巨噬细胞对新生隐球菌主要毒性基因表达的影响

目的研究巨噬细胞对新生隐球菌B3501标准株的主要毒性基因表达影响。方法将对数生长期的J774．16巨噬细胞分别与新生隐球菌野生株B3501共孵育4h,收集被J774．16吞噬的B3501作为实验组,提取实验组和在37℃条件下5％二氧化碳单独培养的对照组B3501的RNA,采用实时荧光定量PCR技术,检测J774．16细胞内和对照组B3501的CNLAC1、CAP60、URE1、NMT表达的差异。结果实验组中新生隐球菌的CAP6,CNLAC1,NMT及URE1基因的mRNA在每百万看家基因（GAPDH基因）中的平均含量分别为（2．698±0．084）×10^4,（1．806±0．322）×10^4,（2．267±0．074）×10^4和（4．041±0．271）×10^4;而对照组这4种毒性因子基因含量分别为：（1．139±0．183）×10^6,（9．324±5．028）×10^3,（1．326±0．028）×10^6和（1．307±0．001）×10^6,均远比实验组高,其中以NMT最为明显。结论新生隐球菌被巨噬细胞吞噬后,主要的毒性因子基因表达下降,其中以NMT最为明显,而CNLAC1下降幅度最小。     

Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms

Cryptococcus neoformans is the causative agent of cryptococcosis and cryptococcal meningitis, which are serious pathological conditions affecting up to 10% of patients with AIDS. Mechanisms of pathogenicity of C. neoformans and the host defenses against this fungus are reviewed, incorporating recent data and perspectives.     

Melanin and virulence in Cryptococcus neoformans

Melanin synthesis has been associated with virulence for the human pathogenic fungus Cryptococcus neoformans. Recent evidence indicates that C. neoformans cells synthesize melanin during infection and that this pigment protects the fungus against immune defense mechanisms.     

Viral protease inhibitors affect the production of virulence factors in Cryptococcus neoformans

The effects of the protease inhibitors saquinavir, darunavir, ritonavir, and indinavir on growth inhibition, protease and phospholipase activities, as well as capsule thickness of Cryptococcus neoformans were investigated. Viral protease inhibitors did not reduce fungal growth when tested in concentrations ranging from 0.001 to 1.000 mg/L. A tendency toward increasing phospholipase activity was observed with the highest tested drug concentration in a strain-specific pattern. However, these drugs reduced protease activity as well as capsule production. Our results confirm a previous finding that antiretroviral drugs affect the production of important virulence factors of C. neoformans.     

'Ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi--the Cryptococcus neoformans paradigm

Environmental pathogenic fungi present a paradox in that they are virulent in animals without requiring animal hosts for replication or survival, a phenomenon we call 'ready-made' virulence. In the human pathogenic fungus Cryptococcus neoformans, the capacity for virulence in animals may originate from environmental selective pressures imposed by such organisms as amoeboid and nematode predators. Many C. neoformans virulence factors appear to have 'dual use' capabilities that confer survival advantages in both animal hosts and in the environment. The findings with C. neoformans may provide a paradigm for understanding the origin and maintenance of virulence in other pathogenic environmental fungi.     

Beyond the big three: systematic analysis of virulence factors in Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans has three major virulence factors: growth at 37 degrees C, capsule synthesis, and melanin formation. Recently in Cell, Liu et al. (2008) employed signature tagged mutagenesis to systematically identify virulence genes. Remarkably, mutations in many of these genes did not influence the major virulence traits.     

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.     

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.     

Intersection of fungal fitness and virulence in Cryptococcus neoformans

The use of insertional mutagenesis to discover genes that impact laccase activity has resulted in the identification of multiple cellular processes that affect the fitness of Cryptococcus neoformans. Fitness has been defined as the ability of an organism to propagate and evolve within a given environment. Because the human host is an evolutionary dead-end for an opportunistic pathogen, we have defined pathogenic fitness here as the capability to successfully propagate within the stressful environment of the host, causing disease by expression of virulence traits that damage the host. In this review, laccase-deficient insertional mutants will be highlighted in terms of the basic biological processes in which they are involved. The impact of laccase-associated cellular functions on fitness and virulence will be discussed, as will the mutants' potential as therapeutic targets. Vacuolar function, copper homeostasis, mitochondrial function and carbon repression are covered.     

