The Siderophore Transporter Sit1 Determines Susceptibility to the Antifungal VL-2397.

VL-2397 (previously termed ASP2397) is an antifungal, aluminum-chelating cyclic hexapeptide with a structure analogous to that of ferrichrome-type siderophores, whereby replacement of aluminum by iron was shown to decrease the antifungal activity of this compound. Here, we found that inactivation of an importer for ferrichrome-type siderophores, termed Sit1, renders Aspergillus fumigatus resistant to VL-2397. Moreover, expression of the endogenous sit1 gene under the control of a xylose-inducible promoter (to uncouple sit1 expression from iron repression) combined with C-terminal tagging with a fluorescent protein demonstrated localization of Sit1 in the plasma membrane and xylose-dependent VL-2397 susceptibility. This underlines that Sit1-mediated uptake is essential for VL-2397 susceptibility. Under xylose-induced sit1 expression, VL-2397 also retained antifungal activity after replacing aluminum with iron, which demonstrates that VL-2397 bears antifungal activity independent of cellular aluminum importation. Analysis of sit1 expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of sit1 expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to A. fumigatus and Candida glabrata, Saccharomyces cerevisiae displays intrinsic resistance to VL-2397 antifungal activity. However, expression of sit1 from A. fumigatus, or its homologue from C. glabrata, resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of S. cerevisiae is based on lack of uptake and that A. fumigatus, C. glabrata, and S. cerevisiae share an intracellular target for VL-2397.

Expression, Purification, and Biochemical Characterization of Human Afamin.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.

Evaluation of sugar-cane vinasse treated with Pleurotus sajor-caju utilizing aquatic organisms as toxicological indicators.

Toxicity tests with aquatic organisms constitute an effective tool in the evaluation, prediction and detection of the potential effect of pollutants from environmental samples in living organisms. Vinasse, a highly colored effluent, is a sub-product rich in nutrients, mainly organic matter, with high pollutant potential when disposed in the environment. Assays for vinasse decolorization were performed using the fungus Pleurotus sajor-caju CCB020 in vinasse biodegradation study, were occurred reductions of 82.8% in COD, 75.3% in BOD, 99.2% in the coloration and 99.7% in turbidity. The vinasse toxicity reduction was determined by the exposition to the following organisms: Pseudokirchneriella subcapitata, Daphnia magna, Daphnia similis and Hydra attenuata. This work concluded that the systematic combination of P. sajor-caju and vinasse can be applied in the bioprocess of color reduction and degradation of complex vinasse compounds, with reduction in the toxicity and improving its physical-chemical properties.

Sequential degradation of raw vinasse by a laccase enzyme producing fungus Pleurotus sajor-caju and its ATPS purification.

This study evaluated simultaneously the raw vinasse degradation, an effluent from the sugar-alcohol industry, the laccase production by Pleurotus sajor-caju and its purification using aqueous two-phase systems (ATPS). To improve laccase production, different concentrations of inducers (ethanol and CuSO4) were added. The higher laccase production promoted an increase of 4-fold using 0.4 mM of CuSO4 as inducer, with maximum enzymatic activity of 539.3 U/L on the 3rd day of fermentation. The final treated vinasse had a decolorization of 92% and turbidity removal of 99% using CuSO4. Moreover, the produced laccase was then purified by ATPS in a single purification step, reaching 2.9-fold and recovered ≈ 99,9 %, in the top phase (PEG-rich phase) using 12 wt% of PEG 1500 + 20 wt% of citrate buffer + enzyme broth + water, at 25 °C. Thus, an integrated process of vinasse degradation, laccase production and purification with potential industrial application was proposed.

Pulp wash: a new source for production of ligninolytic enzymes and biomass and its toxicological evaluation after biological treatment.

Pulp wash was used as substrate for the activity of ligninolytic enzymes of the fungus Pleurotus sajor-caju. Activity of laccase (Lac) and manganese peroxidase (MnP) as well as fungal biomass occurred under four conditions: different pulp wash concentrations, pH variation at the optimal pulp wash concentration, different glucose concentrations, and different concentrations of ammonium nitrate. The best enzyme activity and biomass production were obtained with in natura pulp wash and pH corrected to 5.0 (4884 IU/L Lac; 82 IU/L MnP; 25 g/100 mL biomass). However, the addition of glucose and ammonium nitrate to the pulp wash was not necessary for increasing the enzyme activity and biomass production. Efficient removal of pulp wash chemical oxygen demand (99.66%) and biochemical oxygen demand (83.27%) occurred after the mycoremediation with P. sajor-caju in the optimized conditions. Lactuca sativa L. seeds germination bioassay showed a four-fold reduction in the residue toxicity (EC50 28.72%) after the treatment with the fungus. Our findings are consistent with the notion that pulp wash is an excellent substrate for inducing the activity of ligninolytic enzymes and producing fungal biomass, and that the biological treatment is efficient to reduce effluent toxicity.