David A. Fitzpatrick

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis

BackgroundTo date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available.ResultsA dataset of 345,829 genes was extracted from 42 publicly available fungal genomes. Supertree methods were employed to derive phylogenies from 4,805 single gene families. We found that the average consensus supertree method may suffer from long-branch attraction artifacts, while matrix representation with parsimony (MRP) appears to be immune from these. A genome phylogeny was also reconstructed from a concatenated alignment of 153 universally distributed orthologs. Our MRP supertree and concatenated phylogeny are highly congruent. Within the Ascomycota, the sub-phyla Pezizomycotina and Saccharomycotina were resolved. Both phylogenies infer that the Leotiomycetes are the closest sister group to the Sordariomycetes. There is some ambiguity regarding the placement of Stagonospora nodurum, the sole member of the class Dothideomycetes present in the dataset.Within the Saccharomycotina, a monophyletic clade containing organisms that translate CTG as serine instead of leucine is evident. There is also strong support for two groups within the CTG clade, one containing the fully sexual species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus. The second major clade within the Saccharomycotina contains species whose genomes have undergone a whole genome duplication (WGD), and their close relatives. We could not confidently resolve whether Candida glabrata or Saccharomyces castellii lies at the base of the WGD clade.ConclusionWe have constructed robust phylogenies for fungi based on whole genome analysis. Overall, our phylogenies provide strong support for the classification of phyla, sub-phyla, classes and orders. We have resolved the relationship of the classes Leotiomyctes and Sordariomycetes, and have identified two classes within the CTG clade of the Saccharomycotina that may correlate with sexual status.

Horizontal gene transfer in fungi

Horizontal gene transfer (HGT) is frequently observed in prokaryotes and until recently was assumed to be of limited importance to eukaryotes. However, there is an increasing body of evidence to suggest that HGT is an important mechanism in eukaryotic genome evolution, particularly in unicellular organisms. The transfer of individual genes, gene clusters or entire chromosomes can have significant impacts on niche specification, disease emergence or shift in metabolic capabilities. In terms of genomic sequencing, the fungal kingdom is one of the most densely sampled eukaryotic lineages and is at the forefront of eukaryote comparative genomics and enables us to use fungi to study eukaryotic evolutionary mechanisms including HGT. This review describes the bioinformatics-based methodologies commonly used to locate HGT in fungal genomes and investigates the possible mechanisms involved in transferring genetic material laterally into fungal species. I will highlight a number of fungal HGT events and discuss the impact they have played on fungal evolution and discuss the implications HGT may have on the fungal tree of life.

Does a tree-like phylogeny only exist at the tips in the prokaryotes?

The extent to which prokaryotic evolution has been influenced by horizontal gene transfer (HGT) and therefore might be more of a network than a tree is unclear. Here we use supertree methods to ask whether a definitive prokaryotic phylogenetic tree exists and whether it can be confidently inferred using orthologous genes. We analysed an 11–taxon dataset spanning the deepest divisions of prokaryotic relationships, a 10–taxon dataset spanning the relatively recent ?–proteobacteria and a 61–taxon dataset spanning both, using species for which complete genomes are available. Congruence among gene trees spanning deep relationships is not better than random. By contrast, a strong, almost perfect phylogenetic signal exists in ?–proteobacterial genes. Deep–level prokaryotic relationships are difficult to infer because of signal erosion, systematic bias, hidden paralogy and/or HGT. Our results do not preclude levels of HGT that would be inconsistent with the notion of a prokaryotic phylogeny. This approach will help decide the extent to which we can say that there is a prokaryotic phylogeny and where in the phylogeny a cohesive genomic signal exists.

Fatty acid biosynthesis in Mycobacterium tuberculosis: lateral gene transfer, adaptive evolution, and gene duplication.

Mycobacterium tuberculosis is a high GC Gram-positive member of the actinobacteria. The mycobacterial cell wall is composed of a complex assortment of lipids and is the interface between the bacterium and its environment. The biosynthesis of fatty acids plays an essential role in the formation of cell wall components, in particular mycolic acids, which have been targeted by many of the drugs used to treat M. tuberculosis infection. M. tuberculosis has ≈250 genes involved in fatty acid metabolism, a much higher proportion than in any other organism. In silico methods have been used to compare the genome of M. tuberculosis CDC1551 to a database of 58 complete bacterial genomes. The resulting alignments were scanned for genes specifically involved in fatty acid biosynthetic pathway I. Phylogenetic analysis of these alignments was used to investigate horizontal gene transfer, gene duplication, and adaptive evolution. It was found that of the eight gene families examined, five of the phylogenies reconstructed suggest that the actinobacteria have a closer relationship with the α-proteobacteria than expected. This is either due to either an ancient transfer of genes or deep paralogy and subsequent retention of the genes in unrelated lineages. Additionally, adaptive evolution and gene duplication have been an influence in the evolution of the pathway. This study provides a key insight into how M. tuberculosis has developed its unique fatty acid synthetic abilities.

