PilY1 Promotes Legionella pneumophila Infection of Human Lung Tissue Explants and Contributes to Bacterial Adhesion, Host Cell Invasion, and Twitching Motility

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

FKBPs in bacterial infections.

FK506-binding proteins (FKBPs) contain a domain with peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and bind the immunosuppressive drugs FK506 and rapamycin. FKBPs belong to the immunophilin family and are found in eukaryotes and bacteria

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

Legionella-protozoa-nematode interactions in aquatic biofilms and influence of Mip on Caenorhabditis elegans colonization

International audienceLegionella pneumophila, the causative agent of Legionnairesi disease, is naturally found in aquatic habitats. The intracellular life cycle within protozoa pre-adapted the "accidental" human pathogen to also infect human professional phagocytes like alveolar macrophages. Previous studies employing the model organism Caenorhabditis elegans suggest that also nematodes might serve as a natural host for L pneumophila. Here, we report for the first time from a natural co-habitation of L. pneumophila and environmental nematode species within biofilms of a warm water spring. In addition, we identified the protozoan species Oxytricha bifaria, Stylonychia mytilus, Ciliophrya sp. which have never been described as potential interaction partners of L. pneumophila before. Modeling and dissection of the Legionella-protozoa-nematode interaction revealed that C elegans ruptures Legionella-infected amoebal cells and by this means incorporate the pathogen. Further infection studies revealed that the macrophage infectivity potentiator (Mip) protein of L. pneumophila, which is known to bind collagen IV during human lung infection, promotes the colonization of the intestinal tract of L4 larvae of C. elegans and negatively influences the life span of the worms. The Mip-negative L. pneumophila mutant exhibited a 32-fold reduced colonization rate of the nematodes after 48 h when compared to the wild-type strain. Taken together, these studies suggest that nematodes may serve as natural hosts for L pneumophila, promote their persistence and dissemination in the environment, and co-evolutionarily pre-adapt the pathogen for interactions with extracellular constituents of human lung tissue

Influence of end groups variation of self assembled monolayers on performance of planar perovskite solar cells by interface regulation

WOS:000604244800006We present the synthesis and application of new class of self-assembled monolayer molecules (SAMs) for acquiring feasible interfacial engineering in inverted type perovskite solar cells (PSCs). The proposed SAMs bearing different electron donating terminal groups have been utilized to tune the work function of indium tin oxide (ITO) electrodes. Fine-tuning of terminal groups of the SAMs allows us to compare relationship between molecular structures and device parameters. Moreover, ionic and hybrid nature of perovskite enables forming various chemical interactions with terminal groups of SAMs. Employed SAMs has resulted in permanent increase in work function of ITO, increase power conversion efficiency (PCE) of the cells and passivation of trap states at the interface between electrode and perovskite layer. The present study provides new insights into correlation between molecular engineering and solar cell performance through treating holistic comparison of synthesized molecules at the interface of ITO and perovskite layer. © 2020 Elsevier Lt

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

Therapeutic efficacy and gastrointestinal biodistribution of polycationic nanoparticles for oral camptothecin delivery in early and late-stage colorectal tumor-bearing animal model.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world and is the second leading cause of cancer related deaths. New cases are increasingly diagnosed every day, but current therapeutic options are still insufficient for an effective treatment. In CRC treatment, there is a significant need for alternative treatment approaches that can both prevent relapse and provide strong antimetastatic effects as the intestines and colon are prone to metastasis to neighboring organs and tissues as well as the liver and the lung. In this study, optimized polycationic cyclodextrin (CD) nanoparticles for oral Camptothecin (CPT) delivery were comprehensively examined for in vivo performance in early and late stage tumor bearing mouse model in terms of antitumoral and antimetastatic efficacy of CPT bound to polycationic CD nanoparticles in comparison to free CPT. In addition, the gastrointestinal localization of a single administration of fluorescent dye loaded pol-ycationic CD nanoparticles in the gastrointestinal tract at the end of 24 h after oral administration was also imaged and evaluated by in vivo imaging system against fluorescent dye intensity. Results showed that survival percentage was significantly improved in CRC-bearing mice compared to oral CPT solution, with significantly reduced colorectal tumor masses and number of liver metastatic foci (p < 0.05). It was also possible to differ-entiate between the effectiveness of nanoparticles in early or late stages of CRC. In vivo imaging studies have also confirmed that polycationic CD nanoparticles are able to deliver the therapeutic load up to the colon and tend to accumulate especially in tumor foci, indicating an effective local treatment strategy. In addition number of liver metastases were significantly decreased with the CPT-loaded polycationic CD nanoparticle formulation in both early and late stage tumor models. These findings indicated that CPT-loaded polycationic CD nanoparticles could be an efficient oral nanocarrier formulation for anticancer molecules that have limited application because of oral bioavailability and stability problems