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The lung environment is defined by unique biological boundary conditions, including complex alveolar geometry, extracellular matrix composition and mechanical forces generated during respiration. These factors were shown to regulate alveolar permeability, surfactant secretion, cell contractility and apoptosis, but their role in fungal infections remains unknown. is a critical fungal pathogen that causes severe pulmonary infections in immunocompromised individuals. Our study addresses a knowledge gap by investigating how boundary conditions affect conidia interactions with alveolar epithelial cells. We applied micropatterned substrates to confine cells into defined shapes and densities, allowing precise control over geometric conditions and extracellular matrix composition. Using cell line stably expressing the phagolysosomal protein Lamp1-NeonGreen and multiplane fluorescent microscopy, we evaluated conidia binding and internalization efficiency. We observed significantly faster and more efficient conidia internalization in cells confined on micropatterns compared to previously reported studies using cell monolayers. Altering cell geometry, density and extracellular matrix composition strongly affected conidia binding and localization to Lamp1 phagolysosomes. Cells on X-shaped or multicellular micropatterns showed higher internalization rates, particularly at the periphery, suggesting spatial heterogeneity in pathogen uptake. Additionally, changes in extracellular matrix composition influenced the intracellular trafficking of conidia. Our findings emphasize the essential role that local mechanical and biochemical cues play in shaping the interactions between fungal pathogens and alveolar cells. Understanding how lung boundary conditions change in disease states will provide important insights into fungal infection outcomes.

Background The discovery of CRISPR/Cas9 and its subsequent accessibility in daily research initiated a new era in genome editing. This game-changing genetic instrument enabled a vast array of challenging applications requiring site-specific genome engineering as well as applications involving the equipment of cells with additional genetic traits. Despite the undisputed benefits of this technology, for facile and efficient selection of successfully manipulated cells selectable markers remain indispensable. Over the past years endogenous counter-selectable markers have come into focus in antifungal research enabling site-directed integration of multiple genes into the genome of the human mold pathogen Aspergillus fumigatus . However, gene cassettes had to be transformed in a consecutive manner keeping multigene integrations laborious and time-consuming. Results In this work, we coupled the use of CRISPR/Cas9 with endogenous counter-selectable markers to achieve the simultaneous integration of multiple expression cassettes. The three markers used in this work included the herein employed azgA and the previously identified fcyB and cntA , responsible for 8-azaguanine, 5-fluorocytosine and 5-fluorouridine uptake, respectively. Exploiting their role in uptake of different selective agents, a triple selective transformation procedure and genomic integration of three expression cassettes in A. fumigatus was successfully accomplished. In addition to three distinct cellular reporters, we introduced strain-specific fluorescent reporters into four isolates displaying different levels of antifungal azole resistance to subsequently visualize and monitor their growth patterns in the same growth environment. Conclusions The technology described in this study significantly streamlines the genetic manipulation process, reducing both time and labor associated with sequential transformations. By enabling the introduction of multiple genetic traits in a single transformation event, this strategy provides a flexible and efficient platform for a wide range of applications. As such, it enhances the potential for rapid and effective multigene integration, advancing the field of genetic engineering in fungi.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes serious lung diseases in immunocompromised patients. The lung surfactant produced by alveolar type II and Clara cells in the lungs is an important line of defense against A. fumigatus. The surfactant consists of phospholipids and surfactant proteins (SP-A, SP-B, SP-C and SP-D). The binding to SP-A and SP-D proteins leads to the agglutination and neutralization of lung pathogens as well as the modulation of immune responses. SP-B and SP-C proteins are essential for surfactant metabolism and can modulate the local immune response; however, the molecular mechanisms remain unclear. We investigated changes in the SP gene expression in human lung NCI-H441 cells infected with conidia or treated with culture filtrates obtained from A. fumigatus. To further identify fungal cell wall components that may affect the expression of SP genes, we examined the effect of different A. fumigatus mutant strains, including dihydroxynaphthalene (DHN)-melanin-deficient ΔpksP, galactomannan (GM)-deficient Δugm1 and galactosaminogalactan (GAG)-deficient Δgt4bc strains. Our results show that the tested strains alter the mRNA expression of SP, with the most prominent and consistent downregulation of the lung-specific SP-C. Our findings also suggest that secondary metabolites rather than the membrane composition of conidia/hyphae inhibit SP-C mRNA expression in NCI-H441 cells.

