A. fumigatus DAL (wild-type, CBS 144.89) strain was cultured in filter-cap cell culture bottles on sabouraud 4% glucose agar (15 g/L agar, 40 g/L D (+)-glucose, 10 g/L peptone, Sigma-Aldrich, 1.06404, Austria) at 37°C for 10 days until full maturation of conidia was observed. Conidia were collected by plate flooding using sterile spore buffer (0.01% Tween, Fisher Scientific, 10113103, Germany, 0.9% NaCl, Carl Roth, 8986.1, Germany), centrifugated at 4000 rpm for 5 minutes and resuspended in sterile spore buffer.

Human A549 cell line stably expressing Lamp1-NeonGreen (Schiefermeier-Mach et al., 2021) were cultured in a growth medium consisting of RPMI-1640 (Capricorn, RPMI-XA, Germany), 10% fetal bovine serum (FBS, Capricorn Scientific, FBS-11A, Germany), 2 µl/ml puromycin, (Carl Roth, 0240.2, Germany) 1% penicillin/streptomycin (Capricorn Scientific, PS-B, Germany) and 1% L-glutamine (Capricorn Scientific, GLN-B, Germany).

Micropatterns were generated with deep-ultraviolet (UV) lithography on polyethylene glycol (PEG)-coated glass coverslips. Glass coverslips were incubated in a 1 mM solution of a linear PEG-silane (Rapp Polymere – reference 122000-71, Germany) in dry toluene for 20 h at 80°C under inert atmosphere, in order to covalently immobilize a monolayer of PEG (Azioune et al., 2010; Clausen et al., 2023). The substrates were removed, rinsed intensively with isopropanol, methanol and water, and dried with nitrogen. A PEG-coated glass coverslip and a chromium-coated quartz photomask (Compugraphics, Jena) were assembled with a vacuum on a holder, which was immediately exposed to deep ultraviolet (UV) light using a low-pressure mercury lamp (NIQ 60/35 XL longlife lamp, quartz tube, 60 W from Heraeus Noblelight, Germany) at 5 cm distance for 5 min. Deep UV light with a wavelength of 185 nm oxidizes PEG, significantly impairs PEG antifouling properties and allows ECM protein adsorption. The patterned substrates were subsequently incubated with 150 μl of fibronectin (10 μg/ml, Sigma Aldrich, 341631, Austria) in PBS or vitronectin (3.3 µl/ml, ProSci, 91-362, USA) at 4°C overnight and washed once with PBS. 1 ml of growth medium containing 10⁵ cells/ml (for smaller micropatterns) or 10⁶ cells/ml (for bigger micropatterns) was added and incubated in growth medium with reduced FCS (1%) for 2 hours at 37°C to allow adhesion. Cells were further carefully washed with warm PBS to remove unattached cells and further incubated in growth medium with reduced FCS (1%) overnight.

For infection experiments, cells were grown in 35 mm culture dishes on fibronectin- or vitronectin-coated micropatterns in serum-reduced medium and incubated for 24 hours. After the incubation time, the serum-reduced medium was carefully replaced with fresh RPMI-1640 containing 10% FBS without antibiotics. Cells were infected with 10⁶ cfu/ml A. fumigatus conidia. Infected cells were incubated for 1 and 3 hours at 37°C prior to fixation.

Cells infected with A. fumigatus conidia were fixed as described before with slight modifications (Scheffler et al., 2014). In short, 8% paraformaldehyde (PFA, Sigma Aldrich, 104005, Austria) in PBS was slowly added directly to the cells in cell medium in proportion 1:1 and incubated for 15 minutes at 37°C. Then, medium-PFA mix was carefully replaced with 4% PFA, incubated further 15 minutes and washed with PBS. Cells were further incubated with a mixture of EasyProbe ™ ActinRed 555 to visualize actin (THP Medical Products, FP032, Austria), Dapi (Sigma-Aldrich, D9542-5MG, Austria, 1:4000) for nuclei staining and 0.05% saponin in PBS for 1 hour at room temperature following washing in PBS and mounting on glass objective slides with Mowiol (Sigma-Aldrich, C9368, Austria).

