- Volume 1, Issue 9, 2019

Chitin is an essential structural polysaccharide component of the cell walls of fungi. Because chitin is not found in human cells it also represents an attractive target for antifungal therapy. Recent reports have suggested that Candida cells can resist killing by the antifungal echinocandin both in vitro and in vivo by up-regulating chitin synthesis and thereby sustaining cell wall integrity (Lee et al., 2012). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of echinocandins often have supra-high rather than high chitin levels, and therefore having too much chitin in the cell wall may be detrimental for viability. Furthermore, mutants in the Ca2+/calcineurin pathway are associated with appearance of viable super-high chitin cells following cell wall stress, suggesting suggest that this pathway may negatively regulate other chitin stimulation pathways. We, therefore, propose that C. albicans has evolved a mechanism to maintain their chitin content high enough to protect cells against cell wall damage, but not so high that will negatively affect cell viability. In our model, the Ca2+/calcineurin pathway acts as a buffering system or attenuator in maintaining viable high chitin cells by coordinating both chitin upregulation and modulation of the cell wall integrity pathway, through negative regulation of the MAPK Mkc1 pathway. In summary, we have uncovered a novel mechanism used by C. albicans to survive cell wall stress imposed by echinocandin treatment.

Singer Instruments have been embedded in the yeast academic community since 1982, and had supported the wider scientific community for 85 years. Traditionally, the role of our lab has been testing prototypes, engaging in the work of R&D for the production and improvement of robotic products. For the first time, in collaboration with the University of Manchester, Singer have recruited a PhD student to actively engaged in independent research. This has required quickly changing tack from a prototype and software development test bed, to a lab capable of outputting meaningful data to collaborators within the Aromagenesis project. Collaboration with local beverage producers has allowed a transitional generation of impactful data, that is in the process of informing experimentation into their own brewing processes. Colonies are picked using the newly developed PIXL, and strains arrayed via the ROTOR in excess of 1536 colonies per plate, colony growth data was analysed using the imaging device PhenoBooth. This has allowed rapid generation of data on numerous media types in the projects relatively short time span. The effects of in-house research are already been realised, with innovative changes to products, and detailed requests for making software more helpful to scientists. With a greater automation capacity than is currently required by our own research, the ability to engage in collaborative projects and for Singer to provide a service to the yeast research community is very much increasing.

The rapid acquisition of drug resistance by Candida glabrata has placed an increased burden on health systems. Combination therapies, which apply multiple known compounds, can thwart the compensatory behaviours that lead to drug resistance and can open therapeutic avenues that were not previously available. Genetic interactions, in which the loss of a pair of genes has a stronger effect than the loss of either of the genes alone, can be useful in identifying targets for combination therapy. However, the number of possible interactions is immense while experimentally establishing a genetic interaction is non-trivial. We are developing a method for predicting genetic interactions in C. glabrata as a means to prioritize the experimental validation of interactions and combination therapies. The method applies machine-learning techniques to infer genetic interactions from diverse data in C. glabrata and from other yeast species, such as coexpression, protein-protein interactions and coevolution. We are establishing the method by building a model for Saccharomyces cerevisiae to validate against the large-scale genetic interaction data available for that species. This model will then integrated with C. glabrata-specific data, validated against known genetic interactions, and used to produce novel predictions for further characterisation in the lab.

Candida species are important pathogens of humans and are the fourth most commonly isolated pathogen from nosocomial blood stream infections. Although Candida albicans is the principle causative agent of invasive candidiasis, the incidence of C. glabrata infection has grown rapidly. The reason for this increase is not fully understood but it is clear that the species has a higher innate tolerance to commonly administered azole antifungals, in addition to being highly tolerance to combinatorial stresses. Using S. cerevisiae, as a model due to its intrinsic sensitivity to combinatorial stress and hypothesizing that the expression of mediators of Candida glabrata combinatorial stress resistance (CSR) in S. cerevisiae would lead to induced resistance. To test this we transformed, en-masse, the Candida glabrata ORFeome into S. cerevisiae. This resulted in 1,500 CSR colonies and recovered plasmids of 118 ORFS. Sanger sequencing of these plasmids revealed a total of 29 different C. glabrata ORFS. The recovery of genes encoding known stress protectant proteins e.g. GPD1, GPD2 and TRX3 was predicted and validated the integrity of the screen. Through this screen we identified four C. glabrata unique ORFs that confer CSR and we are in the process of characterising.

