Search

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506‐binding proteins (FKBPs), plays a major role in the proliferation of the gram‐negative bacterium in host organisms. In this work, we test our library of >1000 FKBP‐focused ligands for inhibition of LpMip. The [4.3.1]‐bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non‐toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection‐assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]‐bicyclic sulfonamides as anti‐legionellal agents, although their anti‐infective properties cannot be explained by inhibition of LpMip alone.

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506‐binding proteins (FKBPs), plays a major role in the proliferation of the gram‐negative bacterium in host organisms. In this work, we test our library of >1000 FKBP‐focused ligands for inhibition of LpMip. The [4.3.1]‐bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non‐toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection‐assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]‐bicyclic sulfonamides as anti‐legionellal agents, although their anti‐infective properties cannot be explained by inhibition of LpMip alone.

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506‐binding proteins (FKBPs), plays a major role in the proliferation of the gram‐negative bacterium in host organisms. In this work, we test our library of >1000 FKBP‐focused ligands for inhibition of LpMip. The [4.3.1]‐bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non‐toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection‐assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]‐bicyclic sulfonamides as anti‐legionellal agents, although their anti‐infective properties cannot be explained by inhibition of LpMip alone.

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506‐binding proteins (FKBPs), plays a major role in the proliferation of the gram‐negative bacterium in host organisms. In this work, we test our library of >1000 FKBP‐focused ligands for inhibition of LpMip. The [4.3.1]‐bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non‐toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection‐assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]‐bicyclic sulfonamides as anti‐legionellal agents, although their anti‐infective properties cannot be explained by inhibition of LpMip alone.

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506‐binding proteins (FKBPs), plays a major role in the proliferation of the gram‐negative bacterium in host organisms. In this work, we test our library of >1000 FKBP‐focused ligands for inhibition of LpMip. The [4.3.1]‐bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non‐toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection‐assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]‐bicyclic sulfonamides as anti‐legionellal agents, although their anti‐infective properties cannot be explained by inhibition of LpMip alone.

The pathogenicity of L. pneumophila, the causative agent of Legionnaires’ disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted “protein sociology” of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

The pathogenicity of L. pneumophila, the causative agent of Legionnaires’ disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted “protein sociology” of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires’ disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella’s major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella–nematode–amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators., Deutsche Forschungsgemeinschaft (DFG), Peer Reviewed

The pathogenicity of L. pneumophila, the causative agent of Legionnaires’ disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted “protein sociology” of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

The pathogenicity of L. pneumophila, the causative agent of Legionnaires’ disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted “protein sociology” of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

The pathogenicity of L. pneumophila, the causative agent of Legionnaires’ disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted “protein sociology” of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

The peptidyl-prolyl-cis/trans-isomerase (PPIase) macrophage infectivity potentiator (Mip) contributes to the pathogenicity and fitness of L. pneumophila, the causative agent of Legionnaires’ disease. Here, we identified the stringent starvation protein SspB, hypothetical protein Lpc2061, and flagellin FlaA as bacterial interaction partners of Mip. The macrolide FK506, which inhibits the PPIase activity of Mip, interfered with the binding of Lpc2061. Moreover, we demonstrated that the N-terminal dimerization region and amino acid Y185 in the C-terminal PPIase domain of Mip are required for the binding of Lpc2061 and FlaA. The modeling of the interaction partners and global docking with Mip suggested nonoverlapping binding interfaces, and a molecular dynamic simulation predicted an increased stability for the tripartite interaction of Lpc2061, Mip, and FlaA. On the functional level, we demonstrated that Mip promotes L. pneumophila flagellation, which is positively influenced by the binding of Lpc2061 and reduced by FK506. Also, L. pneumophila mutants expressing the Y185A or the monomeric Mip variant, which bind less Lpc2061, were nonmotile, were less flagellated, and yielded less FlaA when quantified. To our knowledge, this is the first report in which a PPIase and its bacterial interaction partners were demonstrated to influence flagellation.

