Project description:The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of posttranscriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs. Two-color experimental design comparing cells with a double-knockout of the CCR4 genes to cells with a reintegrated CCR4 gene.
Project description:The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of posttranscriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.
Project description:Eliminating drug-tolerant persister (DTP) cells remain a significant challenge in overcoming resistance to tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung adenocarcinoma. Utilizing single-cell RNA sequencing, we identify a novel RGS5+MYL9+ cancer associated fibroblasts (CAFs) subset. This subset is linked to TKI resistance and correlates with poorer prognosis. The RGS5+MYL9+ CAFs are spatially positioned close to DTP cells, a proximity facilitated by CCL11 recruitment from DTP cells. TKIs induced mitochondria ROS activates Rho GTPase 1 (Miro1) and RhoA of tumor cells, subsequently promoting the formation of tunneling nanotube connections with neighboring RGS5+MYL9+ CAFs. These CAFs play a protective role for DTP cells, aiding in the transfer of damaged mitochondria from tumor cells through these nanotubes. By employing the Rho kinase inhibitor Fasudil to block this transfer of damaged mitochondria, we observed a significant reduction in persistent tolerance to Osimertinib in a xenograft mouse model. Our study highlights the critical role of RGS5+MYL9+ CAFs in mediating resistance to EGFR-TKIs and suggests potential therapeutic strategies for overcoming this challenge.
Project description:With the growing limitations on arable land, alfalfa (a widely cultivated, low-input forage) is now being selected to extend cultivation into saline lands for low-cost biofeedstock purposes. Here, minerals and transcriptome profiles were compared between two new salinity-tolerant North American alfalfa breeding populations and a more salinity-sensitive Western Canadian alfalfa population grown under hydroponic saline conditions. All three populations accumulated two-fold higher sodium in roots than shoots as a function of increased electrical conductivity. At least 50% of differentially expressed genes (p < 0.05) were down-regulated in the salt-sensitive population growing under high salinity, while remaining unchanged in the saline-tolerant populations. In particular, most reduction in transcript levels in the salt-sensitive population were observed in genes specifying cell wall structural components, lipids, secondary metabolism, auxin and ethylene hormones, development, transport, signalling, heat shock, proteolysis, pathogenesis-response, abiotic stress, RNA processing, and protein metabolism. Transcript diversity for transcription factors, protein modification, and protein degradation genes was also more strongly affected in salt-tolerant CW064027 than in salt-tolerant Bridgeview and salt-sensitive Rangelander, while both saline-tolerant populations showed more substantial up-regulation in redox-related genes and B-ZIP transcripts. The report highlights the first use of bulked genotypes as replicated samples to compare the transcriptomes of obligate out-cross breeding populations in alfalfa. Three lines of Alfalfa (salt-tolerant CW064027, salt-tolerant Bridgeview, salt-sensitive Rangelander) were grown on 3 different concentrations of salt. For each cultivar-salt condition, 3 biological replicates were collected for a total of 27 samples.