Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.     

Characterisation and expression of phospholipases B from the opportunistic fungus Aspergillus fumigatus

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (afplb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.     

Role and mechanism of phosphatidylinositol-specific phospholipase C in survival and virulence of Cryptococcus neoformans

Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans . To investigate the role of phosphatidylinositol-specific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii ( PLC1 and PLC2 ), and created Δ plc1 and Δ plc2 deletion mutants. In Δ plc1 , which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37°C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Δ plc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Δ plc2 , which was as virulent as WT, Δ plc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25°C, demonstrating that mechanism(s) independent of the 37°C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.     

Phenotype characterization of environmental Cryptococcus neoformans isolates

Cryptococcosis is caused by the three varieties of C. neoformans with physiological and virulence differences, some of which have been studied to determine biological aspects of this microorganism. The phenotypical aspects of environmental isolates from varieties grubii and gattii were evaluated to establish differences associated with their life cycle and virulence. To this end, 28 and 31 strains of C. neoformans serotypes A (var. grubii) and C (var. gattii) were studied. The microscopic and macroscopic morphology on Sabouraud agar and soils, growth rate at 37 degrees C, production of 22 extracellular enzymes, haploid fructification, mating type, killer toxin sensitivity patterns and virulence in BALB/c mice were evaluated. No differences were observed between the two varieties regarding microscopic and macroscopic morphology or growth at 37 degrees C (p > 0.05). However, a decrease in the cellular and capsular sizes of yeast in soil, as compared to Sabouraud, was observed (p < 0.05). Additionally, higher enzimatic activity of proteases, phospholipases, phenoloxidase and beta-glucosidase was observed in var. grubii isolates as compared to var. gattii (p < 0.05). In both varieties, structures related with haploid fruitification were observed and all isolates were mating type alpha. Killer toxin sensitivity patterns of the isolates of var. grubii were I and II; in contrast, in var. gattii, seven different patterns were found: I, V, IX-XIII. In the animal model we found that 12 of 22 (54.5%) isolates of var. grubii caused the death of the mice during the observation period, while none of the 14 var. gattii isolates caused it. The decrease in capsular and cellular sizes of the yeast in soil and the frequency of mating type alpha with structures related to haploid fructification suggest an important mechanism of production of infectious particles in nature. Additionally, greater enzimatic activity of var. grubii can be associated with the virulence in the animal model.     

Comparison of phospholipase production in shape Cryptococcus neoformans isolates from AIDS patients and bird droppings

Secreted phospholipase has been recently proposed as a virulence determinant in Cryptococcus neoformans as well as Candida albicans. This issue of cryptococcal phospholipase requires screening of phospholipase production in a larger number of isolates from clinical and environmental sources. In this study we examined phospholipase production in a total of 67 C. neoformans isolates from AIDS patients and bird droppings by using the egg-yolk plate method. Phenoloxidase activity, capsule size and growth at 37 °C were also measured in these strains in order to observe a possible relationship between phospholipase production of different C. neoformans strains and its virulence. Four of the 21 AIDS strains at 28 °C and 1 at 37 °C did not produce phospholipase, respectively. In contrast, 38 and 34 of the 46 bird dropping strains were negative for phospholipase production at 28, and 37 °C, respectively. Statistical analysis revealed a significant difference in phospholipase production, capsule size and growth ability at 37 °C, but not phenoloxidase activity, between the AIDS and the bird dropping strains. The highly prevalent distribution of phospholipase activity in the AIDS strains suggests a role of the enzyme in invading the host. This revised version was published online in June 2006 with corrections to the Cover Date.     

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirão Preto, São Paulo, Brazil

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeir?o Preto, S?o Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors.     

Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1

Acidification of vesicular compartments plays an important role in a number of cellular transport processes, including protein secretion, metal cofactor insertion, glycosylation and pH stability. In the present study, we identify and characterize a component of the vesicular proton pump, Vph1p, to determine its role in the virulence of the AIDS-related fungal pathogen Cryptococcus neoformans. Insertional mutagenesis and plasmid rescue were used to identify the VPH1 gene by screening for mutants defective in laccase activity. Disruption of VPH1 resulted in defects in three virulence factors (capsule production, laccase and urease expression), as well as a growth defect at 37 degrees C, but only a small growth reduction at 30 degrees C. These effects were duplicated by the vacuolar (H+)-ATPase inhibitor bafilomycin A1. Furthermore, the vph1 insertional mutant was also avirulent in a mouse meningo-encephalitis model. Complementation of the insertional mutant with wild-type VPH1 resulted in a recovery of virulence factor expression, normal growth at 37 degrees C and restoration of full virulence. These studies establish the importance of the VPH1 gene and vesicular acidification in the virulence of C. neoformans.     

In vitro determination of virulence factors activity associated with several Cryptococcus neoformans clinical isolates

Different phenotypic characteristics associated with virulence of the Cryptococcus neoformans species complex have shown an important role in their pathogenicity. In this study we have determined the role of phenotypically and genotypically factors of some virulence factors from clinical isolates in the two species of the complex; 35 C. neoformans and 19 Cryptococcus gattii. Growth at 37 degrees C, macroscopic and microscopic morphology, switching phenomenon, activity of 23 extracellular enzymes, variability of the colonies in agar with phloxin B; phospholipase B gene, and the mating type were determined by PCR; the molecular pattern was determined by URA5 RFLP. All isolates grew at 37 degrees C, the capsular size was greater in C. gattii (1.87 microm -/+1.47 microm) than in C. neoformans (0.83 microm -/+0.15 microm). Switching was observed mainly in isolates of C. gattii. All isolates expressed the enzyme urease, a lower activity of the proteases (Pz= 0.54), but a higher activity of the phospholipase (Pz=0.43) and phenoloxidase (Pz=0.003) was determined for C. gattii.     

