Cryptococcal Phospholipase B1 Is Required for Intracellular Proliferation and Control of Titan Cell Morphology during Macrophage Infection

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of fungal-infection-related fatalities, especially in immunocompromised hosts. Several virulence factors are known to play a major role in the pathogenesis of cryptococcal infections, including the enzyme phospholipase B1 (Plb1). Compared to other well-studied Cryptococcus neoformans virulence factors such as the polysaccharide capsule and melanin production, very little is known about the contribution of Plb1 to cryptococcal virulence. Phospholipase B1 is a phospholipid-modifying enzyme that has been implicated in multiple stages of cryptococcal pathogenesis, including initiation and persistence of pulmonary infection and dissemination to the central nervous system, but the underlying reason for these phenotypes remains unknown. Here we demonstrate that a Δplb1 knockout strain of C. neoformans has a profound defect in intracellular growth within host macrophages. This defect is due to a combination of a 50% decrease in proliferation and a 2-fold increase in cryptococcal killing within the phagosome. In addition, we show for the first time that the Δplb1 strain undergoes a morphological change during in vitro and in vivo intracellular infection, resulting in a subpopulation of very large titan cells, which may arise as a result of the attenuated mutant's inability to cope within the macrophage

A deletion in the first cysteine-rich repeat of the low-density-lipoprotein receptor leads to the formation of multiple misfolded isomers

The ligand-binding domain of the low-density-lipoprotein (LDL) receptor comprises seven cysteine-rich repeats, each approximately 40 amino acids long. The deletion of two amino acids (Asp26 and Gly27) from the first of these repeats (LB1), leads to a defective LDL receptor, and the clinical syndrome of familial hypercholesterolemia [Leitersdorf, E., Hobbs, H. H., Fourie, A. M., Jacobs, M., van der Westhuyzen, D. R. and Coetzee, G.A. (1988) Proc. Natl. Acad. Sci. USA 85, 7912-7916]. Receptors which reach the cell surface fail to bind IgG-C7, a conformation-specific monoclonal antibody directed to LB1. To determine the effects of the two-amino-acid deletion on the folding of the LB1 of the LDL receptor, we have expressed LB1 and the mutant repeat, des-Asp26, Gly27-LB1, as recombinant (rLB1 and des Asp26, Gly27-rLB1) peptides, and have determined their ability to fold in vitro. Unlike rLB1, which folded into a single isomer that was recognized by IgG-C7 and had three disulfide bonds, des-Asp26, Gly27-rLB1 folded into an equilibrium mixture of four isomers. Each of-these isomers contained three disulfide bonds, but none were recognized by IgG C7. We suggest that mutant LDL receptors in the endoplasmic reticulum (ER) of the cell also fold into an equilibrium mixture of distinct receptor molecules, each with an abnormally folded isomer of des-Asp26, Gly27-LB1, and that the retarded transport of receptors to the cell surface arises because only a subset of the isomers reaches the cell surface

Three-Dimensional Structure of the Second Cysteine-Rich Repeat from the Human Low-Density Lipoprotein Receptor

The ligand-binding domain of the low-density lipoprotein receptor comprises seven cysteinerich repeats, which have been highly conserved through evolution. This domain mediates interactions of the receptor with two lipoprotein apoproteins, apo E and apo B-100, putatively through a calcium-dependent association of the ligands with a cluster of acidic residues on the receptor. The second repeat (rLB2) of the receptor binding domain has been expressed as a thrombin-cleavable GST fusion protein, cleaved, and purified. On oxidation the protein refolded to give a single peak on reverse-phase HPLC. The aqueous solution structure of rLB2 has been determined using two-dimensional H NMR spectroscopy. In contrast to the amino-terminal repeat, rLBl, rLB2 has a very flexible structure in water. However, the conformation of rLB2 is markedly more ordered in the presence of a 4-fold molar excess of calcium chloride; the proton resonance dispersion and the number of NOESY cross-peaks are greatly enhanced. The threedimensional structure of rLB2, obtained from the NMR data by molecular geometry and restrained molecular dynamics methods, parallels that of rLBl, with an amino-terminal hairpin structure followed by a succession of turns. However, there are clear differences in the backbone topology and structural flexibility. As for rLBl, the acidic residues are clustered on one face of the module. The side chain of Asp 37, which is part of a completely conserved SDE sequence thought to be involved in ligand binding, is buried, as is its counterpart (Asp 36) in rLB 1. These results provide the first experimental support for the hypothesis that each of the repeats in the ligand-binding domain has a similar global fold but also highlight significant differences in structure and internal dynamics

Lipid Rafts in Cryptococcus neoformans Concentrate the Virulence Determinants Phospholipase B1 and Cu/Zn Superoxide Dismutase

