Inhibition of <i>A. fumigatus</i> internalization by PLD chemical inhibitors.

<p>A549 cells were incubated for 30 min with 1% (v/v) 1-butanol or tert-butanol (A, B), 2 nM VU0359595 (PLD1-specific inhibitor), 100 nM VU0285655-1 (PLD2-specific inhibitor), or both (C, D). Subsequently, the cells were infected with <i>A. fumigatus</i> 13073 swollen conidia at an MOI of 10. <i>A. fumigatus</i> internalization was analyzed by the nystatin protection assay (A, C) and PLD activities (B, D) were measured. Differences in [³H] PtdEtOH formation or <i>A. fumigatus</i> internalization between the untreated (control) cells and inhibitor-pretreated cells were compared. Data are represented as the mean ± SE (n = 3–4). *P<0.05.</p

β-1,3-glucan induces PLD activity in A549 cells.

<p>A549 cells were stimulated with the indicated concentrations of β-1,3-glucan for 30 min (A) or for the indicated duration of time with 50 µg/mL of β-1,3-glucan (B). Thereafter, the PLD activity was determined and differences in [³H] PtdEtOH formation between the control (indicated by “0”) and other groups were compared. In C and D, A549 cells were first incubated with HBSS (Control), HBSS containing 5 µg/mL of isotype control antibody and HBSS containing 5 µg/mL of anti-dectin-1 mAb GE2 (ab82888) for 30 min, respectively. Then, the cells were infected with swollen conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 (C) or stimulated by HBSS and HBSS containing 50 µg/mL of β-1,3-glucan for 30 min, respectively (D). <i>A. fumigatus</i> internalization was analyzed by the nystatin protection assay (C) and the PLD activity (D) was measured. Differences in <i>A. fumigatus</i> internalization and [³H] PtdEtOH formation between the untreated cells (Control) and antibody-treated cells were compared. In E and F, A549 cells were infected with swollen conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Subsequently, the cells were stained with isotype (IC) antibody or the primary anti-dectin-1 mAb GE2 (ab82888) and analyzed by FACS Calibur flow cytometer. The geometric mean fluorescence intensity was determined by Cell Quest Pro software. Differences between uninfected cells (Control) and conidia-infected cells were compared. G. A549 cells were incubated with swollen conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Cells were analyzed for dectin-1 expression by immunoblotting using an anti-dectin-1 antibody (sc-26094). FACS profiles and immunoblots shown here are characteristic of 3 independent experiments. Data are represented as mean ± SE (n = 3–4). *P<0.05.</p

<i>A. fumigatus</i> stimulates PLD activity during its internalization into A549 cells.

<p>A. A549 cells were prelabeled with [³H] oleic acid and infected with the resting conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 for the indicated time periods. Then, ethanol was added to determine the PLD activity. B. A549 cells were infected with the resting conidia of <i>A. fumigatus</i> 13073 at an MOI of 10 for the indicated time periods, and the internalization of <i>A. fumigatus</i> was analyzed by the nystatin protection assay. Differences in [³H] PtdEtOH formation between the 0 h time point and the other time points (A) and differences in the internalization of <i>A. fumigatus</i> between the 2 h time point and the other time points (B) were compared. In parallel, the cells were lysed for immunoblotting with the indicated antibody (C) and the densitometric analysis of immunoblots for three independent experiments is shown (D). Data are represented as the mean ± SE (n = 3–4), and the blots are characteristic of 3 independent experiments. *P<0.05.</p

Heat-killed swollen conidia stimulate PLD activity and internalize in A549 cells.

<p>A549 cells were infected with heat-killed (HK) resting conidia, HK swollen conidia (germinated for 6 h), and HK hyphae (germinated for 12 h) of <i>A. fumigatus</i> 13073 (at an MOI of 10 each). The PLD activities were measured (A) and <i>A. fumigatus</i> internalization was determined by immunofluorescent staining (B). Differences in [³H] PtdEtOH formation and in the internalization indices between groups were compared as indicated in the figure. Data are represented as the mean ± SE (n = 3–4). *P<0.05.</p

Interference of endogenous PLD host cell expression reduces the internalization of <i>A. fumigatus</i>.

<p>A549 cells were transfected with non-specific small interfering RNAs (siRNAs) (Control), PLD1-specific siRNAs (A, C), or PLD2-specific siRNAs (B, D). After 48 h, cells were infected with the swollen conidia of <i>A. fumigatus</i> 13073 at an MOI of 10. <i>A. fumigatus</i> internalization was analyzed by the nystatin protection assay (A, B) and PLD activities (C, D) were measured. Differences in [³H] PtdEtOH formation and <i>A. fumigatus</i> internalization between the untransfected cells (Control) and PLD-silenced cells were compared. Data are represented as the mean ± SE (n = 3–4). *P<0.05. The immunoblots represent PLD1 and PLD2 expression in A549 cell lysates.</p

Host cell PLD activity is stimulated by swollen conidia, but not resting conidia.

<p><b>A.</b> A549 cells were infected with the live resting conidia, swollen conidia (germinated for 6 h), and hyphae (germinated for 12 h) of <i>A. fumigatus</i> 13073 at an MOI of 10 for 30 min. B. A549 cells were infected with the swollen conidia of <i>A. fumigatus</i> 13073 for 30 min at the indicated MOI. C. A549 cells were infected with live resting conidia, swollen conidia and hyphae of <i>A. fumigatus</i> AF293 at an MOI of 10 for 30 min. Thereafter, the PLD activity in the A549 cells was measured, and the differences in [³H] PtdEtOH formation between uninfected group (Control) and infected group or between groups were compared as indicated in the figure (A, B, C). D. A549 cells were infected with resting conidia (germinating time = 0), or conidia of <i>A. fumigatus</i> 13073 germinated for the indicated time periods at an MOI of 10 for 60 min. <i>A. fumigatus</i> internalization was determined by immunofluorescent staining. Differences in the internalization index between resting conidia and germinated conidia were compared. Data are represented as the mean ± SE (n = 3–4). *P<0.05.</p