Cryptococcus neoformans is internalized by receptor-mediated or 'triggered' phagocytosis, dependent on actin recruitment.

Caroline Rezende Guerra,Sergio Henrique Seabra,Wanderley De Souza,Sonia Rozental,Oscar Zaragoza

doi:10.1371/journal.pone.0089250

Abstract

Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans 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 C. neoformans 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 Cryptococcus uptake by host cells.

Highlights

The encapsulated yeast Cryptococcus neoformans is responsible for human cryptococcosis, affecting mostly immunocompromised individuals
We focused on the initial steps of the interaction between C. neoformans and peritoneal macrophages, using confocal fluorescence microscopy, electron microscopy and flow cytometry techniques to address different aspects of the internalization processes, including the participation of the cytoskeleton and the mode of yeast phagocytosis by macrophages
Flow cytometry analysis showed that both compounds decreased the internalization of all four C. neoformans strains by macrophages, with corresponding increases in the number of attached yeast cells (Figure 1, A–B)

Summary

Introduction

The encapsulated yeast Cryptococcus neoformans is responsible for human cryptococcosis, affecting mostly immunocompromised individuals. Capsule production is important for host immune system avoidance by interfering, for example, with cytokine secretion and providing resistance to phagolysosomal enzymes [5,6], C. neoformans displays capsule-independent mechanisms of escape from immune system cells, such as the expression of anti-phagocytic proteins [7,8]. These mechanisms do not fully prevent phagocytosis through antibody or complement opsonization, or via direct GXM recognition by host cell membrane receptors [9,10]

Methods

Results

Conclusion