Prp8 intein in fungal pathogens: target for potential antifungal drugs

Inteins are self-splicing intervening sequences in proteins, and inteins of pathogenic organisms can be attractive drug targets. Here, we report an intein in important fungal pathogens including Aspergillus fumigatus, Aspergillus nidulans, Histoplasma capsulatum, and different serotypes of Cryptococcus neoformans. This intein is inside the extremely conserved and functionally essential Prp8 protein, and it varies in size from 170 aa in C. neoformans to 819 aa in A. fumigatus, which is caused by the presence or absence of an endonuclease domain and a putative tongs subdomain in the intein. Prp8 inteins of these organisms were demonstrated to do protein splicing in a recombinant protein in Escherichia coli. These findings revealed Prp8 inteins as attractive targets for potential antifungal drugs to be identified using existing selection and screening methods.     

Laboratory Examination of Organic Fungicides Against Zoopathogenic Fungi in Soil

Seven commercial and five experimental organic fungicides were tested against Histoplasma capsulatum, Cryptococcus neoformans, Allescheria boydii, and Sporotrichum schenckii in a series of laboratory studies. Preliminary agar dilution plate tests indicated that Lanstan, DAC 649, DAC 469, DAC 2787, He 3944, maneb, and nabam, in order of decreasing activity, inhibited all test fungi. Terraclor, Dexon, and He 13274 were active only in high concentration. The best fungicidal properties were exhibited by Lanstan, Vapam, and DAC 649; maneb, nabam, DAC 469, and DAC 2787 were fungicidal but to a lesser degree than the former compounds. In subsequent tests against H. capsulatum and C. neoformans in artificially infested soil or by use of the buried plug technique, Lanstan, Vapam, and DAC 649 showed good activity and merit further study.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

氯喹与氟康唑联合对新生隐球菌的体外药敏实验

目的研究氯喹与氟康唑体外联合应用对隐球菌生长作用的影响。方法参考M27-A方案,检测10μmol/L,100μmol/L与1 000μmol/L 3种浓度氯喹与氟康唑联合后对20株菌株的氟康唑对隐球菌最小抑菌浓度与最小杀菌浓度的变化。结果与10μmol/L氯喹组相比,100μmol/L以上浓度的氯喹组可以显著降低氟康唑对隐球菌的M IC与MFC。结论氯喹在体外可以提高氟康唑抗隐球菌的作用,与氟康唑具有协同抗隐球菌的作用。     

Amphotericin Pharmacophobia

Five cases are described in which fear of the possibly hazardous effects of giving amphotericin to patients with kidney disease resulted in death from progressive infection by an amphotericin-sensitive fungus (Cryptococcus neoformans in three cases, Blastomyces dermatitidis in one case, and Histoplasma capsulatum in one case).     

Effect of antilymphocyte serum on animals experimentally infected with Histoplasma capsulatum or Cryptococcus neoformans

The anti-mouse and anti-guinea pig antilymphocyte sera (ALS) prepared for this study were shown to contain cytoxic and leucoagglutinating antibodies, and were capable of producing severe lymphopenia in these animals. Guinea pigs treated weekly with ALS were more susceptible to development of fatal infection when inoculated with Histoplasma capsulatum. No fatalities occurred in guinea pigs infected with equal doses of H. capsulatum but treated with normal rabbit serum (NRS) or saline. The time necessary to reach 50% fatality in mice infected with Cryptococcus neoformans was greatly reduced by pretreatment with ALS in comparison with infected controls treated with NRS or saline. When low dosages were used (0.1 ld(50)), the effect was even more pronounced. Spleen homogenates from mice infected with equal dosages of H. capsulatum and treated with ALS or NRS were cultured. More than 150 times as many organisms were present in the spleens of the ALS-treated group. Similar results were obtained from culturing the lungs and liver. Delayed hypersensitive skin reactions were radically decreased or abrogated in H. capsulatum-infected guinea pigs inoculated intraperitoneally with ALS 12 hr before skin testing with histoplasmin. When ALS was given weekly, the influence on skin reactivity was less notable. Given intradermally, ALS was shown to inhibit the delayed reaction to histoplasmin within a radius of 40 mm.     

