<i>Cryptococcus neoformans</i> Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

<div><p>Cryptococcosis by the encapsulated yeast <i>Cryptococcus neoformans</i> affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of <i>Cryptococcus</i> yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of <i>Cryptococcus</i> internalization by host cells remain poorly understood. Here, we investigate the mechanism of <i>Cryptococcus neoformans</i> phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of <i>C. neoformans</i> are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of <i>Cryptococcus</i> uptake by host cells.</p></div

Actin recruitment is inhibited by cytochalasin D.

<p>Confocal laser scanning microscopy of <i>C. neoformans</i> capsular strain H99 interacting with macrophages (single confocal plane). DIC showing internalized yeasts (arrows); and confocal images showing actin filaments (red), microtubules (green), yeast (blue) and host DNA (blue, indicated by ‘n’). Actin is recruited to the site of phagocytosis in untreated cells (A), and actin recruitment was inhibited by 0.5 µM cytochalasin D (B). In contrast, treatment with 5 µM nocodazole (C) or with a combination of nocodazole and cytochalasin D (D) did not inhibit actin recruitment to the phagosome area. Scale bars, 5 µm.</p

Uptake of <i>Cryptococcus</i> strains by trigger-like and zipper-like structures.

<p>Scanning electron microscopy of <i>C. neoformans</i> capsular strain H99 (A–E) and acapsular strain CAP59 (F–G) interacting with peritoneal macrophages. Improved preservation of macrophage membranes was obtained with routine SEM fixation (A–B; F–G), although post-fixation in the presence of sucrose provided better capsule preservation and allowed visualization of direct interactions between the capsule and host cell membranes, prior to internalization (C–E). Both trigger-like (arrow in A and F) and zipper-like (arrow-head in B and G) uptake structures were observed. Scale bars, 1 µm (A–C; F–G) and 0.5 µm (D–E).</p

Actin is recruited to the phagosome area during <i>C. neoformans</i> internalization.

<p>Confocal laser scanning microscopy (z-stack series of confocal planes) of interacting macrophages and <i>C. neoformans</i> yeast cells from strains H99 (A and B) and CAP59 (C and D). Internalized yeasts identified by DIC (arrows in A and C) can be visualized in the context of host cell actin (red) and microtubule (green) cytoskeletons (B and D). Host cell DNA is labeled with DAPI (blue, indicated by the letter ‘N’) and yeast is labeled with calcofluor (blue, indicated by arrows). Actin, but not tubulin, is recruited to sites of yeast internalization. Scale bars, 5 µm.</p

Treatment with both cytochalasin D and nocodazole did not increase the inhibitory effect.

<p>Quantification of the internalization (A) and the attachment (B) to macrophages of <i>C. neoformans</i> yeast cells from capsular (H99 and B3501) and acapsular (CAP67 and CAP 59) strains, in the absence of cytoskeletal inhibitors or in the presence of cytochalasin D and nocodazole. The metabolic viability of <i>C. neoformans</i> strains H99 and CAP59 was measured using the FUN®-1 dye (C) and the metabolic viability of macrophages was measured by MTS/PMS (D) after incubation with cytoskeletal inhibitors for 2 h. Yeast cells fixed with 70% ethanol, and macrophages with 4% formaldehyde, were used as a positive control for the loss of cell viability in each method. Graphs show normalized mean values and standard deviation from three experiments (A–B) and mean and standard deviation from absolute values of fluorescence intensity (C) and absorbance (D).*p<0.05; **p<0.01; ***p<0.001.</p

Involvement of the cytoskeleton in the yeast-macrophage interaction.

<p>Scanning electron microscopy of membrane extracted macrophages interacting with <i>C. neoformans</i> strains H99 (A) and CAP59 (B and C), showing cytoskeletal filaments associated with yeasts in untreated samples (A–B). After 5 µm nocodazole treatment (C) the area surrounding yeast cells appeared mostly devoid of cytoskeletal components but association with yeast still occurred (inset in C). Scale bars, 2 µm.</p

Effect of aspartyl PIs on the viability and killing activity of macrophage cells (RAW264.7 lineage).

