Abstract
Cryptococcal meningoencephalitis, the most common form of cryptococcosis, is caused by the opportunistic fungal pathogen, Cryptococcus neoformans. Molecular strategies used by C. neoformans to invade the central nervous system (CNS) have been the focus of several studies. Recently, the role of a novel secreted metalloprotease (Mpr1) in the pathogenicity of C. neoformans was confirmed by studies demonstrating that Mpr1 mediated the migration of fungal cells into the CNS. Given this central function, the aim here was to identify the molecular determinants of Mpr1 activity and resolve their role in the migration of cryptococci across the blood-brain barrier (BBB). The Mpr1 protein belongs to an understudied group of metalloproteases of the M36 class of fungalysins unique to fungi. They are generally synthesized as propeptides with fairly long prodomains and highly conserved regions within their catalytic core. Through structure-function analysis of Mpr1, our study identified the prodomain cleavage sites of Mpr1 and demonstrated that when mutated, the prodomain appears to remain attached to the catalytic C-terminus of Mpr1 rendering a nonfunctional Mpr1 protein and an inability for cryptococci to cross the BBB. We found that proteolytic activity of Mpr1 was dependent on the coordination of zinc with two histidine residues in the active site of Mpr1, since amino acid substitutions in the HExxH motif abolished Mpr1 proteolytic activity and prevented the migration of cryptococci across the BBB. A phylogenetic analysis of Mpr1 revealed a distinct pattern likely reflecting the neurotropic nature of C. neoformans and the specific function of Mpr1 in breaching the BBB. This study contributes to a deeper understanding of the molecular regulation of Mpr1 activity and may lead to the development of specific inhibitors that could be used to restrict fungal penetration of the CNS and thus prevent cryptococcal meningoencephalitis-related deaths.
Highlights
Cryptococcus neoformans is a fungal pathogen that primarily affects individuals with a compromised immune system [1, 2]
Mpr1 is a member of the M36 class of fungal metalloproteases and like other proteases within this group the predicted structure of Mpr1 from C. neoformans includes the typical hallmarks such as a signal sequence, a prodomain and a catalytic region at the C-terminus (Fig 2A)
We demonstrated previously that Mpr1 was required for C. neoformans to cross the blood-brain barrier (BBB) and enter the central nervous system but whether the proteolytic activity of Mpr1 played a direct role in promoting the migration of fungal cells across the BBB was not known.[7, 30]
Summary
Cryptococcus neoformans is a fungal pathogen that primarily affects individuals with a compromised immune system [1, 2]. Development of fungal meningoencephalitis causes high morbidity and accounts for 15% of AIDS-related deaths worldwide [3, 4]. Movement across the blood-brain barrier (BBB) is highly restricted in order to protect the central nervous system (CNS), but cryptococci can invade the CNS by breaching brain capillary endothelial cells, which constitute the BBB.[5] Several studies have demonstrated that freely moving cryptococci in the bloodstream can cross the BBB via a transcellular pathway through brain capillary endothelial cells and cause disruption and remodeling of tight junction proteins suggesting a paracellular path.[6,7,8,9,10] In addition, studies have demonstrated that cryptococci make use of a stealthy mechanism that involves penetrating the BBB by hiding within monocytes, referred to as the Trojan horse mechanism [11,12,13,14]
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