Production and action of an Aspergillus phoenicis enzymatic pool using different carbon sources

Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with several applications in the food industry due to its great hydrolytic potential. In this work, the fungus produced high enzymatic levels when cultivated on inexpensive culture media consisting of flakes from different origins such as cassava flour, wheat fibre, crushed soybean, agro-industrial wastes, starch, glucose or maltose. Several enzymatic systems were produced from these carbon sources, but amylase was the most evident, followed by pectinase and xylanase. Traces of CMCases, avicelase, lipase, β-xylosidase, β-glucosidase and α-glucosidase activities were also detected. Amylases were produced on rye flakes, starch, oat flakes, corn flakes, cassava flour and wheat fibre. Significant amylolytic levels were produced in the culture medium with glucose or when this sugar was exhausted, suggesting an enzyme in the constitutive form. Cassava flour, rye, oats, barley and corn flakes were also used as substrates in the hydrolytic reactions, aiming to verify the liberation potential of reducing sugars. Corn flakes induced greater liberation of reducing sugars as compared to the others. Thin layer chromatography of the reaction end products showed that the hydrolysis of cassava flour liberated maltooligosaccharides, but cassava flour and corn, rye, oats and barley flakes were hydrolyzed to glucose. These results suggested the presence of glucoamylase and α-amylase as part of the enzymatic pool of A. phoencis.

Identification of Aspergillus flavus isolates as potential biocontrol agents of aflatoxin contamination in crops

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops

The microtubule plus-end localization of Aspergillus dynein is important for dynein-early-endosome interaction but not for dynein ATPase activation

Cytoplasmic dynein in filamentous fungi accumulates at microtubule plus-ends near the hyphal tip, which is important for minus-end-directed transport of early endosomes. It was hypothesized that dynein is switched on at the plus-end by cargo association. Here, we show in Aspergillus nidulans that kinesin-1-dependent plus-end localization is not a prerequisite for dynein ATPase activation. First, the Walker A and Walker B mutations in the dynein heavy chain AAA1 domain implicated in blocking different steps of the ATPase cycle cause different effects on dynein localization to microtubules, arguing against the suggestion that ATPase is inactive before arriving at the plus-end. Second, dynein from kinA (kinesin 1) mutant cells has normal ATPase activity despite the absence of dynein plus-end accumulation. In kinA hyphae, dynein localizes along microtubules and does not colocalize with abnormally accumulated early endosomes at the hyphal tip. This is in contrast to the colocalization of dynein and early endosomes in the absence of NUDF/LIS1. However, the Walker B mutation allows dynein to colocalize with the hyphal-tip-accumulated early endosomes in the kinA background. We suggest that the normal ability of dyenin to interact with microtubules as an active minus-end-directed motor demands kinesin-1-mediated plus-end accumulation for effective interactions with early endosomes.

Role of complement and Fc{gamma} receptors in the protective activity of the long pentraxin PTX3 against Aspergillus fumigatus

Pentraxin 3 (PTX3) is a soluble pattern recognition molecule playing a nonredundant role in resistance against Aspergillus fumigatus. The present study was designed to investigate the molecular pathways involved in the opsonic activity of PTX3. The PTX3 N-terminal domain was responsible for conidia recognition, but the full-length molecule was necessary for opsonic activity. The PTX3-dependent pathway of enhanced neutrophil phagocytic activity involved complement activation via the alternative pathway; Fc receptor (Fc R) IIA/CD32 recognition of PTX3-sensitized conidia and complement receptor 3 (CR3) activation; and CR3 and CD32 localization to the phagocytic cup. Gene targeted mice (ptx3, FcR common chain, C3, C1q) validated the in vivo relevance of the pathway. In particular, the protective activity of exogenous PTX3 against A fumigatus was abolished in FcR common chain-deficient mice. Thus, the opsonic and antifungal activity of PTX3 is at the crossroad between complement, complement receptor 3-, and Fc R-mediated recognition. Because short pentraxins (eg, C-reactive protein) interact with complement and Fc R, the present results may have general significance for the mode of action of these components of the humoral arm of innate immunity.

Parapharyngeal abscess by Aspergillus flavus in a neutropenic patient with myelogenous leukaemia

This is the first description of an Aspergillus infection of the parapharyngeal space. A high degree of clinical suspicion may be needed due to the non-specific signs and symptoms and repeated tissue biopsies for microbiological and histopathological work up may be warranted.

Calcineurin inhibition or mutation enhances cell wall inhibitors against Aspergillus fumigatus

Calcineurin mutation or inhibition enhanced the antifungal morphological effect of cell wall inhibitors caspofungin or nikkomycin Z against Aspergillus fumigatus. Quantification of 1,3-beta-d-glucan revealed decreased amounts in the calcineurin A (DeltacnaA) mutant. Calcineurin can be an excellent adjunct therapeutic target in combination with other cell wall inhibitors against A. fumigatus.

Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DeltanudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DeltanudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.