In vitro interactions between Blastomyces dermatitidis and other zoopathogenic fungi

The results of in vitro interactions between colonies of Blastomyces dermatitidis and six other zoopathogenic fungi are reported. The interactions were found to range from neutral with Histoplasma capsulatum and Candida albicans to strongly antagonistic with Microsporum gypseum, Pseudallescheria boydii, and Sporothrix schenckii, and including lysis by Cryptococcus neoformans. These observations suggest that interactions between zoopathogenic fungi may be one of the biotic factors likely to influence the occurrence of B. dermatitidis in natural systems.     

Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes

Vesicular secretion of macromolecules has recently been described in the basidiomycete Cryptococcus neoformans , raising the question as to whether ascomycetes similarly utilize vesicles for transport. In the present study, we examine whether the clinically important ascomycete Histoplasma capsulatum produce vesicles and utilized these structures to secrete macromolecules. Transmission electron microscopy (TEM) shows transcellular secretion of vesicles by yeast cells. Proteomic and lipidomic analyses of vesicles isolated from culture supernatants reveal a rich collection of macromolecules involved in diverse processes, including metabolism, cell recycling, signalling and virulence. The results demonstrate that H. capsulatum can utilize a trans-cell wall vesicular transport secretory mechanism to promote virulence. Additionally, TEM of supernatants collected from Candida albicans , Candida parapsilosis , Sporothrix schenckii and Saccharomyces cerevisiae documents that vesicles are similarly produced by additional ascomycetes. The vesicles from H. capsulatum react with immune serum from patients with histoplasmosis, providing an association of the vesicular products with pathogenesis. The findings support the proposal that vesicular secretion is a general mechanism in fungi for the transport of macromolecules related to virulence and that this process could be a target for novel therapeutics.     

Characterization of monoclonal antibody MEST-2 specific to glucosylceramide of fungi and plants

An IgG2a monoclonal antibody anti-glucosylceramide was established and termed MEST-2. High performance thin layer chromatography immunostaining, and solid-phase radioimmunoassay showed that MEST-2 reacts with glucosylceramide from yeast and mycelium forms of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Sporothrix schenckii; from hyphae of Aspergillus fumigatus; and from yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. Studies on the fine specificity of MEST-2 showed that it recognizes the beta-D-glucose residue, and that the 2-hydroxy group present in the fatty acid is an important auxiliary feature for the antibody binding. It was also demonstrated that phosphatidylcholine and ergosterol modulate MEST-2 reactivity to glucosylceramide, by solid-phase radioimmunoassay. Indirect immunofluorescence showed that MEST-2 reacts with the surface of yeast forms of P. brasiliensis, H. capsulatum and S. schenckii. Weak staining of mycelial forms of P. brasiliensis and hyphae of A. fumigatus was also observed. The availability of a monoclonal antibody specific to fungal glucosylceramide, and its potential use in analyzing biological roles attributed to glucosylceramide in fungi are discussed.     

Mammalian model hosts of cryptococcal infection

The rising incidence of serious fungal diseases represents a growing threat to human health. Cryptococcus neoformans, an encapsulated yeast saprophyte with global distribution, has been recognized as an important emerging pathogen. Humans frequently develop asymptomatic or mild infection with C. neoformans, but individuals with impaired host defense systems may develop severe pneumonia and potentially fatal meningoencephalitis. Insight into the biology and virulence of C. neoformans is advancing rapidly and will be propelled even further by the recently completed and published genome sequences for two related strains of C. neoformans serotype D. Several mammalian model hosts including the guinea pig, rabbit, rat, and mouse have been developed for the study of cryptococcosis. The combination of microbial genomics with well-characterized model hosts that are amenable to immunologic and genetic manipulation represents a powerful resource for comprehensive study of cryptococcal disease pathogenesis as well as vaccine and antifungal drug therapy. This review provides an introduction to each mammalian model host and briefly highlights the advantages, limitations, and potential of each system for future research involving cryptococci.     

