Biology subjects

Cuomo, C. A.

Publications and source records attributed to Cuomo, C. A..

12 recordsLinked to original sources

bioRxiv2018

Phenotypic variability correlates with clinical outcome in Cryptococcus isolates obtained from Botswanan HIV/AIDS patients

Pathogenic species of Cryptococcus cause hundreds of thousands of deaths annually. Considerable phenotypic variation is exhibited during infection, including increased capsule size, capsule shedding, giant cells ([≥] 15 m) and micro cells ([≤] 1 m). We examined 70 clinical isolates of Cryptococcus neoformans and Cryptococcus tetragattii from HIV/AIDS patients in Botswana to determine if the capacity to produce morphological variants was associated with clinical parameters. Isolates were cultured under conditions designed to simulate in vivo stresses. Substantial variation was seen across morphological and clinical data. Giant cells were more common in C. tetragattii, while micro cells and shed capsule occurred in C. neoformans only. Phenotypic variables fell into two groups associated with differing symptoms. The production of \"large\" phenotypes (greater cell and capsule size and giant cells) was associated with higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting these are induced in early-stage infection. \"Small\" phenotypes (micro cells and shed capsule) were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators and positively correlated with intracranial pressure indicators, suggesting they are produced later during infection and may contribute to immune suppression and promote proliferation and dissemination. These trends persisted at the species level, indicating that they were not driven by association with particular Cryptococcus species. Isolates possessing giant cells, micro cells, and shed capsule were rare, but strikingly were associated with patient death (p=0.0165). Our data indicate that pleomorphism is an important driver in Cryptococcus infection.\n\nImportanceCryptococcosis results in hundreds of thousands of deaths annually, predominantly in sub-Saharan Africa. Cryptococcus is an encapsulated yeast, and during infection cells have the capacity for substantial morphological changes, including capsule enlargement and shedding, and variations in cell shape and size. In this study we examined 70 Cryptococcus isolates causing meningitis in HIV/AIDS patients in Botswana in order to look for associations between phenotypic variation and clinical symptoms. Four variant phenotypes were seen across strains: giant cells [≥] 15 m, micro cells [≤] 1 m, shed extracellular capsule, and irregularly shaped cells. We found \"large\" and \"small\" phenotypes were associated with differing disease symptoms, indicating that their production may be important during the disease process. Overall, our study indicates that Cryptococcus strains that can switch on cell types under different situations may be more able to sustain infection and resist the host response.

microbiology↗

bioRxiv2018

Transcriptional heterogeneity of Cryptococcus gattii VGII compared with non-VGII lineages underpins key pathogenicity pathways

Cryptococcus gattii is a pathogenic yeast of humans and other animals, which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual RNA-seq experiment for four lineages of C. gattii (VGI-IV) interacting with mouse macrophages at 1hr, 3hr and 6hr post infection. Comparison of in vitro to ex vivo gene expression indicates lineage VGII is transcriptionally divergent to non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment and ergosterol production. Various paralogs demonstrate sub-functionalisation between lineages including an upregulation of capsule biosynthesis-related gene CAP2, and downregulation of CAP1 in VGIII. Isolates also compensate for lineage-specific gene-losses by over-expression of genetically similar paralogs, including an over-expression of capsule gene CAS3 in VGIV having lost CAS31. Differential expression of one in five C. gattii genes was detected following co-incubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and a downregulation of capsule attachment genes. We also show that VGII switches expression of two laccase paralogs (from LAC1 to LAC2) during co-incubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of C. gattii by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This study highlights the evolutionary breadth of expression profiles amongst the lineages of C. gattii and the diversity of transcriptional responses at this host-pathogen interface.\n\nImportanceThe transcriptional profiles of related pathogens and their response to host induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of C. gattii in rich media. Our analyses identified key processes including cell capsule, ergosterol production and melanin that are differentially expressed between lineages, and we find that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone sub-functionalisation for various paralogs including capsule biosynthesis and attachment genes. Most genes appeared down-regulated during co-incubation with macrophages, with the largest decrease observed for capsule attachment genes, which appears coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched expression of the laccase LAC1 to LAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow derived macrophages, marking a role in the host response to C. gattii. This work highlights the dynamic roles of key C. gattii virulence genes in response to macrophages.

genomics↗

bioRxiv2018

Antagonistic paralogs control a switch between growth and pathogen resistance in C. elegans

