磷矿石的微生物风化作用——以一株黑曲霉（Aspergillus niger）为例

以直接和间接作用的方式研究培养基中黑曲霉Aspergillus niger对磷矿粉的风化作用。在装有100目磷矿石粉的液体培养基中接入该菌研究其对磷矿石粉的直接风化作用;同时将装有100目磷矿石粉的透析袋放入液体培养基中再接入该菌研究其对磷矿石粉的间接风化作用。按不同时间取培养液上清液,用电感耦合等离子体-发射光谱仪测定Ca2+、Mg2+、Al3+、Fe3+、K+和Mn2+浓度,用离子色谱法测定H2PO4-、SO42-和Cl-浓度。此外,黑曲霉风化作用后的矿物残渣用电子探针作表面微观形态分析和XRD矿物物相分析。结果表明:黑曲霉对磷矿石粉风化作用的直接作用强度大于间接作用;

Aspergillus e Aspergilose

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex.

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-POLYGALACTURONASE FROM ASPERGILLUS NIGER

A pectinase was identified and isolated from a commercial Aspergillus niger pectinase preparation. The crude enzyme preparation, which was prepared by precipitation of the water extract of the culture of A. niger with ammonium sulfate, was further fractionated by three steps of chromatography, i. e., cation exchange, hydrophobic interaction and onion exchange, to obtain an electrophoretically homogeneous pectinase. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be about 40.4 kDa under both nonreducing and reducing conditions, with the optimum pH at 5.0 and the optimum temperature at 36C. The enzyme was stable at temperatures below 35C. The partial N-terminal ammo acid sequence data analysis of the first 19 amina acids of the obtained pectinase revealed 94.7% and 89.5% homology with two reported pectinases from A. niger.

Effect of Aspergillus fumigatus and Candida albicans on pro-inflammatory response in cystic fibrosis epithelium.

Background
The identification of filamentous fungi and/or yeasts in the airway secretions of individuals with cystic fibrosis (CF) is becoming increasingly prevalent; yet the importance of these organisms in relation to underlying inflammation is poorly defined.

Methods
Cystic fibrosis bronchial epithelial cells (CFBE) and human bronchial epithelial cells (HBE) were co-incubated with Candida albicans whole cells or Aspergillus fumigatus conidia for 24 h prior to the measurement of pro-inflammatory cytokines IL-6 and IL-8 by ELISA.

Results
Treatment of HBE or CFBE with C. albicans whole cells did not alter cytokine secretion. However treatment of CFBE with A. fumigatus conidia resulted in a 1.45-fold increase in IL-6 and a 1.65-fold increase in IL-8 secretion in comparison to basal levels; in contrast there was far less secretion from HBE cells.

Conclusion
Our data indicate that A. fumigatus infection modulates a pro-inflammatory response in CF epithelial cells while C. albicans does not.

A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae

Identification of Rhizoctonia solani, R. oryzae and R. oryzae-sativae, components of the rice sheath disease complex, is extremely difficult and often inaccurate and as a result may hinder the success of extensive breeding programmes throughout Asia. In this study, primers designed from unique regions within the rDNA internal transcribed spacers have been used to develop a rapid PCR-based diagnostic test to provide an accurate identification of the species on rice. Tests on the specificity of the primers concerned showed that they provide the means for accurate identification of the Rhizoctonia species responsible for sheath diseases in rice.

Lovastatin production by Aspergillus terreus using lignocellulose biomass in large scale packed bed reactor

The effect of superficial air velocity on lovastatin production by Aspergillus terreus PL10 using wheat bran and wheat straw was investigated in a 7 l and a 1200 l packed bed reactor. Mass transfer and reaction limitations on bioconversion in the 1200 l reactor was studied based on a central composite design of experiments constructed using the superficial air velocity and solid substrate composition as variables and lovastatin production as response.
The surface response prediction showed a maximum lovastatin production of 1.86 mg g-1 dry substrate on day 5 of the bioconversion process when the reactor was operated using 0.19 vvm airflow rate (23.37 cm min-1 superficial air velocity) and 54% substrate composition (wC). Lovastatin production did not increase significantly with superficial air velocity in the 7 l reactor. Variation in temperature and exit CO2 composition was recorded, and the Damköhler number was calculated for lovastatin production at these two scales. The results showed that in larger reactors mass transfer limitation controlled bioconversion while in smaller reactors bioconversion was controlled by reaction rate limitations. In addition, mass transfer limitations in larger reactors reduced the rate of metabolic heat removal, resulting in hot spots within the substrate bed.

A decrease in bulk water and mannitol and accumulation of trehalose and trehalose-based oligosaccharides define a two-stage maturation process towards extreme stress resistance in ascospores of Neosartorya fischeri (Aspergillus fischeri)

Aspergillus infection in pulmonary cavitating lesions with right atrial myxoma

Cardiac myxomas are rare primary tumors with varied clinical presentations that may pose a diagnostic challenge. Here, we describe the case of a 21-year-old man with multiple cavitating lung lesions with aspergillosis and underlying right atrial myxoma, who presented with hemoptysis and weight loss. He was successfully treated with right atrial myxoma resection and antifungal agents, with no recurrence or complications after one year of follow-up.