The proteasome regulator Rpn4 controls antifungal drug tolerance by coupling protein homeostasis with metabolic responses to drug stress.

Fungal pathogens overcome antifungal drug therapy by classic resistance mechanisms, such as increased efflux or changes to the drug target. However, even when a fungal strain is susceptible, trailing or persistent microbial growth in the presence of an antifungal drug can contribute to therapeutic failure. This trailing growth is caused by adaptive physiological changes that enable the growth of a subpopulation of fungal cells in high drug concentrations, in what is described as drug tolerance. Mechanistically, antifungal drug tolerance is incompletely understood. Here we report that the transcriptional activator Rpn4 is important for drug tolerance in the human fungal pathogen Candida albicans. Deletion of RPN4 eliminates tolerance to the commonly used antifungal drug fluconazole. We defined the mechanism and show that Rpn4 controls fluconazole tolerance via two target pathways. First, Rpn4 activates proteasome gene expression, which enables sufficient proteasome capacity to overcome fluconazole-induced proteotoxicity and the accumulation of ubiquitinated proteins targeted for degradation. Consistently, inhibition of the proteasome with MG132 eliminates fluconazole tolerance and resistance, and phenocopies the rpn4Δ/Δ mutant for loss of tolerance. Second, Rpn4 is required for wild type expression of the genes required for the synthesis of the membrane lipid ergosterol. Our data indicates that this function of Rpn4 is required for mitigating the inhibition of ergosterol biosynthesis by fluconazole. Based on our findings, we propose that Rpn4 is a central hub for fluconazole tolerance in C. albicans by coupling the regulation of protein homeostasis (proteostasis) and lipid metabolism to overcome drug-induced proteotoxicity and membrane stress.

Cold plasma effect on the proteome of Pseudomonas aeruginosa - Role for bacterioferritin.

Cold atmospheric-pressure plasma (CAP) is a relatively new method used for bacterial inactivation. CAP is ionized gas that can be generated by applying an electric current to air or a feeding gas. It contains reactive species and emits UV radiation, which have antibacterial activity. Previous data suggests that CAP is effective in microbial inactivation and can decontaminate and sterilize surfaces, but its exact mode of action is still under debate. This study demonstrates the effect of CAP on the whole proteome of Pseudomonas aeruginosa PAO1 biofilms, which is a dominant pathogen in cystic fibrosis and medical device-related infections. Liquid chromatography-mass spectrometry (LC-MS) was used to identify differentially regulated proteins of whole cell P. aeruginosa extracts. A total of 16 proteins were identified to be affected by plasma treatment compared to the control. Eight of the identified proteins have functions in transcription and translation and their expression changes are likely to be part of a general physiological response instead of a CAP-specific adaptation. However, CAP also affected bacterioferritin (Bfr), Isocitrate dehydrogenase (Idh), Trigger factor (Tig) and a chemotaxis protein, which may be involved in P. aeruginosa's specific response to CAP. We confirm that bacterioferritin B plays a role in the bacterial response to CAP because ΔbfrB mutants of both PAO1 and PA14 are more susceptible to plasma-induced cell-death than their corresponding wild-type strains. To our knowledge, this is the first study showing the effect of plasma on the whole proteome of a pathogenic microorganism. It will help our understanding of the mode of action of CAP-mediated bacterial inactivation and thus support a safe and effective routine use of CAP in clinical and industrial settings.