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.     

Calcineurin is required for virulence of Cryptococcus neoformans.

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.     

Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91%) killed 50% or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37oC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.     

The temperature-sensitive role of Cryptococcus neoformans ROM2 in cell morphogenesis

ROM2 is associated with Cryptococcus neoformans virulence. We examined additional roles of ROM2 in C. neoformans and found that ROM2 plays a role in several cell functions specifically at high temperature conditions. Morphologically rom2 mutant cells demonstrated a "tear"-like shape and clustered together. A sub-population of cells had a hyperelongated phenotype at restrictive growth conditions. Altered morphology was associated with defects in actin that was concentrated at the cell periphery and with abnormalities in microtubule organization. Interestingly, the ROM2 associated defects in cell morphology, location of nuclei, and actin and microtubule organization were not observed in cells grown at temperatures below 37 degrees C. These results indicate that in C. neoformans, ROM2 is important at restrictive temperature conditions and is involved in several cell maintenance functions.     

Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans

The cell wall of pathogenic fungi such as Cryptococcus neoformans , provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild-type strains containing empty plasmid, but a complete absence in the i SEC6 strain. In addition, a green fluorescent protein–laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in i SEC6 strains. In contrast, i SEC6 strains retained normal growth at 37°C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans .     

Pathogenetic significance of the psychrophilia of Yersinia pseudotuberculosis

The work presents the data indicating that the temperature of Y. pseudotuberculosis cultivation is very important in regulating the activity of pathogenicity factors, necessary for the initiation of the pathogenic process in the cells of the macroorganism. Low temperature (8-10 degrees C), necessary for the growth of Y. pseudotuberculosis, facilitates the activation of invasive and toxic pathogenicity factors. At a growth temperature of 37 degrees C the inhibition of such necessary attributes of virulence as adhesion and invasion into epithelial cells occurs in Y. pseudotuberculosis, which decreases the capacity of these bacteria for inducing the infectious process. The virulence of Y. pseudotuberculosis population, lost as the result of its cultivation in synthetic culture media at a temperature of 37 degrees C, has been found to be restored at low temperature.     

Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca2+- calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37 degrees C and virulence of C.neoformans. We found that calcineurin is also required for mating. FK506 blocks mating of C.neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATalpha cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, our studies suggest a second link between calcineurin and virulence of C.neoformans. Calcine urin regulates filamentation and 37 degrees C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, our findings indicate that calcineurin plays a conserved role in the control of cell morphology.     

Urease testing and yeast taxonomy

When urease production was assayed by the hydrolysis of [14C]urea, all basidiomycetous yeasts tested, including the Cryptococcus vishniacii complex (previously reported urease negative), produced significant amounts of 14CO2. The Schizosaccharomycetaceae were the only urease-positive ascomycetous yeasts tested. Yarrowia lipolytica was urease negative. The stoichiometry of [14C]urea hydrolysis paralleled by Roberts' rapid urea hydrolysis (RUH) test indicated that causes of anomalous results in conventional urease testing include acidification and alkalinization of the test medium by products of endogenous metabolism and autolysis rather than urease activity. Anomalous results also occurred when cells were grown on media containing the chelating agent ethylenediaminetetraacetic acid (EDTA) prior to RUH. The addition of EDTA to a complex natural medium inhibited urease production in all yeasts reportedly growing at 35 degrees C (and all other yeasts tested), except Filobasidiella (Cr.) neoformans var. neoformans (NIH 12). The RUH test could differentiate at the varietal level: Fil. (Cr.) neoformans var. neoformans was about 10 times more resistant to EDTA in media used for the growth of cells prior to RUH testing than was Fil. neoformans var. bacillispora (Cr. neoformans var. gattii) (NIH 191). Urease production by Fil. neoformans var. bacillispora was specifically restored to half maximal activity by the addition of 22 microM Ni+2 (as NiCl2) to a growth medium containing 0.100 mM EDTA.     

Cch1 mediates calcium entry in Cryptococcus neoformans and is essential in low-calcium environments

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37 degrees C to 39 degrees C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low ( approximately 50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise ( approximately 1 to 10 microM) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca(2+)-permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca(2+)](extracellular), approximately 100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca(2+)](extracellular) ( approximately 100 nM) became more severe with increasing temperature (25 degrees C to 38.5 degrees ), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca(2+)](extracellular), approximately 0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.     

Isolation and characterisation of the phospholipase B gene of Cryptococcus neoformans var. gattii

Cryptococcus neoformans var. gattii (serotypes B and C) is a human pathogen, ecologically, biochemically, clinically and genetically different from C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D). The phospholipase B (PLB1) gene from serotypes B and C was isolated and characterised. It resembled the serotype A and D genes, with an overall sequence homology of more than 85%. The respective open reading frames were 2236 bp (serotype B) and 2239 bp (serotype C) in length. Each contained six introns and encoded a 68-kDa protein destined for secretion. PLB1 was located on the second smallest chromosome in both serotypes. Gene expression, measured as mRNA, was not regulated by temperature, pH or exogenous nutrients.