Genomic interrogation of Candida albicans with relation to reproductive health and fertility

Candida albicans is a commensal yeast that can colonize a variety of host-associated niches including the human urogenital tract. It is the most common cause of fungal infections both superficial and systemic. Fungal infections, including vulvovaginal candidiasis, have been heavily implicated as a multifaceted cause in human infertility with host immune effects and microbiome alterations being other influencing factors. Previous work investigated the prevalence and diversity of a number of Candida albicans isolates sourced from individuals with differing fertility statuses using MLST-based methods. This current study aimed to use comparative genomic methods to investigate at whole genome level the previously described isolates in combination with database genomes to identify ifgenes or genetic variants display an association with the ability to colonize certain niches. Pangenomeconstruction and enrichment analysis of database C. albicans assemblies showed an enrichment ofvirulence genes with the core genome. A genome wide association study of the Swansea isolates and a large dataset originating from NCBI’s sequence read archive (SRA) identified 35 variants significantly associated with isolation from the female reproductive tract which. These variants presented enrichment for functions related to antifungal resistance and hyphal growth. Together, these variants may influence the ability for a strain to persist within the female reproductive tract and to be capable of causing recurring vulvovaginal candidiasis thus potentially influencing fertility. These results offer ideal targets for further study from a genomic perspective to explore their ecological presence within the organism’s natural environment and further as targets for phenotypic investigations. The outcomes of which can be used to better our understanding of how C. albicans can influence reproductive health and wellbeing

Curr Clin Microbiol Rep

Purpose of review:Fungi represent a central yet often overlooked domain of clinically relevant pathogens that have become increasingly important in human disease. With unique adaptive lifestyles that vary widely across species, human fungal pathogens show remarkable diversity in their virulence strategies. The majority of these fungal pathogens are opportunistic, primarily existing in the environment or as commensals that take advantage of immunocompromised hosts to cause disease. In addition, many fungal pathogens have evolved from non-pathogenic lifestyles. The extent of genetic diversity and heritability of virulence traits remains poorly explored in human fungal pathogens.Recent findings:Genetic variation caused by mutations, genomic rearrangements, gene gain or loss, changes in ploidy, and sexual reproduction have profound effects on genetic diversity. These mechanisms contribute to the remarkable diversity of fungal genomes and have large impacts on their prevalence in human disease, virulence, and resistance to antifungal therapies.Summary:Here, we focus on the genomic structure of the most common human fungal pathogens and the aspects of genetic variability that contribute to their dominance in human disease.R01 AI137418/AI/NIAID NIH HHSUnited States/U19 AI166059/AI/NIAID NIH HHSUnited States/U54 CK000603/CK/NCEZID CDC HHSUnited States

Comparative genomics and epidemiology of the amphibian-killing fungus Batrachochytrium dendrobatidis

The primary aim of this thesis was to study the population structure and epidemiology of the fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd). I have addressed these questions by collecting and isolating Bd from multiple infected host species. I have then extracted and sequenced whole nuclear and mitochondrial genomes for 50 isolates of Bd using the ABI SOLiD 3 and Illumina HiSeq 2000 platforms. The first aspect of the analysis was to tailor a new method for identifying variant sites amongst the isolates, as well as verifying the accuracy of the alignment and SNP-calling methods. Next, using a number of phylogenetic methods, I identified a population split into at least three deeply divergent lineages. Two of these lineages were found in multiple continents and are associated with known introductions by anthropogenic means. Isolates belonging to one clade, which we named the Global Panzootic Lineage (BdGPL), have emerged across at least five continents and are associated with the onset of epizootics in all five continents we tested. Dating the divergence between BdGPL isolates suggested a recent common ancestor in the 20th Century, and that the widespread trade of amphibians is an important mechanism of transmission. In contrast, BdGPL diverged from the other two lineages approximately 1000 years ago, clearly refuting a single emergence hypothesis. The two newly identified divergent lineages were the Cape lineage (BdCAPE) that appeared to have originated from the Cape Province in South Africa and a Swiss lineage (BdCH) comprised of a single isolate from a pond in Gamlikon, Switzerland. The secondary aim of this thesis was to identify and compare virulence determinants and other genomic features responsible for known differences in phenotypes. Using a variety of statistical and computational methods, I identified compelling evidence for genetic recombination targeting virulence factors, selection of those and other virulence factors, and rapid changes in ploidy and aneuploidy amongst the isolates of all three lineages. These genomic features shed light on the emergence, patterns of global spread, and modes of evolution in the pathogen(s) responsible for contemporary disease-driven losses in amphibian biodiversity. Finally, I discuss how these findings update our understanding of Bd and the importance for tracking and understanding the dynamics of other current emerging pathogens in an increasingly globalised habitat.Open Acces

