- Volume 1, Issue 9, 2019
Volume 1, Issue 9, 2019
- Abstracts from the British Yeast Group Meeting 2019
-
- Oral Abstract
-
-
The AP-2 endocytic adaptor complex in Candida albicans morphology and virulence
More LessFungi such as Candida species are a major cause of hospital-acquired infections especially in immuno-compromised patients, and invasive candidiasis is associated with a high mortality rate. Central to Candida albicans virulence is its ability to switch between budding (yeast) and filamentous (hyphal) growth, and to colonise different body niches. In each distinct environment it must remodel its cell surface to ensure appropriate levels of transporters and cell wall biosynthesis enzymes. Endocytosis is known to be a critical pathway in surface remodelling, allowing cells to internalise proteins that are no longer needed at the plasma membrane. Endocytosis is also crucial to highly polarised hyphal growth, where endocytic recycling of key membrane proteins is essential to maintain their polarised location at the hyphal tip. The aim of this study is to investigate the role of the AP-2 endocytic adaptor complex in endocytosis within C. albicans. Homozygous deletions were generated in an essential subunit of the AP-2 complex. The deletion did not affect rates of cell growth or fluid phase endocytosis, but using fluorescence and electron microscopy, defects were observed in hyphal polarisation and in cell wall organisation. We have shown that the AP-2 complex is required for the recycling of the key cell wall biosynthesis enzyme Chs3, via its Yxxφ internalisation motif(s). We demonstrate this interaction is critical for correct cell wall deposition and polarised growth. Thus, AP-2 mediated endocytic recycling is a key step in the regulation of the fungal cell wall.
-
-
-
The chitin attenuator: Cross-talk between calcineurin and the cell wall salvage pathways prevents chitin overexpression and loss of fungal viability
More LessChitin 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.
-
-
-
High-throughput growth and data with ‘impact’: Converting the Singer testing fleet into a lab
More LessSinger 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.
-
-
-
Data-driven prediction of genetic interactions in Candida glabrata
More LessThe 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.
-
-
-
Functional characterisation of novel oxidative stress protection proteins in the pathogenic yeast Candida glabrata
More LessCandida 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.
-
-
-
Engineering synthetic mini-chromosomes to study chromosome segregation in budding yeast
More LessChromosomes 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.
-
-
-
Mec1ATM/ATR is a multifaceted protein kinase promoting cellular response to genotoxic stress and perturbation in protein homeostasis
More LessATM 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.
-
-
-
Using Saccharomyces cerevisiae as a tool for mapping genetic variants underlying differences in response to anti-cancer agents
More LessBackgroundThere are variations in response to anti-cancer agents amongst individuals because they are complex traits controlled by multiple genes and environmental factors. Here, we aimed to decipher the genes controlling the variations in response to aspirin, metformin, disulfiram and 5-fluorouracil using quantitative trait loci (QTL) mapping in yeast before translating findings to humans. This approach was feasible due to the conservation of genes between these two organisms.
MethodA panel of 111 F12 meiotic segregants generated from a four-parent S. cerevisiae cross were genotyped by whole genome sequencing. Segregants growth in different treatments were phenotyped using PHENOS. Subsequently, linkage-based fine QTL mapping was performed to locate regions of the genome and the identification of causative genes.
ResultsLinkage analysis has mapped hundreds of genetic loci in the yeast genome responsible for the variations in response to the agents tested. Conserved homologs to human genes were identified. Some hits have been previously supported in the literature such as the effect of aspirin and metformin on MTOR thus validating this screening approach. Novel genes identified and pathway enrichment revealed mechanisms by which these agents may exhibit their anti-cancer properties.
ConclusionDetection of genetic variants influencing the differences in drug response could help identify individuals at risk or benefit of using anti-cancer agents. Current work includes validating the alleles of causative genes (Rad57 and VMA22) in yeast by reciprocal hemizygosity and allele swapping. This study could aid the development of biomarkers for drug response and validate the repurposing of drugs for cancer prevention.
-
-
-
The BioScreening Facility at Newcastle University
More LessThe 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.
-
-
-
Fungal Transformers: Tracking a Moving Target
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.
