Strong antagonism of an endophyte of Fraxinus excelsior towards the ash dieback pathogen, Hymenoscyphus fraxineus, is mediated by the antifungal secondary metabolite PF1140.

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4-6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent.IMPORTANCEEnvironmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

Exposure of honey bees to mixtures of microbial biopesticides and their effects on bee survival under laboratory conditions.

Biopesticides, having as active ingredients viruses, bacteria, or fungi, are developed to substitute or reduce the use of chemical plant protection products in different agrosystems. Though the application of mixtures containing several products is a common practice, interactions between microbial biopesticides and related effects on bees as non-target organisms have not been studied yet. In the current study, we exposed winter bees to five different microbial-based products and their combinations at the maximum recommended application rate to assess their responses. Laboratory oral exposure tests (acute/chronic) to single or binary products were conducted. Survival and food consumption of the tested bees were evaluated over the experimental duration. Our results show that some product combinations have potential additive or synergistic effects on bees, whereas others did not affect the bee's survival compared to the control. Exposure of tested bees to the most critical combination of products containing Bacillus thuringiensis aizawai ABTS-1857 and B. amyloliquefaciens QST 713 strongly resulted in a median lifespan of 4.5 days compared to 8.0 and 8.5 days after exposure to the solo products, respectively. The exposure to inactivated microorganisms by autoclaving them did not differ from their respective uncontaminated negative controls, indicating effects on bee mortality might originate in the treatment with the different microorganisms or their metabolites. Further investigations should be conducted under field conditions to prove the magnitude of observed effects on bee colonies and other bee species.

Structured illumination ptychography and at-wavelength characterization with an EUV diffuser at 13.5†nm wavelength.

Structured illumination is essential for high-performance ptychography. Especially in the extreme ultraviolet (EUV) range, where reflective optics are prevalent, the generation of structured beams is challenging and, so far, mostly amplitude-only masks have been used. In this study, we generate a highly structured beam using a phase-shifting diffuser optimized for 13.5 nm wavelength and apply this beam to EUV ptychography. This tailored illumination significantly enhances the quality and resolution of the ptychography reconstructions. In particular, when utilizing the full dynamics range of the detector, the resolution has been improved from 125 nm, when using an unstructured beam, to 34 nm. Further, ptychography enables the quantitative measurement of both the amplitude and phase of the EUV diffuser at 13.5 nm wavelength. This capability allows us to evaluate the influence of imperfections and contaminations on its "at wavelength" performance, paving the way for advanced EUV metrology applications and highlighting its importance for future developments in nanolithography and related fields.

Color Routing of the Emission from Magnetic and Electric Dipole Transitions of Eu3+ by Broken-Symmetry TiO2 Metasurfaces.

Manipulation of magnetic dipole emission with resonant photonic nanostructures is of great interest for both fundamental research and applications. However, obtaining selective control over the emission properties of magnetic dipole transitions is challenging, as they usually occur within a manifold of spectrally close emission lines associated with different spin states of the involved electronic levels. Here we demonstrate spectrally selective directional tailoring of magnetic dipole emission using designed photonic nanostructures featuring a high quality factor. Specifically, we employ a hybrid nanoscale optical system consisting of a Eu3+ compound coupled to a designed broken-symmetry TiO2 metasurface to demonstrate directional color routing of the compound's emission through its distinct electric and magnetic-dominated electronic transition channels. Using low numerical aperture collection optics, we achieve a fluorescence signal enhancement of up to 33.13 for the magnetic-dominated dipole transition at 590 nm when it spectrally overlaps with a spectrally narrow resonance of the metasurface. This makes the, usually weak, magnetic dipole transition the most intense spectral line in our recorded fluorescence spectra. By studying the directional emission properties for the coupled system using Fourier imaging and time-resolved fluorescence measurements, we demonstrate that the high-quality-factor modes in the metasurface enable free-space light routing, where forward-directed emission is established for the magnetic-dominated dipole transition, whereas the light emitted via the electric dipole transition is mainly directed sideways. Our results underpin the importance of magnetic light-matter interactions as an additional degree of freedom in photonic and optoelectronic systems. Moreover, they facilitate the development of spectrometer-free and highly integrated nanophotonic imaging, sensing, and probing devices.

Legionella pneumophila Presence in Dental Unit Waterlines: A Cultural and Molecular Investigation in the West Bank, Palestine.