Chromosomes need to be replicated and partitioned into daughter cells during cellular division. Failure to do so leads to aneuploidy, which has been linked to medical conditions such as cancer. Cohesin, a ring-shaped protein complex, holds the replicated sister chromatids together to ensure their accurate segregation. In Saccharomyces cerevisiae a large pool of cohesin is loaded at centromeres. Introducing the centromeric sequence to circular and linear DNA has been exploited to allow propagation of extra-chromosomal DNA. However, these extra-chromosomal DNA do not segregate as well as endogenous chromosomes and the reasons for this are unknown. Interestingly, small circular chromosomes are more stable than their linear counterparts. Furthermore, the stability of linear chromosomes increases with chromosome length. The DNA sequence itself appears to be important as linear chromosomes containing endogenous DNA are more stable than chromosomes derived from artificial DNA of similar lengths. During our project, we sought to identify cis-acting DNA elements, other than the centromere, that are important for chromosome segregation.

We have developed a synthetic biology approach to engineer a library of synthetic circular and linear chromosomes of various lengths containing unnatural DNA (designed to lack features of known elements such as transcription units). These chromosomes act as a “blank canvas” where candidate DNA elements are introduced to test their functionality. Our design, build and test cycle has allowed us to determine how chromosome length, circularization and DNA sequence influence chromosome segregation and the minimal requirements for building the optimal synthetic chromosome. Our latest findings will be presented.

ATM and ATR are master regulators of the DNA damage response linked to cancer, neurodegeneration, and accelerated ageing. We find that inactivation of Mec1, an essential budding yeast ATM/ATR protein, leads to widespread protein aggregation and cell death in response to three different types of proteotoxic stresses; heat, Huntingtin (HTT), the aggregation prone Huntington’s disease protein, andazetidine 2 carboxylic acid (AZC), a proline analogue that induces protein misfolding. Conditions that activate protein catabolism (e.g. activation of autophagy) or impede protein anabolism (e.g. cycloheximide [CHX] or deletion of genes involved translation) rescues the lethality via aggregate-resolution. Inactivation of Rad53- or Dun1- kinases, the two key components of the Mec1 DNA damage checkpoint response, confers distinct sensitivity profiles: rad53K277A confers sensitivity to AZC, HTT, and CHX; in contrast, dun1Δconfers sensitive only to AZC and HTT but robust resistance to CHX. We also find that Sml1, an inhibitor of ribonucleotide reductase (RNR), which undergoes Mec1-Rad53-Dun1 dependent degradation in response to DNA damage is maintained in response to proteotoxic stress. Taken together, these results unveil a new function of Mec1 in mediating cellular response to perturbation in protein homeostasis. We propose that Mec1 is a versatile signal transduction protein that promotes resistance to both genotoxic and proteotoxic stresses via distinct mechanisms.

The BioScreening facility based within the Medical School at Newcastle University combines robotics, high throughput reagents and expertise to provide a resource to academics throughout the UK. The facility was created in 2011 using technology within the laboratory of Professor David Lydall and has been run since this date by Dr Peter Banks. The facility caters for a range of microbial organisms but yeast screening has for many years been a core part of the BioScreening Facility. The services offered for yeast screening include genomic screening of deletion, over expression and temperature sensitive collections, protein interaction screening and histone mutant collection screening. Compound screening is also offered in collaboration with the life sciences charity LifeArc. The BioScreening facility offers a fully comprehensive yeast screening service from the initial experiment to final data analysis and presentation if required. However, we also welcome requests for training on robotic systems, high throughput experimental design and analysis and advice on large scale data handling.

