Proteomic analysis of dietary restriction in yeast reveals a role for Hsp26 in replicative lifespan extension.

Dietary restriction (DR) has been shown to increase lifespan in organisms ranging from yeast to mammals. This suggests that the underlying mechanisms may be evolutionarily conserved. Indeed, upstream signalling pathways, such as TOR, are strongly linked to DR-induced longevity in various organisms. However, the downstream effector proteins that ultimately mediate lifespan extension are less clear. To shed light on this, we used a proteomic approach on budding yeast. Our reasoning was that analysis of proteome-wide changes in response to DR might enable the identification of proteins that mediate its physiological effects, including replicative lifespan extension. Of over 2500 proteins we identified by liquid chromatography-mass spectrometry, 183 were significantly altered in expression by at least 3-fold in response to DR. Most of these proteins were mitochondrial and/or had clear links to respiration and metabolism. Indeed, direct analysis of oxygen consumption confirmed that mitochondrial respiration was increased several-fold in response to DR. In addition, several key proteins involved in mating, including Ste2 and Ste6, were down-regulated by DR. Consistent with this, shmoo formation in response to α-factor pheromone was reduced by DR, thus confirming the inhibitory effect of DR on yeast mating. Finally, we found that Hsp26, a member of the conserved small heat shock protein (sHSP) family, was up-regulated by DR and that overexpression of Hsp26 extended yeast replicative lifespan. As overexpression of sHSPs in Caenorhabditis elegans and Drosophila has previously been shown to extend lifespan, our data on yeast Hsp26 suggest that sHSPs may be universally conserved effectors of longevity.

CO2 enhances the formation, nutrient scavenging and drug resistance properties of C. albicans biofilms.

C. albicans is the predominant human fungal pathogen and frequently colonises medical devices, such as voice prostheses, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is the elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance, the effect of CO2 on C. albicans biofilm growth has not been investigated to date. Here we show that physiologically relevant CO2 elevation enhances each stage of the C. albicans biofilm-forming process: from attachment through maturation to dispersion. The effects of CO2 are mediated via the Ras/cAMP/PKA signalling pathway and the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown under elevated CO2 conditions also exhibit increased azole resistance, increased Sef1-dependent iron scavenging and enhanced glucose uptake to support their rapid growth. These findings suggest that C. albicans has evolved to utilise the CO2 signal to promote biofilm formation within the host. We investigate the possibility of targeting CO2-activated processes and propose 2-deoxyglucose as a drug that may be repurposed to prevent C. albicans biofilm formation on medical airway management implants. We thus characterise the mechanisms by which CO2 promotes C. albicans biofilm formation and suggest new approaches for future preventative strategies.

Precision Antifungal Treatment Significantly Extends Voice Prosthesis Lifespan in Patients Following Total Laryngectomy.

Indwelling silicone valves called voice prostheses (VPs) are the gold standard for speech rehabilitation in patients with laryngeal cancer following total laryngectomy. Reported VP lifespans amongst these patients are highly variable but when devices fail patients experience loss of voice and an increase risk of chest infection. Early failure of VP is a current clinical concern that is associated with regular hospital visits, reduced quality of life and associated medical cost. Poly-microbial biofilms comprised of both bacterial and fungal microorganisms readily colonize VPs and are linked to loss of device performance and its early failure in addition to providing a reservoir for potential infection. Our detailed analysis of poly-microbial biofilm composition on 159 early failing VPs from 48 total laryngectomy patients confirmed Candida albicans as the predominant fungal species and Staphylococcus aureus as the most common bacterial colonizer within our patient cohort. Using a combination of microbiological analysis, patient data and a high-throughput antifungal test assay mimicking in vivo conditions we established an evidence based precision antifungal treatment approach to VP management. Our approach has allowed us to implement a personalized VP management pathway, which increases device in situ lifespan by an average of 270%. Our study represents a significant step forward in both our understanding of the cause of VP failure and a new effective treatment pathway that offers tangible benefit to patients.

Ras signalling in pathogenic yeasts.

The small GTPase Ras acts as a master regulator of growth, stress response and cell death in eukaryotic cells. The control of Ras activity is fundamental, as highlighted by the oncogenic properties of constitutive forms of Ras proteins. Ras also plays a crucial role in the pathogenicity of fungal pathogens where it has been found to regulate a number of adaptions required for virulence. The importance of Ras in fungal disease raises the possibility that it may provide a useful target for the development of new treatments at a time when resistance to available antifungals is increasing. New findings suggest that important regulatory sequences found within fungal Ras proteins that are not conserved may prove useful in the development of new antifungals. Here we review the roles of Ras protein function and signalling in the major human yeast pathogens Candida albicans and Cryptococcus neoformans and discuss the potential for targeting Ras as a novel approach to anti-fungal therapy.

New links between SOD1 and metabolic dysfunction from a yeast model of amyotrophic lateral sclerosis.

A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast Saccharomyces cerevisiae We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a Caenorhabditis elegans model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.