Mie-resonant high-index dielectric nanoparticles and metasurfaces have been suggested as a viable platform for enhancing both electric and magnetic dipole transitions of fluorescent emitters. While the enhancement of the electric dipole transitions by such dielectric nanoparticles has been demonstrated experimentally, the case of magnetic-dipole transitions remains largely unexplored. Here, we study the enhancement of spontaneous emission of Eu3+ ions, featuring both electric and magnetic-dominated dipole transitions, by dielectric metasurfaces composed of Mie-resonant silicon nanocylinders. By coating the metasurfaces with a layer of an Eu3+ doped polymer, we observe an enhancement of the Eu3+ emission associated with the electric (at 610 nm) and magnetic-dominated (at 590 nm) dipole transitions. The enhancement factor depends systematically on the spectral proximity of the atomic transitions to the Mie resonances as well as their multipolar order, both controlled by the nanocylinder size. Importantly, the branching ratio of emission via the electric or magnetic transition channel can be modified by carefully designing the metasurface, where the magnetic dipole transition is enhanced more than the electric transition for cylinders with radii of about 130 nm. We confirm our observations by numerical simulations based on the reciprocity principle. Our results open new opportunities for bright nanoscale light sources based on magnetic transitions.

Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of effector proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.

Environmental bacteria of the genus naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the -containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for spp., but also useful cellular models for eukaryotic phagocytes. In particular, and emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.

Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of effector proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.

Background: The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires' disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans. Results: We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains. Conclusions: Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

Background: The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires' disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans. Results: We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains. Conclusions: Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

We predict and demonstrate the generation of a plasmonic hot spot on the surface of a metal film by the interference of two Airy surface plasmons. We show that the position of the hot spot can be controlled by the distance between the excitation gratings as well as by the phase front of the initial excitation. The observed effect constitutes a planar analogy to Airy beam autofocusing and offers new opportunities for spatially resolved surface plasmon sensing and optical surface tweezers.

We review our activities on the generation and manipulation of Airy surface plasmons. We demonstrate the excitation of surface Airy plasmon polaritons using a specially designed diffraction pattern. We predict and observe the generation of a plasmonic hot

RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA localizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intracellular replication was significantly enhanced in rpkA(-). The difference between wild type and rpkA(-) was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA(-) we could not detect a difference in endosomal pH but rpkA(-) showed depletion of phosphoinositides (PIP and PIP2) when we compared metabolically labeled phosphoinositides from wild type and rpkA(-). Furthermore rpkA(-) exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes.

RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA localizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intracellular replication was significantly enhanced in rpkA(-). The difference between wild type and rpkA(-) was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA(-) we could not detect a difference in endosomal pH but rpkA(-) showed depletion of phosphoinositides (PIP and PIP2) when we compared metabolically labeled phosphoinositides from wild type and rpkA(-). Furthermore rpkA(-) exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes.

Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

In der vorliegenden Arbeit wurde das Pad-Protein von Legionella pneumophila mit zell- bzw. molekularbiologischen sowie immunochemischen Methoden charakterisiert. Mittels Einbau des mutierten Pad-Locus, kodiert auf dem Plasmid pCS-25, in das Genom der L. pneumophila Stämme WH88 (Serogruppen 6) bzw. WH89 (Serogruppe 10) wurden je drei Pad-negative Mutanten gewonnen. Mit Hilfe der PCR, des Southernblot bzw. ELISA wurde der korrekte Austausch der Wildtypsequenz gegen den mutierten Bereich des Proteins nachgewiesen. Die Infektionsversuche mit den Mutanten bestätigten die Vermutung, dass das Protein eine Rolle an der initialen Kontaktaufnahme von L. pneumophila in den natürlichen Wirt, A. castellanii, hat. Dabei wurden signifikant niedrigere Aufnahmeraten der Mutanten im Vergleich zu den beiden Wildtypen beobachtet. Durch Kultivierung und Infektion des Ursprungstammes L. pneumophila Corby, der pad-negativen Mutante CP7 und der Pad-komplementierten Mutante bei 25°C, 30°C bzw. 37°C wurde der Einfluss der Temperatur auf die Funktion des Proteins untersucht. Die Ergebnisse zeigten dabei einen signifikanten Anstieg der Aufnahmeraten bei Vergleich von 25°C und 30°C bzw. 25°C und 37°C. Keine Signifikanz trat zwischen 30°C und 37°C auf. Ein zweiter Aspekt dieses Versuchs war die Prüfung der Infektionsrate in Abhängigkeit von der Proteinexpression unter dem Einfluss der Wachstumsphase. Dabei wurden durch vergleichende Infektion von A. castellanii mit exponentiellen (EP) bzw. stationäre Phase-Kulturen (SP) in diesem Versuch signifikante Unterschiede zwischen dem Wildtyp Corby und der Mutante CP7 bzw. zwischen der Mutante CP7 und der Pad+-Komplementante bei den jeweils untersuchten Temperaturen beobachtet. Keine signifikanten Unterschiede waren zwischen Wildtyp und Pad-Komplementante feststellbar. Zur Speziesübergreifenden Charakterisierung des Proteins Pad wurde das Wildtyplocus-kodierende Plasmid (pCS31) in die fünf non-pneumophila Stämme L. parisiensis (H962); L. bozemanii (L99-3

In der vorliegenden Arbeit wurde das Pad-Protein von Legionella pneumophila mit zell- bzw. molekularbiologischen sowie immunochemischen Methoden charakterisiert. Mittels Einbau des mutierten Pad-Locus, kodiert auf dem Plasmid pCS-25, in das Genom der L. pneumophila Stämme WH88 (Serogruppen 6) bzw. WH89 (Serogruppe 10) wurden je drei Pad-negative Mutanten gewonnen. Mit Hilfe der PCR, des Southernblot bzw. ELISA wurde der korrekte Austausch der Wildtypsequenz gegen den mutierten Bereich des Proteins nachgewiesen. Die Infektionsversuche mit den Mutanten bestätigten die Vermutung, dass das Protein eine Rolle an der initialen Kontaktaufnahme von L. pneumophila in den natürlichen Wirt, A. castellanii, hat. Dabei wurden signifikant niedrigere Aufnahmeraten der Mutanten im Vergleich zu den beiden Wildtypen beobachtet. Durch Kultivierung und Infektion des Ursprungstammes L. pneumophila Corby, der pad-negativen Mutante CP7 und der Pad-komplementierten Mutante bei 25°C, 30°C bzw. 37°C wurde der Einfluss der Temperatur auf die Funktion des Proteins untersucht. Die Ergebnisse zeigten dabei einen signifikanten Anstieg der Aufnahmeraten bei Vergleich von 25°C und 30°C bzw. 25°C und 37°C. Keine Signifikanz trat zwischen 30°C und 37°C auf. Ein zweiter Aspekt dieses Versuchs war die Prüfung der Infektionsrate in Abhängigkeit von der Proteinexpression unter dem Einfluss der Wachstumsphase. Dabei wurden durch vergleichende Infektion von A. castellanii mit exponentiellen (EP) bzw. stationäre Phase-Kulturen (SP) in diesem Versuch signifikante Unterschiede zwischen dem Wildtyp Corby und der Mutante CP7 bzw. zwischen der Mutante CP7 und der Pad+-Komplementante bei den jeweils untersuchten Temperaturen beobachtet. Keine signifikanten Unterschiede waren zwischen Wildtyp und Pad-Komplementante feststellbar. Zur Speziesübergreifenden Charakterisierung des Proteins Pad wurde das Wildtyplocus-kodierende Plasmid (pCS31) in die fünf non-pneumophila Stämme L. parisiensis (H962); L. bozemanii (L99-3