In human hosts, the opportunistic fungal pathogen Candida albicans primarily proliferates in nutrient diverse niches. Environmental condition sensing regulates several fungal cellular features including, but not limited to, metabolism, cell wall elasticity, and virulence. In addition, yeast cell division exposes pathogen-associated molecular patterns (PAMPs) at the cell surface that are known to be immune-stimulatory (e.g. β-glucan). While various host environmental signals and cell wall stressors have been implicated in PAMP exposure in vitro, little is known about the molecular mechanisms that modulate PAMP exposure. We have shown that lactate, an alternative carbon source present in mucosal niches and produced by activated innate immune cells, acts as a signalling molecule to reduce β-glucan exposure. However, it is unknown whether the reduction in β-glucan exposure is the result of PAMP camouflaging by other cell wall components, PAMP modification, or a combination of both processes. We characterized the downstream effectors affecting PAMP exposure in response to different carbon sources and environmental conditions that C. albicans encounters during transit through host niches. Using proteomics, gene deletion analysis, and pharmacological assays, we identified the downstream effectors involved in evading β-glucan recognition by the host pattern recognition receptor, Dectin-1. We can also show microscopic changes to the overall distribution of Dectin-1-recognised β-glucan on the cell surface in response to masking conditions as well as alterations to the interactions of masked cells with phagocytes. Finally, we are examining the impact of PAMP modulation and its inhibition on disease outcomes.

Our understanding of normal immunity to opportunistic fungal infection is limited by a lack of species-specific commensal/host/pathogen models. There are many species of opportunistic fungal pathogens (such as Candida spp. and Cryptococcus spp.) which exist as commensal organisms or are common in our environment. Therefore, we sought to develop a model of commensal opportunistic infection within zebrafish by identification of a normal commensal fungus that could act as an opportunistic pathogen.

Using dissections of D. rerio guts we isolated a monoculture of a commensal yeast. The strain was identified as Candida famata via DNA sequencing and phenotypic analysis (e.g. halotolerance). C. famata was isolated from six individual animals and three strains (DJ1-3) have been deposited in the CBS collection.

C. famata is a common commensal of animals, including humans and is a rare opportunistic pathogen. Our DJ1 strain was thermotolerant and grows at mammalian body temperature. We found it was easily phagocytosed by mouse macrophages, in some cases to the extent of host cell destruction. Infection of zebrafish larval model of opportunistic infection demonstrated C. famata could act as an opportunistic pathogen of its commensal host.

Thus, we have characterised the first commensal fungus to be isolated from zebrafish and demonstrated that it may be a potential opportunistic pathogen, as in humans. The study of C. famata in zebrafish therefore represents an unparalleled opportunity to understand the evolution of fungal immunology and the switch between commensal and pathogen.

The Saccharomyces complex is a well-studied group of yeasts of considerable academic and industrial importance. Many species within the complex have no genome sequence publicly available and have yet to be formally compared at the genomic level with related species. We have recently sequenced the genomes of 40 species from 11 clades within the Saccharomyces complex. Considerable genomic diversity was observed in this dataset, including varying predicted genome size (9-22Mbp), coding proportion (42-76%) and gene number (4,194-11,001).

We evaluated the completeness of our gene sets and draft genome assemblies using the BUSCO tool, with a few species found to have a large number of duplicated and missing genes. These same species also had larger than average genome sizes and numbers of genes, which could indicate a duplication or hybridisation event. The total GC content was also found to vary significantly across the dataset, from Hanseniaspora uvarum (31.3%) to Lachancea thermotolerans (46%). GC content in the coding regions versus the whole genome was also observed, which could indicate differences in evolutionary pressures between species according to their environmental niches.

Here, we highlight some of the key differences found within this fascinating dataset, investigating the origins of some of the more extreme results and touching upon the opportunities and challenges they present in investigating the evolution of these yeasts.

