Intracellular crystal formation as a mechanism of cytotoxicity in murine pulmonary Cryptococcus neoformans infection

Abstract

Rod-like crystalline structures formed during eosinophilic Cryptococcus neoformans pneumonia in C57BL/6 mice. Crystals were found associated with yeast cells and free in host cell cytoplasm. The crystals apparently formed because of the interaction of a host protein with the cryptococcal polysaccharide. Crystal formation likely contributes to pathogenesis by causing cellular damage.

FIG. 1

(A) Eosinophils recruited to the site of infection discharge electron-dense granular contents at the surface of an extracellular Cryptococcus, organism, allowing contact of cryptococcal polysaccharide with granule proteins (day 14; magnification, ×3,000). (B) The location of the yeast cell and crystals within a large multinucleated cell at a magnification lower than that for panel A. An electron-dense rim forms around the yeast cell, and crystals subsequently polymerize in contact with yeast and in the cytoplasm (day 14; magnification, ×2,000). (C) Budding intracellular C. neoformans in murine lung tissue 14 days after infection with electron-dense rim surrounding the polysaccharide capsule. Crystals formed in association with the yeast cell (magnification, ×4,000). (D) On day 28 after infection, large numbers of crystals were seen inside multinucleated cells, some of which were dying (magnification, ×2,000). (E) Crystals disrupted host cell membranes and became extracellular (day 28 after infection; magnification, ×3,000). (F) Higher magnification of the area enclosed in the boxed area in panel E demonstrates membrane disruption (magnification, ×20,000). (G and H) On day 28 after infection, crystals disrupted the bronchial epithelium, with loss of cilia. Epithelial cell debris and larger extracellular crystals were seen inside bronchi (magnification, ×1,000).

FIG. 2

Light microscopic staining of intracellular crystals (arrows). (A) Crystals from a 1-μm-thick section of mouse lung 14 days after infection stained with eosin. (B) Staining of a 5-μm-thick section of mouse lung 28 days after infection with hematoxylin and eosin showing a macrophage containing a large number of intracellular crystals. Magnification, ×1,000 (panels A and B).

FIG. 3

Immunoelctron microscopy shows the presence of gold label for cryptococcal polysaccharide surrounding the outer membrane of the crystals but not over the crystals. The asterisk is located on a cryptococcal capsule. The arrow points to artifactual contraction of the capsule from the edge of a phagosome. magnification, ×15,000).

FIG. 4

Crystals formed after incubation of C. neoformans strain 24067 with rat peritoneal inflammatory cells in vitro for 3 days. Bar, 1 μm.