-
-
-
Investigating a novel commensal strain of Candida famata within Daniorerio
More LessOur 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.
-
- Poster Presentation
-
-
Assessing the ecological role of yeasts in the human gut
More LessBackground:The influence of gut microorganisms in health and disease is well documented, however, the role of yeasts has not been as widely studied as bacteria, despite associations with gastrointestinal disorders, such as Irritable Bowel Syndrome (IBS). For instance, Candida albicans is an opportunistic pathogen that has been linked to gastrointestinal symptoms.
Methods:Urine, stool and blood samples will be collected from 40 healthy controls, 40 IBS and 40 inflammatory bowel disease (IBD) patients for microbial and metabolic analysis using primarily flow cytometry-fluorescence in situ hybridisation (FC-FISH) and proton nuclear magnetic resonance (1H-NMR) spectroscopy, respectively. Calprotectin and anti-Saccharomyces cerevisiae antibodies (ASCA) levels will assess gastrointestinal inflammation. Statistical analysis will identify yeasts and their metabolites associated with the cohorts of interest.
Preliminary results:Preliminary tests to assess the presence and functionality of yeasts in healthy and IBS faecal samples using in vitro batch culture fermentation highlighted significant metabolic differences between the phenotypes: Trimethylamine (TMA), short chain fatty acids (SCFA) and ethanol predominantly distinguished in the IBS metabolic profile, compared to gamma-amino-N-butyrate (GABA) in the healthy donor. The addition of C. albicans shifted the healthy phenotype to resemble the IBS donor, whereas nystatin shifted the IBS phenotype towards the healthy metabolic profile.
Conclusion:This study will further define the preliminary results that indicate the implication of yeasts in IBS pathogenesis. Understanding the role of yeasts within the healthy and diseased human gut is necessary to develop targeted therapies to improve patient’s quality of lives and relieve burdens on the economy and healthcare systems.
-
-
-
Comparative Genomics of the Saccharomyces complex
More LessThe 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.
-
-
-
Hog1 signalling in the emerging multidrug resistant fungal pathogen Candida auris
More LessThe 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.
-
-
-
Evaluating the use of variation graphs for the characterisation of yeast rDNA arrays
More LessOver 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.
-
-
-
Development of a new high-throughput method for screening large yeast libraries for use in the beverage industry
More LessHigh-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.
-
-
-
The activity of S. pombe LTR retrotransposons is activated in response to rapamycin
More LessLong 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.
-
-
-
Dissecting the role of Peroxiredoxins in regulating conserved ROS-activated kinases
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.
-
-
-
Inhibition of Drug-Resistant Candida glabrata by Killer Toxins Produced by Saccharomyces cerevisiae
More LessCombatting 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.
-
-
-
Drug resistant Candida colonization among ICU patients in a Nigerian teaching hospital
More LessIntroduction:Colonisation of body surfaces with resistant strains is an initial step in the pathogenesis of drug resistant invasive Candida infections. Nigeria has little data on the species distribution and resistance pattern of colonising Candida species in intensive care unit(ICU) patients. This study set out to investigate the burden of Candida colonisation in the ICU of a tertiary health institution in Ile-Ife, Nigeria.
Methodology:At admission, 72 hours and 7 days, swabs of skin, oropharynx, perianal region, surgical wounds, tracheal aspirates and urine samples taken from adult ICU patients were cultured on media selective for fungi. Candida species were identified by standard microbiological methods. Sensitivity to fluconazole, voriconazole and caspofungin was determined by disc diffusion according to CLSI guidelines. Strain interrelatedness was investigated by Randomly Amplified Polymorphic DNA (RAPD).
Results:Of 744 samples from 110 patients, 142 samples (18.3%) yielded Candida. The average colonisation index increased with duration of admission (p=0.001). Most frequently colonized were the oropharynx (39.5%) and the perianal region (22.4%). Species isolated were C. albicans 97 (68.3%), C. tropicalis 20 (14.1%), C. glabrata 12 (8.5%), C. parapsilosis 7 (4.9%) and C. krusei 6 (4.2%). About 1 in every 3 isolates was resistant to fluconazole (42/142; 29.6%). In addition, resistance to voriconazole was 12/142 (8.5%) while 10/142 (7%) were resistant to caspofungin. RAPD showed identical strains across several body sites of many patients.
