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

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.

In Vitro activity of novel glycopolymer against clinical isolates of multidrug-resistant Staphylococcus aureus.

The incidence of multidrug-resistant (MDR) organisms, including methicillin-resistant Staphylococcus aureus (MRSA), is a serious threat to public health. Progress in developing new therapeutics is being outpaced by antibiotic resistance development, and alternative agents that rapidly permeabilize bacteria hold tremendous potential for treating MDR infections. A new class of glycopolymers includes polycationic poly-N (acetyl, arginyl) glucosamine (PAAG) is under development as an alternative to traditional antibiotic strategies to treat MRSA infections. This study demonstrates the antibacterial activity of PAAG against clinical isolates of methicillin and mupirocin-resistant Staphylococcus aureus. Multidrug-resistant S. aureus was rapidly killed by PAAG, which completely eradicated 88% (15/17) of all tested strains (6-log reduction in CFU) in ≤ 12-hours at doses that are non-toxic to mammalian cells. PAAG also sensitized all the clinical MRSA strains (17/17) to oxacillin as demonstrated by the observed reduction in the oxacillin MIC to below the antibiotic resistance breakpoint. The effect of PAAG and standard antibiotics including vancomycin, oxacillin, mupirocin and bacitracin on MRSA permeability was studied by measuring propidium iodide (PI) uptake by bacterial cells. Antimicrobial resistance studies showed that S. aureus developed resistance to PAAG at a rate slower than to mupirocin but similar to bacitracin. PAAG was observed to resensitize drug-resistant S. aureus strains sampled from passage 13 and 20 of the multi-passage resistance study, reducing MICs of mupirocin and bacitracin below their clinical sensitivity breakpoints. This class of bacterial permeabilizing glycopolymers may provide a new tool in the battle against multidrug-resistant bacteria.

The blood-brain barrier internalises Cryptococcus neoformans via the EphA2-tyrosine kinase receptor.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis most commonly in populations with impaired immunity. Here, we resolved the transcriptome of the human brain endothelium challenged with C. neoformans to establish whether C. neoformans invades the CNS by co-opting particular signalling pathways as a means to promote its own entry. Among the 5 major pathways targeted by C. neoformans, the EPH-EphrinA1 (EphA2) tyrosine kinase receptor-signalling pathway was examined further. Silencing the EphA2 receptor transcript in a human brain endothelial cell line or blocking EphA2 activity with an antibody or chemical inhibitor prevented transmigration of C. neoformans in an in vitro model of the blood-brain barrier (BBB). In contrast, treating brain endothelial cells with an EphA2 chemical agonist or an EphA2 ligand promoted greater migration of fungal cells across the BBB. C. neoformans activated the EPH-tyrosine kinase pathway through a CD44-dependent phosphorylation of EphA2, promoting clustering and internalisation of EphA2 receptors. Moreover, HEK293T cells expressing EphA2 revealed an association between EphA2 and C. neoformans that boosted internalisation of C. neoformans. Collectively, the results suggest that C. neoformans promotes EphA2 activity via CD44, and this in turn creates a permeable barrier that facilitates the migration of C. neoformans across the BBB.

Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11.

Cryptococcus gattii isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattii is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37 °C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 µg/ml for the wild-type isolate to <0.03 µg/ml for the pdr11Δ strain and reverted to 32 µg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattii clinical isolates should be assessed further in order to identify other potential mechanisms of resistance.

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

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 (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. 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.

Activity of the calcium channel pore Cch1 is dependent on a modulatory region of the subunit Mid1 in Cryptococcus neoformans.

Calcium (Ca(2+))-mediated signaling events in fungal pathogens such as Cryptococcus neoformans are central to physiological processes, including those that mediate stress responses and promote virulence. The Cch1-Mid1 channel (CMC) represents the only high-affinity Ca(2+) channel in the plasma membrane of fungal cells; consequently, cryptococci cannot survive in low-Ca(2+) environments in the absence of CMC. Previous electrophysiological characterization revealed that Cch1, the predicted channel pore, and Mid1, a binding partner of Cch1, function as a store-operated Ca(2+)-selective channel gated by depletion of endoplasmic reticulum (ER) Ca(2+) stores. Cryptococci lacking CMC did not survive ER stress, indicating its critical role in restoring Ca(2+) homeostasis. Despite the requirement for Mid1 in promoting Ca(2+) influx via Cch1, identification of the role of Mid1 remains elusive. Here we show that the C-terminal tail of Mid1 is a modulatory region that impinges on Cch1 channel activity directly and mediates the trafficking of Mid1 to the plasma membrane. This region consists of the last 24 residues of Mid1, and the functional expression of Mid1 in a human embryonic cell line (HEK293) and in C. neoformans is dependent on this domain. Substitutions of arginine (R619A) or cysteine (C621A) in the modulatory region failed to target Mid1 to the plasma membrane and prevented CMC activity. Interestingly, loss of a predicted protein kinase C (PKC)-phosphorylated serine residue (S605A) had no effect on Mid1 trafficking but did alter the kinetics of Cch1 channel activity. Thus, establishment of Ca(2+) homeostasis in C. neoformans is dependent on a modulatory domain of Mid1.

The role of protective lead clothing in reducing radiation exposure rates to personnel during equine bone scintigraphy.

Bone scintigraphy is often used in horses because of its sensitivity and noninvasive nature. A 99mTc labeled radiopharmaceutical is injected at a dose of between 5.7 and 7.3GBq. Images are acquired immediately postinjection and 2-4h post. People are often in the room with the horse during the acquisition process. Objectives of this study were to (a) document the radiation exposure rates at different distances from various sites of the horse at varying times post injection and (b) study the usefulness of wearing lead aprons to reduce exposure rates to personnel. Radiation exposure rates were measured in at three distances (at skin surface and at 30 and 100 cm from the skin) from three sites (shoulder, thorax, and pelvis) in 19 horses. Exposure rates were measured with and without shielding by a 0.5-mm lead equivalent apron during both the pool and delayed phases. A 0.5mm equivalent lead apron significantly decreases radiation exposure (P<0.05) at these three distances from the three sites during both image acquisition phases. Mean dose reduction factors from the lead apron range from 3.6 to 5.7.

Evaluation and treatment of decreased libido associated with painful lumbar lesions in two bulls.

Painful lesions of the vertebral column may cause decreased libido in bulls. Radiographic evaluation of vertebral skeletal problems in mature bulls is limited because of high body mass. Two breeding bulls with signs of decreased libido and spermatozoa production were evaluated. Initial systemic medical treatment for the conditions had not focused on localized lesions and was unsuccessful. Nuclear scintigraphy was performed in both bulls to determine the location of vertebral column lesions and facilitate localized treatment. Localized medical treatment was successful and resulted in decreased signs of pain and increased spermatozoa production in both bulls.