<p>Initially, the macrophages (1×10⁵ cells) were incubated in a 96-well plate for 24 hours in the absence or in the presence of different concentrations (3.125, 6.25, 12.5, 25 and 50 µM) of the following PIs: indinavir, saquinavir, ritonavir and nelfinavir. After this period, the viability of the macrophage cells was determined spectrophotometrically at 490 nm by means of MTT assay. The dotted line separates the graphic in two portions: minor than and equal or major than 90% of macrophage viability (A). After that, we studied the influence of aspartyl PIs on the killing activity of macrophages. In this context, we repeated the interaction of conidia with macrophage (ratio of 10∶1, respectively) for 1 hour, followed by exhaustive washing in PBS and then incubated the interaction for additional 24 hours in the absence (control) or in the presence of indinavir, nelfinavir and ritonavir at 6.25 µM, concentration in which more than 90% of the macrophage cells were viable as demonstrated in (A). Finally, the adhered macrophages were washed, lysed with sterile cold water, and the resulting suspension was plated on Czapek solid medium to count the number of CFU (B).</p

Effect of aspartyl HIV PIs on the interaction between <i>Fonsecaea pedrosoi</i> conidia and epithelial cells (CHO).

<p>After growth in Czapek medium for 5 days at room temperature, the conidia were harvested, washed with PBS and 1×10⁶ cells were incubated for 1 hour in the absence or in the presence of different concentrations (50, 100 and 200 µM) of the following PIs: indinavir, saquinavir, ritonavir and nelfinavir. After that, the conidial cells were washed in PBS and the interaction process was performed as described in Experimental Procedures section. The viability of the conidial cells was not affected by the PIs treatment used in this set of experiments as judged by propidium iodide staining (data not shown). Adhesion (A) and endocytic (B) indices of the interaction after 2 h were shown. The values represent the mean±standard deviation of three independent experiments performed in triplicate. Symbols denote systems treated with PIs that had an interaction index significantly different from the control (♦, <i>P</i><0.05; ★, <i>P</i><0.001; Student's t test). Representative images of attached (a₁ and a₂) and ingested (b₁ and b₂) fungi are shown by means of light (a₁ and b₁) and transmission electron (a₂ and b₂) microscopy analyses. Note that ingested conidia are located within vacuole (b₁ and b₂). Arrows show the conidia attached (a₁) or ingested (b₁) by CHO cells. Scale bars: (a₁ and b₁), 10 µm; (a₂ and b₂), 1 µm.</p

Effect of aspartyl HIV PIs on the <i>F. pedrosoi</i> morphological transition (conidia into mycelia transformation) after the interaction with epithelial cells (CHO).

<p>In this set of experiment, conidia of <i>F. pedrosoi</i> were initially untreated (a and b) or treated (c) with 100 µM of HIV aspartyl PIs for 1 hour, washed in PBS and then interacted with CHO cells for 4 hours. Optical microscopy of the interaction showed that in the control system, some conidial cells transform in hyphae, as indicated by the arrows, showing the well-known differentiation process triggered by the contact of conidia with host cells. Conversely, when conidia were pre-treated with aspartyl PIs, especially saquinavir (c), the number of hyphae is drastically diminished. Arrowheads show the conidia. Scale bars: (a, c), 10 µm; (b), 1 µm.</p

Study of the possible synergistic effect between aspartyl HIV PI and antifungal compounds against <i>F. pedrosoi</i> development.

<p>In this experiments, conidia (1×10³ cells) were treated or not with a sub-inhibitory concentration of both PIs (saquinavir and nelfinavir at 25 µM) and antifungal drugs (itraconazol at 0.3 µg/ml and amphotericin B at 3 µg/ml), alone or in combination for 1 hour. Afterward, the conidial cells were washed in PBS and inoculated in a fresh solid Czapek medium to measure the CFU. The values represent the mean±standard deviation of three independent experiments performed in triplicate. Symbol denotes system that had a growth rate significantly different from the control (★, <i>P</i><0.001; Student's t test).</p