Conclusion:Candida species with high rates of resistance to the first line antifungal agent colonise and are transmitted among ICU patients. These can potentially cause difficult to treat invasive infections, underscoring a need for better infection control practices.
-
-
-
Modification of Candida albicans cell wall by commensal gut bacteria
More LessThe human gut is populated with a vast community of microbes, the microbiota. Fungi comprise 0.1% of the total gut microbiota. Some of these fungi exist as benign members, however others such Candida albicans can undergo a pathogenic switch causing disease. The fungal cell wall is the first target for immune system recognition. Recent studies have suggested that Candida is decorated with different cell wall epitopes within different physiological niches, due to the impact of carbon source and oxygen availability on cell wall remodelling. Here we hypothesize that resident gut bacteria also play a major role in fungal cell wall remodelling and immune recognition. Data from our lab has shown that a common bacterium from the gut, Bacteroides thetaiotaomicron (Bt), produces an extensive repertoire of degradative enzymes to breakdown the Candida cell wall. Recently, we have identified novel enzymes in Bt from the glycoside hydrolase family 130 (GH130), which specifically target β1,2-linked mannan, a unique feature of Candida mannan. We have deleted multiple fungal mannan specific loci in Bt and examined the ability of deletion strains to utilise Candida mannan as a carbon source. These data suggest that Bt contains multiple pathways to degrade the Candida cell wall. Now we are systematically dissecting the impact of mannan degradation on the physiology of the fungus. This will provide insights into how two prominent members of the gut microbiota interact with each other, how the Candida cell wall is modified in the anaerobic environment of the gut, and the importance of this in promoting immune homeostasis.
-
- Research Article
-
-
-
Middle East respiratory coronavirus (MERS-CoV) spike (S) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies
Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic coronavirus that was identified in 2012. MERS-CoV infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. The MERS-CoV spike (S) protein is a major target for neutralizing antibodies in infected patients. The MERS-CoV microneutralization test (MNt) is the gold standard method for demonstrating prior infection. However, this method requires the use of live MERS-CoV in biosafety level 3 (BSL-3) containment. The present work describes the generation and validation of S protein-bearing vesicular stomatitis virus (VSV) pseudotype particles (VSV-MERS-CoV-S) in which the VSV glycoprotein G gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect MERS-CoV neutralizing antibodies under BSL-2 conditions. Using a panel of human sera from confirmed MERS-CoV patients, the VSV-MERS-CoV particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live MERS-CoV. The results suggest that the VSV-MERS-CoV-S pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect MERS-CoV neutralizing antibodies in human sera.
-
-
-
-
Analysis of CRISPR gene drive design in budding yeast
More LessControl of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology – a gene drive – has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems in vivo, including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in Saccharomyces cerevisiae that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of ‘resistant’ clones that still harboured both the drive and target selection markers following expression of Streptococcus pyogenes Cas9; de novo mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.
-
-
-
Shiga toxin-producing Escherichia coli O26:H11 associated with a cluster of haemolytic uraemic syndrome cases in South Africa, 2017
Introduction . Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that may cause diarrhoeal outbreaks and occasionally are associated with haemolytic-uraemic syndrome (HUS). We report on STEC O26:H11 associated with a cluster of four HUS cases in South Africa in 2017.
Methodology . All case-patients were female and aged 5 years and under. Standard microbiological tests were performed for culture and identification of STEC from specimens (human stool and food samples). Further analysis of genomic DNA extracted from bacterial cultures and specimens included PCR for specific virulence genes, whole-genome sequencing and shotgun metagenomic sequencing.
Results. For 2/4 cases, stool specimens revealed STEC O26:H11 containing eae, stx2a and stx2b virulence genes. All food samples were found to be negative for STEC. No epidemiological links could be established between the HUS cases. Dried meat products were the leading food item suspected to be the vehicle of transmission for these cases, as 3/4 case-patients reported they had eaten this. However, testing of dried meat products could not confirm this.
