Fig 1: Growth inhibition of A. fumigatus by SP-D upon replenishment, and kinetics of growth inhibition. (A) rfhSP-D (15 μg/mL) was added to conidia (1x105/mL) in RPMI + 3.45% MOPS + 2% glucose and incubated at 37°C. At 8 h-intervals, culture medium was aspirated, fresh medium with rfhSP-D (10 μg/mL) was added, incubation continued and the growth at 24 h was monitored under light microscope (scale bar = 100 μm). (B) Screen shots of live microscopy at 3 h, 6 h and 8 h of co-incubation of conidia and rfhSP-D. Starting from 6 h, the hyphal length of untreated control was longer than rfhSP-D-treated hyphae. (C) The hyphal length at 8 h was quantified by Fiji. The hyphal germination starting time was not significantly different between control (201 ± 51 min) and rfhSP-D (207 ± 56 min) treated conidia. (D) The control hyphae were significantly longer (199.9 ± 81.2 µm) than the rfhSP-D-treated hyphae (56.2 ± 32.7 µm) at the end of the incubation (8 h). Quantification was performed for six independent experiments (number of quantified conidia = 60). The bars and the error bars indicate the means and standard error of the mean, respectively. Students’ t-test was performed to examine the difference between control and rfhSP-D-treated samples (ns – not significant, ****p<0.0001).
Fig 2: SP-D gets deposited on the A. fumigatus germ tubes/hyphae during the course of germination. A. fumigatus germ tubes were prepared on a 8-well Lab-Tek chamber slide well by incubating conidia (1x105/mL, 200 μL/well) in RPMI + 3.45% MOPS + 2% glucose with/without FL-SP-D or rfhSP-D (30 μg/mL) at 37°C for 9-16 h. SP-D treated germ-tubes were then PFA fixed and immunolabelled upon sequential incubation with primary rabbit polyclonal anti-SP-D antibodies (serum) or mouse monoclonal anti-SP-D antibodies and secondary rabbit Alexa Fluor 633 conjugated IgG or TRITC conjugated mouse IgG, respectively. (A) and (B) are immunolabelling of 9 h and 16 h grown germ tubes for SP-D. Positive control: SP-D untreated germ tubes that were PFA fixed and treated with FL-SP-D (30 μg/mL) followed by immunolabelling with primary and secondary antibodies. Negative control: SP-D untreated germ tubes, PFA fixed and treated with primary and secondary antibodies.
Fig 3: Growth inhibition of A. fumigatus by SP-D. (A) A. fumigatus conidia (1x105/mL) were incubated with FL-SP-D/rfhSP-D (30 μg/mL) in RPMI + 3.45% MOPS + 2% glucose at 37oC. The fungal growth was monitored under light microscope after 16-24 h (scale bar = 100 μm). FL-SP-D exhibited stronger growth inhibitory effect than rfhSP-D. (B) Growth inhibitory effect of SP-D was tested against another clinical isolate of A. fumigatus, CNRMA 15.354 and using other source of SP-D (ab152069, Abcam) against A. fumigatus isolate CBS 144-89. (C) Growth inhibition by SP-D on A. fumigatus and A. flavus was examined by resazurin method using 1% yeast-extract + 2% glucose medium with a range of rfhSP-D concentration (error bars, mean ± SEM); after co-incubation of the contents at 37°C for 24 h, optical density was measured at 569 nm. (D) A. fumigatus growth inhibition by a humoral component, ficolin-2 (FCN2; 5 μg/mL (>physiological concentration in human alveolar fluid) by resazurin method, performed in 1% yeast-extract+2% glucose medium) was examined after 24 h of co-incubation of the contents at 37°C, and reading the optical density at 569 nm.
Fig 4: SP-D binds to A. fumigatus cell wall polysaccharides (A) galactosaminogalactan (GAG) and (B) galactomannan (GM). FL-SP-D/rfhSP-D suspended in 50 mM carbonate buffer (pH 9.6) was coated on the microtiter plate wells (500 ng/well) and added with GAG or GM (500 ng/well) in PBS containing 3% BSA-5 mM CaCl2. Interaction between polysaccharides and SP-D was examined by sequential incubation with monoclonal anti-GAG/-GM antibodies, peroxidase-conjugated secondary antibody and O-phenylenediamine as the substrate (with PBS washings between each step). The absorbance of the colour developed was read at 492 nm. Students’ t-test was performed to evaluate the differential binding of FL-SP-D and rfhSP-D with GM/GAG (**p<0.005, ****p<0.0001). (C) Following the same protocol, the binding efficiencies of FL-SP-D and rfhSP-D with deacetylated (deacetyl), native and acetylated (Acetyl) GAGs were checked.
Fig 5: (A) Incubation with SP-D increases the permeability of A. fumigatus germ tubes. Control and rfhSP-D-treated (30 μg/mL) germ tubes were prepared on 8-well Lab-Tek chamber slide well in 1% yeast-extract medium at 37°C for 16 h. Following, they were stained with Calcofluor white (5 μg/mL) for 15 min, washed with and suspended in PBS and observe under confocal microscope. (B) SP-D treatment facilitated the antifungal efficacy of voriconazole towards A. fumigatus. Control and rfhSP-D-treated (10 μg/mL) germ tubes were prepared in 1% yeast-extract medium at 37°C for 9 h and then treated with voriconazole (at two-fold dilutions, between 1.6-0.0015625 μg/mL) in 1% yeast-extract medium containing resazurin (0.03%). After incubating the contents at 37oC for 24 h, the optical densities were measured at 569 nm using a Tecan plate reader. The percent growth inhibition of voriconazole and rfhSP-D + voriconazole treated hyphae was calculated considering optical density of both rfhSP-D and voriconazole untreated hyphal growth as 100%.
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