A Genome based approach to characterize genes involved in yeast adaptation to Sherry-like wines’ biological ageing

Wine fermentation and flor ageing are performed by two different lineages of yeast strains, with very different lifestyles. In this thesis we have studied the genome of flor yeast in comparison to wine yeast in order to unravel their specificities. We have first developed a set of haploid flor strains for the molecular evaluation of different targets, and developed as well a synthetic media mimicking wine for that purpose. From the genome sequence of 16 strains (8 wine and 8 flor) from France, Hungary, Italy and Spain we have drawn a phylogeny that showed that flor yeast represent a specific group of yeast different from wine and identified divergent regions. These regions contain genes involved in key functions and several associated with velum growth. Remarkably, many genes involved in FLO11 regulation such as MAP kinase, or Ras/PKA pathways were mutated among flor strains and many variations were encountered in genes involved with metal homeostasis such as zinc and divalent metal transporters. The impact of allelic variation of several genes has been evaluated for: the phosphatidylinositol 4-kinase PIK1 possibly involved in pseudohyphal growth, the high affinity zinc transporter ZRT1, and of the major pyruvate decarboxylase PDC1 in order to assess their role in the flor phenotype

Génomique des populations et adaptation des champignons pathogènes responsables de la maladie hollandaise de l'orme

La Maladie Hollandaise de l'Orme (MHO) est causée par des champignons du genre Ophiostoma. Ceux-ci sont responsables de la mort de plusieurs centaines de milliers d'ormes adultes en Europe ainsi qu'en Amérique du Nord, modifiant de manière drastiques les paysages forestiers et urbains. L'étude de la MHO a permis de caractériser deux espèces différentes, O. ulmi et O. novo-ulmi, qui présentent des phénotypes différents en terne de virulence et de croissance. L'analyse de données de séquençage à haut débit (génomique) associée à l'utilisation de données phénotypiques s'est répandue ces dernières décennies dans le domaine de la phytopathologie et permet de comprendre plus en détails la structure des populations ainsi que les gènes et mécanismes impliqués dans l'adaptation chez les champignons pathogènes. Dans le premier chapitre, nous comparons les caractéristiques évolutives des champignons phytopathogènes des cultures et des forêts. Nous contrastons l'impact des différents degrés de domestication et de gestion des milieux agricoles et forestiers sur ces populations de pathogènes. Les milieux agricoles et les forêts présentent des caractéristiques très différentes, comme le temps de génération ou le niveau de domestication. Cependant, nous trouvons que les mécanismes modelant les populations de pathogènes restent similaires, comme l'hybridation, les sauts d'hôtes, la sélection, la spécialisation et l'expansion clonale. Dans un second temps nous faisons un bilan des méthodes et techniques disponibles pour la gestion et l'amélioration des plantes de ces systèmes afin de prévenir ou lutter contre de futures épidémies. Dans le second chapitre, nous avons utilisé des données de génomiques pour examiner la structure génétique des populations des champignons responsables de la Maladie Hollandaise de l'Orme (MHO) Ophiostoma ulmi et Ophiostoma novo-ulmi. Nous quantifions et caractérisons la diversité génétique au sein des quatre lignées génétiques, ainsi que la divergence et la phylogénie entre chaque taxon. Nous décrivons le rôle de l'hybridation et de l'introgression dans l'histoire évolutive de ces pathogènes comme étant le mécanisme principal générant de la diversité