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

UnlabelledCryptococcus 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.ImportanceCryptococcus 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

Cryptococcal Meningitis and Anti-virulence Therapeutic Strategies

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

Macropinocytosis as a potential mechanism driving neurotropism of Cryptococcus neoformans

Cryptococcus neoformans can invade the central nervous system by crossing the blood-brain barrier via a transcellular mechanism that relies on multiple host factors. In this narrative, we review the evidence that a direct interplay between C. neoformans and brain endothelial cells forms the basis for invasion and transmigration across the brain endothelium. Adherence and internalization of C. neoformans is dependent on transmembrane proteins, including a hyaluronic acid receptor and an ephrin receptor tyrosine kinase. We consider the role of EphA2 in facilitating the invasion of the central nervous system by C. neoformans and highlight experimental evidence supporting macropinocytosis as a potential mechanism of internalization and transcytosis. How macropinocytosis might be conclusively demonstrated in the context of C. neoformans is also discussed

Examination of Fluconazole-Induced Alopecia in an Animal Model and Human Cohort.

Fluconazole-induced alopecia is a significant problem for patients receiving long-term therapy. We evaluated the hair cycle changes of fluconazole in a rat model and investigated potential molecular mechanisms. Plasma and tissue levels of retinoic acid were not found to be causal. Human patients with alopecia attributed to fluconazole also underwent detailed assessment and in both our murine model and human cohort fluconazole induced telogen effluvium. Future work further examining the mechanism of fluconazole-induced alopecia should be undertaken

Time- and region-dependent blood-brain barrier impairment in a rat model of organophosphate-induced status epilepticus

Acute organophosphate (OP) intoxication can trigger seizures that progress to status epilepticus (SE), and survivors often develop chronic morbidities, including spontaneous recurrent seizures (SRS). The pathogenic mechanisms underlying OP-induced SRS are unknown, but increased BBB permeability is hypothesized to be involved. Previous studies reported BBB leakage following OP-induced SE, but key information regarding time and regional distribution of BBB impairment during the epileptogenic period is missing. To address this data gap, we characterized the spatiotemporal progression of BBB impairment during the first week post-exposure in a rat model of diisopropylfluorophosphate-induced SE, using MRI and albumin immunohistochemistry. Increased BBB permeability, which was detected at 6 h and persisted up to 7 d post-exposure, was most severe and persistent in the piriform cortex and amygdala, moderate but persistent in the thalamus, and less severe and transient in the hippocampus and somatosensory cortex. The extent of BBB leakage was positively correlated with behavioral seizure severity, with the strongest association identified in the piriform cortex and amygdala. These findings provide evidence of the duration, magnitude and spatial breakdown of the BBB during the epileptogenic period following OP-induced SE and support BBB regulation as a viable therapeutic target for preventing SRS following acute OP intoxication

Antifungal activity of 6-substituted amiloride and hexamethylene amiloride (HMA) analogs

Fungal infections have become an increasing threat as a result of growing numbers of susceptible hosts and diminishing effectiveness of antifungal drugs due to multi-drug resistance. This reality underscores the need to develop novel drugs with unique mechanisms of action. We recently identified 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of human Na+/H+ exchanger isoform 1, as a promising scaffold for antifungal drug development. In this work, we carried out susceptibility testing of 45 6-substituted HMA and amiloride analogs against a panel of pathogenic fungi. A series of 6-(2-benzofuran)amiloride and HMA analogs that showed up to a 16-fold increase in activity against Cryptococcus neoformans were identified. Hits from these series showed broad-spectrum activity against both basidiomycete and ascomycete fungal pathogens, including multidrug-resistant clinical isolates

The electrophysiological characterization of plant calcium channels and their role in calcium signaling

grantor: University of TorontoThe identification and characterization of Ca\sp{2+} channels in the vacuolar and the plasma membrane of higher plants was accomplished through the use of conventional patch clamp techniques. Intact vacuoles from sugar beet cells were isolated and used to identify Ca\sp{2+} currents at the whole vacuole level and outside-out patches of membrane were used for single channel analysis. Permeability and selectivity studies strongly suggested that one of the channels identified in the vacuolar membrane could efficiently and selectively release Ca\sp{2+} from the vacuole resulting in increased levels of cytosolic Ca\sp{2+}. A second channel co-residing with the vacuolar-release channel is the SV (slow-vacuolar type) channel. Although this channel is active at non-physiological potentials, the Ca\sp{2+} permeability of the SV channel suggests that it could be involved in the sequestration of Ca\sp{2+} following an increase in cytosolic Ca\sp{2+} levels. The proposed role of the SV channel in Ca\sp{2+}-induced Ca\sp{2+}-release is also discussed. Stimulus-induced fluctuations in cytosolic Ca\sp{2+} are mediated by Ca\sp{2+} channels located in both the vacuolar and plasma membrane. The role of a plasma membrane Ca\sp{2+} channel in stimulus-response coupling and the mechanisms of channel regulation were investigated by exposing protoplasts isolated from tomato cell near-isogenic for the resistance gene Cf5 to elicitor preparations containing the avr5 gene from race 4 (IF\sb4) of the fungus Cladosporium fulvum. The stimulation of Ca\sp{2+} channels by IF\sb4, was up-regulated by phosphorylation mediated by the action of a G protein. The concerted action of Ca\sp{2+} channels in the vacuolar and plasma membrane in maintaining Ca\sp{2-} homeostasis and the mechanisms by which plant cells generate and perceive a stimulus-specific Ca\sp{2+} signal is discussed.Ph.D

Elongation Factor 3, EF3, Associates with the Calcium Channel Cch1 and Targets Cch1 to the Plasma Membrane in Cryptococcus neoformans▿

Ca2+-mediated signaling events in eukaryotic cells are initiated by Ca2+ channels located in the plasma membranes and endomembranes. Cch1, a high-affinity Ca2+ channel in the plasma membranes of Cryptococcus neoformans and other fungi, plays a role in many different cellular processes, but the mechanisms that regulate Cch1 are not well understood. A Ras recruitment two-hybrid screen was used to identify protein partners of Cch1 as a means of identifying possible mechanisms of channel regulation. Here, we show that Cch1 specifically associates with a cytoplasmic protein known as elongation factor 3 (EF3). The robust interaction between the cytosolic C terminus of the Cch1 protein and EF3 shown here was confirmed by demonstrating that Cch1 could coimmunoprecipitate with EF3 in yeast lysates. To examine the effects of EF3 on Cch1 behavior, we altered the EF3 gene function by constructing a C. neoformans antisense EF3 repression strain. Our results show that the repression of EF3 led to the mislocalization of Cch1, suggesting a role for EF3 in targeting Cch1 to the plasma membrane of C. neoformans. Consistent with this notion, the antisense EF3 repression strain displayed a growth defect under conditions of limited extracellular Ca2+. Collectively, these results suggest that EF3 and Cch1 are functionally coupled and that EF3 has a function apart from its role in the protein translation cycle