Analysis of gene evolution and metabolic pathways using the Candida Gene Order Browser

BackgroundCandida species are the most common cause of opportunistic fungal infection worldwide. Recent sequencing efforts have provided a wealth of Candida genomic data. We have developed the Candida Gene Order Browser (CGOB), an online tool that aids comparative syntenic analyses of Candida species. CGOB incorporates all available Candida clade genome sequences including two Candida albicans isolates (SC5314 and WO-1) and 8 closely related species (Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Lodderomyces elongisporus, Debaryomyces hansenii, Pichia stipitis, Candida guilliermondii and Candida lusitaniae). Saccharomyces cerevisiae is also included as a reference genome.ResultsCGOB assignments of homology were manually curated based on sequence similarity and synteny. In total CGOB includes 65617 genes arranged into 13625 homology columns. We have also generated improved Candida gene sets by merging/removing partial genes in each genome. Interrogation of CGOB revealed that the majority of tandemly duplicated genes are under strong purifying selection in all Candida species. We identified clusters of adjacent genes involved in the same metabolic pathways (such as catabolism of biotin, galactose and N-acetyl glucosamine) and we showed that some clusters are species or lineage-specific. We also identified one example of intron gain in C. albicans.ConclusionsOur analysis provides an important resource that is now available for the Candida community. CGOB is available at http://cgob.ucd.ie.

Evidence of recent interkingdom horizontal gene transfer between bacteria and Candida parapsilosis

BackgroundTo date very few incidences of interdomain gene transfer into fungi have been identified. Here, we used the emerging genome sequences of Candida albicans WO-1, Candida tropicalis, Candida parapsilosis, Clavispora lusitaniae, Pichia guilliermondii, and Lodderomyces elongisporus to identify recent interdomain HGT events. We refer to these as CTG species because they translate the CTG codon as serine rather than leucine, and share a recent common ancestor.ResultsPhylogenetic and syntenic information infer that two C. parapsilosis genes originate from bacterial sources. One encodes a putative proline racemase (PR). Phylogenetic analysis also infers that there were independent transfers of bacterial PR enzymes into members of the Pezizomycotina, and protists. The second HGT gene in C. parapsilosis belongs to the phenazine F (PhzF) superfamily. Most CTG species also contain a fungal PhzF homolog. Our phylogeny suggests that the CTG homolog originated from an ancient HGT event, from a member of the proteobacteria. An analysis of synteny suggests that C. parapsilosis has lost the endogenous fungal form of PhzF, and subsequently reacquired it from a proteobacterial source. There is evidence that Schizosaccharomyces pombe and Basidiomycotina also obtained a PhzF homolog through HGT.ConclusionOur search revealed two instances of well-supported HGT from bacteria into the CTG clade, both specific to C. parapsilosis. Therefore, while recent interkingdom gene transfer has taken place in the CTG lineage, its occurrence is rare. However, our analysis will not detect ancient gene transfers, and we may have underestimated the global extent of HGT into CTG species.

Genomic and Proteomic Dissection of the Ubiquitous Plant Pathogen, Armillaria mellea: Toward a New Infection Model System

Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the A. mellea genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (n = 629 unique) and secreted (n = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (n = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the A. mellea proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that A. mellea exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive A. mellea proteins (n = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms.

The copper regulon of the human fungal pathogen Cryptococcus neoformans H99.

Cryptococcus neoformans is a human fungal pathogen that is the causative agent of cryptococcosis and fatal meningitis in immuno‐compromised hosts. Recent studies suggest that copper (Cu) acquisition plays an important role in C. neoformans virulence, as mutants that lack Cuf1, which activates the Ctr4 high affinity Cu importer, are hypo‐virulent in mouse models. To understand the constellation of Cu‐responsive genes in C. neoformans and how their expression might contribute to virulence, we determined the transcript profile of C. neoformans in response to elevated Cu or Cu deficiency. We identified two metallothionein genes (CMT1 and CMT2), encoding cysteine‐rich Cu binding and detoxifying proteins, whose expression is dramatically elevated in response to excess Cu. We identified a new C. neoformans Cu transporter, CnCtr1, that is induced by Cu deficiency and is distinct from CnCtr4 and which shows significant phylogenetic relationship to Ctr1 from other fungi. Surprisingly, in contrast to other fungi, we found that induction of both CnCTR1 and CnCTR4 expression under Cu limitation, and CMT1 and CMT2 in response to Cu excess, are dependent on the CnCuf1 Cu metalloregulatory transcription factor. These studies set the stage for the evaluation of the specific Cuf1 target genes required for virulence in C. neoformans.

Universally distributed single-copy genes indicate a constant rate of horizontal transfer

Single copy genes, universally distributed across the three domains of life and encoding mostly ancient parts of the translation machinery, are thought to be only rarely subjected to horizontal gene transfer (HGT). Indeed it has been proposed to have occurred in only a few genes and implies a rare, probably not advantageous event in which an ortholog displaces the original gene and has to function in a foreign context (orthologous gene displacement, OGD). Here, we have utilised an automatic method to identify HGT based on a conservative statistical approach capable of robustly assigning both donors and acceptors. Applied to 40 universally single copy genes we found that as many as 68 HGTs (implying OGDs) have occurred in these genes with a rate of 1.7 per family since the last universal common ancestor (LUCA). We examined a number of factors that have been claimed to be fundamental to HGT in general and tested their validity in the subset of universally distributed single copy genes. We found that differing functional constraints impact rates of OGD and the more evolutionarily distant the donor and acceptor, the less likely an OGD is to occur. Furthermore, species with larger genomes are more likely to be subjected to OGD. Most importantly, regardless of the trends above, the number of OGDs increases linearly with time, indicating a neutral, constant rate. This suggests that levels of HGT above this rate may be indicative of positively selected transfers that may allow niche adaptation or bestow other benefits to the recipient organism.