Invasion of the central nervous system by Cryptococcus neoformans requires a secreted fungal metalloprotease

Abstract

Cryptococcus spp. cause life-threatening fungal infection of the central nervous system (CNS), predominantly in patients with a compromised immune system. Why Cryptococcus neoformans has this remarkable tropism for the CNS is not clear. Recent research on cerebral pathogenesis of C. neoformans revealed a predominantly transcellular migration of cryptococci across the brain endothelium; however, the identities of key fungal virulence factors that function specifically to invade the CNS remain unresolved. Here we found that a novel, secreted metalloprotease (Mpr1) that we identified in the extracellular proteome of C. neoformans (CnMpr1) is required for establishing fungal disease in the CNS. Mpr1 belongs to a poorly characterized M36 class of fungalysins that are expressed in only some fungal species. A strain of C. neoformans lacking the gene encoding Mpr1 (mpr1Δ) failed to breach the endothelium in an in vitro model of the human blood-brain barrier (BBB). A mammalian host infected with the mpr1Δ null strain demonstrated significant improvement in survival due to a reduced brain fungal burden and lacked the brain pathology commonly associated with cryptococcal disease. The in vivo studies further indicate that Mpr1 is not required for fungal dissemination and Mpr1 likely targets the brain endothelium specifically. Remarkably, the sole expression of CnMPR1 in Saccharomyces cerevisiae resulted in a robust migration of yeast cells across the brain endothelium, demonstrating Mpr1's specific activity in breaching the BBB and suggesting that Mpr1 may function independently of the hyaluronic acid-CD44 pathway. This distinct role for Mpr1 may develop into innovative treatment options and facilitate a brain-specific drug delivery platform.

Importance: Cryptococcus neoformans is a medically relevant fungal pathogen causing significant morbidity and mortality, particularly in immunocompromised individuals. An intriguing feature is its strong neurotropism, and consequently the hallmark of cryptococcal disease is a brain infection, cryptococcal meningoencephalitis. For C. neoformans to penetrate the central nervous system (CNS), it first breaches the blood-brain barrier via a transcellular pathway; however, the identities of fungal factors required for this transmigration remain largely unknown. In an effort to identify extracellular fungal proteins that could mediate interactions with the brain endothelium, we undertook a proteomic analysis of the extracellular proteome and identified a secreted metalloprotease (Mpr1) belonging to the M36 class of fungalysins. Here we found that Mpr1 promotes migration of C. neoformans across the brain endothelium and into the CNS by facilitating attachment of cryptococci to the endothelium surface, thus underscoring the critical role of M36 proteases in fungal pathogenesis.

FIG 1

A novel metalloprotease (Mpr1) is required for the migration of Cryptococcus neoformans across the brain endothelium. (A) A schematic representation of the in vitro model of the BBB. A monolayer of hCMEC/D3 cells grown on transwell inserts submerged in medium such that the luminal and abluminal sides represented the blood and brain sides, respectively. Cryptococci were added to the luminal side and collected from the abluminal side approximately 18 h later for CFU determination. (B) Strains of C. neoformans lacking the genes encoding various proteases (H99 serotype A background; see Materials and Methods) were tested for their ability to transmigrate the brain endothelium in the in vitro model of the BBB. The strain D (mpr1Δ null strain, H99 serotype A background) was defective in crossing the endothelial barrier (*, P = 0.009) (n = 9). (C) CFUs representing cryptococci of the wild-type (H99) and mpr1Δ strains, equally able to proliferate in hCMEC/D3 growth medium and conditions in the absence of endothelial cells (n = 9). (D) Mpr1 is an extracellular protein. An MPR1-dsRED fusion protein was expressed in the mpr1Δ background strain to assess its localization. Confocal microscopy of an MPR1-dsRED fusion protein under the control of an ACTIN promoter revealed a surface localization (scale bar = 10 µm). (E) A schematic diagram of the predicted structure of Mpr1 illustrates the N-terminal signal sequence, the predicted prodomain (FTP region, Pfam), and the peptidase M36 domain with the conserved Zn²⁺-binding site.

FIG 2

Mpr1 promotes the adherence of Cryptococcus neoformans to brain endothelial cells. Shown are CFUs representing the number of cryptococci adhering to brain endothelial cells following 20 min or 8 h of coincubation (A) or a 2-h incubation (B) of H99 or the MPR1 (mpr1Δ::MPR1; reconstituted strain) and mpr1Δ mutant strains with the endothelium in the in vitro model of the BBB. The mpr1Δ mutant strain did not adhere to brain endothelial cells (***, P = 0.002) (n = 9). (C) The CFUs representing cryptococci that transmigrated to the abluminal (brain) side of the endothelium following a prolonged incubation with the brain endothelium revealed that the mpr1Δ mutant strain did not transmigrate. (D and E) Wild-type (H99) and mpr1Δ cells were incubated with the macrophage cell line J774A.1 to analyze intracellular survival. Following a 1-h coincubation at 37°C in 5% CO₂, extracellular cryptococci were removed and the cocultures were either lysed and plated for CFU analysis (D) or replenished with fresh medium and incubated for an additional 23 h (overnight) at 37°C in 5% CO₂ to determine intracellular viability of cryptococci (E). Following either a 1-h or overnight coincubation, cryptococci were collected from macrophages, plated on YPD medium, and incubated at 30°C for 2 days for CFU determination. The two strains were similar in their ability to survive within macrophages (n = 9). Statistical significance was determined by an unpaired t test with Welch’s correction (GraphPad Prism5 software).

