Physiological adventures in Candida albicans: farnesol and ubiquinones.

SUMMARYFarnesol was first identified as a quorum-sensing molecule, which blocked the yeast to hyphal transition in Candida albicans, 22 years ago. However, its interactions with Candida biology are surprisingly complex. Exogenous (secreted or supplied) farnesol can also act as a virulence factor during pathogenesis and as a fungicidal agent triggering apoptosis in other competing fungi. Farnesol synthesis is turned off both during anaerobic growth and in opaque cells. Distinctly different cellular responses are observed as exogenous farnesol levels are increased from 0.1 to 100 µM. Reported changes include altered morphology, stress response, pathogenicity, antibiotic sensitivity/resistance, and even cell lysis. Throughout, there has been a dearth of mechanisms associated with these observations, in part due to the absence of accurate measurement of intracellular farnesol levels (Fi). This obstacle has recently been overcome, and the above phenomena can now be viewed in terms of changing Fi levels and the percentage of farnesol secreted. Critically, two aspects of isoprenoid metabolism present in higher organisms are absent in C. albicans and likely in other yeasts. These are pathways for farnesol salvage (converting farnesol to farnesyl pyrophosphate) and farnesylcysteine cleavage, a necessary step in the turnover of farnesylated proteins. Together, these developments suggest a unifying model, whereby high, threshold levels of Fi regulate which target proteins are farnesylated or the extent to which they are farnesylated. Thus, we suggest that the diversity of cellular responses to farnesol reflects the diversity of the proteins that are or are not farnesylated.

Farnesol as an antifungal agent: comparisons among MTLa and MTLα haploid and diploid Candida albicans and Saccharomyces cerevisiae.

Aims: Farnesol was identified 20 years ago in a search for Candida albicans quorum sensing molecules (QSM), but there is still uncertainty regarding many aspects of its mode of action including whether it employs farnesol transport mechanisms other than diffusion. Based on the structural similarity between farnesol and the farnesylated portion of the MTL a pheromone, we explored the effects of ploidy and mating type locus (MTL) on the antifungal activity of exogenous farnesol. Methods and results: We approached this question by examining five MTL a and five MTLα haploid strains with regard to their farnesol sensitivity in comparison to six heterozygous MTL a/ α diploids. We examined the haploid and diploid strains for percent cell death after exposure of exponentially growing cells to 0-200 µM farnesol. The heterozygous (MTL a/α) diploids were tolerant of exogenous farnesol whereas the MTL a and MTLα haploids were on average 2- and 4-times more sensitive, respectively. In the critical range from 10-40 µM farnesol their cell death values were in the ratio of 1:2:4. Very similar results were obtained with two matched sets of MAT a, MATα, and MAT a/α Saccharomyces cerevisiae strains. Conclusion: We propose that the observed MTL dependence of farnesol is based on differentially regulated mechanisms of entry and efflux which determine the actual cellular concentration of farnesol. The mechanisms by which pathogens such as C. albicans tolerate the otherwise lethal effects of farnesol embrace a wide range of physiological functions, including MTL type, ubiquinone type (UQ6-UQ9), energy availability, and aerobic/anaerobic status.

Transcriptional regulation of the synthesis and secretion of farnesol in the fungus Candida albicans: examination of the Homann transcription regulator knockout collection.

Candida albicans is an efficient colonizer of human gastrointestinal tracts and skin and is an opportunistic pathogen. C. albicans exhibits morphological plasticity, and the ability to switch between yeast and filamentous morphologies is associated with virulence. One regulator of this switch is the quorum sensing molecule farnesol that is produced by C. albicans throughout growth. However, the synthesis, secretion, regulation, and turnover of farnesol are not fully understood. To address this, we used our improved farnesol assay to screen a transcription regulator knockout library for differences in farnesol accumulation in whole cultures, pellets, and supernatants. All screened mutants produced farnesol and they averaged 9.2× more farnesol in the pellet than the supernatant. Nineteen mutants had significant differences with ten mutants producing more farnesol than their SN152+ wild-type control strain while nine produced less. Seven mutants exhibited greater secretion of farnesol while two exhibited less. We examined the time course for farnesol accumulation in six mutants with the greatest accumulation differences and found that those differences persisted throughout growth and they were not time dependent. Significantly, two high-accumulating mutants did not exhibit the decay in farnesol levels during stationary phase characteristic of wild-type C. albicans, suggesting that a farnesol modification/degradation mechanism is absent in these mutants. Identifying these transcriptional regulators provides new insight into farnesol's physiological functions regarding cell cycle progression, white-opaque switching, yeast-mycelial dimorphism, and response to cellular stress.

Quantitative assay for farnesol and the aromatic fusel alcohols from the fungus Candida albicans.

