The structure-function analysis of the Mpr1 metalloprotease determinants of activity during migration of fungal cells across the blood-brain barrier

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.

Zinc Finger Proteins in the Human Fungal Pathogen Cryptococcus neoformans

“…Strikingly, the sole expression of cryptococcal MPR1 in S. cerevisiae endowed the yeast cells with the ability to cross the BBB, which demonstrated that Mpr1 is specific in breaching the BBB and suggested that Mpr1 may function independently of the hyaluronic acid-CD44 pathway in breaching the brain endothelium [88]. Further structure-function analysis of Mpr1 showed that both mutations in the cleavage sites of the prodomain and amino acid substitutions in the HExxH motif resulted in the failure of cryptococci to cross the BBB [91]. The proteolytic activity assay showed that the implementation of Mpr1 s complete function was dependent on the coordination of zinc with the HExxH motif in the active site of Mpr1 [91].…”

“…Further structure-function analysis of Mpr1 showed that both mutations in the cleavage sites of the prodomain and amino acid substitutions in the HExxH motif resulted in the failure of cryptococci to cross the BBB [91]. The proteolytic activity assay showed that the implementation of Mpr1 s complete function was dependent on the coordination of zinc with the HExxH motif in the active site of Mpr1 [91].…”

How Environmental Fungi Cause a Range of Clinical Outcomes in Susceptible Hosts

“…Mpr1 is sufficient for crossing in vitro blood brain barrier models. Expression of cryptococcal MPR1 in Saccharomyces cerevisiae allowed the species to cross an in vitro blood brain model, when it is normally incapable of doing so [392,393]. Mpr1 may facilitate transversal of the blood-brain barrier by stimulating cytoskeletal reorganization through interactions with Annexin A2 [393].…”

An Antivirulence Approach for Preventing Cryptococcus neoformans from Crossing the Blood-Brain Barrier via Novel Natural Product Inhibitors of a Fungal Metalloprotease

“…Previous studies demonstrated that Mpr1 is not required for fungal viability/growth, but rather, it functions as a major virulence factor by promoting Cn across the BBB (23,32,33). We propose that Mpr1 is a high-value target based on studies that found significantly less brain fungal burden in mice that had been inoculated with a strain of Cn lacking Mpr1 (32).…”

“…In the case of Cn, only one M36 fungalysin, MPR1, is present in its genome (31,32). These secreted metalloproteases have a similar overall structure consisting of a signal peptide, a prodomain, and a catalytic domain that coordinates zinc and H 2 O for activity (33). So far, evidence for these metalloproteases support a role in fungal pathogenesis, but in most cases, their targets have not been identified (25).…”