In der vorliegenden Arbeit wurde das Pad-Protein von Legionella pneumophila mit zell- bzw. molekularbiologischen sowie immunochemischen Methoden charakterisiert. Mittels Einbau des mutierten Pad-Locus, kodiert auf dem Plasmid pCS-25, in das Genom der L. pneumophila Stämme WH88 (Serogruppen 6) bzw. WH89 (Serogruppe 10) wurden je drei Pad-negative Mutanten gewonnen. Mit Hilfe der PCR, des Southernblot bzw. ELISA wurde der korrekte Austausch der Wildtypsequenz gegen den mutierten Bereich des Proteins nachgewiesen. Die Infektionsversuche mit den Mutanten bestätigten die Vermutung, dass das Protein eine Rolle an der initialen Kontaktaufnahme von L. pneumophila in den natürlichen Wirt, A. castellanii, hat. Dabei wurden signifikant niedrigere Aufnahmeraten der Mutanten im Vergleich zu den beiden Wildtypen beobachtet. Durch Kultivierung und Infektion des Ursprungstammes L. pneumophila Corby, der pad-negativen Mutante CP7 und der Pad-komplementierten Mutante bei 25°C, 30°C bzw. 37°C wurde der Einfluss der Temperatur auf die Funktion des Proteins untersucht. Die Ergebnisse zeigten dabei einen signifikanten Anstieg der Aufnahmeraten bei Vergleich von 25°C und 30°C bzw. 25°C und 37°C. Keine Signifikanz trat zwischen 30°C und 37°C auf. Ein zweiter Aspekt dieses Versuchs war die Prüfung der Infektionsrate in Abhängigkeit von der Proteinexpression unter dem Einfluss der Wachstumsphase. Dabei wurden durch vergleichende Infektion von A. castellanii mit exponentiellen (EP) bzw. stationäre Phase-Kulturen (SP) in diesem Versuch signifikante Unterschiede zwischen dem Wildtyp Corby und der Mutante CP7 bzw. zwischen der Mutante CP7 und der Pad+-Komplementante bei den jeweils untersuchten Temperaturen beobachtet. Keine signifikanten Unterschiede waren zwischen Wildtyp und Pad-Komplementante feststellbar. Zur Speziesübergreifenden Charakterisierung des Proteins Pad wurde das Wildtyplocus-kodierende Plasmid (pCS31) in die fünf non-pneumophila Stämme L. parisiensis (H962); L. bozemanii (L99-3

Sponges (Porifera) are filter feeders that take up microorganisms from seawater and digest them by phagocytosis. At the same time, many sponges are known to harbor massive consortia of symbiotic microorganisms, which are phylogenetically distinct from those in seawater, within the mesohyl matrix. In the present study, feeding experiments were performed to investigate whether phylogenetically different bacterial isolates, hereafter termed “food bacteria,” microbial seawater consortia, and sponge symbiont consortia are taken up and processed differently by the host sponge. Aplysina aerophoba retained high numbers of bacterial isolates and microbial seawater consortia with rates of up to 2.76 × 106 bacteria (g sponge wet weight)–1 h–1, whereas the retention of sponge symbionts was lower by nearly two orders of magnitude [5.37 × 104 bacteria (g sponge wet weight)−1 h–1]. In order to visualize the processing of a food bacterium within sponge tissues, the green fluorescent protein-labeled Vibrio strain MMW1, which had originally been isolated from A. aerophoba, was constructed. Incubation of this strain with A. aerophoba and subsequent visualization in tissue cryosections showed its presence in the choanocytes and/or endopinacocytes lining the canals but, unlike latex beads, not in deeper regions of the mesohyl, which suggests digestion of the bacteria upon contact with the host. Denaturing gradient gel electrophoresis (DGGE) was performed on the incubation seawater to monitor the changes in phylogenetic composition after incubation of the sponge with either seawater or sponge symbiont consortia. However, the DGGE experiment provided no evidence for selective processing of individual lineages by the host sponge. In conclusion, this study extends early studies by Wilkinson et al. (Proc R Soc London B 220:519–528, 1984) that sponges, here A. aerophoba, are able to differentiate between food bacteria and their own bacterial symbionts.