A Legionella pneumophila bacterium is ubiquitous in water distribution systems, including dental unit waterlines (DUWLs). Legionellosis is atypical pneumonia, including Legionnaires' disease (LD) and the less acute form of Pontiac fever. Legionellosis occurs as a result of inhalation/aspiration of aerosolized Legionella-contaminated water by susceptible patients, health workers, and dentists. In this study, we undertook to determine the prevalence of Legionella in water and biofilm samples from Tap and DUWLs collected from five sites of dental clinics and faculties across the West Bank. Water samples were tested for physical and chemical parameters. The study samples included 185 samples, 89 (48%) water samples, and 96 (52%) biofilm swabs, which were analyzed by cultivation-dependent analysis (CDA) and by the cultivation-independent technique (CIA). Also, partial sequencing of the 16S rRNA gene for fifteen L. pneumophila isolates was performed for quality assurance and identification. L. pneumophila was isolated from 28 (15%) of 185 samples using CDA and was detected in 142 (77%) of 185 samples using CIA. The abundance of culturable L. pneumophila was low in DUWL of the sampling sites (range: 27-115 CFU/Liter). PCR was 5× more sensitive than the culture technique. L. pneumophila Sg 1 was detected in (75%) of the isolates, while (25%) isolates were L. pneumophila Sg 2-14. All fifteen sequenced Legionella isolates were identified as L. pneumophila ≥ 94.5%. The analysis of phylogenetic tree showed that L. pneumophila branch clearly identified and distinguished from other branches. These results show that DUWLs of the examined dental clinics and faculties are contaminated with L. pneumophila. This finding reveals a serious potential health risk for infection of immunocompromised patients and dentists' post-exposure.

Bioactive Compounds from an Endophytic Pezicula sp. Showing Antagonistic Effects against the Ash Dieback Pathogen.

A fungal endophyte originating from the Canary Islands was identified as a potent antagonist against the fungal phytopathogen Hymenoscyphus fraxineus, which causes the devastating ash dieback disease. This endophyte was tentatively identified as Pezicula cf. ericae, using molecular barcoding. Isolation of secondary metabolites by preparative high-performance liquid chromatography (HPLC) yielded the known compounds CJ-17,572 (1), mycorrhizin A (3) and cryptosporioptides A-C (4-6), besides a new N-acetylated dihydroxyphenylalanin derivative 2, named peziculastatin. Planar structures were elucidated by NMR and HRMS data, while the relative stereochemistry of 2 was assigned by H,H and C,H coupling constants. The assignment of the unknown stereochemistry of CJ-17,572 (1) was hampered by the broadening of NMR signals. Nevertheless, after semisynthetic conversion of 1 into its methyl derivatives 7 and 8, presumably preventing tautomeric effects, the relative configuration could be assigned, whereas comparison of ECD data to those of related compounds determined the absolute configuration. Metabolites 1 and 3 showed significant antifungal effects in vitro against H. fraxineus. Furthermore, 4-6 exhibited significant dispersive effects on preformed biofilms of S. aureus at concentrations up to 2 µg/mL, while the biofilm formation of C. albicans was also inhibited. Thus, cryptosporioptides might constitute a potential source for the development of novel antibiofilm agents.

Influence of virus abundances in donor colonies and nurse hives on queens of Apis mellifera during the rearing process.

Background: Honeybees are one of the three most important animals for mankind. In order to be safe and increase number of bee colonies for pollination, the breeding of queens is necessary. For several decades, bees were selected on economic and behavioral aspects. With the appearance of the neozootic mite Varroa destructor beekeepers were forced to adapt their methods. Varroa destructor can act as a vector for many different bee pathogenic viruses and by this potentiates its devastating impact. Aim: Methods of rearing queens were not evaluated since the mites' appearance. Besides scientific approaches, viruses received too little attention in regard to the rearing process of honeybee queens. Herein, we present a detailed analysis of virus abundances [Aparavirus, acute bee paralysis virus (ABPV); Triatovirus, black queen cell virus (BQCV); Cripavirus, chronic bee paralysis virus (CBPV); and Iflaviruses, deformed wings virus (DWV), Sacbrood virus (SBV), VDV-1] in breeding hives, donating first instar larvae, hives that are nursing these larvae until the pupa stage, and on queens of Apis mellifera in a breeding apiary. Methods: Nurse and donor colonies of the queen-rearing process were sampled in the year 2020 and analyzed by RT qPCR. Virus quantifications were correlated with queen mortalities and seasonal effects. Results: Virus detections increased in reared queens, however, the elevated virus titers did not increase the mortality of the queens until their exclosure. Moreover, we observed a lower interrelation between virus abundance in queens and their original donor colonies, than between nurse hives and their nursed queens. Conclusion: The bee pathogenic viruses ABPV, BQCV, CBPV, DWV, SBV, and VDV-1 do not influence the mortality of bee queens during the rearing process. Whether respective virus loads result in sublethal or long-term effects remains to be elucidated.

