An objective approach to model reduction: Application to the Sirius wheat model.

An existing simulation model of wheat growth and development, Sirius, was evaluated through a systematic model reduction procedure. The model was automatically manipulated under software control to replace variables within the model structure with constants, individually and in combination. Predictions of the resultant models were compared to growth analysis observations of total biomass, grain yield, and canopy leaf area derived from 9 trials conducted in the UK and New Zealand under optimal, nitrogen limiting and drought conditions. Model performance in predicting these observations was compared in order to evaluate whether individual model variables contributed positively to the overall prediction. Of the 1 1 1 model variables considered 16 were identified as potentially redundant. Areas of the model where there was evidence of redundancy were: (a) translocation of biomass carbon to grain; (b) nitrogen physiology; (c) adjustment of air temperature for various modelled processes; (d) allowance for diurnal variation in temperature; (e) vernalisation (f) soil nitrogen mineralisation (g) soil surface evaporation. It is not suggested that these are not important processes in real crops, rather, that their representation in the model cannot be justified in the context of the analysis. The approach described is analogous to a detailed model inter-comparison although it would be better described as a model intra-comparison as it is based on the comparison of many simplified forms of the same model. The approach provides automation to increase the efficiency of the evaluation and a systematic means of increasing the rigour of the evaluation.

Identification and characterization of an SKN7 homologue in Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated fungal pathogen that primarily infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis. The capacity of C. neoformans to subvert host defenses and disseminate by intracellular parasitism of alveolar macrophages in the immune-compromised host has led to studies to evaluate genes associated with C. neoformans resistance to oxidative stress. In the present study, we identify and characterize a C. neoformans homologue to SKN7, a transcription factor in Saccharomyces cerevisiae that regulates the oxidative stress response, cell cycle, and cell wall biosynthesis. To examine the contribution of SKN7 in the pathogenesis of fungal infections, we created skn7 mutants via targeted disruption. The skn7 mutants were observed to be more susceptible to reactive oxygen species in vitro and were significantly less virulent than the wild-type strain and a reconstituted strain as measured by cumulative survival in the mouse inhalational model. The Skn7 protein was observed to be important for expression of thioredoxin reductase in response to oxidative challenge. Interestingly, skn7 mutants were also observed to flocculate following in vitro culture, a novel phenotype not observed in skn7 mutants derived from other fungi. These findings demonstrate that SKN7 contributes to the virulence composite but is not required for pathogenicity in C. neoformans. In addition, flocculation of C. neoformans skn7 mutants suggests a potentially unique function of SKN7 not previously observed in other cryptococcal strains or skn7 mutants.

The impact of culture isolation of Aspergillus species: a hospital-based survey of aspergillosis.

The term "aspergillosis" comprises several categories of infection: invasive aspergillosis; chronic necrotizing aspergillosis; aspergilloma, or fungus ball; and allergic bronchopulmonary aspergillosis. In 24 medical centers, we examined the impact of a culture positive for Aspergillus species on the diagnosis, risk factors, management, and outcome associated with these diseases. Most Aspergillus culture isolates from nonsterile body sites do not represent disease. However, for high-risk patients, such as allogeneic bone marrow transplant recipients (60%), persons with hematologic cancer (50%), and those with signs of neutropenia (60%) or malnutrition (30%), a positive culture result is associated with invasive disease. When such risk factors as human immunodeficiency virus infection (20%), solid-organ transplantation (20%), corticosteroid use (20%), or an underlying pulmonary disease (10%) are associated with a positive culture result, clinical judgment and better diagnostic tests are necessary. The management of invasive aspergillosis remains suboptimal: only 38% of patients are alive 3 months after diagnosis. Chronic necrotizing aspergillosis, aspergilloma, and allergic bronchopulmonary aspergillosis have variable management strategies and better short-term outcomes.

Fungal phospholipase activity and susceptibility to lipid preparations of amphotericin B.

It has been postulated that phospholipases of fungal origin can affect in vitro susceptibility testing of amphotericin B lipid complex (ABLC). We used specific phospholipase-deficient mutants of Candida albicans and Cryptococcus neoformans in susceptibility testing and demonstrated that extracellular fungal phospholipase activity does not influence the in vitro susceptibilities of these two fungi to ABLC.

Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans.

Cyclophilin A is the target of the immunosuppressant cyclosporin A (CsA) and is encoded by a single unique gene conserved from yeast to humans. In the pathogenic fungus Cryptococcus neoformans, two homologous linked genes, CPA1 and CPA2, were found to encode two conserved cyclophilin A proteins. In contrast to Saccharomyces cerevisiae, in which cyclophilin A mutations confer CsA resistance but few other phenotypes, cyclophilin A mutations conferred dramatic phenotypes in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth, mating, virulence and CsA toxicity. The Cpa1 and Cpa2 proteins also have divergent functions. cpa1 mutants are inviable at 39 degrees C and attenuated for virulence, whereas cpa2 mutants are viable at 39 degrees C and fully virulent. cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence. Cyclophilin A active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures, suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function.

Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity.

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

The use of flow cytometry as a tool for monitoring filament formation of fungi.

Flow cytometry (FC) has the ability to discriminate a variety of cell parameters including cell size and complexity, and fluorescence intensity. As yeast cells or fungal spores germinate they undergo a morphological transformation from round oval shaped cells to elongate filamentous forms. To date, monitoring these events has been performed using microscopic examination. Microscopic examination is a labor intensive process that examines a very small percentage of the total cell population. We have developed a method using FC that is rapid, simple to perform, and reproducible. The major advantages of FC include analysis of a larger number of cells, increased objectivity due to nonselective measurements of all cells in the population studied, and the computer related data analysis capability of the flow cytometer.

A new dominant selectable marker for use in Cryptococcus neoformans.

Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections. There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetyltransferase gene (nat1) from Streptomyces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of natl with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1,000 transformants microg(-1) of DNA and 20 transformants microg(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.

Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans.

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

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.

Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus.

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. C. neoformans occurs in four predominant serotypes (A, B, C, and D), which represent different varieties or species. Rare clinical and environmental isolates with an unusual AD serotype have been reported and suggested to be diploid. We found by fluorescence-activated cell sorter analysis that serotype AD strains are aneuploid or diploid. PCR analysis with primers specific for serotype A or D alleles of the CNA1, CLA4, and GPA1 genes revealed that both alleles are often present in serotype AD strains. PCR analysis with primers specific for genes in the MATa or MATalpha mating-type loci revealed that serotype AD strains are heterozygous for the mating-type locus. Interestingly, in several serotype AD strains, the MATalpha locus was derived from the serotype D parent and the MATa locus was inherited from a serotype A parent that has been thought to be extinct. Basidiospores from a self-fertile serotype AD strain bearing the putative serotype A MATa locus showed a very low viability ( approximately 5%), and no fertile serotype A MATa strain could be recovered. Serotype AD strains were virulent in a murine model. Hybrid AD strains could readily be isolated following a laboratory cross between a serotype A strain and a serotype D strain. In summary, serotype AD strains of C. neoformans are unusual aneuploid or diploid strains that result from matings between serotype A and D strains. Self-fertile isolates fail to undergo normal meiosis because of genetic divergence. Our findings further suggest that serotype A MATa strains may exist in nature.

Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving mating between haploid MATa and MATalpha cells. Here we describe the isolation of part of the MATa mating-type locus encoding a Ste20 kinase homolog, Ste20a. We show that the STE20a gene cosegregates with the MATa mating type in genetic crosses, maps within the mating-type locus on a 1.8-Mb chromosome, and is allelic with the MATalpha locus. We identify the first MATa isolate of the most common pathogenic variety of C. neoformans (serotype A, variety grubii) which had been thought to be extinct. This serotype A MATa strain is sterile, fails to produce mating pheromone, and is less virulent than a serotype A MATalpha strain in an animal model. Our studies illustrate an association of mating type with virulence and suggest that, like Candida albicans, pathogenic isolates of C. neoformans may be largely asexual.

Syringe exchanges: a public health response to problem drug use.

This paper presents the findings of the first ever Irish follow-up study to establish the effectiveness of syringe exchanges as a harm reduction strategy in the context of public health. The study was conducted in collaboration with 370 injecting drug users who attended the Merchant's Quay Project's Health Promotion Unit between May 1st 1997 and October 31st 1998. Data was collected from respondents by means of a structured interview at first visit and three-months after initial contact. Evidence from the study demonstrates that syringe exchanges play an important contributing role in significantly reducing the numbers reporting both the use of heroin as a primary drug (n=41; McNemar x2=10.1;p<0.01), and its frequency of use (n=70; McNemar x2=4.13;p<0.05) at follow-up. Moreover, there were significant reductions in the reported borrowing (n=60; McNemar x2=10.1;p<0.01) and lending (n=42: McNemar x2=7.9;p<0.01) of used injecting equipment. There was however no significant change in the reported sharing of injecting paraphernalia or in levels of reported condom use. The results of this study illustrate that low threshold services such as the Health Promotion Unit have the ability to attract injecting drug users not in contact with other drug treatment services. Furthermore, this type of service has been shown to be effective in maintaining contact with injecting drug users and promoting safer drug use among attendees. Even though the distribution of sterile injecting equipment reduces the situational sharing, this is hampered by the limited availability of sterile injecting equipment in Dublin. There is a need to continue to develop strategies to prevent HIV and hepatitis C transmission. One such approach would be to develop effective outreach as a means of reaching greater numbers of drug users earlier in their injecting histories. Other areas for future HIV and HCV prevention include discouraging the transition from smoking to injecting, and among those who inject, encouraging further reductions in sharing, sustaining risk reduction over time, and encouraging the adoption of safer sexual behaviour.

Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans.