génétique. La production de données phénotypiques nous permet également de caractériser l'impact de l'introgression sur l'adaptation de ces espèces. Dans le troisième chapitre, nous avons utilisé une approche « GWAS » (Genome Wide Analysis Study) pour révéler les marqueurs impliqués dans l'adaptation à la température et à un composé de défense de l'hôte chez O. ulmi et O. novo-ulmi. Nous trouvons d'importants gènes et familles de gènes associés avec les phénotypes de croissance et de virulence comme des transporteurs, des cytochromes, des protéines de choc thermique ou des protéines impliquées dans le système d'incompatibilité végétative qui pourraient jouer un rôle dans la protection contre les virus.Dutch Elm Disease (DED) is a highly destructive tree disease caused by fungi from the Ophiostoma genus. These fungi are responsible for the deaths of hundreds of thousands of mature elm trees both in Europe and in North America. Studies on DED allowed the characterization of two disctinct species, O. ulmi and O. novo-ulmi, that exhibit different virulence and growth phenotypes. Global pathogen genomics data including population genomics and high-quality reference assemblies are crucial for understanding the evolution and adaptation of pathogens. In a first chapter, we review crops and forest pathosystems with remarkably different characteristics, such as generation time and the level of domestication. They also have different management systems for disease control which is more intensive in crops than forest trees. By comparing and contrasting results from pathogen population genomic studies done on widely different agricultural and forest production systems, we can improve our understanding of pathogen evolution and adaptation to different selective pressures. We find that despite these differences, similar processes such as hybridization, host jumps, selection, specialization, and clonal expansion are shaping the pathogen populations in both crops and forest trees. We propose some solutions to reduce these impacts and to lower the probability of global pathogen outbreaks so that we can envision better management strategies to sustain global food production as well as ecosystem services. In a second chapter, we investigate how hybridization and the resulting introgression can drive the success of DED fungi via the rapid acquisition of adaptive traits. Using whole-genome sequences and growth phenotyping of a worldwide collection of isolates, we show that introgression has been the main driver of genomic diversity and that it impacted fitness-related traits. Introgressions contain genes involved in host-pathogen interactions and reproduction. Introgressed isolates have enhanced growth rate at high temperature and produce different necrosis sizes on an in vivo model for pathogenicity. In addition, lineages diverge in many pathogenicity-associated genes and exhibit differential mycelial growth in the presence of a proxy of a host defence compound, implying an important role of host trees in the molecular and functional differentiation of these pathogens. In the third chapter, we performed the identification of O. ulmi and O. novo-ulmi genes potentially associated with virulence and growth using Genome-Wide Association (GWA) analysis. We measured necrosis size induced on apples as a proxy for fungal virulence and measured growth rates at three different temperatures and two different media. We found several candidate genes for virulence, such as a CFEM domain containing protein and a HC-toxin efflux carrier. For growth, we identify several important gene families such as ABC and MFS transporters, cytochromes, transcription factors and proteins from the vegetative incompatibility complex