The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila.

Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires' disease (LD). The bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD.

[4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip-Dependent and LpMip-Independent Mechanisms.

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.

Gas Transport Mechanisms through Molecular Thin Carbon Nanomembranes.

Molecular thin carbon nanomembranes (CNMs) synthesized by electron irradiation induced cross-linking of aromatic self-assembled monolayers (SAMs) are promising 2D materials for the next generation of filtration technologies. Their unique properties including ultimately low thickness of ≈1 nm, sub-nanometer porosity, mechanical and chemical stability are attractive for the development of innovative filters with low energy consumption, improved selectivity, and robustness. However, the permeation mechanisms through CNMs resulting in, e.g., an ≈1000 times higher fluxes of water in comparison to helium have not been yet understood. Here, a study of the permeation of He, Ne, D2 , CO2 , Ar, O2 and D2 O using mass spectrometry in the temperature range from room temperature to ≈120 °C is studied. As a model system, CNMs made from [1″,4',1',1]-terphenyl-4-thiol SAMs are investigated. It is found out that all studied gases experience an activation energy barrier upon the permeation which scales with their kinetic diameters. Moreover, their permeation rates are dependent on the adsorption on the nanomembrane surface. These findings enable to rationalize the permeation mechanisms and establish a model, which paves the way toward the rational design not only of CNMs but also of other organic and inorganic 2D materials for energy-efficient and highly selective filtration applications.

Legionella pneumophila and Free-Living Nematodes: Environmental Co-Occurrence and Trophic Link.

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.

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.

Comparative Genomics of Legionella pneumophila Isolates from the West Bank and Germany Support Molecular Epidemiology of Legionnaires' Disease.

Legionella pneumophila is an environmental bacterium and clinical pathogen that causes many life-threating outbreaks of an atypical pneumonia called Legionnaires' disease (LD). Studies of this pathogen have focused mainly on Europe and the United States. A shortage in L. pneumophila data is clearly observed for developing countries. To reduce this knowledge gap, L. pneumophila isolates were studied in two widely different geographical areas, i.e., the West Bank and Germany. For this study, we sequenced and compared the whole genome of 38 clinical and environmental isolates of L. pneumophila covering different MLVA-8(12) genotypes in the two areas. Sequencing was conducted using the Illumina HiSeq 2500 platform. In addition, two isolates (A194 and H3) were sequenced using a Pacific Biosciences (PacBio) RSII platform to generate complete reference genomes from each of the geographical areas. Genome sequences from 55 L. pneumophila strains, including 17 reference strains, were aligned with the genome sequence of the closest strain (L. pneumophila strain Alcoy). A whole genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using the ParSNP software v 1.0. The reference genomes obtained for isolates A194 and H3 consisted of circular chromosomes of 3,467,904 bp and 3,691,263 bp, respectively. An average of 36,418 SNPs (min. 8569, max. 70,708 SNPs) against our reference strain L. pneumophila str. Alcoy, and 2367 core-genes were identified among the fifty-five strains. An analysis of the genomic population structure by SNP comparison divided the fifty-five L. pneumophila strains into six branches. Individual isolates in sub-lineages in these branches differed by less than 120 SNPs if they had the same MLVA genotype and were isolated from the same location. A bioinformatics analysis identified the genomic islands (GIs) for horizontal gene transfer and mobile genetic elements, demonstrating that L. pneumophila showed high genome plasticity. Four L. pneumophila isolates (H3, A29, A129 and L10-091) contained well-defined plasmids. On average, only about half of the plasmid genes could be matched to proteins in databases. In silico phage findings suggested that 43 strains contained at least one phage. However, none of them were found to be complete. BLASTp analysis of proteins from the type IV secretion Dot/Icm system showed those proteins highly conserved, with less than 25% structural differences in the new L. pneumophila isolates. Overall, we demonstrated that whole genome sequencing provides a molecular surveillance tool for L. pneumophila at the highest conceivable discriminatory level, i.e., two to eight SNPs were observed for isolates from the same location but several years apart.

Whole-genome sequencing of the clinical isolate of Legionella pneumophila ALAW1 from the West Bank allows high-resolution typing and determination of pathogenicity mechanisms.