FIG 3

Expression of CnMPR1 cDNA in Saccharomyces cerevisiae (Sc) resulted in the attachment to and migration of S. cerevisiae across the brain endothelium. (A) Reverse transcriptase PCR confirmed the expression of the CnMPR1 transcript in S. cerevisiae. The band corresponding to the MPR1 transcript is absent in the mpr1Δ and S. cerevisiae (wild-type) strains, as expected. Actin is shown as an internal control for H99 given the typical faint appearance of the MPR1 transcript in H99. cDNA of CnMPR1 was subcloned adjacent to a GDP promoter in a yeast episomal 2µ expression vector. (B) A recombinant CnMpr1-His-tagged protein isolated via nickel chromatography from a yeast S. cerevisiae strain transformed with CnMPR1-HIS cDNA (Sc<CnMPR1-HIS>) retained proteolytic activity (n = 3). Trypsin activity is shown as a positive control for the proteolytic assay. (C) A 4-h coincubation of the Sc<CnMPR1> strain with brain endothelial cells in the in vitro BBB model resulted in a significant increase in the ability of yeast cells to associate with the surface of endothelial cells following a 4-h coincubation (*, P = 0.0294; n = 9). (D) The Sc<CnMPR1> strain migrated across the brain endothelial cells following an overnight coincubation. Transformed yeast cells (Sc<CnMPR1>) were added to the luminal side of the in vitro model of the BBB and collected from the abluminal side for CFU determination (*, P = 0.009; n = 9). Shown are C. neoformans strains (CnWT and Cnmpr1Δ) and S. cerevisiae strains (ScWT and Sc<CnMPR1>: yeast strain transformed with cDNA of MPR1 from C. neoformans).

FIG 4

Mpr1 alters the extracellular environment of the fungus-endothelium interface. Scanning electron micrographs of cryptococcal cells from H99 and mpr1Δ strains reveal physical differences at the brain endothelium-fungus interface. In the absence (A and B) or presence (C to F) of hCMED/D3 cells (brain endothelial cells), the mpr1Δ null cells displayed a more porous surface than H99 cells (indicated by arrows). (C and E) Exposure of hCMED/D3 cells to H99 revealed extensive microvillus-like structures that appeared to be less abundant upon coincubation with mpr1Δ cells (D and F) (indicated by arrows). The microvillus-like protrusions were not observed on the surface of hCMED/D3 in the absence of C. neoformans (G) (scale bar = 2 µm unless noted differently in the figure).

FIG 5

The murine inhalation model inoculated with a strain of Cryptococcus neoformans lacking Mpr1 showed significant improvement in survival due to a lack of fungal burden in the brain. (A) Shown is percent survival of mice inoculated via the nares, mimicking the natural route of infection, with the H99 (wild type), mpr1Δ, and MPR1 (mpr1Δ::MPR1; reconstituted strain) strains. Mpr1Δ strain-infected mice showed a significant improvement in survival (P = 0.001, Wilcoxon test, sample size determined by power analysis [GPower3.1]) (B and C) CFUs representing the fungal burden at the time of death in lungs (B) or brains (C) of mice inoculated with H99, the mpr1Δ mutant, and the MPR1-reconstituted strains revealed significantly less fungal burden in brains from mice infected with the mpr1Δ mutant strain (**, P = 0.009). (D) Following 10 days p.i. (postinfection), mpr1Δ strain-infected mice and H99-infected mice showed similar fungal burdens in lungs, kidneys, spleen, and heart, suggesting that Mpr1 is not required for dissemination out of the lung and Mpr1 does not play a role in breaching other vascular endothelia (P > 0.05 for all tissues).

FIG 6

Association of cryptococci with the brain endothelium requires Mpr1 in the tail-vein murine model. (A) The percent survival of mice inoculated via a more direct route to the brain (tail vein) with the H99 and mpr1Δ strains revealed a dramatic improvement in survival of mice infected with the mpr1Δ strain. (B and C) CFUs representing the fungal burden in brains of mice following 12 h, 24 h, and 48 h (B) or 16 days (C) postinfection with the H99 and mpr1Δ strains revealed significantly less fungal burden in brains of mice infected by the mpr1Δ null strain. (D) The number of cryptococci that survived within brain parenchyma of mice following inoculation by direct intracranial injection of the wild-type and mpr1Δ strains revealed no significant differences between strains. (Asterisks correspond to P values. **, P = 0.008; ***, P < 0.001.)

FIG 7

Brains from mice inoculated via the tail vein with the strain lacking Mpr1 revealed a complete lack of brain pathology, suggesting that Mpr1 is required for fungal invasion of the CNS. Mice were inoculated by tail-vein injection, and brains were examined at 12 h, 24 h, and 48 h p.i. by taking 20 consecutive, longitudinal brain sections and staining with mucicarmine (specific for cryptococci) or H&E. Histological analysis of brain slices from brains of mice inoculated with the H99 (A, C, E, and G) revealed lesions filled with cryptococci at various stages of cell division (arrows point to cryptococci within lesions), consistent with fungal disease of the CNS (n = 12). However, brain sections from mice infected with the mpr1Δ mutant strain (B, D, F, and H) at 48 h p.i. (A and D), 24 h p.i. (F), or 12 h p.i. (H) revealed a complete absence of cryptococcus-filled lesions, indicating no brain pathology (n = 12). Brain sections were stained with mucicarmine (A, B, E, F, G, and H) or H&E (C and D).