The emerging fungal pathogen Candida auris is responsible for recent global outbreaks caused by genetically divergent clades that, alarmingly, display multi-drug resistance to currently available antifungals. Although not much is known about the pathobiology of this emerging pathogen, we recently found that the Hog1 stress-activated protein kinase (SAPK) in C. auris promotes stress resistance and virulence. SAPKs are evolutionary conserved pathways employed by fungi to promote survival in hostile environments. The aim of this project is to characterise the upstream signalling proteins that relay stress signals to C. auris Hog1. In all eukaryotes, SAPK pathways are comprised of three tiers of kinases with upstream MAPKKK and MAPKKs relaying signals to the terminal SAPK. However, unique to fungi and plants, is an upstream two-component related signalling cascade that transmits signals to the SAPK module, comprising of a histidine kinase, a phosphorelay protein and a response regulator. In C. auris, the proteins that regulate Hog1 activation are unknown. However, bioinformatics analyses of the C. auris genome has allowed for the identification of putative homologues of key Hog1 regulatory proteins including the Pbs2 MAPKK, the Ssk2 MAPKKK, the Ssk1 response regulator, the Ypd1 phosphorelay protein and the Sln1 and Nik1 histidine kinases. Mutational analyses is currently underway to dissect the role of these proteins in Hog1 regulation. Understanding how the Hog1 SAPK is regulated in C. auris may facilitate the identification of fungal specific anti-fungal targets that can be exploited to tackle the threat of this emerging drug resistant pathogen.

Over the past decade, we have gained considerable insight into the identification of sequence variation within the rDNA array of Saccharomyces cerevisiae and its closest wild relative, Saccharomyces paradoxus. Yet considerable challenges remain in the computational characterisation of this complex genomic region. This study aimed to evaluate the use of variation graphs for this purpose, formally comparing their effectiveness with traditional linear approaches.

Specifically, we aimed to identify both partial and fixed variants (i.e. pSNPs, SNPs, pINDELs and INDELs) in the rDNA arrays of 10 diverse, haploid Saccharomyces cerevisiae strains with high quality genomic datasets. We constructed two computational pipelines using two highly different approaches. The first pipeline used the BWA read mapper and the BCFtools variant caller to identify variants against the linear S288c reference, with the second pipeline using the vg tool to call variants against a graphical reference (either based on a graphical representation of the S288c genome or a Saccharomyces cerevisiae pan-genome).

The results showed that the graph-based pipeline was able to identify more variants than the linear pipeline, and in particular partial variants, while also missing some key variants identified by BWA/BCFtools. A major discrepancy between the two pipelines was found in the read coverage at loci where the vg pipeline identified variants. In the coming months, we aim to investigate the cause of these differences and to develop a new graph-based computational pipeline that can accurately identify the full range of sequence and copy number variation within this key genomic region.

High-throughput techniques have become crucial for the discovery of new strains suitable for the beverage industry. These approaches allow assessment of critical traits for fermented beverages such as; stress tolerance, growth, viability, vitality and aromatic compound production in a fast and effective manner.

The growth rate of 24 Saccharomyces and non-Saccharomyces yeast strains were evaluated in industrially relevant media. High-throughput robotic platforms, the ROTOR HDA and PIXL, were used to pin colonies on solidified synthetic apple juice (SAJ), malt extract (ME), apple juice (AJ) and wort (WO) media. During incubation at 20 and 25 °C for up to 240 hours, the plates were imaged, and analysed via PhenoBooth and the ggplot2 R package. The results were corroborated via liquid growth.

Pichia scullata Y-7663 and Hanseniaspora vineae Y-17530 showed the highest growth values in ME, WO and AJ; with final colony diameters double that of the Saccharomyces cerevisiae ESM356-1 control. However, the former two strains displayed defective growth in SAJ. Kluyveromyces lactis Y-1140 exhibited high growth performance, especially in ME and WO. Interestingly, the ale strain S. cerevisiae Y-11875, showed significantly higher growth in SAJ and AJ medium than in ME and WO. Better adaptation to growth in wort conditions was expected.

This trial shows that the fitness of yeast strains can be assessed in solid media representing industrial conditions. The method can be up-scaled to a wider format, opening the possibility of screening a thousand-fold libraries of industrially relevant and/or novel yeast isolates in a fast, cost-effective manner.