The dimorphic fungus Candida albicans is a commensal and opportunistic fungal pathogen of humans. It secretes at least four small lipophilic molecules, farnesol and three aromatic fusel alcohols. Farnesol has been identified as both a quorum sensing molecule (QSM) and a virulence factor. Our gas chromatography (GC)-based assay for these molecules exhibits high throughput, prevention of analyte loss by avoiding filtration and rotary evaporation, simultaneous cell lysis and analyte extraction by ethyl acetate, and the ability to compare whole cultures with their cell pellets and supernatants. Farnesol synthesis and secretion were separable phenomena and pellet:supernatant ratios for farnesol were high, up to 12:1. The assay was validated in terms of precision, specificity, ruggedness, accuracy, solution stability, detection limits (DL), quantitation limits (QL), and dynamic range. The DL for farnesol was 0.02 ng/µl (0.09 µM). Measurement quality was assessed by the relative error of the whole culture versus the sum of pellet and supernatant fractions (WPS). C. albicans strain SC5314 grown at 30 °C in complex and defined media (YPD and mRPMI) was assayed in biological triplicate 17 times over 3 days. Farnesol and the three aromatic fusel alcohols can be measured in the same assay. The levels of all four are greatly altered by the growth medium chosen. Significantly, the three fusel alcohols are synthesized during stationary phase, not during growth. They are secreted quickly without being retained in the cell pellet and may accumulate up to mM concentrations. KEY POINTS: • Quantitative analysis of both intra- and extracellular farnesol, and aromatic fusel oils. • High throughput, whole culture assay with simultaneous lysis and extraction. • Farnesol secretion and synthesis are distinct and separate events.

Changes in lipid profiles of epileptic mouse model.

INTRODUCTION: Approximately 1% of the world's population is impacted by epilepsy, a chronic neurological disorder characterized by seizures. One-third of epileptic patients are resistant to AEDs, or have medically refractory epilepsy (MRE). One non-invasive treatment that exists for MRE includes the ketogenic diet, a high-fat, low-carbohydrate diet. Despite the KD's success in seizure attenuation, it has a few risks and its mechanisms remain poorly understood. The KD has been shown to improve metabolism and mitochondrial function in epileptic phenotypes. Potassium channels have implications in epileptic conditions as they have dual roles as metabolic sensors and control neuronal excitation. OBJECTIVES: The goal of this study was to explore changes in the lipidome in hippocampal and cortical tissue from Kv1.1-KO model of epilepsy. METHODS: FT-ICR/MS analysis was utilized to examine nonpolar metabolome of cortical and hippocampal tissue isolated from a Kv1.1 channel knockout mouse model of epilepsy (n = 5) and wild-type mice (n = 5). RESULTS: Distinct metabolic profiles were observed, significant (p < 0.05) features in hippocampus often being upregulated (FC ≥ 2) and the cortex being downregulated (FC ≤ 0.5). Pathway enrichment analysis shows lipid biosynthesis was affected. Partition ratio analysis revealed that the ratio of most metabolites tended to be increased in Kv1.1-/-. Metabolites in hippocampal tissue were commonly upregulated, suggesting seizure initiation in the hippocampus. Aberrant mitochondrial function is implicated by the upregulation of cardiolipin, a common component in the mitochondrial membrane. CONCLUSION: Generally, our study finds that the lipidome is changed in the hippocampus and cortex in response to Kv1.1-KO indicating changes in membrane structural integrity and synaptic transmission.

Aberrant energy metabolism and redox balance in seizure onset zones of epileptic patients.

Epilepsy is a disorder that affects around 1% of the population. Approximately one third of patients do not respond to anti-convulsant drugs treatment. To understand the underlying biological processes involved in drug resistant epilepsy (DRE), a combination of proteomics strategies was used to compare molecular differences and enzymatic activities in tissue implicated in seizure onset to tissue with no abnormal activity within patients. Label free quantitation identified 17 proteins with altered abundance in the seizure onset zone as compared to tissue with normal activity. Assessment of oxidative protein damage by protein carbonylation identified additional 11 proteins with potentially altered function in the seizure onset zone. Pathway analysis revealed that most of the affected proteins are involved in energy metabolism and redox balance. Further, enzymatic assays showed significantly decreased activity of transketolase indicating a disruption of the Pentose Phosphate Pathway and diversion of intermediates into purine metabolic pathway, resulting in the generation of the potentially pro-convulsant metabolites. Altogether, these findings suggest that imbalance in energy metabolism and redox balance, pathways critical to proper neuronal function, play important roles in neuronal network hyperexcitability and can be used as a primary target for potential therapeutic strategies to combat DRE. SIGNIFICANCE: Epileptic seizures are some of the most difficult to treat neurological disorders. Up to 40% of patients with epilepsy are resistant to first- and second-line anticonvulsant therapy, a condition that has been classified as refractory epilepsy. One potential therapy for this patient population is the ketogenic diet (KD), which has been proven effective against multiple refractory seizure types However, compliance with the KD is extremely difficult, and carries severe risks, including ketoacidosis, renal failure, and dangerous electrolyte imbalances. Therefore, identification of pathways disruptions or shortages can potentially uncover cellular targets for anticonvulsants, leading to a personalized treatment approach depending on a patient's individual metabolic signature.

Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast.

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6h. Exposure of an LC50 dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by ICP-MS. Measurement of the relative ATP and ADP concentrations indicates an initial 3-fold depletion of ATP suggesting an alteration in the ATP:ADP ratio. At the 6h time point the lidocaine exposed population contained ATP concentrations roughly 85% that of the negative control suggesting the surviving population adapted its metabolic pathways to, at least partially restore cellular bioenergetics. Metabolite analysis indicates an increase of intermediates in the pentose phosphate pathway, the preparatory phase of glycolysis, and NADPH. Oxidative stress produced by lidocaine exposure targets aconitase decreasing its activity with an observed decrease in isocitrate and an increase citrate. Similarly, increases in α-ketoglutarate, malate, and oxaloacetate imply activation of anaplerotic reactions. Antioxidant molecule glutathione and its precursor amino acids, cysteine and glutamate were greatly increased at later time points. Phosphatidylserine externalization suggestive of early phase apoptosis was also observed. Genetic studies using metacaspase null strains showed resistance to lidocaine induced cell death. These data suggest lidocaine induces perpetual mitochondrial depolarization, ROS/RNS generation along with increased glutathione to combat the oxidative cellular environment, glycolytic to PPP cycling of carbon generating NADPH, obstruction of carbon flow through the TCA cycle, decreased ATP generation, and metacaspase dependent apoptotic cell death.

A proteomic approach to identify metalloproteins and metal-binding proteins in liver from diabetic rats.

Proteins play crucial roles in biological systems, thus studies comparing the protein pattern present in a healthy sample with an affected sample have been widely used for disease biomarker discovery. Although proteins containing metal ions constitute only a small proportion of the proteome, they are essential in a multitude of structural and functional processes. The correct association between metal ions and proteins is essential because this binding can significantly interfere with normal protein function. Employment of a metalloproteomic study of liver samples from diabetic rats permitted determination of the differential abundance of copper-, selenium-, zinc- and magnesium-associated proteins between diabetic, diabetic treatment with insulin and non-diabetic rats. Proteins were detected by ESI-MS/MS. Seventy-five different proteins were found with alterations in the metal ions of interest. The most prominent pathways affected under the diabetic model included: amino-acid metabolism and its derivates, glycogen storage, metabolism of carbohydrates, redox systems and glucose metabolism. Overall, the current methods employed yielded a greater understanding of metal binding and how type 1 diabetes and insulin treatment can modify some metal bonds in proteins, and therefore affect their mechanism of action and function.

Liver Proteome in Diabetes Type 1 Rat Model: Insulin-Dependent and -Independent Changes.

Diabetes mellitus type 1 (DM1) is a major public health problem that continues to burden the healthcare systems worldwide, costing exponentially more as the epidemic grows. Innovative strategies and omics system diagnostics for earlier diagnosis or prognostication of DM1 are essential to prevent secondary complications and alleviate the associated economic burden. In a preclinical study design that involved streptozotocin (STZ)-induced DM1, insulin-treated STZ-induced DM1, and control rats, we characterized the insulin-dependent and -independent changes in protein profiles in liver samples. Digested proteins were subjected to LC-MSE for proteomic data. Progenesis QI data processing and analysis of variance were utilized for statistical analyses. We found 305 proteins with significantly altered abundance among the control, DM1, and insulin-treated DM1 groups (p < 0.05). These differentially regulated proteins were related to enzymes that function in key metabolic pathways and stress responses. For example, gluconeogenesis appeared to return to control levels in the DM1 group after insulin treatment, with the restoration of gluconeogenesis regulatory enzyme, FBP1. Insulin administration to DM1 rats also restored the blood glucose levels and enzymes of general stress and antioxidant response systems. These observations are crucial for insights on DM1 pathophysiology and new molecular targets for future clinical biomarkers, drug discovery, and development. Additionally, we underscore that proteomics offers much potential in preclinical biomarker discovery for diabetes as well as common complex diseases such as cancer, dementia, and infectious disorders.

Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE, quantitative proteomics, and bioinformatics.

Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death.