Chloroquine, a lysosomotropic agent, inhibits zygote formation in yeast

Haploid cells of opposite mating type of Saccharomyces cerevisiae conjugate to form zygote. During the conjugation process, the degradation or reorganization of the cell wall and the fusion of the two plasma membranes take place. Since chloroquine inhibits cellular events associated with the reorganization of the plasma membrane, the effect of the drug on conjugation was studied. Chloroquine at a concentration, at which cell growth was not retarded, inhibited zygote formation, while it did not affect other mating functions, such as sexual agglutination, production of and response to mating pheromone. Cells in a mating culture containing chloroquine formed no "prezygote" suggesting that they were not prepared for entering into fusion process. The inhibitory effect of chloroquine was reversible as cells formed zygote when they were washed after treatment with chloroquine. Zygote formation was unaffected in cells possessing chloroquine within vacuoles after incubation with the drug in complete medium (YPD) at pH 7.5, followed by washing. This suggests that chloroquine inhibits zygote formation by adsorbing to the plasma membrane of S. cerevisiae.     

Antibody-mediated protective immunity in fungal infections

The host response to fungal infection is the result of a complex interaction between the pathogen and the host's innate and adaptive immune system. Cell-mediated immunity is widely considered to be critical for the successful outcome of fungal infections. However, in recent years numerous studies have established that certain antibodies may play an important role in host immunoprotection against pathogenic fungi, through interaction with different cellular targets, such as mannans, heat shock proteins, capsular polysaccharides, surface proteins, and yeast killer toxin receptors, with mechanisms of action sometimes still undefined. This review summarizes the latest findings on the role of different types of antibodies in the antifungal defense against infections caused by epidemiologically important fungi, such as Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and others. New perspectives of antibody-mediated therapy, based on the availability of monoclonal and recombinant antibodies as well as genetically engineered antibody fragments of defined specificity, will be also envisaged and discussed.     

Ocular Complications of Chloroquine Therapy

Ocular complications of long-term chloroquine therapy were observed in 18 of 45 patients so treated. This therapy was used in patients with rheumatoid arthritis, lupus erythematosus, sarcoidosis, discoid lupus and other chronic “collagen disease”. Thirteen patients had reversible corneal opacifications, and seven had irreversible retinal changes, with visual loss and visual field defects. Pathological evidence of chloroquine retinopathy was obtained in one patient. Physicians are therefore warned to use this drug only after careful consideration. If it is used, repeated ocular examinations should include assessment of visual acuity, visual fields on a tangent screen and fundus examination through a dilated pupil.     

Inter-individual variation in the metabolic activation of the antimalarial biguanides

The aryl-biguanides proguanil and chlorproguanil were developed as part of a collaborative programme between ICI and the Liverpool School of Tropical Medicine during the 1940s. The compounds were characterized by their absence of host toxicity. However, the rapid development of parasite resistance to the actions of these drugs and the development of the 4-aminoquinoline, chloroquine, severely limited their use. The subsequent widespread development of parasite resistance to chloroquine, together with the observations that the magnitude of dihydrofolate reductase inhibitor resistance (the site of action of the biguanides) developed to pyrimethamine is not directly correlated with biguanide resistance(1,2). has resulted in renewed interest in these drugs. In particular, proguanil is now the drug of choice for malaria prophylaxis, in combination with chloroquine; used in combination with a suitable sulphonamide, it may be of value in malaria therapy.     

