细胞周期、免疫反应、DNA修复以及囊泡转运等。鉴于泛素化对于生命活动的重要性,病原菌在与宿主细胞的长期进化过程中衍生出一系列针对宿主泛素化过程的效应蛋白质,调控宿主体内泛素化过程,从而构建有利于病原菌自身生长繁殖的内环境。嗜肺军团菌是一种革兰氏阴性菌,是军团菌肺炎的致病菌,能够引起发热和肺部感染,重型病死率高达15%~30%。Dot/Icm Ⅳ型分泌系统是嗜肺军团菌侵染过程中最主要的毒力系统。在侵染宿主细胞的过程中,嗜肺军团菌利用该分泌系统,分泌超过330种效应蛋白质,协助细菌在宿主胞内生存、增殖和逃逸。多种嗜肺军团菌效应蛋白质通过直接或者间接的方式对宿主泛素化过程进行调控。近年的研究发现,多种效应蛋白质可以介导不同于真核生物经典泛素化的新型泛素化过程。本文介绍了嗜肺军团菌效应蛋白质介导的新型泛素化过程的最新研究进展,为理解泛素化过程在嗜肺军团菌致病过程中的重要作用提供参考依据。     

Immunogenicity and adjuvanticity of lipopolysaccharide from Legionella pneumophila

Lipopolysaccharide isolated from Legionella pneumophila was found to be a potent antigen and inducer of antibody with strong adjuvant activity for related and unrelated antigens such as sheep erythrocytes by in vivo and in vitro systems. The LPS was also a potent stimulator of blastogenic responses by spleen cells from normal mice as well as from mice immunized with inactivated whole cells of Legionella. It strongly stimulated production of interferon and interleukin 1. These results indicate that the LPS of Legionella may be an important immune regulator in the host response.     

IcmR-regulated membrane insertion and efflux by the Legionella pneumophila IcmQ protein

Legionella pneumophila proliferates within alveolar macrophages as a central property of Legionnaires' disease. Intracellular growth involves formation of a replicative phagosome, which requires the bacterial Dot/Icm system, a multiprotein secretion apparatus that translocates proteins from the bacterium across the macrophage plasma membrane. Two components of this system, IcmR and IcmQ, are proposed to exhibit a chaperone/substrate relationship similar to that observed in other protein translocation systems. We report here that IcmQ inserts into lipid membranes and forms pores that allow the efflux of the dye calcein but not Dextran 3000. Both membrane insertion and pore formation were inhibited by IcmR. Trypsin digestion mapping demonstrated that IcmQ is subdivided into two functional domains. The N-terminal region of IcmQ was necessary and sufficient for insertion into lipid membranes and calcein efflux. The C-terminal domain was necessary for efficient association of the protein with lipid bilayers. IcmR was found to bind to the N-terminal portion of the protein thus providing a mechanism for its ability to inhibit IcmQ pore-forming activity. Localization of IcmQ on the surface of the L. pneumophila shortly after infection as well as its pore-forming capacities suggest a role for IcmQ in forming a channel that leads translocated effectors out of the bacterium.     

Cytolytic activity of Legionella pneumophila

The properties of cytolysin and metalloproteinase purified by different methods have been studied. The physico-chemical properties of these proteins, including their molecular weight, immunodiffusion patterns, the degree of inhibition by EDTA and diethyl pyrocarbonate, amino acid composition, cytolytic and proteolytic activity, have proved to be similar. We have come to the conclusion that cytolysin and metalloproteinase have similar composition and metalloproteinase activity determines the cytolytic and necrotic activity of the above-mentioned cytolysin.     

Necrotrophic growth of Legionella pneumophila

This study examined whether Legionella pneumophila is able to thrive on heat-killed microbial cells (necrotrophy) present in biofilms or heat-treated water systems. Quantification by means of plate counting, real-time PCR, and flow cytometry demonstrated necrotrophic growth of L. pneumophila in water after 96 h, when at least 100 dead cells are available to one L. pneumophila cell. Compared to the starting concentration of L. pneumophila, the maximum observed necrotrophic growth was 1.89 log units for real-time PCR and 1.49 log units for plate counting. The average growth was 1.57 +/- 0.32 log units (n = 5) for real-time PCR and 1.14 +/- 0.35 log units (n = 5) for plate counting. Viability staining and flow cytometry showed that the fraction of living cells in the L. pneumophila population rose from the initial 54% to 82% after 96 h. Growth was measured on heat-killed Pseudomonas putida, Escherichia coli, Acanthamoeba castellanii, Saccharomyces boulardii, and a biofilm sample. Gram-positive organisms did not result in significant growth of L. pneumophila, probably due to their robust cell wall structure. Although necrotrophy showed lower growth yields compared to replication within protozoan hosts, these findings indicate that it may be of major importance in the environmental persistence of L. pneumophila. Techniques aimed at the elimination of protozoa or biofilm from water systems will not necessarily result in a subsequent removal of L. pneumophila unless the formation of dead microbial cells is minimized.