Protein sociology of ProA, Mip and other secreted virulence factors at the Legionella pneumophila surface

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

Functional, molecular and structural characterization of the zinc metalloprotease ProA from Legionella pneumophila

Die Legionärskrankheit ist eine schwere Pneumonie, die durch Inhalation des humanen Lungenpathogens Legionella pneumophila ausgelöst wird. Durch eine umfassende Charakterisierung auf verschiedenen Organisationsebenen konnte in dieser Arbeit eine vielseitige Rolle der bakteriellen Zink-Metalloprotease ProA als Virulenzfaktor aufgezeigt und spezifiziert werden. Infektionsstudien mit proA-Mutagenesestämmen offenbarten, dass die Protease maßgeblich zur Vermehrung von L. pneumophila in humanen Lungengewebsexplantaten (HLTEs) beiträgt. Die intrazelluläre Replikation in Makrophagen und Epithelzelllinien ließ dabei vornehmlich auf extrazelluläre Effekte schließen. Erstmals konnte in histologischen Untersuchungen eine Verdickung der Alveolarsepten infolge ProA-vermittelter Degradation von Kollagen IV quantifiziert und eine verbesserte bakterielle Ausbreitung durch die Gewebeschädigung beobachtet werden. Darüber hinaus legte eine erhöhte Sensitivität der proA-defizienten Mutante gegenüber humanem Serum eine zentrale Rolle der Protease im Schutz vor der humoralen Immunantwort nahe. Auch das Zusammenspiel von ProA und bakteriellem FlaA ermöglicht dem Pathogen eine verbesserte Evasion. So wurde in einem HEK-Blue hTLR5-Modellsystem deutlich, dass die Flagellin-vermittelte Stimulation des TLR5-NF-κB-Signalwegs im Wirt durch einen Abbau der FlaA-Monomere über ProA reduziert wird. Eine Mutagenese und phänotypische Charakterisierung weiterer lasB-annotierter Proteasen von L. pneumophila Corby enthüllte einzelne synergistische Funktionen hinsichtlich der Vermehrung in HLTEs, der Tensidfilmsezernierung sowie der Serumresistenz des Pathogens. Eine Kristallisation und Strukturaufklärung von ProA bei 1,48 Å zeigte zudem Übereinstimmung mit homologen M4-Proteasen anderer Pathogene wie Pseudolysin von Pseudomonas aeruginosa. Unterschiede wurden insbesondere in äußeren Loopstrukturen wie der Substratbindestelle S1' deutlich. Die Substratdeterminierung verschiedener Vertreter der Enzymfamilie scheint daher vorwiegend von spezifischen Aminosäuren in definierten Bindungsmotiven und weniger von allgemeinen strukturellen Ähnlichkeiten abhängig zu sein. Insgesamt demonstriert die vorliegende Arbeit eine elementare Bedeutung von ProA für die Pathogenese der Legionärskrankheit durch Zerstörung pulmonalen Gewebes, Hemmung der Wirtsabwehrmechanismen und Unterstützung der Vermehrung und Ausbreitung von L. pneumophila in der menschlichen Lunge.Legionnaires' disease is a severe pneumonia caused by inhalation of the human lung pathogen Legionella pneumophila. By a comprehensive characterization at different organizational levels, this thesis showed and specified a versatile role of the bacterial zinc metalloprotease ProA as an important virulence factor. Infection studies with proA mutagenesis strains revealed that the protease contributes significantly to the proliferation of L. pneumophila in human lung tissue explants (HLTEs). The intracellular replication in macrophages and epithelial cell lines particularly indicated extracellular effects. For the first time, thickening of alveolar septa as a consequence of ProA-mediated degradation of collagen IV was quantified in histopathological analyses, and improved bacterial dissemination due to tissue damage was observed. Furthermore, an increased susceptibility of the proA-deficient mutant to human serum suggested a central role of the protease in antagonizing the humoral immune response. The interplay of ProA and bacterial FlaA also improves evasion for the pathogen. Hence, in a HEK-Blue hTLR5 model system, it became evident that flagellin-mediated stimulation of the TLR5-NF-κB signaling pathway in the host is reduced by degradation of FlaA monomers via ProA. Mutagenesis and phenotypical characterization of other lasB-annotated proteases from L. pneumophila Corby revealed individual synergistic functions regarding the replication in HLTEs, secretion of surfactants and serum resistance of the pathogen. Crystallization and structure determination of ProA at 1.48 Å also showed high conformity with homologous M4 proteases of other pathogens such as pseudolysin from Pseudomonas aeruginosa. Differences were especially evident in outer loop structures like the substrate binding pocket S1'. Substrate determination of different members of the enzyme family therefore seems to depend primarily on specific amino acids in defined binding motifs and less on general structural similarities. Overall, the present thesis demonstrates crucial importance of ProA for the pathogenesis of Legionnaires' disease by destroying pulmonary tissue, inhibiting host defense mechanisms and promoting proliferation and dissemination of L. pneumophila in the human lung

Protein sociology of ProA, Mip and other secreted virulence factors at the Legionella pneumophila surface

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

Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin

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

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

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