The Efficacy of a Novel Microbial 6-Phytase Expressed in Aspergillus oryzae on the Performance and Phosphorus Utilization of Cold- and Warm-Water Fish: Rainbow Trout, Oncorhynchus mykiss , and Nile Tilapia, Oreochromis niloticus

The efficacy and tolerance of a novel microbial 6-phytase were investigated in rainbow trout, Oncorhynchus mykiss, and Nile tilapia, Oreochromis niloticus. Reference diets were sufficient in available phosphorus (P). The test diet limiting in available P was supplemented with phytase at 500, 1000, or 2000 phytase units/kg feed. The enzyme was effective in increasing total P apparent digestibility coefficient in relation to increasing the dose of phytase in rainbow trout and Nile tilapia. Zinc apparent digestibility improved in relation to phytase supplementation in rainbow trout. P release due to phytase supplementation ranged from 0.06 to 0.18% P/kg feed in rainbow trout and from 0.13 to 0.26% P/kg feed in Nile tilapia. A 58-d performance trial was conducted to evaluate tolerance of fish to phytase supplementation. Dietary treatments consisted of a basal diet without phytase or supplemented with 2000 and 200,000 phytase units/kg feed. Results indicate that this novel microbial 6-phytase is well tolerated by fish. Significant improvements for growth as well as feed conversion ratio were observed when the phytase was fed at 2000 phytase units/kg feed. This phytase is proven efficient in releasing P from phytate and could be added when plants are used for fish meal replacement in diets for salmonid and omnivorous fish.

Comprehensive environmental study in high risk areas for cancer patients comparing air and water Aspergillus species samples

Background. Nosocomial invasive aspergillosis (a highly fatal disease) is an increasing problem for immunocompromised patients. Aspergillus spp. can be transmitted via air (most commonly) and by water. ^ The hypothesis for this prospective study was that there is an association between patient occupancy, housekeeping practices, patients, visitors, and Aspergillus spp. loading. Rooms were sampled as not terminally cleaned (dirty) and terminally cleaned (clean). The secondary hypothesis was that Aspergillus spp. positive samples collected from more than one sampling location within the same patient room represent the same isolate. ^ Methods. Between April and October 2004, 2873 environmental samples (713 air, 607 water, 1256 surface and 297 spore traps) were collected in and around 209 “clean” and “dirty” patient rooms in a large cancer center hospital. Water sources included aerosolized water from patient room showerheads, sinks, drains, and toilets. Bioaerosol samples were from the patient room and from the running shower, flushing toilet, and outside the building. The surface samples included sink and shower drains, showerheads, and air grills. Aspergillus spp. positive samples were also sent for PCR, molecular typing (n = 89). ^ Results. All water samples were negative for Aspergillus spp. There were a total of 130 positive culturable samples (5.1%). The predominant species found was Aspergillus niger. Of the positive culturable samples, 106 (14.9%) were air and 24 (3.8%) were surface. There were 147 spore trap samples, and 49.5% were positive for Aspergillus/Penicillum spp. Of the culturable positive samples sent for PCR, 16 were indistinguishable matches. There was no significant relationship between air and water samples and positive samples from the same room. ^ Conclusion. Primarily patients, visitors and staff bring the Aspergillus spp. into the hospital. The high number of A. niger samples suggests the spores are entering the hospital from outdoors. Eliminating the materials brought to the patient floors from the outside, requiring employees, staff, and visitors to wear cover up over their street clothes, and improved cleaning procedures could further reduce positive samples. Mold strains change frequently; it is probably more significant to understand pathogenicity of viable spores than to commit resources on molecular strain testing on environmental samples alone. ^

NOVEL INDUCERS OF GLIOTOXIN PRODUCTION IN ASPERGILLUS FUMIGATUS

Secondary metabolites are produced by numerous organisms and can either be benign to humans or harmful. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often located in subtelomeric regions of the chromosome. These clusters are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Secondary metabolites are also regulated by a variety of factors, including nutritional factors, environmental factors and developmental processes. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal organisms, although no study has demonstrated the direct binding of any protein to a promoter region in the gliotoxin cluster. I report here two novel proteins, GipA, a C2H2 transcription factor and GipB, a hybrid sensor kinase, which are involved in regulating the gliotoxin biosynthetic cluster. GipA plays an important role in gliotoxin production, as high-copy expression of gipA induces gliotoxin biosynthesis and loss of gipA reduces gliotoxin biosynthesis by 50%. GipB is also involved in regulating gliotoxin production, as high-copy expression of gipB induces gliotoxin biosynthesis, but only during certain stages of asexual development. Furthermore, loss of gipB reduces gliotoxin biosynthesis by 10%. Based on data obtained from this project, I propose a model for the regulation of gliA, the efflux pump of the gliotoxin cluster, which involves GipB signaling through both GliZ and GipA. I propose that GliZ and GipA are interdependent, as mutation of the GipA DNA binding site in the gliA promoter negatively affects both GliZ-mediated and GipA-mediated induction of gliA. This is further supported by the fact that GliZ cannot fully induce gliA in the absence of GipA and vice versa. This is the first time that anyone has shown evidence of a protein directly binding to the gliotoxin cluster. Even though biosynthetic clusters are often coordinately regulated, my model raises the possibility that gliA is independently regulated, as the layout of the binding site in the gliA promoter is not present upstream of any other genes in the gliotoxin cluster, except for gliZ.

A protein encoded by a group I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endonuclease

Some group I introns self-splice in vitro, but almost all are thought to be assisted by proteins in vivo. Mutational analysis has shown that the splicing of certain group I introns depends upon a maturase protein encoded by the intron itself. However the effect of a protein on splicing can be indirect. We now provide evidence that a mitochondrial intron-encoded protein from Aspergillus nidulans directly facilitates splicing in vitro. This demonstrates that a maturase is an RNA splicing protein. The protein-assisted reaction is as fast as that of any other known group I intron. Interestingly the protein is also a DNA endonuclease, an activity required for intron mobilization. Mobile elements frequently encode proteins that promote their propagation. Intron-encoded proteins that also assist RNA splicing would facilitate both the transposition and horizontal transmission of introns.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.