Gene_locus Report for: legpn-i7i328

The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal alpha/beta-hydrolase domain expanded by two noncanonical two-stranded beta-sheets, beta-6/beta-7 and beta-9/beta-10. The C-terminal domain reveals a fold displaying a bilobed beta-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. deltabeta-9/beta-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.

The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 mum where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells. The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L. pneumophila are known to contribute to this ability. By screening a genomic library of L. pneumophila, we found an additional L. pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence. Moreover, Escherichia coli harboring the L. pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphospho- and lysophospholipids, demonstrating that it encodes a phospholipase A. It has been reported that culture supernatants and cell lysates of L. pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level. We therefore generated isogenic L. pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages. Compared to wild-type L. pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity. We conclude that L. pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity. On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.

The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal alpha/beta-hydrolase domain expanded by two noncanonical two-stranded beta-sheets, beta-6/beta-7 and beta-9/beta-10. The C-terminal domain reveals a fold displaying a bilobed beta-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. deltabeta-9/beta-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.

The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 mum where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.

We report here the genomic sequence of Legionella pneumophila strain LPE509 from the water distribution system of a hospital in Shanghai, China. This is the first complete genome sequence of an environmental L. pneumophila isolate. Genomic analyses identified approximately 600 genes unique to LPE509 compared to those of the 7 available L. pneumophila genomes.

We present the genomic sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H (ATCC 43290), a clinical isolate from the Colorado Department of Health, Denver, CO. This is the first example of a genome sequence of L. pneumophila from a serogroup other than serogroup 1. We highlight the similarities and differences relative to six genome sequences that have been reported for serogroup 1 strains.

BACKGROUND: Amoebae are phagocytic protists where genetic exchanges might take place between amoeba-resistant bacteria. These amoebal pathogens are able to escape the phagocytic behaviour of their host. They belong to different bacterial phyla and often show a larger genome size than human-infecting pathogens. This characteristic is proposed to be the result of frequent gene exchanges with other bacteria that share a sympatric lifestyle and contrasts with the genome reduction observed among strict human pathogens.
RESULTS: We sequenced the genome of a new amoebal pathogen, Legionella drancourtii, and compared its gene content to that of a Chlamydia-related bacterium, Parachlamydia acanthamoebae. Phylogenetic reconstructions identified seven potential horizontal gene transfers (HGTs) between the two amoeba-resistant bacteria, including a complete operon of four genes that encodes an ABC-type transporter. These comparisons pinpointed potential cases of gene exchange between P. acanthamoebae and Legionella pneumophila, as well as gene exchanges between other members of the Legionellales and Chlamydiales orders. Moreover, nine cases represent possible HGTs between representatives from the Legionellales or Chlamydiales and members of the Rickettsiales order.
CONCLUSIONS: This study identifies numerous gene exchanges between intracellular Legionellales and Chlamydiales bacteria, which could preferentially occur within common inclusions in their amoebal hosts. Therefore it contributes to improve our knowledge on the intra-amoebal gene properties associated to their specific lifestyle.

BACKGROUND: Legionella pneumophila subsp. pneumophila is a gram-negative gamma-Proteobacterium and the causative agent of Legionnaires' disease, a form of epidemic pneumonia. It has a water-related life cycle. In industrialized cities L. pneumophila is commonly encountered in refrigeration towers and water pipes. Infection is always via infected aerosols to humans. Although many efforts have been made to eradicate Legionella from buildings, it still contaminates the water systems. The town of Alcoy (Valencian Region, Spain) has had recurrent outbreaks since 1999. The strain "Alcoy 2300/99" is a particularly persistent and recurrent strain that was isolated during one of the most significant outbreaks between the years 1999-2000. RESULTS: We have sequenced the genome of the particularly persistent L. pneumophila strain Alcoy 2300/99 and have compared it with four previously sequenced strains known as Philadelphia (USA), Lens (France), Paris (France) and Corby (England).Pangenome analysis facilitated the identification of strain-specific features, as well as some that are shared by two or more strains. We identified: (1) three islands related to anti-drug resistance systems; (2) a system for transport and secretion of heavy metals; (3) three systems related to DNA transfer; (4) two CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, known to provide resistance against phage infections, one similar in the Lens and Alcoy strains, and another specific to the Paris strain; and (5) seven islands of phage-related proteins, five of which seem to be strain-specific and two shared. CONCLUSIONS: The dispensable genome disclosed by the pangenomic analysis seems to be a reservoir of new traits that have mainly been acquired by horizontal gene transfer and could confer evolutionary advantages over strains lacking them.

Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity.

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates as an intracellular parasite of amoebae and persists in the environment as a free-living microbe. Here we have analyzed the complete genome sequences of L. pneumophila Paris (3,503,610 bp, 3,077 genes), an endemic strain that is predominant in France, and Lens (3,345,687 bp, 2,932 genes), an epidemic strain responsible for a major outbreak of disease in France. The L. pneumophila genomes show marked plasticity, with three different plasmids and with about 13% of the sequence differing between the two strains. Only strain Paris contains a type V secretion system, and its Lvh type IV secretion system is encoded by a 36-kb region that is either carried on a multicopy plasmid or integrated into the chromosome. Genetic mobility may enhance the versatility of L. pneumophila. Numerous genes encode eukaryotic-like proteins or motifs that are predicted to modulate host cell functions to the pathogen's advantage. The genome thus reflects the history and lifestyle of L. pneumophila, a human pathogen of macrophages that coevolved with fresh-water amoebae.

We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells. The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L. pneumophila are known to contribute to this ability. By screening a genomic library of L. pneumophila, we found an additional L. pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence. Moreover, Escherichia coli harboring the L. pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphospho- and lysophospholipids, demonstrating that it encodes a phospholipase A. It has been reported that culture supernatants and cell lysates of L. pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level. We therefore generated isogenic L. pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages. Compared to wild-type L. pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity. We conclude that L. pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity. On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.