A Cytochrome B5-Like Heme/Steroid Binding Domain Protein, PlCB5L1, Regulates Mycelial Growth, Pathogenicity and Oxidative Stress Tolerance in Peronophythora litchii

As an electron transport component, cytochrome b5 is an essential component of the Class II cytochrome P450 monooxygenation system and widely present in animals, plants, and fungi. However, the roles of Cyt-b5 domain proteins in pathogenic oomycetes remain unknown. Peronophythora litchii is an oomycete pathogen that causes litchi downy blight, the most destructive disease of litchi. In this study, we identified a gene, designated PlCB5L1, that encodes a Cyt-b5 domain protein in P. litchii, and characterized its function. PlCB5L1 is highly expressed in the zoospores, cysts, germinated cysts, and during early stages of infection. PlCB5L1 knockout mutants showed reduced growth rate and β-sitosterol utilization. Importantly, we also found that PlCB5L1 is required for the full pathogenicity of P. litchii. Compared with the wild-type strain, the PlCB5L1 mutants exhibited significantly higher tolerance to SDS and sorbitol, but impaired tolerance to cell wall stress, osmotic stress, and oxidative stress. Further, the expression of genes involved in oxidative stress tolerance, including peroxidase, cytochrome P450, and laccase genes, were down-regulated in PlCB5L1 mutants under oxidative stress. This is the first report that a Cyt-b5 domain protein contributes to the development, stress response, and pathogenicity in plant pathogenic oomycetes.

Up-Regulated Galectin-1 in Angiostrongylus Cantonensis L5 Reduces Body Fat and Increases Oxidative Stress Tolerance

Background: Angiostrongylus cantonensis L5, parasitizing in human cerebrospinal fluid, leads to eosinophilic meningitis, which is attributed to tissue inflammatory responses caused primarily by high percentage of eosinophils. Eosinophils are also involved in helminthic killing, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival. In previous study, we have demonstrated the extracellular function of Acan-Gal-1 in inducing the apoptosis of macrophages. And here, the intracellular functions of Acan-Gal-1 were investigated with the aim to further reveal the mechanism of A. cantonensis L5 worms surviving in the central nervous system of human from inflammatory responses. Methods: Bioinformatics were used to analyse the structural characterisation of Acan-Gal-1; qRT-PCR and microinjection were performed to detect the expression patterns of Acan-gal-1; microinjection was performed to construct transgenic worms; oxidative stress assay and Oil Red O fat staining were used to determine the functions of Acan-Gal-1.Results: The results showed that Acan-Gal-1 was expressed ubiquitously and mainly localized in cuticle, and it was up-regulated in both L5 and adult worm. N2 worms expressing pCe-Acan-gal-1::Acan-gal-1::rfp, with lipid deposition reduced, were significantly resistant to oxidative stress. lec-1 mutant worms, with lipid deposition increased, showed susceptible to oxidative stress, and this phenotype could be rescued by expressing pCe-Acan-gal-1::Acan-gal-1::rfp. And fat-6;fat-7 double-mutant worms expressing pCe-Acan-gal-1::Acan-gal-1::rfp showed no significant changes in oxidative stress tolerance.Conclusion: In C. elegans worms, up-regulated Acan-Gal-1 plays a defensive role against damage due to oxidative stress for worm survival through reducing fat deposition. And this might indicate the mechanism of A. cantonensis L5 worms, with Acan-Gal-1 up-regulated, surviving in the central nervous system of human from immune attack of Eosinophil.

Oxidative stress tolerance contributes to heterologous protein production in Pichia pastoris

Background Pichia pastoris (syn. Komagataella phaffii) is an important yeast system for heterologous protein expression. A robust P. pastoris mutant with oxidative and thermal stress cross-tolerance was acquired in our previous study. The robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions. Results In this study, we found that the robust mutant not only can express a high level of lipase, but also can express a high level of other heterogeneous proteins (e.g., green fluorescence protein) under methanol induction conditions. Additionally, the intracellular reactive oxygen species (ROS) levels in the robust mutant were lower than that in the WT under methanol induction conditions. To figure out the difference of cellular response to methanol between the WT and the robust mutant, RNA-seq was detected and compared. The results of RNA-seq showed that the expression levels of genes related to antioxidant, MAPK pathway, ergosterol synthesis pathway, transcription factors, and the peroxisome pathway were upregulated in the robust mutant compared to the WT. The upregulation of these key pathways can improve the oxidative stress tolerance of strains and efficiently eliminate cellular ROS. Hence, we inferred that the high heterologous protein expression efficiency in the robust mutant may be due to its enhanced oxidative stress tolerance. Promisingly, we have indeed increased the expression level of lipase up to 1.6-fold by overexpressing antioxidant genes in P. pastoris. Conclusions This study demonstrated the impact of methanol on the expression levels of genes in P. pastoris and emphasized the contribution of oxidative stress tolerance on heterologous protein expression in P. pastoris. Our results shed light on the understanding of protein expression mechanism in P. pastoris and provided an idea for the rational construction of robust yeast with high expression ability.