Capsule structure changes associated with Cryptococcus neoformans crossing of the blood-brain barrier

Abstract

Cryptococcus neoformans is a yeast responsible for disseminated meningoencephalitis in patients with cellular immune defects. The major virulence factor is the polysaccharide capsule. We took advantage of a relevant murine model of disseminated meningoencephalitis to study the early events associated with blood-brain barrier (BBB) crossing. Mice were sacrificed at 1, 6, 24, and 48 hours post-intravenous inoculation, and classical histology, electron microscopy, and double immunofluorescence were used to study tissues and yeasts. Crossing of the BBB occurred early after inoculation, did not involve the choroid plexus but instead occurred at the level of the cortical capillaries, and caused early and severe damage to the structure of the microvessels. Seeding of the leptomeninges was not the primary event but occurred secondary to leakage of cortical pseudocysts. Organ invasion was associated with changes in cryptococcal capsule structure and cell size, which differed in terms of magnitude and kinetics, depending on both the organs involved, and potentially, on the bed structure of the local capillary. The rapid changes in capsule structure could contribute to inability of the host immune response to control cryptococcal infection in extrapulmonary spaces.

Figure 1

Changes in fungal load with time after intravenous inoculation of 2 × 10⁴ C. neoformans strain H99. Outbred mice were sacrificed at various times after inoculation and the CFU were enumerated in brain homogenates (gray bars) and in the blood (white bars). Results expressed as logCFU/g of organ or ml of blood are the mean ± SD from 3 to 11 mice for each time point.

Figure 2

Fungal load in various organs after inoculation of 10⁷ C. neoformans strain H99 cells at 1 hour (white bars) and 24 hours (gray bars). Results at each time are the mean ± SD from three mice.

Figure 3

Brain sections showing the localization of yeast cells at various time intervals after inoculation of 10⁷ C. neoformans strain H99 cells. Tissue sections from mice sacrificed 5 minutes, 6 hours, 24 hours, and 48 hours after inoculation were stained with Alcian blue (pH 2.5) (left) and stained in red with anti-collagen IV antibody and TRITC-labeled reagent and in green with anti-capsular polysaccharide antibodies by direct (labeled FITC-E1) or indirect (CRND-8 followed by FITC-labeled anti-mouse IgM) labeling. Nuclei are labeled with DAPI (blue) on immunofluorescence (magnification, ×1000).

Figure 4

Cortical section of a mouse brain inoculated 48 hours before with 10⁷ C. neoformans strain H99 cells (methenamine silver staining). The pseudocyst appears ready to extend into the leptomeningeal area.

Figure 5

Evaluation of the blood-brain barrier leakage by horseradish peroxidase (HRP) extravasation in mice inoculated with 10⁷ C. neoformans strain H99 cells. Brain section of mice sacrificed 1 hour (A), 6 hours (B), 24 hours (C), and 48 hours (D) after inoculation. Extravasation of HRP (B, C, and D) can be seen together with intact capillaries (all panels) (magnification, ×400). Brain tissue sections from control mice or mice sacrificed 5 minutes after inoculation appear as in A.

Figure 6

Electron microscopy of brain tissue from mice inoculated intravenously with C. neoformans H99. Left: One yeast filled the lumen of a cerebral capillary in the brain of a mouse inoculated 5 minutes before sacrifice (magnification, ×10,000). Right: Disruption of the vessel wall with neuropil edema around the yeast in the brain of a mouse sacrifice 24 hours post-inoculation (magnification, ×4000).

Figure 7

Comparison of the yeast sizes in tissue section according to the time of sacrifice. Tissue sections from two mice inoculated with 10⁷ C. neoformans strain H99 cells were subjected to silver staining, and yeast size including the capsule was determined. Each dot represents the value obtained from each of the 30 yeasts measured, the bar represents the median value.

Figure 8

Comparison of the capsule antigenic structure over time as demonstrated by immunofluorescence studies using double-labeling with two monoclonal antibodies specific for different epitopes on the cryptococcal capsule. Brain tissue sections obtained 1, 6, 24 and 48 hours after inoculation with 10⁷ C. neoformans strain H99 cells (serotype A) were sequentially incubated with CRND-8 (a murine monoclonal IgM antibody that recognizes sugar epitopes mainly found on serotype D cells), a TRITC-labeled anti-mouse IgM and FITC-labeled E1 (a murine monoclonal IgG1 antibody specific for sugar epitopes mainly found on serotype A cells) (magnification, ×1000).

Figure 9

Comparison of the changes in cryptococcal capsular antigenic structure in various tissues as demonstrated by the binding of two monoclonal antibodies specific for different epitopes on the cryptococcal capsule. The mean percentage of yeasts (± SD) that were stained only with E1 (black bars), only with CRND-8 (white bars) or with both antibodies (striped bars) are recorded. Results were obtained after analysis of tissue sections prepared from three mice infected during three independent experiments.

Figure 10

Double-staining of a yeast observed in the brain of a mouse inoculated 48 hours before with C. neoformans H99. The thin green ring corresponds to capsule structures recognized by E1 whereas the red staining corresponds to capsule structures recognized by CRND-8. Note the large granules present inside the yeast cytoplasma suggesting active synthesis of new material.