Antifungal activity of Saccharomyces cerevisiae and assessment of ochratoxigenic load on currants by means of Real Time PCR.

Currants are prone to contamination by ochratoxin during cultivation, processing and storage conditions. Saccharomyces cerevisiae is considered to be among the main species of grape yeast flora able to control antagonistic fungi. In this study, the potential of S. cerevisiae Y33 was investigated to inhibit the growth of several fungal species indigenous to the microbiota of grapes. Moreover, the efficacy of this yeast species was investigated to inhibit OTA by toxin producing fungi both in vitro and in situ. For this purpose thirty-five different fungal species, belonging to the genera Aspergillus, Penicillium, Cladosporium, Fusarium and Alternaria interacted in vitro with S. cerevisiae on Malt Extract agar plates, stored at 25 °C for 14 days. Results showed that the highest OTA producer A. carbonarius F71 was inhibited more than 99% from day 7, in contrast to A. niger strains that presented enhanced OTA production at day 14 due to interaction with S. cerevisiae Y33. Additionally, the antifungal potential of the selected yeast was also studied in situ on currants subjected to different treatments and stored at 25 °C for 28 days. Microbiological analysis was undertaken for the enumeration of the bacterial and fungal flora, together with OTA determination at 7 and 21 days. To quantify A. carbonarius on all treated currant samples, molecular analysis with Real Time PCR was employed. A standard curve was prepared with A. carbonarius DNA. The efficiency of the curve was estimated to 10.416, the slope to -3.312 and the range of haploid genome that could be estimated was from 1.05 to 105∙105. The amount of A. carbonarius DNA in all treated currants samples, where the fungus was positively detected, ranged from as low as 0.08 to 562 ng DNA/g currants. The antifungal activity of S. cerevisiae Y33 was observed in all studied cases, causing inhibition of fungal growth and OTA production.

Assessment of the pulmonary adaptive immune response to Cladosporium cladosporioides infection using an experimental mouse model.

Cladosporium cladosporioides causes asthma and superficial and deep infections, mostly in immunodeficient individuals and animals. This study aimed to investigate whether C. cladosporioides spores can enter the lungs through pulmonary circulation and influence pulmonary immune response. We intravenously injected mice with C. cladosporioides spore suspension and conducted several assays on the lungs. Pulmonary hemorrhage symptoms and congestion were most severe on days 1, 2, and 3 post-inoculation (PI). Extensive inflammatory cell infiltration occurred throughout the period of infection. More spores and hyphae colonizing the lungs were detected on days 1, 2, and 3 PI, and fewer spores and hyphae were observed within 21 d of infection. Numerous macrophages, dendritic cells, and neutrophils were observed on day 5 PI, along with upregulation of CD54, an intercellular adhesion molecule. Th1 and Th2 cells increased after infection; specifically, Th2 cells increased considerably on day 5 PI. These results suggest that days 2 and 5 PI represent the inflammatory peak in the lungs and that the Th2 and Th1 signaling pathways are potentially involved in pulmonary immune responses. In conclusion, the further adaptive immune responses played important roles in establishing effective pulmonary immunity against C. cladosporioides systemic infections based on innate immune responses.

Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris.

Cost effective 13C/15N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The 13C/15N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut+) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (MutS) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for 13C-isotope labeling can be substantially reduced using the Mut+ strain compared to the MutS strain, as no 13C3-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for 15N-labeling by an optimal supply of 10 g/L (15NH4)2SO4 every 24 h.