BackgroundThe fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface.MethodologyThe invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen.ConclusionsThe results of this study provide evidence for the cooperative role of multiple virulence determinants in C. neoformans pathogenesis and suggest new avenues for the development of anti-infective agents in the prevention of fungal tissue invasion.

Calcineurin (PP3/PP2B) is a serine-threonine-specific phosphatase of the protein phosphatase (PP) family (EC 3.1.3.16), which also includes PP1, PP2A,. Phosphatases share a highly conserved primary structure in eukaryotes and were originally grouped based on their preferential dephosphorylation of the alpha subunit (PP2) or beta subunit (PP1) of phosphorylase kinase and the sensitivity (PP1) or insensitivity (PP2) of phosphatase activity to heat-stable cytosolic proteins known as inhibitor 1 and inhibitor 2. Calcineurin differs from other phosphatases in metal ion requirements, drug sensitivity, range of substrate specificity, and cellular regulation. These differential properties arise out of the unique structural aspects of calcineurin that confer its complex regulation in eukaryotic cells. Calcineurin exists as a heterodimer of 57-to 71 kDa catalytic subunits (CnA) and 18-to 20-kDa regulatory subunits (CnB), in which primary sequence and higher-order structural features define the relatively narrow substrate range characteristic of this enzyme and facilitate key regulatory aspects of its cellular function ( Fig. 1) (18, 40,104). Additionally, the exclusive binuclear Fe 2ϩ /Zn 2ϩ metal center of calcineurin ( Fig. 1) catalyzes phosphoester hydrolysis by a two-step metal-activated process requiring reduced iron, linking the functional activation of calcineurin with changes in cellular redox chemistry (74,87).The CnA subunit contains the catalytic domain and three regulatory elements, which include the CnB-binding, the calmodulin-binding, and the autoinhibitory domains, located toward the carboxyl terminus (60,76). In resting cells, the autoinhibitory domain blocks the catalytic center and thus provides an intrinsic mechanism for the coordination of calcineurin phosphatase activity with changes in the cellular activation state. Intrinsic structural features that confer the self-regulation of calcineurin are not found in PP1 and PP2A, which exist as free catalytic subunits that are constitutively active in the absence of allosteric inhibitors (25, 69). The CnB-and calmodulin-binding domains of calcineurin work in concert with autoregulatory mechanisms to coordinate phosphatase activity with the modulation of intracellular calcium ion homeostasis and associated changes in the cellular activation state. CnB is an essential structural component of calcineurin that is required for both CnA stabilization and Ca 2ϩ /calmodulin activation of calcineurin (61,91,93,109,119). Calmodulin and CnB, though functionally dissimilar, are structurally conserved and 35% identical in primary sequence (3, 72). In each, Ca 2ϩ binding activity is mediated through four EF-hand Ca 2ϩ -binding loops similarly positioned at opposite ends of a symmetrical dumbbell-shaped tertiary structure (3). The appropriate cellular functions of calmodulin and CnB depend on their relative affinity for Ca 2ϩ , which allows for the structural stability of CnB at basal Ca 2ϩ levels present in resting cells and facilitates the inducible activation of calmodu...

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni2+-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45–75 kDa species (peak Mr ∼60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of Mr ∼40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting ∼60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337–346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.