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.     

Molecular characterization and evaluation of virulence factors of Cryptococcus laurentii and Cryptococcus neoformans strains isolated from external hospital areas

Cryptococcosis is a common opportunistic fungal infection that is mainly caused by the species Cryptococcus neoformans and Cryptococcus gattii, but there have recently been several reports of infection by non-neoformans Cryptococcus species. The aims of this study were to genetically characterize Cryptococcus spp. isolated from external hospital areas in Minas Gerais State, Brazil, and to evaluate their pathogenic potential, analyzing their phospholipase and melanin production and the capacity for capsule enlargement. Seventy-three different samples were collected: 62 from bird droppings and 11 from tree detritus. C.?neoformans alone was isolated from 43.8% of the samples, Cryptococcus laurentii alone from 23.3% and both fungi were found together in 10.9%. C. laurentii was exclusively isolated from 45% (5/11) of the tree samples (Anacardium occidentale, Guazuma ulmifolia, Mangifera indica and Ficus benjamina). Among the 51 C. neoformans isolates, 47 were classified as type VNI and four as type VNII. All of the C. neoformans isolates were of MATα type. Among the 21 isolates of C. laurentii genotyped using the URA5-RFLP technique, 16 amplified a 1.6kb amplicon which produced a specific restriction profile in 15 isolates. In C.?neoformans, 76.4% of the isolates were capable of capsule enlargement in the induction medium and 92.1% were phospholipase producers. In C. laurentii, 7.4% of the isolates were capable of capsule enlargement and 85.1% were phospholipase producers. Characterization of the genotypes and the pathogenic potential of the Cryptococcus spp. isolates studied may contribute towards better understanding of the epidemiology of cryptococcosis and the ecology of agents causing this disease in our region.     

The F-Box protein Fbp1 regulates sexual reproduction and virulence in Cryptococcus neoformans

Cryptococcus neoformans is the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Although fbp1 mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, as fbp1 mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating between fbp1 mutants, even though normal dikaryotic hyphae were observed during mating. In vitro assays of stress responses revealed that fbp1 mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in both Saccharomyces cerevisiae and C. neoformans via its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence in C. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.     

Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice

A double mutant of Cryptococcus neoformans which lacked the ability to produce melanin (Mel-) on media containing diphenols and failed to grow at 37 degrees C (temperature sensitive, Tem-) was obtained by UV irradiation and subsequent cloning. The mutant showed two lesions in melanogenesis in that it lacked the active transport system for diphenolic compounds and also lacked phenoloxidase. Ultrastructures of the mutant and wild-type cells grown on a medium with or without L-dopa showed that only the wild-type cells grown on L-dopa medium formed a dark cell wall layer, presumably containing melanin. The mutant was crossed with a wild type, and the phenotypes of the progeny were analyzed. The analysis showed no linkage between the mating type and either Mel or Tem loci, but loose linkage was seen between Mel and Tem loci. The progeny, Mel+ Tem+, Mel+ Tem-, Mel- Tem+, and Mel- Tem-, were studied for their virulence in mice. Only Mel+ Tem+ types killed mice with an inoculum of 5 X 10(5) cells within 50 days.     

Multiple roles of Ypd1 phosphotransfer protein in viability, stress response, and virulence factor regulation in Cryptococcus neoformans

Ypd1 is a key phosphorelay protein that controls eukaryotic two-component systems, but its function in Cryptococcus neoformans is not known. Here, we report that Ypd1 is required for the viability of C. neoformans via the Hog1 mitogen-activated protein kinase (MAPK) pathway but plays multiple cellular roles in both a Hog1-dependent and -independent manner.     

Self-aggregation of Cryptococcus neoformans capsular glucuronoxylomannan is dependent on divalent cations

The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca(2+) in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules.     

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.     

Role of laccase in the biology and virulence of Cryptococcus neoformans

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.