Cryptococcal lipid metabolism: phospholipase B1 is implicated in transcellular metabolism of macrophage-derived lipids.

Cryptococci survive and replicate within macrophages and can use exogenous arachidonic acid for the production of eicosanoids. Phospholipase B1 (PLB1) has a putative, but uninvestigated, role in these processes. We have shown that uptake and esterification of radiolabeled arachidonic, palmitic, and oleic acids by the Cryptococcus neoformans var. grubii H99 wild-type strain and its PLB1 deletion mutant strain (the Deltaplb1 strain) are independent of PLB1, except under hyperosmolar stress. Similarly, PLB1 was required for metabolism of 1-palmitoyl lysophosphatidylcholine (LysoPC), which is toxic to eukaryotic cell membranes, under hyperosmolar conditions. During both logarithmic and stationary phases of growth, the physiologically relevant phospholipids, dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine, were taken up and metabolized via PLB1. Exogenous DPPC did not enhance growth in the presence of glucose as a carbon source but could support it for at least 24 h in glucose-free medium. Detoxification of LysoPC by reacylation occurred in both the H99 wild-type and the Deltaplb1 strains in the presence of glucose, but PLB1 was required when LysoPC was the sole carbon source. This indicates that both energy-independent (via PLB1) and energy-dependent transacylation pathways are active in cryptococci. Phospholipase A(1) activity was identified by PLB1-independent degradation of 1-palmitoyl-2-arachidonoyl phosphatidylcholine, but the arachidonoyl LysoPC formed was not detoxified by reacylation. Using the human macrophage-like cell line THP-1, we demonstrated the PLB1-dependent incorporation of macrophage-derived arachidonic acid into cryptococcal lipids during cryptococcus-phagocyte interaction. This pool of arachidonate can be sequestered for eicosanoid production by the fungus and/or suppression of host phagocytic activity, thus diminishing the immune response.

Phospholipase B activity enhances adhesion of Cryptococcus neoformans to a human lung epithelial cell line.

Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.

Hexadecylphosphocholine (miltefosine) has broad-spectrum fungicidal activity and is efficacious in a mouse model of cryptococcosis.

The alkyl phosphocholine drug miltefosine is structurally similar to natural substrates of the fungal virulence determinant phospholipase B1 (PLB1), which is a potential drug target. We determined the MICs of miltefosine against key fungal pathogens, correlated antifungal activity with inhibition of the PLB1 activities (PLB, lysophospholipase [LPL], and lysophospholipase-transacylase [LPTA]), and investigated its efficacy in a mouse model of disseminated cryptococcosis. Miltefosine inhibited secreted cryptococcal LPTA activity by 35% at the subhemolytic concentration of 25 microM (10.2 microg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA2). At 250 microM, cytosolic PLB, LPL, and LPTA activities were inhibited by 25%, 51%, and 77%, respectively. The MICs at which 90% of isolates were inhibited (MIC90s) against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Fusarium solani, Scedosporium prolificans, and Scedosporium apiospermum were 2 to 4 microg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 microg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 microg/ml (MIC90), and those against Aspergillus flavus (n = 8) were 2 to 16 microg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 microM (2.8 microg/ml). Miltefosine given orally to mice on days 1 to 5 after intravenous infection with C. neoformans delayed the development of illness and mortality and significantly reduced the brain cryptococcal burden. We conclude that miltefosine has broad-spectrum antifungal activity and is active in vivo in a mouse model of disseminated cryptococcosis. The relatively small inhibitory effect on PLB1 enzyme activities at concentrations exceeding the MIC by 2 to 20 times suggests that PLB1 inhibition is not the only mechanism of the antifungal effect.

In vitro antifungal activities of inhibitors of phospholipases from the fungal pathogen Cryptococcus neoformans.

Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein. These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA). Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy. We determined in C. neoformans var. grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated. Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes. Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors. These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D). The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test. Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A(2) at 0.25 micro M. Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal. We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal. They also provide tools for assessing the relative importance of the various enzyme activities in virulence. Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.