All images were taken using an inverted wide-field IX83 Olympus Microscope (Olympus Austria) equipped with UPlanXApo 60x, 1.42 N.A, PH3 objective and 2x digital zoom. Images were recorded in fluorescence (Dapi, TRITC, FITC) and phase contrast (PH3) channels as multiplane Z-stacks with a step size set for all images at 0,5 µm. A resolution of 2048 x 2048 and a 16-bit-grayscale were used. Images were further de-convoluted using cellSens software (Olympus, deconvolution parameters: nearest neighbor 50%). For TRITC/FITC fluorescence channels exposure times of 120-150 ms, for Dapi 8 ms and phase contrast (PH3) 60ms were applied. 50 micropatterns from 3 biological repetitions were analyzed for each data set. Manual analysis of images was performed independently by two trained scientists using the “Point tool” and “Analyze-Measure” functions in Fiji software (RRID: SCR_002285). Quantifications of the center of mass of fungal conidia, as well as Lamp1⁺ and a subpopulation of Lamp1⁺Actin⁺ were saved as X, Y-coordinates. Z-stacks were examined in parallel: Dapi staining was used to count the cell number, a multi-channel merged fluorescence image of FITC/TRITC channels was used to identify Lamp1⁺ and Lamp1⁺Actin⁺ vesicles, phase contrast images were used to quantify the total number of conidia and for the identification of co-localization with Lamp1-NeonGreen and actin.

Analysis was performed in GraphPad Prism 10.1.2 (RRID: SCR_002798, USA) and figures were prepared in Fiji and Adobe Photoshop (RRID: SCR_014199). Tests used: descriptive statistics, ordinary one-way ANOVA (multiple comparisons), correlation analysis (correlation matrix, with Pearson coefficients, two-tailed, 95% confidence interval). The statistical significance of the data was determined by p-values.

To investigate the influence of cell confinement on the internalization of A. fumigatus conidia, human A549 cells stably expressing Lamp1-NeonGreen were cultured on fibronectin-coated circular micropatterns of 28 µm and 60 µm in diameter. We used circular patterns of different sizes to minimize cell contractility in contrast to non-micropatterned substrates and X-shaped patterns. By increasing pattern diameter, cell density was increased and cell area decreased, leading further to changes in cell contractility. To obtain a reproducible cell density, suspended single cells were seeded on patterns for only 2 hours to avoid the formation of cellular aggregates and multilayers on individual patterns. Upon washing unbound cells, adhered cells were further cultured overnight to give them time to form a homogeneous monolayer, similar to an epithelial monolayer.

Cells were seeded at various densities to achieve 1 and 2 cells on smaller micropatterns and 10-12 cells on bigger patterns ( Figure 1A ). The defined cell geometry or grouping of cells on each micropattern facilitated the quantification and mapping of host-pathogen interaction events by overlaying conidia coordinates from 50 identical micropatterns. Lamp1-NeonGreen was used as a marker of conidia internalization into phagolysosomes (Schiefermeier-Mach et al., 2021). Infection of cells with dormant A. fumigatus conidia for 1 and 3 hours resulted in specific conidia adhesion to confined cells without binding to the PEG-coated surrounding area. The average total number of detected conidia was significantly higher on big micropatterns with multicellular islands in comparison to smaller ones and strongly increased over time. However, there were no differences for 28 µm-micropattern with 1 or 2 cells ( Figure 1B ).

Multiplane imaging of cells by phase contrast and fluorescence microscopy revealed that some conidia were internalized into phagolysosomes (Lamp1⁺ vesicles) already after 1 hour, ( Figure 1C ; Supplementary Video S1 ). Since cells on micropatterns of different sizes can bind different numbers of conidia per pattern, we further normalized all values to the “total number of conidia” for each individual micropattern condition for better comparisons. Quantification of conidia in Lamp1⁺ vesicles as a part of all observed conidia showed a higher percentage on multicellular islands of 60 µm in diameter as compared to cells on 28 µm-patterns. Moreover, this percentage increased over time. Interestingly, not only the micropattern size but the number of cells per micropattern correlated with the number of phagolysosomes. There were significantly more conidia in Lamp1⁺ vesicles when 2 cells were confined to 28 µm-patterns in comparison to 1 cell ( Figure 1D ).

Further analysis of conidia distribution revealed that binding and phagolysosomal internalization of conidia were heterogeneously distributed across the micropattern ( Figure 1E ). Conidia mapping and their radial distribution showed that their binding and internalization at the outer edge of the 60 µm-patterns is favored. A similar trend for 28 µm-patterns with 2 cells was also observed. The lowest number of conidia was detected in the center of all micropatterns independent of size and cell number ( Figures 1E, F ).