New approaches to characterise viral pathogens in aquaculture

Aquaculture is the fastest growing food-producing sector in the world and of considerable economic, cultural and environmental relevance. This sector will be vital to achieving future food security demands, but its continued sustainable expansion is severely threatened by infectious diseases, with viral diseases amongst the most problematic to control. Unlike farmed livestock, fish are generally reared in open systems with constant circulation between farms and the natural aquatic environment. This routinely exposes the animals to naturally occurring viruses in the water, both pathogenic and non-pathogenic, which are generally uptaken through mucosal surfaces (i.e. gills and gut surfaces). However, with the increase in globalisation, aquatic species are frequently farmed in non-native habitats, thus exposing them not only to the pathogens present in wild relatives of the same species, but to pathogens of other species in their introduced habitat. Moreover, wild fish are threatened by viral disease outbreaks on fish farms due to the high density of individuals available to carry and transmit the pathogen. Characterising viral infections is therefore important to support the prevention and control of disease outbreaks, as understanding the disease agent enables both fish farmers and regulating agencies to tailor appropriate mitigation strategies. The routine use of whole genome sequencing to screen infected animals is not yet commonplace in the aquaculture industry, where genetic screening of viruses is largely done using PCR for 1 or 2 marker genes. However, the ‘genomic surveillance’ approach has been used to great effect in cases of disease outbreaks relevant to human health, and could be applied in aquaculture to enhance the resolution of molecular epidemiology investigations and diagnostic tests. Moreover, with the under-researched genetic diversity of aquatic viruses, significant advances in the understanding of host-pathogen interactions could be achieved with a denser and better curated genomic database of viruses. To address these knowledge gaps, I have developed and optimised several approaches to characterise aquatic viruses up-taking various sequencing methods depending on the resolution required for the specific study, using salmonid alphavirus (SAV) as a primary study system. To rapidly and accurately generate consensus-level genomes of specific pathogenic viruses, I developed a targeted PCR approach using overlapping long amplicons tiled across the SAV genome for full coverage. These amplicons are sequenced on the Oxford Nanopore Technologies MinION long-read platform. An analysis workflow was then optimised to generate consensus genomes while maintaining capability to discover SAV subtype-level co-infections by simultaneously mapping to multiple reference sequences. This approach can generate highly accurate consensus sequences (as judged by independent Sanger sequencing) and detect co-infections of strains with ≥ 95% pairwise identity over a 2kb region, even when minor infecting strains are present at just 5% frequency. This approach was used to investigate the population dynamics and phylogeography of the SAV3 epidemic in Norwegian aquaculture, revealing repeated seedings of SAV3 from ‘source’ to ‘sink’ counties. To characterise viral genetic diversity within a host, I applied a targeted sequence capture strategy to obtain SAV genomes at high coverage (using Illumina technology) from infected fish using both pooled and individual tissue samples. This approach utilises RNA baits to capture and enrich for specific DNA (or cDNA) strands in a sample, and allows for greater sequencing efficiency. These baits, while designed from specific templates, are less specific than PCR primers and can tolerate a certain amount of template mismatches, thus capturing all genetic variation of a specific viral species within a sample. This approach was used to compare the genetic diversity of SAV in farmed Atlantic salmon and rainbow trout, in addition to two wild flatfish species, sampled from multiple regions in Scottish and Irish waters. In the same study, I provided evidence of complex infections on single fish farms, and for co-infections within single wild fish. Finally, I developed a pipeline to detect viral infections in metagenomics samples, which can be applied even when the infectious agent is unknown. This involves an optional step of mapping to the host reference genome to increase efficiency of later steps, assembly of the remaining reads with a transcriptome assembler, and identifying viral transcripts using homology-based tools. Before implementation, this pipeline was benchmarked against several datasets, including a simulated virome and a simulated co-infection of two strains of the same virus. It was also tested against datasets with known pathogens, resulting in similar efficiencies of detection as a mapping-based approach. Finally the pipeline was used on datasets with unknown viromes to demonstrate its applicability to detect novel viral species. Overall, my research has led to the development of several cutting-edge approaches for the genomic analysis of aquatic viruses and other pathogens, and helps clarify which approach is most useful in different epidemiological settings. I also demonstrate that genome-wide analyses of viral pathogens impacting salmonid aquaculture generates valuable additional information on viral diversity compared to standard surveillance methods using particular marker genes, advocating for route use of genomic approaches in this sector

Phased Diploid Genome Assemblies for Three Strains of Candida albicans from Oak Trees

Although normally a harmless commensal, Candida albicans, it is also one of the most common causes of bloodstream infections in the U.S. Candida albicans has long been considered an obligate commensal, however, recent studies suggest it can live outside animal hosts. Here, we have generated PacBio sequences and phased genome assemblies for three C. albicans strains from oak trees (NCYC 4144, NCYC 4145, and NCYC 4146). PacBio datasets are high depth (over 400 fold coverage) and more than half of the sequencing data are contained in reads longer than 15 kb. Primary assemblies showed high contiguity with several chromosomes for each strain recovered as single contigs, and greater than half of the alternative haplotype sequence was assembled in haplotigs at least 174 kb long. Using these assemblies we were able to identify structural polymorphisms, including a polymorphic inversion over 100 kb in length. These results show that phased de novo diploid assemblies for C. albicans can enable the study of genomic variation within and among strains of an important fungal pathogen