Calcineurin is the conserved target of the immunosuppressants cyclosporin A and FK506. Using the yeast two-hybrid system, we identified a novel calcineurin binding protein, CBP1, from the pathogenic fungus Cryptococcus neoformans. We show that CBP1 binds to calcineurin in vitro and in vivo, and FKBP12-FK506 inhibits CBP1 binding to calcineurin. Cryptococcus neoformans cbp1 mutant strains exhibit modest defects in growth under stress conditions and virulence, similar to but less severe than the phenotypes of calcineurin mutants. Saccharomyces cerevisiae mutants lacking the CBP1 homolog RCN1 are, like calcineurin mutants, sensitive to lithium cation stress. CBP1 shares a central peptide sequence motif, SPPxSPP, with related proteins in S.CEREVISIAE:, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans and humans, and peptides containing this motif altered calcineurin activity in vitro. Interestingly, the human CBP1 homolog DSCR1 is encoded by the Down's syndrome candidate region interval on chromosome 21, is highly expressed in the heart and central nervous system, and may play a role in calcineurin functions in heart development, neurite extension and memory.

Synthesis of polymerized melanin by Cryptococcus neoformans in infected rodents.

The ability of Cryptococcus neoformans to synthesize polymerized melanin in vitro has been associated with virulence, but it is unclear whether this fungus synthesizes polymerized melanin during infection. To study this question, we used two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunohistochemical studies of C. neoformans-infected rodents, and the other sought to isolate fungal melanin from infected tissues. Digestion of in vitro-melanized C. neoformans cells with proteases, denaturant, and hot concentrated acid yields melanin particles that retain the shape of fungal cells and are therefore called melanin ghosts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluorescence analyses of lung and brain tissues of rodents infected with wild-type melanin-producing (Mel(+)) C. neoformans strains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections were performed with mutant albino (Mel(-)) C. neoformans strains. Particles with striking similarity to melanin ghosts were recovered after digestion of lung and brain tissues from Mel(+) C. neoformans-infected rodents and were reactive with the MAbs to melanin. No particles were recovered from tissues infected with Mel(-) C. neoformans. A Mel(+) C. neoformans strain grown on lung or brain homogenate agar became lightly pigmented and also yielded particles similar to melanin ghosts upon digestion, providing additional evidence that lung and brain tissues contain substrate for C. neoformans melanization. These results demonstrate that C. neoformans synthesizes polymerized melanin during infection, which has important implications for pathogenesis and antifungal drug development.

Urease as a virulence factor in experimental cryptococcosis.

Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.

Comparison of the roles of calcineurin in physiology and virulence in serotype D and serotype A strains of Cryptococcus neoformans.

The calcineurin gene was cloned and disrupted in serotype D strains of Cryptococcus neoformans. Serotype A and serotype D calcineurin mutants were inviable at 37 degrees C and avirulent in mice, whereas only serotype A mutants were cation stress sensitive. Thus, calcineurin plays conserved and divergent roles in serotype A and serotype D strains.

The STE12alpha homolog is required for haploid filamentation but largely dispensable for mating and virulence in Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised hosts. The organism has a known sexual cycle, and strains of the MATalpha mating type are more virulent than isogenic MATa strains in mice, and they are more common in the environment and infected hosts. A C. neoformans homolog of the STE12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae and Candida albicans was identified previously, found to be encoded by a novel region of the MATalpha mating type locus, and shown to enhance filamentous growth when overexpressed. We have disrupted the C. neoformans STE12 gene in a pathogenic serotype A isolate. ste12 mutant strains exhibit a severe defect in filamentation and sporulation (haploid fruiting) in response to nitrogen starvation. In contrast, ste12 mutant strains have only modest mating defects and are fully virulent in two animal models compared to the STE12 wild-type strain. In genetic epistasis experiments, STE12 functions in a MAP kinase cascade to regulate fruiting, but not mating. Thus, the C. neoformans STE12alpha transcription factor homolog plays a specialized function in haploid fruiting, but it is dispensable or redundant for mating and virulence. The association of the MATalpha locus with virulence may involve additional genes, and other transcription factors that regulate mating and virulence remain to be identified.

Association of plasma levels of human immunodeficiency virus type 1 RNA and oropharyngeal Candida colonization.

The pathophysiology of oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV) type 1 is poorly understood. Association between oropharyngeal yeast carriage and various clinical factors in HIV-1-infected patients was studied in 83 patients with no clinical evidence of thrush and no recent antifungal use. Of the clinical factors measured, the only correlate of yeast colonization was with plasma HIV-1 RNA levels (P=.001), whereas the correlation with CD4 cell count was poor (P=.36). By multivariable regression modeling, plasma HIV-1 RNA was the only parameter that correlated with the extent of colonization with Candida infection (P=.003). These data indicate that the presence and amount of asymptomatic oropharyngeal yeast carriage in persons with HIV-1 infection is more significantly correlated with plasma HIV-1 RNA levels than with CD4 cell count. Further studies on the effect of HIV-1 on oropharyngeal yeast colonization, infection, and local immunity are warranted.