Aspergillus terreus is an opportunistic fungal pathogen associated with high mortality rates and intrinsic resistance to amphotericin B. Its ability to persist within host tissues without inducing strong immune responses was suggested to contribute to poor clinical outcomes. The cellular mechanisms underlying A. terreus interactions with host cells remain largely unexplored. In this study, we have used a micropattern-based infection model to investigate the early interactions between A. terreus conidia and alveolar epithelial cells, focusing on the role of Arp2/3-dependent actin remodeling. This system allows quantitative analysis of conidia-cell interactions under defined spatial conditions. We show that A. terreus conidia rapidly bind to micropatterned A549 cell islands, with conidial numbers increasing over time. Conidia were found in actin- and Lamp1-positive vesicles already after one hour of infection. Inhibition of the Arp2/3 complex significantly impaired conidial binding and disrupted the formation of actin-positive vesicles, confirming the essential role of Arp2/3-mediated actin remodeling in early stages of conidial uptake. A subset of conidia was localized to Lamp1-positive phagolysosomes and accumulated over time. Interestingly, we have identified a small but consistent population of Lamp1-positive vesicles decorated with actin structures, potentially resembling actin flashes. These structures were entirely abolished upon Arp2/3 inhibition, indicating active cytoskeletal remodeling at the phagolysosomal interface. Our findings provide the first mechanistic insights into A. terreus internalization by alveolar epithelial cells and establish Arp2/3-mediated actin dynamics as a key process in early host-pathogen interactions. This cellular pathway may further contribute to intracellular trafficking and help understand the delayed onset of A. terreus infections.

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes serious lung diseases in immunocompromised patients. The lung surfactant produced by alveolar type II and Clara cells in the lungs is an important line of defense against A. fumigatus. The surfactant consists of phospholipids and surfactant proteins (SP-A, SP-B, SP-C and SP-D). The binding to SP-A and SP-D proteins leads to the agglutination and neutralization of lung pathogens as well as the modulation of immune responses. SP-B and SP-C proteins are essential for surfactant metabolism and can modulate the local immune response; however, the molecular mechanisms remain unclear. We investigated changes in the SP gene expression in human lung NCI-H441 cells infected with conidia or treated with culture filtrates obtained from A. fumigatus. To further identify fungal cell wall components that may affect the expression of SP genes, we examined the effect of different A. fumigatus mutant strains, including dihydroxynaphthalene (DHN)-melanin-deficient ΔpksP, galactomannan (GM)-deficient Δugm1 and galactosaminogalactan (GAG)-deficient Δgt4bc strains. Our results show that the tested strains alter the mRNA expression of SP, with the most prominent and consistent downregulation of the lung-specific SP-C. Our findings also suggest that secondary metabolites rather than the membrane composition of conidia/hyphae inhibit SP-C mRNA expression in NCI-H441 cells.

We present a new tree boosting algorithm designed for the measurement of parameters in the context of effective field theory (EFT). To construct the algorithm, we interpret the optimized loss function of a traditional decision tree as the maximal Fisher information in Poisson counting experiments. We promote the interpretation to general EFT predictions and develop a suitable boosting method. The resulting ``Boosted Information Tree'' algorithm approximates the score, the derivative of the log-likelihood function with respect to the parameter. It thus provides a sufficient statistic in the vicinity of a reference point in parameter space where the estimator is trained. The training exploits per-event information of likelihood ratios for different theory parameter values available in the simulated EFT data sets.

