Formation of <i>in vitro</i> biofilm of <i>A</i>. <i>fumigatus</i>.

<p>(<b>A</b>) Kinetics of biofilm formation visualized in CLSM: 8 h (1), 12 h (2) and 24 h (3) after inoculation. (<b>B</b>) 24 h–old biofilm in SEM: general aspect of Af biofilm (1), hyphae embedded in ECM and presence of conidia (2), ECM with holes (3). ECM = extracellular matrix, C = conidia.</p

TNFα production or expression by macrophages (isolated from uninfected immunocompetent mice) upon interaction with resting conidia of parental (<i>ku80</i>) and <i>ags</i>Δ_<i>5T</i> strains or <i>ags</i>Δ_<i>5T</i> conidial NaCl extract (3.2 µg proteins) respectively.

<p>(A) TNFα was quantified after 5 h incubation of the conidia with macrophages; (B) Relative expression of TNFα assessed by real time RT-PCR in total RNA from macrophages after 5 h incubation of the <i>ags</i>Δ_<i>5T</i> conidial NaCl extract with macrophages. NaCl supernatant from <i>ku80</i> resting conidia incubated for 2 h in 0.5M NaCl was used as a control. NS: Non-stimulated. *, P<0.05.</p

Conidiocidal activity of macrophages isolated from uninfected p47<i>^phox</i>^−/− mice against resting conidia of <i>ags</i>Δ_<i>5T</i> and parental (<i>ku80</i>) strains.

<p>Conidiocidal activity is expressed in percentage of CFU inhibition after 2 and 6 h incubation of the conidia with macrophages. Data are representative from at least three independent experiments. *, P<0.05.</p

ConA-FITC labeling of <i>ags</i>Δ_<i>5T</i> mutant and parental strain (<i>ku80</i>) resting conidia.

<p>Note the increase in the ConA labeling on the <i>ags</i>Δ_<i>5T</i> mutant conidial surface. Histograms represent the calculated fluorescence intensity of the corresponding images, expressed in Einstein per seconds.</p

Formation of <i>in vitro</i> mixed biofilm of <i>S</i>. <i>maltophilia</i> and <i>A</i>. <i>fumigatus</i>.

<p>(<b>A</b>) Kinetics of mixed biofilm formation visualized in CLSM: 8 h (1), 12 h (2) and 24 h (3) after inoculation. (<b>B</b>) 24 h–old biofilm in SEM: general aspect of mixed biofilm (1), bacteria covering <i>A</i>. <i>fumigatus</i> hyphae and embedded in ECM (2), bacteria between hyphae and embedded in ECM (3). ECM = extracellular matrix.</p

Labeling of the surfaces of <i>ags</i>Δ_<i>5T</i> and parental strain swollen conidia by WGA and the β(1,3)-glucan receptor GNBP3.

<p>The surfaces of the swollen conidia were labeled by WGA-FITC (A) and GNBP3 (B) as described in material and methods. (C, D) Histograms represented the calculated fluorescence intensity of the corresponding images (A, B respectively), expressed in Einstein per seconds.</p

Growth of <i>S</i>. <i>maltophilia</i> and <i>A</i>. <i>fumigatus</i> in the single and mixed biofilms.

<p>Data are expressed in log of CE or BE/mL as measured by qPCR over 24h and presented in the form of mean±SE. Sm = <i>S</i>. <i>maltophilia</i>, Af = <i>A</i>. <i>fumigatus</i>. The experiment was repeated 3 times, using 3 wells per biofilm. Results are expressed in mean±SE, * p < 0.05 compared with the single biofilms.</p

Surface analysis of resting conidia of <i>ags</i>Δ_<i>5T</i> mutant and parental (<i>ku80</i>) strains.

<p>(A): height images (z-range = 1 µm; recorded in water with silicon nitride tips). Atomic Force Microscopy (AFM) images showing the amorphous surface without the rodlet layer on the triple <i>ags</i>Δ_<i>5T</i> mutant conidia whereas the rodlet are observed on the parental strain conidial surface. (B): TEM observations. Note the presence of an extracellular material on the surface of the <i>ags</i>Δ_<i>5T</i> conidia (arrow); CW: cell wall. (C): SDS-PAGE (15% gel) of Hydrofluoric acid (HF) extracts of rodlets from resting conidia showing the two bands, 16 kDa and 14.5 kDa of RodAp classically seen from HF treatment of the conidia <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003716#ppat.1003716-Aimanianda1" target="_blank">[10]</a>. Data are representative of at least three independent experiments.</p

Cell wall thickness of <i>A</i>. <i>fumigatus</i> in the single and mixed biofilms.

<p>(<b>A</b>) Observation on 24 h–old single <i>A</i>. <i>fumigatus</i> biofilm (1–2) and mixed biofilm (3–4) by TEM (<b>B</b>) Cell wall thickness of <i>A</i>. <i>fumigatus</i> measured on TEM images of the single and mixed biofilms. H = hyphae, B = bacteria, CW = cell wall, ECM = extracellular matrix, Sm = <i>S</i>. <i>maltophilia</i>, Af = <i>A</i>. <i>fumigatus</i>. For each biofilm, approximately 15 measurements on 27 hyphae were taken. Results are expressed in mean±SE, * <i>p</i> < 0.0001.</p

Formation of <i>in vitro</i> biofilm of <i>S</i>. <i>maltophilia</i> observed by SEM.

<p>(<b>A-B</b>) Single biofilm of <i>S</i>. <i>maltophilia</i> after 24 h of culture. ECM = extracellular matrix.</p