No abstract

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 diacylphosphoand 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.Legionella pneumophila is an inhabitant of fresh water, where it intracellularly colonizes protozoa (20). When bacteria-laden aerosols are inhaled by humans, L. pneumophila exploits alveolar macrophages and epithelial cells for its multiplication, leading to a severe pneumonia characterized by destruction of alveolar cells (60). The cytopathology of Legionnaires' disease involves several cytotoxic or hemolytic factors produced by L. pneumophila, for example, the zinc metalloprotease ProA, the legiolysin Lly, and several pore-forming toxins, one of which is an RTX (repeats in structural toxin) protein (12,26,31,35,36,47,61). The zinc metalloprotease ProA is the major extracellular protease of L. pneumophila and its export depends on the L. pneumophila type II protein secretion system (28, 37). The enzyme hydrolyzes a broad spectrum of protein substrates and confers hemolytic as well as cytolytic activities (47, 53). Additionally, ProA has been shown to contribute to bacterial pathogenesis in a guinea pig model of pneumonia (38). Another hemolytic, but not cytotoxic, protein is the L. pneumophila legiolysin Lly, which is also responsible for color production and fluorescence of the bacterium (61). Pore-forming activities of L. pneumophila confer contact-dependent hemolytic and cytotoxic activities toward a variety of cells, especially at high bacterial nu...

Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.Legionella pneumophila is a gram-negative bacterium which is found in freshwater environments, where it associates with amoebae. The inhalation of Legionella-containing aerosols can lead to an acute pneumonia, Legionnaires' disease. In the human lung the bacteria are able to replicate within alveolar macrophages and epithelial cells, causing tissue damage and eventually leading to lung failure. Some of the factors which promote intracellular replication are exported by or depend on other factors exported by the type II secretion system Lsp or the type IVB secretion system Dot/Icm (6,22,32,39,40). The type II secretion system is responsible for translocation of a variety of hydrolytic activities, because L. pneumophila type II secretion mutants show considerably reduced protease, acid phosphatase, lipase, lysophospholipase A (LPLA), phospholipase A (PLA), p-nitrophenylphosphorylcholine (p-NPPC) hydrolase, and nuclease activities in their culture supernatants (22,39). Accordingly, substrates of the type II secretion system include the major zinc metalloprotease ProA or Msp, the major acid phosphatase Map, the lipase LipA, and the major secreted LPLA PlaA (3,4,17,22). Studies with knockout mutants indicate that none of these proteins are essential...

The study identifies a sterol- and oxysterol binding protein (OSBP)-regulated phosphatidylinositol 4-kinase that regulates ceramide transport protein (CERT) activity and sphingomyelin (SM) synthesis. RNA interference silencing experiments identify PI4KIIα; as the mediator of Golgi recruitment of CERT, providing a potential mechanism for coordinating assembly of SM and cholesterol in the Golgi or more distal compartments.

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Uptake and discharge of ballast water by ocean-going ships contribute to the worldwide spread of aquatic invasive species, with negative impacts on the environment, economies, and public health. The International Ballast Water Management Convention aims at a global answer. The agreed standards for ballast water discharge will require ballast water treatment. Systems based on various physical and/or chemical methods were developed for on-board installation and approved by the International Maritime Organization. Most common are combinations of high-performance filters with oxidizing chemicals or UV radiation. A well-known problem of oxidative water treatment is the formation of disinfection by-products, many of which show genotoxicity, carcinogenicity, or other long-term toxicity. In natural biota, genetic damages can affect reproductive success and ultimately impact biodiversity. The future exposure towards chemicals from ballast water treatment can only be estimated, based on land-based testing of treatment systems, mathematical models, and exposure scenarios. Systematic studies on the chemistry of oxidants in seawater are lacking, as are data about the background levels of disinfection by-products in the oceans and strategies for monitoring future developments. The international approval procedure of ballast water treatment systems compares the estimated exposure levels of individual substances with their experimental toxicity. While well established in many substance regulations, this approach is also criticised for its simplification, which may disregard critical aspects such as multiple exposures and long-term sub-lethal effects. Moreover, a truly holistic sustainability assessment would need to take into account factors beyond chemical hazards, e.g. energy consumption, air pollution or waste generation.

Background: PlaC is a GDSL enzyme and the major GCAT secreted by Legionella pneumophila. Results: The zinc metalloproteinase ProA processes and activates PlaC. Deletion of regions within a disulfide loop increased GCAT activity. Conclusion: Inhibitory disulfide loop reduction/deletion by ProA activates PlaC GCAT. Significance: Here we recognized the postexport GCAT activation mechanism essential for modification of typical eukaryotic sterols.

Salmonella enterica is the second most reported bacterial cause of food-borne infections in europe. therefore molecular surveillance activities based on pathogen subtyping are an important measure of controlling Salmonellosis by public health agencies. In Germany, at the federal level, this work is carried out by the national Reference center for Salmonella and other Bacterial enteric pathogens (nRc). With rise of next generation sequencing techniques, the NRC has introduced whole-genome-based typing methods for S. enterica in 2016. In this study we report on the feasibility of genome-based in silico serotyping in the German setting using raw sequence reads. We found that SeqSero and seven gene MLST showed 98% and 95% concordance, respectively, with classical serotyping for the here evaluated serotypes, including the most common German serotypes S. enteritidis and S. typhimurium as well as less frequently found serotypes. the level of concordance increased to >99% when the results of both in silico methods were combined. However, both tools exhibited misidentification of monophasic variants, in particular monophasic S. Typhimurium and therefore need to be fine-tuned for reliable detection of this epidemiologically important variant. We conclude that with adjustments Salmonella genomebased serotyping might become the new gold standard.Subtyping of bacterial enteric pathogens, such as Salmonella enterica, traditionally relies on serotyping. The species Salmonella enterica is divided into six subspecies and consists of more than 2600 serovars, which are classified according to the White-Kauffmann-Le Minor Scheme 1 . Serotyping is based on determination of somatic O antigens and flagellin H antigens by reaction with specific antisera. Most S. enterica serovars have two alternately expressed H antigens, also referred to as 'phases' . The phase-1 and phase-2 flagellin proteins are encoded by fliC and fljB, respectively. The phase switch is regulated by the invertase hin and the fliC repressor gene fljA 2 . Therefore, the specific antigenic formula consists of three positions: the first position represents the O antigens, the second and third positions the two different flagellin H antigens. Each antigen position is separated by a colon, i.e. O:H1:H2. The antigenic formula for S. Typhimurium for example is accordingly 1,4,[5],12:i:1,2. There are variants of S. Typhimurium, which express only one flagellin and which therefore are referred to as monophasic S. Typhimurium. S. Enteritidis on the other hand does not possess a second flagellin per se, which is reflected in the antigenic formula: 1,9,12:g,m:-. It should be noted that some serovars share the same antigenic formula and require additional testing for unambiguous identification, e.g. the clinically important serovar S. Chloeraesuis shares its antigenic formula 6,7:c:1,5 with serovars S. Paratyphi C and S. Typhisuis. A differentiation is possible based on biochemical characteristics or PCR 3 .With rise of next generation sequencing (NGS) techniques, genomic typing ...