RESUMO
ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.
Assuntos
Proteínas de Bactérias/metabolismo , Colágeno Tipo IV/metabolismo , Legionella pneumophila/enzimologia , Legionella pneumophila/patogenicidade , Pulmão/microbiologia , Metaloendopeptidases/metabolismo , Alvéolos Pulmonares/patologia , Fatores de Virulência/metabolismo , Células A549 , Proteínas de Bactérias/química , Atividade Bactericida do Sangue , Humanos , Legionella pneumophila/crescimento & desenvolvimento , Pulmão/patologia , Metaloendopeptidases/química , Proteólise , Alvéolos Pulmonares/metabolismo , Células THP-1 , Virulência , Fatores de Virulência/químicaRESUMO
The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires' disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-BlueTM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion.
Assuntos
Legionella pneumophila , Doença dos Legionários , Animais , Flagelina , Humanos , Legionella pneumophila/fisiologia , Doença dos Legionários/microbiologia , Mamíferos , Metaloproteases , Receptor 5 Toll-Like/genética , Zinco/farmacologiaRESUMO
Legionnaires' disease is an acute fibrinopurulent pneumonia. During infection Legionella pneumophila adheres to the alveolar lining and replicates intracellularly within recruited macrophages. Here we provide a sequence and domain composition analysis of the L. pneumophila PilY1 protein, which has a high homology to PilY1 of Pseudomonas aeruginosa. PilY1 proteins of both pathogens contain a von Willebrand factor A (vWFa) and a C-terminal PilY domain. Using cellular fractionation, we assigned the L. pneumophila PilY1 as an outer membrane protein that is only expressed during the transmissive stationary growth phase. PilY1 contributes to infection of human lung tissue explants (HLTEs). A detailed analysis using THP-1 macrophages and A549 lung epithelial cells revealed that this contribution is due to multiple effects depending on host cell type. Deletion of PilY1 resulted in a lower replication rate in THP-1 macrophages but not in A549 cells. Further on, adhesion to THP-1 macrophages and A549 epithelial cells was decreased. Additionally, the invasion into non-phagocytic A549 epithelial cells was drastically reduced when PilY1 was absent. Complementation variants of a PilY1-negative mutant revealed that the C-terminal PilY domain is essential for restoring the wild type phenotype in adhesion, while the putatively mechanosensitive vWFa domain facilitates invasion into non-phagocytic cells. Since PilY1 also promotes twitching motility of L. pneumophila, we discuss the putative contribution of this newly described virulence factor for bacterial dissemination within infected lung tissue.
Assuntos
Aderência Bacteriana/genética , Proteínas de Fímbrias/genética , Legionella pneumophila/fisiologia , Doença dos Legionários/microbiologia , Pulmão/microbiologia , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Doença dos Legionários/patologia , Pulmão/patologia , Mutação , Domínios Proteicos , Transporte Proteico , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Legionella are Gram-negative bacteria that are ubiquitously present in natural and man-made water reservoirs. When humans inhale aerosolized water contaminated with Legionella, alveolar macrophages can be infected, which may lead to a life-threatening pneumonia called Legionnaires' disease. Due to the universal distribution of Legionella in water and their potential threat to human health, the Legionella concentration in water for human use must be strictly monitored, which is difficult since the standard detection still relies on lengthy cultivation and analysis of bacterial morphology. In this study, an antibody against L. pneumophila has been generated from the naïve human HAL antibody libraries by phage-display for the first time. The panning was performed on whole bacterial cells in order to select antibodies that bind specifically to the cell surface of untreated Legionella. The bacterial cell wall component lipopolysaccharide (LPS) was identified as the target structure. Specific binding to the important pathogenic L. pneumophila strains Corby, Philadelphia-1 and Knoxville was observed, while no binding was detected to seven members of the families Enterobacteriaceae, Pseudomonadaceae or Clostridiaceae. Production of this antibody in the recombinant scFv-Fc format using either a murine or a human Fc part allowed the set-up of a sandwich-ELISA for detection of Legionella cells. The scFv-Fc construct proved to be very stable, even when stored for several weeks at elevated temperatures. A sensitivity limit of 4,000 cells was achieved. The scFv-Fc antibody pair was integrated on a biosensor, demonstrating the specific and fast detection of L. pneumophila on a portable device. With this system, 10,000 Legionella cells were detected within 35 min. Combined with a water filtration/concentration system, this antibody may be developed into a promising reagent for rapid on-site Legionella monitoring.
Assuntos
Anticorpos Antibacterianos/química , Ensaio de Imunoadsorção Enzimática/métodos , Fragmentos Fc das Imunoglobulinas/química , Legionella pneumophila/isolamento & purificação , Lipopolissacarídeos/imunologia , Anticorpos de Cadeia Única/química , Microbiologia da Água , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/genética , Afinidade de Anticorpos , Especificidade de Anticorpos , Expressão Gênica , Humanos , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/genética , Legionella pneumophila/imunologia , Limite de Detecção , Lipopolissacarídeos/química , Camundongos , Biblioteca de Peptídeos , Lagoas/microbiologia , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genéticaRESUMO
We study the properties of wavefunctions and the wavepacket dynamics in quasiperiodic tight-binding models in one, two, and three dimensions. The atoms in the one-dimensional quasiperiodic chains are coupled by weak and strong bonds aligned according to the Fibonacci sequence. The associated d-dimensional quasiperiodic tilings are constructed from the direct product of d such chains, which yields either the hypercubic tiling or the labyrinth tiling. This approach allows us to consider fairly large systems numerically. We show that the wavefunctions of the system are multifractal and that their properties can be related to the structure of the system in the regime of strong quasiperiodic modulation by a renormalization group (RG) approach. We also study the dynamics of wavepackets to get information about the electronic transport properties. In particular, we investigate the scaling behaviour of the return probability of the wavepacket with time. Applying again the RG approach we show that in the regime of strong quasiperiodic modulation the return probability is governed by the underlying quasiperiodic structure. Further, we also discuss lower bounds for the scaling exponent of the width of the wavepacket and propose a modified lower bound for the absolute continuous regime.