In der vorliegenden Arbeit wurde das Pad-Protein von Legionella pneumophila mit zell- bzw. molekularbiologischen sowie immunochemischen Methoden charakterisiert. Mittels Einbau des mutierten Pad-Locus, kodiert auf dem Plasmid pCS-25, in das Genom der L. pneumophila Stämme WH88 (Serogruppen 6) bzw. WH89 (Serogruppe 10) wurden je drei Pad-negative Mutanten gewonnen. Mit Hilfe der PCR, des Southernblot bzw. ELISA wurde der korrekte Austausch der Wildtypsequenz gegen den mutierten Bereich des Proteins nachgewiesen. Die Infektionsversuche mit den Mutanten bestätigten die Vermutung, dass das Protein eine Rolle an der initialen Kontaktaufnahme von L. pneumophila in den natürlichen Wirt, A. castellanii, hat. Dabei wurden signifikant niedrigere Aufnahmeraten der Mutanten im Vergleich zu den beiden Wildtypen beobachtet. Durch Kultivierung und Infektion des Ursprungstammes L. pneumophila Corby, der pad-negativen Mutante CP7 und der Pad-komplementierten Mutante bei 25°C, 30°C bzw. 37°C wurde der Einfluss der Temperatur auf die Funktion des Proteins untersucht. Die Ergebnisse zeigten dabei einen signifikanten Anstieg der Aufnahmeraten bei Vergleich von 25°C und 30°C bzw. 25°C und 37°C. Keine Signifikanz trat zwischen 30°C und 37°C auf. Ein zweiter Aspekt dieses Versuchs war die Prüfung der Infektionsrate in Abhängigkeit von der Proteinexpression unter dem Einfluss der Wachstumsphase. Dabei wurden durch vergleichende Infektion von A. castellanii mit exponentiellen (EP) bzw. stationäre Phase-Kulturen (SP) in diesem Versuch signifikante Unterschiede zwischen dem Wildtyp Corby und der Mutante CP7 bzw. zwischen der Mutante CP7 und der Pad+-Komplementante bei den jeweils untersuchten Temperaturen beobachtet. Keine signifikanten Unterschiede waren zwischen Wildtyp und Pad-Komplementante feststellbar. Zur Speziesübergreifenden Charakterisierung des Proteins Pad wurde das Wildtyplocus-kodierende Plasmid (pCS31) in die fünf non-pneumophila Stämme L. parisiensis (H962); L. bozemanii (L99-3

Sponges (Porifera) are filter feeders that take up microorganisms from seawater and digest them by phagocytosis. At the same time, many sponges are known to harbor massive consortia of symbiotic microorganisms, which are phylogenetically distinct from those in seawater, within the mesohyl matrix. In the present study, feeding experiments were performed to investigate whether phylogenetically different bacterial isolates, hereafter termed “food bacteria,” microbial seawater consortia, and sponge symbiont consortia are taken up and processed differently by the host sponge. Aplysina aerophoba retained high numbers of bacterial isolates and microbial seawater consortia with rates of up to 2.76 × 106 bacteria (g sponge wet weight)–1 h–1, whereas the retention of sponge symbionts was lower by nearly two orders of magnitude [5.37 × 104 bacteria (g sponge wet weight)−1 h–1]. In order to visualize the processing of a food bacterium within sponge tissues, the green fluorescent protein-labeled Vibrio strain MMW1, which had originally been isolated from A. aerophoba, was constructed. Incubation of this strain with A. aerophoba and subsequent visualization in tissue cryosections showed its presence in the choanocytes and/or endopinacocytes lining the canals but, unlike latex beads, not in deeper regions of the mesohyl, which suggests digestion of the bacteria upon contact with the host. Denaturing gradient gel electrophoresis (DGGE) was performed on the incubation seawater to monitor the changes in phylogenetic composition after incubation of the sponge with either seawater or sponge symbiont consortia. However, the DGGE experiment provided no evidence for selective processing of individual lineages by the host sponge. In conclusion, this study extends early studies by Wilkinson et al. (Proc R Soc London B 220:519–528, 1984) that sponges, here A. aerophoba, are able to differentiate between food bacteria and their own bacterial symbionts.