Non-perturbative gauge-invariance under the strong and the weak interactions dictates that the proton contains a non-vanishing valence contribution from the Higgs particle. By introducing an additional parton distribution function (PDF), we investigate the experimental consequences of this prediction. The Herwig 7 event generator and a parametrized CMS detector simulation are used to obtain predictions for a scenario amounting to the LHC Run II data set. We use those to assess the impact of the Higgs PDF on the pp->ttbar process in the single lepton final state. Comparing to nominal simulation we derive expected limits as a function of the shape of the valence Higgs PDF. We also investigate the process pp->ttZ at the parton level to add further constraints.

Aspergillus terreus is an opportunistic fungal pathogen associated with high mortality rates and intrinsic resistance to amphotericin B. Its ability to persist within host tissues without inducing strong immune responses was suggested to contribute to poor clinical outcomes. The cellular mechanisms underlying A. terreus interactions with host cells remain largely unexplored. In this study, we have used a micropattern-based infection model to investigate the early interactions between A. terreus conidia and alveolar epithelial cells, focusing on the role of Arp2/3-dependent actin remodeling. This system allows quantitative analysis of conidia-cell interactions under defined spatial conditions. We show that A. terreus conidia rapidly bind to micropatterned A549 cell islands, with conidial numbers increasing over time. Conidia were found in actin- and Lamp1-positive vesicles already after one hour of infection. Inhibition of the Arp2/3 complex significantly impaired conidial binding and disrupted the formation of actin-positive vesicles, confirming the essential role of Arp2/3-mediated actin remodeling in early stages of conidial uptake. A subset of internalized conidia was localized to Lamp1-positive phagolysosomes and accumulated over time. Interestingly, we have identified a small but consistent population of phagolysosomes decorated with actin patches, potentially resembling actin flashes. These structures were entirely abolished upon Arp2/3 inhibition, indicating active cytoskeletal remodeling at the phagolysosomal interface. Our findings provide the first mechanistic insights into A. terreus internalization by alveolar epithelial cells and establish Arp2/3-mediated actin dynamics as a key process in early host-pathogen interactions. This cellular pathway may contribute to intracellular persistence and help understand the delayed onset of A. terreus infections.

Abstract Introduction. The lung environment is defined by unique biological boundary conditions, including complex alveolar geometry, extracellular matrix composition and mechanical forces generated during respiration. These factors were shown to regulate alveolar permeability, surfactant secretion, cell contractility and apoptosis, but their role in fungal infections remains unknown. Aspergillus fumigatus is a critical fungal pathogen that causes severe pulmonary infections in immunocompromised individuals. Our study addresses a knowledge gap by investigating how boundary conditions affect A. fumigatus conidia interactions with alveolar epithelial cells. Methods. We applied micropatterned substrates to confine cells into defined shapes and densities, allowing precise control over geometric conditions and extracellular matrix composition. Using cell line stably expressing the phagolysosomal protein Lamp1-NeonGreen and multiplane fluorescent microscopy, we evaluated A. fumigatus conidia binding and internalization efficiency. Results. We observed significantly faster and more efficient A. fumigatus conidia internalization in cells confined on micropatterns compared to previously reported studies using cell monolayers. Altering cell geometry, density, and extracellular matrix composition strongly affected conidia binding and localization to Lamp1+ phagolysosomes. Cells on X-shaped or multicellular micropatterns showed higher internalization rates, particularly at the periphery, suggesting spatial heterogeneity in pathogen uptake. Additionally, changes in extracellular matrix composition influenced the intracellular trafficking of A. fumigatus conidia. Discussion. Our findings emphasize the essential role that local mechanical and biochemical cues play in shaping the interactions between fungal pathogens and alveolar cells. Understanding how lung boundary conditions change in disease states will provide important insights into fungal infection outcomes.Competing Interest StatementThe authors have declared no competing interest.