. The origins and evolution of virulence in amphibian-infecting chytrids
. Batrachochytrium dendrobatidis
. (
. Bd
. ) and
. Batrachochytrium salamandrivorans
. (
. Bsal)
. are largely unknown. Here, we use deep nanopore sequencing of
. Bsal
. and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that
. Bsal
. has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific
. Bd
. with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (
. n
. = 177) in
. Bsal
. most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally,
. Bsal
. has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both
. Bd
. and
. Bsal
. Furthermore, genes with signatures of positive selection in
. Bd
. are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.
.

AbstractThe origins of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. Bsal has the most repeat-rich genome, comprising 40.9% repetitive elements, which has expanded to more than 3X the length of its conspecific Bd. M36 metalloprotease virulence factors are highly expanded in Bsal and 53% of the 177 unique genes are flanked by transposable elements, suggesting repeat-driven expansion. The largest M36 sub-family are mostly (84%) flanked upstream by a novel LINE element, a repeat superfamily implicated with gene copy number variations. We find that Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes occur in gene-rich/repeat-poor compartments. This is a hallmark of two-speed genome evolution. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting they are a cradle for chytrid pathogenicity evolution, and Bd also has a two-speed genome. This is the first evidence of two-speed genomes in any animal pathogen, and sheds new light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

Amphibian declines worldwide are a major threat to biodiversity and contribute to Earth’s sixth mass extinction. Amphibian extinctions and extirpations are caused by a variety of factors, including habitat loss and climate change. However, the chytridiomycosis panzootic and its causative agents, the batrachochytrids Batrachochytrium salamandrivorans (Bsal) and Batrachochytrium dendrobatidis (Bd), are major contributing factors and are together responsible for over 90 extinctions and more than 500 declines. Bsal and Bd’s devastating impact reaches beyond amphibian populations into ecosystem health including directly threatening human health, exemplified by an increase in malaria incidence in Panama and Costa Rica linked to collapses of local amphibian populations. Mitigation of this global threat to biodiversity and human welfare is crucial. Gaining a deeper understanding of the evolution and virulence of the batrachochytrids responsible for the chytridomycosis panzootic is necessary to inform mitigation efforts and has the potential to unveil deeper understanding of how pathogens adapt to a wide range of hosts, how virulence evolves and how host-pathogen interactions shape genomes.

Until recently, the origins of Bsal’s virulence and the evolution of the batrachochytrids were largely unknown, owing in part to a lack of a high-quality reference genome. Indeed, before 2022, only a highly fragmented short-read assembly of Bsal’s genome was available, mainly due to its repeat-richness.

After a literature review in chapter 1 and 2, outlining the motivation and aim of the study in chapter 3, and detailing my research methodology in chapter 4, I describe my strategy and considerations for generating a new assembly based on deep nanopore long-read sequencing in chapter 5. The resulting assembly provides a significant improvement in contiguity and completeness, as well as repeat resolution. I detail the road map to achieving this high-quality assembly of an extremely repeat-rich genome with comprehensive descriptions of how I performed read quality control, trimming and filtering, pre-assembly assessments, assembly benchmarking, polishing and further annotation. For benchmarking, I introduce a summary quality score for easy comparison of different reference-free assemblies, which is based on and adapted from the C-score for reference-based assemblies
(Zhang et al. 2022).

In chapter 6, I describe my analysis of this new assembly to discover that Bsal has the most repeat-rich genome of the 22 Chytridiomycota investigated in this thesis, comprising 40.9% repetitive elements. The Bsal genome appears to have undergone a repeat-driven expansion to more than 3X the length of its closest relative Bd. Autonomous and fully functional transposable elements of the LTR/Gypsy family appear to contribute significantly to the observed expansion. The M36 metalloprotease virulence factors are highly expanded (n = 177) in Bsal, many of which are flanked by transposable elements (TEs), suggesting they have contributed to a repeat-associated expansion of that protein family (described in detail in chapter 8). Three TE families belonging to the superfamily of LINEs, implicated with gene copy number variations, are found to be enriched upstream of M36 metalloprotease genes. This highlights the role of TEs in actively and passively shaping genome architecture and evolution. Unlike other fungi, Bsal and Bd have no RIP (Repeat Induced Point mutation) machinery to silence TE activity, but appear to rely on i.a. RNAi silencing. I also present evidence of increased methylation of TEs compared to other features of the genome, which could represent another avenue for Bsal to control TE proliferation.

In chapter 7, I take a deeper dive into chytrid genome organization, and discover and describe for the first time Bsal’s highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the
evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution which has previously only been described in plant pathogens.

The main findings of my thesis have been recently published in PNAS (Wacker et al. 2023a) in a comprehensive report of the first two-speed genome architecture described for a pathogen of vertebrates, shedding new light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

. The origins and evolution of virulence in amphibian-infecting chytrids
. Batrachochytrium dendrobatidis
. (
. Bd
. ) and
. Batrachochytrium salamandrivorans
. (
. Bsal)
. are largely unknown. Here, we use deep nanopore sequencing of
. Bsal
. and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that
. Bsal
. has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific
. Bd
. with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (
. n
. = 177) in
. Bsal
. most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally,
. Bsal
. has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both
. Bd
. and
. Bsal
. Furthermore, genes with signatures of positive selection in
. Bd
. are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.
.