Conclusion. Since STEC infection does not always lead to severe symptoms, it is possible that many more cases were associated with this cluster and largely went unrecognized.
-
- Short Communication
-
-
-
Stable genetic integration of a red fluorescent protein in a virulent Group A Streptococcus strain
There are several advantages, both in vitro and in vivo, in utilizing bacteria that express a fluorescent protein. Such a protein can be transiently incorporated into the bacteria or integrated within the bacterial genome. The most widely utilized fluorescent protein is green fluorescent protein (GFP), but limitations exist on its use. Additional fluorescent proteins have been designed that have many advantages over GFP and technologies for their incorporation into bacteria have been optimized. In the current study, we report the successful integration and expression of a stable fluorescent reporter, mCherry (red fluorescent protein, RFP), into the genome of a human pathogen, Group A Streptococcus pyogenes (GAS) isolate AP53(S-). RFP was targeted at the atg codon of the fcR pseudogene that is present in the mga regulon of AP53(S-). Transcription of critical bacterial genes was not functionally altered by the genomic integration of mCherry. Host virulence both in vitro (keratinocyte infection and cytotoxicity) and in vivo (skin infection) was maintained in AP53(S-)-RFP. Additionally, survival of mice infected with either AP53(S-) or AP53(S-)-RFP was similar, demonstrating that overall pathogenicity of the AP53(S-) strain was not altered by the expression of mCherry. These studies demonstrate the feasibility of integrating a fluorescent reporter into the bacterial genome of a naturally virulent isolate of Group A S. pyogenes for comparative experimental studies.
-
-
-
-
Complete genome dynamics of a dominant-lineage strain of Xanthomonas oryzae pv. oryzae harbouring a novel plasmid encoding a type IV secretion system
More LessXanthomonas oryzae pv. oryzae (Xoo) is a serious pathogen causing bacterial blight disease in rice. Population genomic studies have revealed that rampant inter-strain rather than inter-lineage differences are contributing to the evolutionary success of this pathogen. Here, we report the complete genome sequence of BXO1, a strain of Xoo belonging to a dominant lineage from India. A complete genome-based investigation revealed the presence of two plasmids, pBXO1-1 (66.7 kb) and pBXO1-2 (25.6 kb). The pBXO1-1 plasmid encodes 71 genes, 38 of which encode hypothetical proteins of unknown function. However, these hypothetical genes possess atypical GC content, pointing towards their acquisition and movement through horizontal gene transfer. Interestingly, pBXO1-2 encodes a type IV secretion system (T4SS), which is known to play an important role in the conjugative transfer of genetic material, and also provides fitness to pathogenic bacteria for their enhanced survival. Neither plasmid has been reported previously in any other complete Xoo genome published to date. Our analysis also revealed that the pBXO1-2 plasmid is present in Xanthomonas albilineans str. GPE PC73, which is known to cause leaf scald, a lethal disease in sugarcane. Our complete genome sequence analysis of BXO1 has provided us with detailed insights into the two novel strain-specific plasmids, in addition to decoding their functional capabilities, which were not assessable when using the draft genome sequence of the strain. Overall, our study has revealed the mobility of a novel T4SS in two pathogenic species of Xanthomonas that infect the vascular tissues of two economically important monocot plants, i.e. rice and sugarcane.
-
- Case Report
-
-
-
Cardiobacterium hominis endocarditis complicated by aortic root abscess: a case report
More LessThe present report describes a case of infective endocarditis complicated with aortic root abscess caused by Cardiobacterium hominis in a 56-year-old man. C. hominis is a microaerophilic, pleomorphic Gram-negative bacillus and member of the Haemophilus species, Aggregatibacter actinomycetemcomitans , C. hominis , Eikenella corrodens and Kingella kingae (HACEK) group, a group of bacteria known to be a rare cause of endocarditis. With prompt diagnosis and initiation of antimicrobial and surgical management, a successful outcome was achieved.