Background: Legionella pneumophila is water-based bacterium causing Legionnaires' disease (LD). We describe the first documented case of nosocomial LD caused by L. pneumophila sequence type (ST) 461 and serogroup 6. The etiology of LD was confirmed by culturing the bronchoalveolar lavage sample retrieving L. pneumophila strain ALAW1. A 7-days treatment of the LD patient with Azithromycin and Levofloxacin allowed complete recovery. Methods: In details, we sequenced the whole genome of the L. pneumophila ALAW1 using Illumina HiSeq platform. The sequence of ALAW1 was aligned with the genome sequence from the closely related reference strain Alcoy 2300/99 and a whole-genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using Parsnp software. Also, the TYGS web-server was used in order to compare the genome with type strain. Results: An analysis of the population structure by SNP and TYGS comparison clustered ALAW1 with the reference genome Alcoy 2300/99. Blastp analysis of the type IV secretion Dot/Icm system genes showed that these genes were highly conserved with (≤25%) structural differences at the protein level. Conclusions: Overall, this study provides insights into detailed genome structure and demonstrated the value of whole-genome sequencing as the ultimate typing tool for Legionella.

Legionella pneumophila PPIase Mip Interacts with the Bacterial Proteins SspB, Lpc2061, and FlaA and Promotes Flagellation.

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.

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.

Material-specific high-resolution table-top extreme ultraviolet microscopy.

Microscopy with extreme ultraviolet (EUV) radiation holds promise for high-resolution imaging with excellent material contrast, due to the short wavelength and numerous element-specific absorption edges available in this spectral range. At the same time, EUV radiation has significantly larger penetration depths than electrons. It thus enables a nano-scale view into complex three-dimensional structures that are important for material science, semiconductor metrology, and next-generation nano-devices. Here, we present high-resolution and material-specific microscopy at 13.5 nm wavelength. We combine a highly stable, high photon-flux, table-top EUV source with an interferometrically stabilized ptychography setup. By utilizing structured EUV illumination, we overcome the limitations of conventional EUV focusing optics and demonstrate high-resolution microscopy at a half-pitch lateral resolution of 16 nm. Moreover, we propose mixed-state orthogonal probe relaxation ptychography, enabling robust phase-contrast imaging over wide fields of view and long acquisition times. In this way, the complex transmission of an integrated circuit is precisely reconstructed, allowing for the classification of the material composition of mesoscopic semiconductor systems.

Streptococcus pneumoniae Affects Endothelial Cell Migration in Microfluidic Circulation.

Bloodstream infections caused by Streptococcus pneumoniae induce strong inflammatory and procoagulant cellular responses and affect the endothelial barrier of the vascular system. Bacterial virulence determinants, such as the cytotoxic pore-forming pneumolysin, increase the endothelial barrier permeability by inducing cell apoptosis and cell damage. As life-threatening consequences, disseminated intravascular coagulation followed by consumption coagulopathy and low blood pressure is described. With the aim to decipher the role of pneumolysin in endothelial damage and leakage of the vascular barrier in more detail, we established a chamber-separation cell migration assay (CSMA) used to illustrate endothelial wound healing upon bacterial infections. We used chambered inlets for cell cultivation, which, after removal, provide a cell-free area of 500 µm in diameter as a defined gap in primary endothelial cell layers. During the process of wound healing, the size of the cell-free area is decreasing due to cell migration and proliferation, which we quantitatively determined by microscopic live cell monitoring. In addition, differential immunofluorescence staining combined with confocal microscopy was used to morphologically characterize the effect of bacterial attachment on cell migration and the velocity of gap closure. In all assays, the presence of wild-type pneumococci significantly inhibited endothelial gap closure. Remarkably, even in the presence of pneumolysin-deficient pneumococci, cell migration was significantly retarded. Moreover, the inhibitory effect of pneumococci on the proportion of cell proliferation versus cell migration within the process of endothelial gap closure was assessed by implementation of a fluorescence-conjugated nucleoside analogon. We further combined the endothelial CSMA with a microfluidic pump system, which for the first time enabled the microscopic visualization and monitoring of endothelial gap closure in the presence of circulating bacteria at defined vascular shear stress values for up to 48 h. In accordance with our CSMA results under static conditions, the gap remained cell free in the presence of circulating pneumococci in flow. Hence, our combined endothelial cultivation technique represents a complex in vitro system, which mimics the vascular physiology as close as possible by providing essential parameters of the blood flow to gain new insights into the effect of pneumococcal infection on endothelial barrier integrity in flow.