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

ABSTRACT: UNLABELLED: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 (mpr1?) failed to breach the endothelium in an in vitro model of the human blood-brain barrier (BBB). A mammalian host infected with the mpr1? 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.

SUBMITTER: Vu K

PROVIDER: S-EPMC4049100 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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

Vu Kiem K Tham Rick R Uhrig John P JP Thompson George R GR Na Pombejra Sarisa S Jamklang Mantana M Bautos Jennifer M JM Gelli Angie A

mBio 20140603 3

<h4>Unlabelled</h4>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 ...[more]

PMID: 24895304

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Project description:Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the in vivo yeast transcriptomes to those from other environmental conditions, including in vitro growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the CMP1 putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease. IMPORTANCE Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. In this study, we captured transcriptomes of yeasts directly out of the human cerebrospinal fluid, the most concerning site of infection. By comparing the RNA transcript levels with other conditions, we gained insights into how the basic machinery involved in metabolism and environmental responses enable this fungus to cause disease at this body site. This approach was applied to clinical isolates with diverse genotypes to begin to establish a genotype-agnostic understanding of how the yeast responds to stress. Based on these results, future studies can focus on how these genes and their pathways and networks can be targeted with new therapeutics and possibly classify yeasts with bad infection outcomes.

Dataset Information

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

Publications

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

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