Fungal heat-shock proteins in human disease

Heat-shock proteins (hsps) have been identified as molecular chaperones conserved between microbes and man and grouped by their molecular mass and high degree of amino acid homology. This article reviews the major hsps of Saccharomyces cerevisiae, their interactions with trehalose, the effect of fermentation and the role of the heat-shock factor. Information derived from this model, as well as from Neurospora crassa and Achlya ambisexualis, helps in understanding the importance of hsps in the pathogenic fungi, Candida albicans, Cryptococcus neoformans, Aspergillus spp., Histoplasma capsulatum, Paracoccidioides brasiliensis, Trichophyton rubrum, Phycomyces blakesleeanus, Fusarium oxysporum, Coccidioides immitis and Pneumocystis jiroveci. This has been matched with proteomic and genomic information examining hsp expression in response to noxious stimuli. Fungal hsp90 has been identified as a target for immunotherapy by a genetically recombinant antibody. The concept of combining this antibody fragment with an antifungal drug for treating life-threatening fungal infection and the potential interactions with human and microbial hsp90 and nitric oxide is discussed.     

Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene,CnAFR1, involved in the resistance to fluconazole

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.     

Ocular Complications of Chloroquine Therapy

Long-term therapy with chloroquine can lead to irreversible retinal damage and serious loss of visual acuity and visual field. Not only are the retinal changes irreversible but they may continue to progress after discontinuance of the drug. Work is proceeding on the development of electrophysiological techniques for early detection of toxic effect before significant retinal damage occurs. Another possibility for minimizing the toxic effect of chloroquine is the use of other drugs which increase the excretion of chloroquine from the body.     

p53 is required for chloroquine-induced atheroprotection but not insulin sensitization

An intact genotoxic stress response appears to be atheroprotective and insulin sensitizing. ATM, mutated in ataxia telangiectasia, is critical for the genotoxic stress response, and its deficiency is associated with accelerated atherosclerosis and insulin resistance in humans and mice. The antimalarial drug chloroquine activates ATM signaling and improves metabolic phenotypes in mice. p53 is a major effector of ATM signaling, but it is unknown if p53 is required for the beneficial effects of chloroquine. We tested the hypothesis that the cardiometabolic effects of chloroquine are p53-dependent. ApoE-null mice with or without p53 were treated with low-dose chloroquine or saline in the setting of a Western diet. After 8 weeks, there was no p53-dependent or chloroquine-specific effect on serum lipids or body weight. Chloroquine reduced plaque burden in mice wild-type for p53, but it did not decrease lesion extent in p53-null mice. However, chloroquine improved glucose tolerance, enhanced insulin sensitivity, and increased hepatic Akt signaling regardless of the p53 genotype. These results indicate that atheroprotection induced by chloroquine is p53-dependent but the insulin-sensitizing effects of this agent are not. Discrete components of the genotoxic stress response might be targeted to treat lipid-driven disorders, such as diabetes and atherosclerosis.     

pH-dependent uptake and macrofilaricidal effects of chloroquine on adult filarial parasites in vitro

Uptake and macrofilaricidal effects of chloroquine (CQ) and other aminoquinolines were found to be highly pH dependent in Brugia pahangi, Acanthocheilonema viteae, Onchocerca volvulus, and Dirofilaria immitis. Using [3H]CQ, it was found that all of the parasites took up more drug under alkaline conditions (RPMI 1640 at pH 8.4) than in neutral (pH 7.4) or acidic (pH 6.8) media. Differences were seen in the amount of drug taken up among the filariae studied. B. pahangi and A. viteae took up 7 times more chloroquine per milligram of tissue than did O. volvulus, and 30 times more than D. immitis during a 60-min incubation period at pH 8.4. Sensitivity to the aminoquinolines also increased with increasing media pH, and was measured using parasite motility as an indicator of drug efficacy. Potency of chloroquine against B. pahangi increased 100-fold at pH 8.4 compared to pH 7.4. A. viteae and O. volvulus showed similar sensitivity to chloroquine compared to B. pahangi; D. immitis was less sensitive. While uptake of chloroquine was linear from pH 6.8 to 8.4, B. pahangi was unaffected by 32 microM of the drug below pH 7.6; at any pH above this, motility of this parasite was completely inhibited. Calculations of the internal pH of this parasite indicated that it shifted upwards significantly with changes in media pH. It was concluded that these shifts in internal pH may render parasites more sensitive to the effects of chloroquine.