Legionella pneumophila is naturally found in fresh water were the bacteria parasitize within protozoa. It also survives planctonically in water or biofilms. Upon aerosol formation via man-made water systems, L. pneumophila can enter the human lung and cause a severe form of pneumonia, called Legionnaires’ disease. The pathogenesis of Legionnaires’ disease is largely due to the ability of L. pneumophila to invade and grow within macrophages. An important characteristic of the intracellular survival strategy is the replication within the host vacuole that does not fuse with endosomes or lysosomes. In recent times a great number of bacterial virulence factors which affect growth of L. pneumophila in both macrophages and protozoa have been identified. The ongoing Legionella genome project and the use of genetically tractable surrogate hosts are expected to significantly contribute to the understanding of bacterium–host interactions and the regulation of virulence traits during the infection cycle. Since person-to-person transmission of legionellosis has never been observed, the measures for disease prevention have concentrated on eliminating the pathogen from water supplies. In this respect detection and analysis of Legionella in complex environmental consortia become increasingly important. With the availability of new molecular tools this area of applied research has gained new momentum.

Legionella pneumophila is naturally found in fresh water were the bacteria parasitize within protozoa. It also survives planctonically in water or biofilms. Upon aerosol formation via man-made water systems, L. pneumophila can enter the human lung and cause a severe form of pneumonia, called Legionnaires’ disease. The pathogenesis of Legionnaires’ disease is largely due to the ability of L. pneumophila to invade and grow within macrophages. An important characteristic of the intracellular survival strategy is the replication within the host vacuole that does not fuse with endosomes or lysosomes. In recent times a great number of bacterial virulence factors which affect growth of L. pneumophila in both macrophages and protozoa have been identified. The ongoing Legionella genome project and the use of genetically tractable surrogate hosts are expected to significantly contribute to the understanding of bacterium–host interactions and the regulation of virulence traits during the infection cycle. Since person-to-person transmission of legionellosis has never been observed, the measures for disease prevention have concentrated on eliminating the pathogen from water supplies. In this respect detection and analysis of Legionella in complex environmental consortia become increasingly important. With the availability of new molecular tools this area of applied research has gained new momentum.

Refers to: Werner Goebel, Roy Gross Intracellular survival strategies of mutualistic and parasitic prokaryotes Trends in Microbiology, Volume 9, Issue 6, 1 June 2001, Pages 267-273 doi:10.1016/S0966-842X(01)02040-6

Refers to: Werner Goebel, Roy Gross Intracellular survival strategies of mutualistic and parasitic prokaryotes Trends in Microbiology, Volume 9, Issue 6, 1 June 2001, Pages 267-273 doi:10.1016/S0966-842X(01)02040-6

Traditionally, symbiotic and pathogenic interactions were considered different manifestations of the bacteria–host interaction. However, the molecular mechanisms that mediate communication between and cellular modulation of the involved partners are quite similar. With this review we aim to contribute to a reduction of the traditional gap between symbiosis and pathogenesis research.

Traditionally, symbiotic and pathogenic interactions were considered different manifestations of the bacteria–host interaction. However, the molecular mechanisms that mediate communication between and cellular modulation of the involved partners are quite similar. With this review we aim to contribute to a reduction of the traditional gap between symbiosis and pathogenesis research.