Lipid rafts have been identified in the membranes of mammalian cells, the yeast Saccharomyces cerevisiae, and the pathogenic fungus Candida albicans. Formed by a lateral association of sphingolipids and sterols, rafts concentrate proteins carrying a glycosylphosphatidylinositol (GPI) anchor. We report the isolation of membranes with the characteristics of rafts from the fungal pathogen Cryptococcus neoformans. These characteristics include insolubility in Triton X-100 (TX100) at 4°C, more-buoyant density within a sucrose gradient than the remaining membranes, and threefold enrichment with sterols. The virulence determinant phospholipase B1 (PLB1), a GPI-anchored protein, was highly concentrated in raft membranes and could be displaced from them by treatment with the sterol-sequestering agent methyl-β-cyclodextrin (MβCD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37°C. Treatment of viable cryptococcal cells in suspension with MβCD also released PLB1 protein and enzyme activity, consistent with localization of PLB1 in plasma membrane rafts prior to secretion. The antioxidant virulence factor Cu/Zn superoxide dismutase (SOD1) was concentrated six- to ninefold in raft membrane fractions compared with nonraft membranes, whereas the cell wall-associated virulence factor laccase was not detected in membranes. We hypothesize that raft membranes function to cluster certain virulence factors at the cell surface to allow efficient access to enzyme substrate and/or to provide rapid release to the external environment

Marsupial and monotreme cathelicidins display antimicrobialactivity, including against methicillin-resistant Staphylococcus

With the growing demand for new antibiotics to combat increasing multi-drug resistance, a family of antimicrobial peptides known as cathelicidins has emerged as potential candidates. Expansions in cathelicidin-encoding genes in marsupials and monotremes are of specific interest as the peptides they encode have evolved to protect immunologically naive young in the harsh conditions of the pouch and burrow. Our previous work demonstrated that some marsupial and monotreme cathelicidins have broad-spectrum antibacterial activity and kill resistant bacteria, but the activity of many cathelicidins is unknown. To investigate associations between peptide antimicrobial activity and physiochemical properties, we tested 15 cathelicidin mature peptides from tammar wallaby, grey short-tailed opossum, platypus and echidna for antimicrobial activity against a range of bacterial and fungal clinical isolates. One opossum cathelicidin ModoCath4, tammar wallaby MaeuCath7 and echidna Taac-CATH1 had broad-spectrum antibacterial activity and killed methicillin-resistant Staphylococcus aureus. However, antimicrobial activity was reduced in the presence of serum or whole blood, and non-specific toxicity was observed at high concentrations. The active peptides were highly charged, potentially increasing binding to microbial surfaces, and contained amphipathic helical structures, which may facilitate membrane permeabilisation. Peptide sequence homology, net charge, amphipathicity and alpha helical content did not correlate with antimicrobial activity. However active peptides contained a significantly higher percentage of cationic residues than inactive ones, which may be used to predict active peptides in future work. Along with previous studies, our results indicate that marsupial and monotreme cathelicidins show potential for development as novel therapeutics to combat increasing antimicrobial resistance

Inositol Polyphosphate Kinases, Fungal Virulence and Drug Discovery

Opportunistic fungi are a major cause of morbidity and mortality world-wide, particularly in immunocompromised individuals. Developing new treatments to combat invasive fungal disease is challenging given that fungal and mammalian host cells are eukaryotic, with similar organization and physiology. Even therapies targeting unique fungal cell features have limitations and drug resistance is emerging. New approaches to the development of antifungal drugs are therefore needed urgently. Cryptococcus neoformans, the commonest cause of fungal meningitis worldwide, is an accepted model for studying fungal pathogenicity and driving drug discovery. We recently characterized a phospholipase C (Plc1)-dependent pathway in C. neoformans comprising of sequentially-acting inositol polyphosphate kinases (IPK), which are involved in synthesizing inositol polyphosphates (IP). We also showed that the pathway is essential for fungal cellular function and pathogenicity. The IP products of the pathway are structurally diverse, each consisting of an inositol ring, with phosphate (P) and pyrophosphate (PP) groups covalently attached at different positions. This review focuses on (1) the characterization of the Plc1/IPK pathway in C. neoformans; (2) the identification of PP-IP5 (IP7) as the most crucial IP species for fungal fitness and virulence in a mouse model of fungal infection; and (3) why IPK enzymes represent suitable candidates for drug development

The Crz1/Sp1 Transcription Factor of <em>Cryptococcus neoformans</em> Is Activated by Calcineurin and Regulates Cell Wall Integrity

<div><p><em>Cryptococcus neoformans</em> survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of <em>C. neoformans</em> calcineurin are largely unknown. In <em>S. cerevisiae</em> and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the <em>C. neoformans</em> Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a <em>bona fide</em> target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δ<em>cna1,</em> and a <em>CRZ1/SP1</em> mutant (Δ<em>crz1</em>) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, <em>CHS6,</em> was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT <em>C. neoformans</em> and <em>Δcna1</em> in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30–37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of <em>C. neoformans</em> Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.</p> </div

FK506 causes exclusion of heat shock-induced Crz1-GFP puncta from the nucleus.

<p><i>C. neoformans</i> cells growing at room temperature (A) were treated with 10 µg/ml FK506 for 1 hour (B) and then exposed to 42°C for 5 min. The fluorescence pattern was compared in FK506-treated and untreated cells within 45 minutes following heat shock (C, D).</p