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.

Aerial warfare: An inducible production of volatile bioactive metabolites in a novel species of Scytinostroma sp.

Antimicrobial volatile organic compounds (VOCs) may provide fungi an advantage over other competing microorganisms. As these defensive metabolites are often produced in response to microbial competitors, they are easily overlooked in axenic cultures. We used media supplemented with spent medium from Candida albicans to induce the expression of a broad-spectrum antimicrobial response in a previously uncharacterised white-rot fungus, Scytinostroma sp. Crude extractions of Scytinostroma sp. metabolites were found to be cytotoxic to fibroblast cells and antimicrobial to filamentous fungi, yeasts and Gram-positive bacteria. Volatile antimicrobial activity was observed for Scytinostroma sp. cultures and metabolite extracts using antimicrobial assays in bi-compartmentalised plates. Culture headspace analysis using solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) revealed a pronounced shift in Scytinostroma sp. VOCs when cultured on media supplemented with C. albicans spent medium. We observed a significant increase in the levels of 45 identified VOCs, including 7 metabolites with reported antimicrobial activity. Using preparative HPLC combined with GC-MS, we determined that isovelleral is likely to be the main broad-spectrum antimicrobial metabolite produced by Scytinostroma sp. Isovelleral is a sesquiterpene dialdehyde with both antibiotic and antifeedant properties, previously detected in fruit bodies of other Basidiomycetes.

Metabolic competition between host and pathogen dictates inflammasome responses to fungal infection.

The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida's ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections.

Quantitative live-cell imaging of Candida albicans escape from immune phagocytes.

Population-level dynamics of host-pathogen interactions can be characterized using quantitative live-cell imaging. Here, we present a protocol for infecting macrophages with the fungal pathogen Candida albicans in vitro and quantitative live-cell imaging of immune and pathogen responses. We describe steps for detailed image analysis and provide resources for quantification of phagocytosis and pathogen escape, as well as macrophage membrane permeabilization and viability. This protocol is modifiable for applications with a range of pathogens, immune cell types, and host-pathogen mechanisms. For complete details on the use and execution of this protocol, please refer to Olivier et al.1.

Candida auris uses metabolic strategies to escape and kill macrophages while avoiding robust activation of the NLRP3 inflammasome response.

Metabolic adaptations regulate the response of macrophages to infection. The contributions of metabolism to macrophage interactions with the emerging fungal pathogen Candida auris are poorly understood. Here, we show that C. auris-infected macrophages undergo immunometabolic reprogramming and increase glycolysis but fail to activate a strong interleukin (IL)-1ß cytokine response or curb C. auris growth. Further analysis shows that C. auris relies on its own metabolic capacity to escape from macrophages and proliferate in vivo. Furthermore, C. auris kills macrophages by triggering host metabolic stress through glucose starvation. However, despite causing macrophage cell death, C. auris does not trigger robust activation of the NLRP3 inflammasome. Consequently, inflammasome-dependent responses remain low throughout infection. Collectively, our findings show that C. auris uses metabolic regulation to eliminate macrophages while remaining immunologically silent to ensure its own survival. Thus, our data suggest that host and pathogen metabolism could represent therapeutic targets for C. auris infections.

The escape of Candida albicans from macrophages is enabled by the fungal toxin candidalysin and two host cell death pathways.

The egress of Candida hyphae from macrophages facilitates immune evasion, but it also alerts macrophages to infection and triggers inflammation. To better define the mechanisms, here we develop an imaging assay to directly and dynamically quantify hyphal escape and correlate it to macrophage responses. The assay reveals that Candida escapes by using two pore-forming proteins to permeabilize macrophage membranes: the fungal toxin candidalysin and Nlrp3 inflammasome-activated Gasdermin D. Candidalysin plays a major role in escape, with Nlrp3 and Gasdermin D-dependent and -independent contributions. The inactivation of Nlrp3 does not reduce hyphal escape, and we identify ETosis via macrophage extracellular trap formation as an additional pathway facilitating hyphal escape. Suppressing hyphal escape does not reduce fungal loads, but it does reduce inflammatory activation. Our findings explain how Candida escapes from macrophages by using three strategies: permeabilizing macrophage membranes via candidalysin and engaging two host cell death pathways, Gasdermin D-mediated pyroptosis and ETosis.