Candida glabrata is the second most common etiological cause of worldwide systemic candidiasis in adult patients. Genome analysis of 68 isolates from 8 hospitals across Scotland, together with 83 global isolates, revealed insights into the population genetics and evolution of C. glabrata. Clinical isolates of C. glabrata from across Scotland are highly genetically diverse, including at least 19 separate sequence types that have been recovered previously in globally diverse locations, and 1 newly discovered sequence type. Several sequence types had evidence for ancestral recombination, suggesting transmission between distinct geographical regions has coincided with genetic exchange arising in new clades. Three isolates were missing MATα1, potentially representing a second mating type. Signatures of positive selection were identified in every sequence type including enrichment for epithelial adhesins thought to facilitate fungal adhesin to human epithelial cells. In patent microevolution was identified from 7 sets of recurrent cases of candidiasis, revealing an enrichment for nonsynonymous and frameshift indels in cell surface proteins. Microevolution within patients also affected epithelial adhesins genes, and several genes involved in drug resistance including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2, the latter coinciding with a marked drop in fluconazole minimum inhibitory concentration. In addition to nuclear genome diversity, the C. glabrata mitochondrial genome was particularly diverse, with reduced conserved sequence and conserved protein-encoding genes in all nonreference ST15 isolates. Together, this study highlights the genetic diversity within the C. glabrata population that may impact virulence and drug resistance, and 2 major mechanisms generating this diversity: microevolution and genetic exchange/recombination.

Aspergillus fumigatus is the leading cause of the fungal invasive disease called aspergillosis, which is associated with a high mortality rate that can reach 50% in some groups of immunocompromised individuals. The increasing prevalence of azole-resistant A. fumigatus isolates, both in clinical settings and the environment, highlights the importance of discovering new fungal virulence factors that can potentially become targets for novel antifungals. Nitronate monooxygenases (Nmos) represent potential targets for antifungal compounds as no orthologs of those enzymes are present in humans. Nmos catalyse the denitrification of nitroalkanes, thereby detoxifying these mediators of nitro-oxidative stress, and therefore we tested whether Nmos provide protection for A. fumigatus against host-imposed stresses at sites of infection. The results of inhibition zone assays indicated that Nmo2 and Nmo5 are not essential for the oxidative stress resistance of A. fumigatus in vitro. In addition, the resazurin-based metabolic activity assay revealed that the growth of mutants lacking the nmo2 or nmo5 genes was only slightly reduced in the presence of 0.05 mM peroxynitrite. Nevertheless, both Nmo2 and Nmo5 were shown to contribute to defense against murine bone marrow-derived macrophages, and this was no longer observed when NADPH oxidase, the main generator of reactive oxygen species during infection, was inhibited in macrophages. Furthermore, we revealed that Nnmos promote the virulence of the fungus in the Galleria mellonella model of infection. Both nmo2 and nmo5 knock-out strains were less virulent than the wild-type control as recorded 72 h post-infection. Our results indicate that Nmos play a role in the virulence of A. fumigatus.

AbstractThe origins of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. Bsal has the most repeat-rich genome, comprising 40.9% repetitive elements, which has expanded to more than 3X the length of its conspecific Bd. M36 metalloprotease virulence factors are highly expanded in Bsal and 53% of the 177 unique genes are flanked by transposable elements, suggesting repeat-driven expansion. The largest M36 sub-family are mostly (84%) flanked upstream by a novel LINE element, a repeat superfamily implicated with gene copy number variations. We find that Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes occur in gene-rich/repeat-poor compartments. This is a hallmark of two-speed genome evolution. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting they are a cradle for chytrid pathogenicity evolution, and Bd also has a two-speed genome. This is the first evidence of two-speed genomes in any animal pathogen, and sheds new light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

(1) Background: Microbial communities in terrestrial, calcifying high-alkaline springs are not well understood. In this study, we investigate the structure and composition of microbial mats in ultrabasic (pH 10-12) serpentinite springs of the Voltri Massif (Italy). (2) Methods: Along with analysis of chemical and mineralogical parameters, environmental DNA was extracted and subjected to analysis of microbial communities based upon next-generation sequencing. (3) Results: Mineral precipitation and microbialite formation occurred, along with mat formation. Analysis of the serpentinite spring microbial community, based on Illumina sequencing of 16S rRNA amplicons, point to the relevance of alkaliphilic cyanobacteria, colonizing carbonate buildups. Cyanobacterial groups accounted for up to 45% of all retrieved sequences; 3-4 taxa were dominant, belonging to the filamentous groups of Leptolyngbyaceae, Oscillatoriales, and Pseudanabaenaceae. The cyanobacterial community found at these sites is clearly distinct from creek water sediment, highlighting their specific adaptation to these environments.