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 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.

Cryptococcus neoformans cells must cross the blood-brain barrier prior to invading the central nervous system. Here we demonstrate that the immortalized human brain endothelial cell line HCMEC/D3 is a useful alternative to primary brain endothelial cells as a model of the blood-brain barrier for studies of central nervous system infection.Cryptococcus neoformans is a fungal human pathogen that causes meningitis in a predominantly immunocompromised population (26). To invade the central nervous system (CNS), cryptococcal cells must cross the blood-brain barrier (BBB) (2). Despite efforts to understand the propensity of this pathogen for the CNS, little progress has been made in the past couple of decades (9,(18)(19)(20). The major reasons for this have been the inability to recapitulate the properties of the BBB in vitro and the many challenges posed by BBB studies using live animals (13,21,22,24,32,35). Although commercially generated primary human brain microvascular endothelial cells (HBMEC) are now available for BBB studies, there are several disadvantages to developing models of the BBB using these cells. Mainly these primary cells are unstable after a limited number of passages, and they can be very expensive. The alternative, obtaining primary HBMEC from discarded brain tissues, is also undesirable since the process is labor-intensive and introduces variability from batch to batch (4,8). To facilitate the study of the BBB in vitro, researchers have tried to develop human brain endothelial cell lines that retain critical features of primary cells, such as the expression of endothelial cell markers, transporters, and tight junctional proteins (1,15,23,25,27,29,30,33,34,36). The recent development of one particular line of immortalized human brain endothelial cells (HCMEC/D3) that recapitulates many of the key characteristics of primary brain endothelial cells without the need to coculture with glial cells is proving to be a promising cell line for in vitro studies of the BBB (36). Indeed, the HCMEC/D3 cell line has already been successfully used as a BBB model in several studies, further attesting to its high quality and its potential to replace primary cells for in vitro BBB studies (10, 11, 12, 14-16, 28, 31, 37).Here we show that the HCMEC/D3 cell line can serve as a useful in vitro model of the BBB to study the mechanisms used by C. neoformans to breach the brain endothelium and enter the CNS. In order to test the feasibility of this cell line as a BBB model to study the migration of C. neoformans across the BBB, a transcytosis assay was used. This assay consisted of a transwell apparatus with endothelial cells growing in rich endothelial growth medium (EGM-2; Lonza) on a collagencoated porous membrane (8 m; Bioscience) (Fig. 1A) (4, 8). The HCMEC/D3 cells used here were between passages 25 and 35. HCMEC/D3 cells were seeded based on the growth area ratio. A confluent monolayer in a culture flask of 25 cm 2 was trypsinized and resuspended in 12 ml of medium. The ratio 12 ml/25 cm 2 (0.5 ml/1 cm 2 ) was ...

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bCryptococcus spp. cause fungal meningitis, a life-threatening infection that occurs predominately in immunocompromised individuals. In order for Cryptococcus neoformans to invade the central nervous system (CNS), it must first penetrate the brain endothelium, also known as the blood-brain barrier (BBB). Despite the importance of the interrelation between C. neoformans and the brain endothelium in establishing CNS infection, very little is known about this microenvironment. Here we sought to resolve the cellular and molecular basis that defines the fungal-BBB interface during cryptococcal attachment to, and internalization by, the human brain endothelium. In order to accomplish this by a systems-wide approach, the proteomic profile of human brain endothelial cells challenged with C. neoformans was resolved using a label-free differential quantitative mass spectrometry method known as spectral counting (SC). Here, we demonstrate that as brain endothelial cells associate with, and internalize, cryptococci, they upregulate the expression of several proteins involved with cytoskeleton, metabolism, signaling, and inflammation, suggesting that they are actively signaling and undergoing cytoskeleton remodeling via annexin A2, S100A10, transgelin, and myosin. Transmission electronic microscopy (TEM) analysis demonstrates dramatic structural changes in nuclei, mitochondria, the endoplasmic reticulum (ER), and the plasma membrane that are indicative of cell stress and cell damage. The translocation of HMGB1, a marker of cell injury, the downregulation of proteins that function in transcription, energy production, protein processing, and the upregulation of cyclophilin A further support the notion that C. neoformans elicits changes in brain endothelial cells that facilitate the migration of cryptococci across the BBB and ultimately induce endothelial cell necrosis.

Fungal infections of the central nervous system are responsible for significant morbidity and mortality. Cryptococcus neoformans ( Cn ) is the primary cause of fungal meningitis. Infection begins in the lung after inhalation of fungal spores but often spreads to other organs, particularly the brain in immunosuppressed individuals. Cn ’s ability to survive phagocytosis and endure the onslaught of oxidative attack imposed by the innate immune response facilitates dissemination to the central nervous system (CNS). Despite the success of Cn at bypassing innate immunity, entry into the heavily protected brain requires that Cn overwhelm the highly restricted blood-brain barrier (BBB). This is a formidable task but mounting evidence suggests that Cn expresses surface-bound and secreted virulence factors including urease, metalloprotease, and hyaluronic acid that can undermine the BBB. In addition, Cn can exploit multiple routes of entry to gain access to the CNS. In this review, we discuss the cellular and molecular interface of Cn and the BBB, and we propose that the virulence factors mediating BBB crossing could be targeted for the development of anti-virulence drugs aimed at preventing fungal colonization of the CNS.

CoO nanoparticles ranging in size from 7 to 21 nm were prepared via precipitation and thermal decomposition methods. The particles had water contents ranging from 2.18 to 0.19 wt %; water content decreased with increasing particle size. All particles also contained a small amount of Co 3 O 4 impurity. The surface enthalpy was determined to be 2.82 ( 0.20 J‚m -2 by acid solution calorimetry at 298 K. Corrections were made for the water and cobalt spinel impurity. Heat capacity measurements using adiabatic (10-320 K) and semi-adiabatic (0.6-40 K) calorimeters were performed on the sample with particle size 7.0 ( 1.0 nm. The nanoparticle heat capacity had a broad anomaly with a rounded maximum at 265 K, a reduction of 23 K from the Ne ´el temperature T N observed as a sharply peaked maximum in the heat capacity of single-crystal CoO. When corrected for water and Co 3 O 4 , the heat capacity of nanophase CoO is greater than that of single-crystal CoO below about 250 K. Above 250 K, the much larger magnetic peak in the heat capacity of the single crystal dominates the heat capacity difference. Thus the excess entropy of the nanoparticles, calculated from the heat capacity difference, has a maximum of 2.4 ( 0.3 J‚K -1 ‚mol -1 at 245 K but drops to 1.5 ( 0.3 J‚K -1 ‚mol -1 at 298 K. The magnetic and surface contributions to the excess entropy cannot be resolved in a definitive manner, but an estimate of the surface entropy, 0.28 ( 0.03 mJ‚K -1 ‚m -2 , is similar to one literature report for MgO, and the excess magnetic entropy is negative, -0.8 ( 0.3 J‚K -1 ‚mol -1 .

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