-
-
-
-
The needs for diagnostic imaging in cases of group A streptococcal meningitis in children: a case report and review of the literature
More LessGroup A streptococcus (GAS) is a rare cause of bacterial meningitis in children and is associated with a high cerebral complication rate. In this case report, we present a 9-year-old girl with GAS meningitis complicated with cerebritis. Clear guidelines about choice of treatment and indications of follow-up by imaging tests are lacking, making GAS meningitis unpredictable and difficult to treat. Eventually, we found 25 paediatric cases of GAS meningitis presented in the literature and reviewed their treatment choices, outcomes and follow-up by imaging tests. Penicillin and ceftriaxone are most preferred for the treatment of GAS meningitis and adding rifampicin to the antibiotic treatment could be of potential benefit. When considering the duration of antibiotic treatment and follow-up by imaging tests, no clear recommendations were found. We found that GAS meningitis is associated with higher mortality and cerebral complication rates compared to other, more common, bacterial causes of meningitis in children. This should alert the clinician to consider imaging tests routinely, even if the patient improves clinically. We advise clinicians to routinely evaluate for possible cerebral complications through magnetic resonance imaging (MRI) scans. When cerebral complications are found, antibiotic treatment should be prolonged and adding rifampicin to the antibiotic regime may be considered.
-
-
-
Oral flora meningoencephalitis diagnosis by next-generation DNA sequencing
Introduction. Standard culture methods may fail to detect the causative agents of bacterial infection for various reasons including specimen collection after antibiotic administration, or when standard techniques or environmental conditions are not appropriate for growth of the microorganisms. Conventional 16S rRNA gene sequencing is sometimes a useful alternative technique for identification of bacteria, but is confounded by polymicrobial infection. We present a case of a patient who developed a serious neurological infection for which causative oral flora organisms were observed by microscopy, failed to culture but were identified by next-generation DNA sequencing.
Case presentation. A male in his forties developed sinus pain and congestion, followed by facial and eye pain, and several weeks later acute-onset confusion and neck stiffness. Cerebrospinal fluid examination revealed pleocytosis and several bacterial morphologies, which were subsequently identified by next-generation sequencing as oral flora constituents Porphyromonas endodontalis , Fusobacterium nucleatum , Streptococcus constellatus , Prevotella species and Parvimonas micra .
Conclusion. Oral flora can cause meningoencephalitis and brain abscess formation if translocation occurs by injury or surgical procedures. Next-generation sequencing is often not available at healthcare facilities, or when available may not have been validated for a wide spectrum of specimen sources, but is available at reference laboratories and should be considered when routine methods fail to provide a diagnosis for serious infections.
-
- Abstracts from the British Yeast Group Meeting 2019
-
- Poster Presentation
-
-
Novel Double-Stranded RNA Viruses Discovered within Saccharomyces cerevisiae
Saccharomyces yeasts harbor many different viruses and parasitic genetic elements, including double-stranded RNA (dsRNA) viruses from the family Totiviridae. The identification of novel dsRNA viruses in yeasts has been constrained by the lack of effective protocols for the unbiased preparation and sequencing of dsRNAs. We have developed a next-generation sequencing method that enables the amplification and sequencing of yeast dsRNAs. Using this method, we have performed a metagenomic screen of more than 600 strains of Saccharomyces cerevisiae for the presence of novel dsRNA viruses. Surprisingly, we have identified several novel bipartite dsRNA viruses from the family Partitiviridae within different strains of S. cerevisiae. Partitiviruses have never been described within Saccharomyces yeasts or within the wider Saccharomycotina taxonomic subdivision of the Ascomycota phylum. We confirmed the presence of these partitivirus dsRNAs using reverse transcriptase-PCR and have been successful in purifying viral particles from infected yeasts. After deep sequencing of purified dsRNAs, we have identified three species of virus that have weak homology (20-35% amino acid identity) to viruses of the genus Cryspovirus, which replicate within the protozoan pathogen Cryptosporidium parvum. Partitiviruses can modulate the virulence and fecundity of plant and human pathogens, respectively. The discovery of partitiviruses in S. cerevisiae will enable the study of how partitiviruses interact with the host cell environment and alter cellular physiology.
-