<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

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

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

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

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

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

Phosphatidylserine (PS)⁺ and PS⁻ subpopulations of <i>Toxoplasma gondii</i> were magnetically separated and analyzed by flow cytometry after annexin-V staining.

<p>(A) PS⁺ subpopulation of <i>T. gondii</i>. (B) PS⁻ subpopulation of <i>T. gondii</i>. The black lines represent control parasites without Annexin – V and the gray lines refer to the isolated subpopulations. Results from one representative experiment out of five.</p

Analysis of <i>Toxoplasma gondii</i> infection <i>in vivo</i>.

<p>C57/BL6 mice were infected with PS⁺, PS⁻ subpopulations or the total population of <i>T. gondii</i>. (A) Survival curve of mice after the infection with <i>T. gondii</i>. Kaplan Meier analysis <i>p</i> = 0.0273. (B) Light microscopy of spleen and liver tissue of C57/BL6 mice after interaction with PS⁺ or PS⁻ subpopulation of <i>T. gondii</i>. Bars = 100 µm. (B1–3) Spleen images after interaction with the PS⁻ (B1), PS⁺ (B2) or the total population of <i>T. gondii</i> (B3). Note the presence of inflammatory cells in B1 (arrows) and the presence of parasites in B2 and B3 (arrows). (B4–6) Liver images after interaction with PS⁻ (B4), PS⁺ (B5) or the total population of <i>T. gondii</i> (B6). Note similar results obtained for the spleen tissue. Inset – Transmission electron microscopy: B2, Bar = 2 µm; B4, Bar = 16 µm. Results are from two independent experiments with 6 animals per group.</p