Long terminal repeat (LTR) retrotransposons are mobile genetic elements that are present in the genomes of most eukaryotes. They are closely related to retroviruses and mobilize through an RNA intermediate. The uncontrolled mobilization of retrotransposons is potentially harmful to the integrity of the genome and so the activity of these elements is subjected to strict host cell controls. We are using the fission yeast, Schizosaccharomyces pombe to study the signalling pathways that regulate the activity ofLTR retrotransposons. We have found that the expression and mobilization of the Tf2 LTR retrotransposons is activated in response to exposure to the immunosuppressant drug rapamycin. Rapamycin binds to the conserved FKBP12 protein (called Fkh1 in S. pombe) and the resulting FKBP12-rapamycin complex inhibits the kinase activity of the conserved the TORC1 complex. This suggests that Tf2 activity is under the control of the TORC1 signalling network which is a master regulator of cellular responses to nutrient and energy availability. However, the inhibition of TORC1 activity using a tor2 temperature sensitive allele or a direct chemical inhibitor (Torin) did not activate either the expression or mobilization of Tf2 elements. Therefore, rapamycin may be controlling Tf2 activity via a TORC1-independent pathway. We are currently defining this pathway and find that it is dependent upon the FKBP12 protein, Fkh1 and the Forkhead transcription factor, Fhl1.

In order to protect against oxidative damage, cells have evolved a host of ROS-detoxifying enzymes. These include peroxiredoxins, a highly conserved family of thioredoxin peroxidases. Unexpectedly, given their role in lowering H2O2levels, peroxiredoxins have been shown to be required for the activation of conserved stress-activated MAPKs in response to ROS in yeast1 and human2 cells. For example, we have previously shown that the single 2-Cys peroxiredoxin in S. pombe, Tpx1, but not its thioredoxin peroxidase activity, is required for the H2O2-induced activation of the p38/JNK-related MAPK, Sty1. Our findings revealed that Tpx1 forms H2O2-induced disulphide bonds with cysteines in Sty11, which suggested that Tpx1 may directly regulate Sty1 through these complexes. However, the mechanisms by which Tpx1-Sty1 disulphide complexes alter Sty1 function have remained unclear. Sty1, like its mammalian counterparts, has a number of important functions, including roles in coordinating cell growth, division, stress resistance and longevity in response to a variety of nutritional and stress stimuli. Our data suggests that disulphide complexes with Tpx1 are important for a subset of these roles. Intriguingly, our proteomic studies have identified multiple protein kinases that form disulphide complexes with Tpx1, these include kinases with established roles in regulating cell division and ageing. Here, we will present data suggesting that interactions with Tpx1 play important roles in regulating the activities of these kinases.

1. Veal et al. (2004) Molecular Cell, 15(1), pp. 129-139.

2. Jarvis et al. (2012) Free Radical Biology and Medicine, 53(7), pp. 1522-1530.

Combatting the spread of drug-resistant microbes requires new antifungal compounds with novel mechanisms of inhibition. Our lab investigates natural, proteinaceous toxins that are coded by double stranded RNA satellites found within Saccharomyces cerevisiae. Commonly known as “killer yeasts”, toxin-producing strains of S. cerevisiae have been found to inhibit the growth of many important fungal pathogens. To assess the value of killer yeasts for medical application we tested over 6,000 interactions between killer yeasts and various important fungal pathogens. To determine the susceptibility of fungi to killer toxins, we competed killer yeasts against a dilute lawn of a fungal pathogen; an active toxin produces a zone of growth inhibition in the lawn around the killer yeast. We determined that Candida glabratais broadly susceptible to killer toxins, while other Candida species showed little or no susceptibility. Of the 90 strains of S. cerevisiae tested against C. glabrata seven were capable of inhibiting all clinical strains of drug-resistant C. glabrata available from the Center for Disease Control and the U.S. Department of Agriculture. In our evaluation of these toxins as potential antifungal therapeutics we confirmed their inhibitory capability against C. glabrata under physiological conditions. Based on the results of these tests and prior knowledge about killer yeasts, we believe that these toxins show a potential as antifungal therapeutic precursors to treat recalcitrant infections caused by C. glabrata.