To further investigate the intracellular trafficking and processing of A. fumigatus conidia, we additionally stained infected cells with an actin probe to detect intracellular actin distribution. We have observed that a small subset of Lamp1⁺ vesicles containing fungal conidia also displayed actin, forming a Lamp1⁺Actin⁺ subpopulation ( Figure 2A ). The proportion of conidia within these double-positive vesicles was relatively small compared to the overall Lamp1⁺ vesicles. However, the percentage of Lamp1⁺Actin⁺ vesicles relative to the total conidia number, showed a significant increase of these vesicles in 28 µm-patterns harboring 2 cells as compared to 1 cell or 10-12 cells on larger micropatterns ( Figure 2B ). Interestingly, in contrast to the population of Lamp1⁺ vesicles, which increased over time, the percentage of conidia in double-positive vesicles remained stable between 1 and 3 hours ( Figure 2B ). Conidia mapping further showed the number and localization of double vesicles with a tendency toward micropattern edges on bigger micropatterns ( Figure 2C ).

Next, we assessed the impact of ECM substrates in our cell model. We analyzed multicellular island constrained on 60 µm - micropattern coated with vitronectin (VN) instead of fibronectin (FN). VN is known from the literature to preferentially recruit ß3 integrins whereas FN recruits more ß1 integrins to coordinate cell adhesion (Schaufler et al., 2016). Analysis of conidia distribution on cells constricted on 60 µm-patterns coated with VN, revealed that binding and phagolysosomal internalization of conidia were similarly to the ones on FN ( Supplementary Figure S1A ). With both coating conditions, more binding and internalization of conidia was observed at the outer edge of the micropattern ( Supplementary Figures S1A, B ). When we quantified the average total number of conidia, we observed a decrease when using VN versus FN ( Figure 2D ). However, the percentage of conidia internalized in the Lamp1⁺ vesicles did not differ from the one observed in cells micropatterned on FN ( Figure 2D ). Interestingly, there was a strong difference between the substrates in terms of the percentage of conidia internalized into the subpopulation of Lamp1⁺Actin⁺ vesicles: this percentage was increased when cells were constrained on VN ( Figure 2D ).

To further investigate the impact of cell geometry on the cell-conidia interactions, we seeded A549-Lamp1-NeonGreen cells on X-shaped patterns with an edge of 28 µm. By presenting edges and adhesion-free areas, X-shaped patterns allowed us to further modify cell contractility and investigate its effect on conidia internalization. By varying the number of cells, we established 3 distinct cell densities: 1, 2 and 4 cells per micropattern, each resulting in characteristic changes in cell morphology due to the shape constraints imposed by the X-pattern ( Figure 3A ). Upon infection with A. fumigatus conidia for 1 and 3 hours, we observed notable differences in conidia internalization across these conditions. Similarly to circular patterns, conidia were heterogeneously distributed across the micropattern with increased binding and internalization at the outer edge ( Figures 3B, C ). After 1 hour of infection, while the average total number of conidia did not differ, there was an increased percentage of conidia in Lamp1⁺ vesicles in the 4-cell configuration in comparison to 1 cell and 2 cells ( Figures 3D, E ). After 3 hours of infection, the average total number of conidia correlated with cell number and was the highest in the 4-cell micropattern. Internalization of conidia in Lamp1⁺ vesicles increased after 3 hours compared to 1 hour and was higher in micropatterns with 2 and 4 cells compared to those with only 1 cell. The percentage of conidia in double Lamp1⁺Actin⁺ vesicles did not differ significantly between the X-micropatterns with different cell numbers and did not change over time.

Interestingly, there were notable differences when comparing X-shaped to circular micropatterns ( Figure 3F ). Conidia were internalized more efficiently by cells constrained on X-micropatterns. Moreover, the percentage of double Lamp1⁺Actin⁺ vesicles was also higher in these cells ( Figure 3F ). To further elucidate the relationship between different parameters and conidia internalization, we performed a correlation analysis using a correlation matrix and Pearson correlation coefficients. This analysis revealed that the percentage of conidia in Lamp1⁺ and Lamp1⁺Actin⁺ vesicles was negatively correlated with cell area at both 1 hour and 3 hours. This negative correlation was moderate after 1 hour (r=-0.447) and strong after 3 hours (r=-0.716). The correlation of cell area with Lamp1⁺Actin⁺ vesicles was negative and moderate both after 1 hour (r=-0.310) and 3 hours (r=-0.299). No other significant correlations with other parameters were found.