Immune genes are under intense pressure from pathogens, which cause these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of \"Intracellular Pathogen Response\" or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. These phenotypic and evolutionary features of pals-22 and pals-25 are strikingly similar to species-specific R gene pairs in plants that control immunity against co-evolved pathogens.

genetics↗

bioRxiv2018

Pathways of pathogenicity: The transcriptional stages of germination in the fatal fungal pathogen Rhizopus delemar

Rhizopus delemar is an invasive fungal pathogen, responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which spores of Rhizopus delemar infect and cause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. Understanding the molecular mechanisms which underpin this transformation may be key to controlling mucormycosis, however the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes which take place over the course of germination. This process is characterised by four distinct stages: dormancy, isotropic swelling, germ tube emergence and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transforming Rhizopus delemar from a single to a multicellular organism.\n\nImportanceGermination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.

microbiology↗

bioRxiv2018

Genomic basis of multidrug-resistance, mating, and virulence in Candida auris and related emerging species

Candida auris is an emergent fungal pathogen of rising public health concern due to increasing reports of outbreaks in healthcare settings and resistance to multiple classes of antifungal drugs. While distantly related to the more common pathogens C. albicans and C. glabrata, C. auris is closely related to three rarely observed and often multidrug-resistant species, C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Here, we generated and analyzed near complete genome assemblies and RNA-Seq-guided gene predictions for isolates from each of the four major C. auris clades and for C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Our analyses mapped seven chromosomes and revealed chromosomal rearrangements between C. auris clades and related species. We found conservation of genes involved in mating and meiosis and identified both MTLa and MTL C. auris isolates, suggesting the potential for mating between clades. Gene conservation analysis highlighted that many genes linked to drug resistance and virulence in other pathogenic Candida species are conserved in C. auris and related species including expanded families of transporters and lipases, as well as mutations and copy number variants in ERG11 that confer drug resistance. In addition, we found genetic features of the emerging species that likely underlie differences in virulence and drug response between these and other Candida species, including genes involved in cell wall structure. To begin to characterize the species-specific genes important for antifungal response, we profiled the gene expression of C. auris in response to voriconazole and amphotericin B and found induction of several transporters and metabolic regulators that may play a role in drug resistance. This study provides a comprehensive view of the genomic basis of drug resistance, potential for mating, and virulence in this emerging fungal clade.

genomics↗

bioRxiv2018

A Global Analysis of Mutations Accompanying Microevolution in the Heterozygous Diploid Pathogen Candida albicans

Candida albicans is a heterozygous diploid yeast that is a commensal of the human gastrointestinal (GI) tract and a prevalent opportunistic pathogen. Here, whole-genome sequencing was performed on multiple C. albicans isolates passaged in different niches to characterize the complete spectrum of mutations arising during microevolution. We reveal that evolution during short time-scales (<600 generations) is driven by both de novo base substitutions and short-tract loss of heterozygosity (LOH) events. In contrast, large-scale chromosomal changes are relatively rare, although chromosome 7 trisomies repeatedly emerged during passaging in one GI colonization model. Both strain background and chromosomal features affected mutational patterns, with mutation rates being greatly elevated in regions adjacent to emergent LOH tracts. Mutation rates were also elevated during host infection where genomes showed strong evidence of purifying selection. These results establish the genetic events driving C. albicans evolution and that this heterozygous diploid is extensively shaped by purifying selection.

genomics↗

bioRxiv2018

Whole genome analysis illustrates global clonal population structure of the ubiquitous dermatophyte pathogen Trichophyton rubrum

Dermatophytes include fungal species that infect humans, as well as those which also infect other animals or only grow in the environment. The dermatophyte species Trichophyton rubrum is a frequent cause of skin infection in immunocompetent individuals. While members of the T. rubrum species complex have been further categorized based on various morphologies, the population structure and ability to undergo sexual reproduction are not well understood. In this study, we analyze a large set of T. rubrum and Trichophyton interdigitale isolates to examine mating types, evidence of mating, and genetic variation. We find that nearly all isolates of T. rubrum are of a single mating type, and that incubation with T. rubrum morphotype megninii isolates of the other mating type failed to induce sexual development. While the region around the mating type locus is characterized by a higher frequency of SNPs compared to other genomic regions, we find that the population is remarkably clonal, with highly conserved gene content, low levels of variation, and little evidence of recombination These results support a model of recent transition to asexual growth when this species specialized to growth on human hosts.\n\nData access: Genome sequence data is available in NCBI under the Umbrella BioProject PRJNA186851.

genetics↗

bioRxiv2017

Rapid genome sequencing for outbreak analysis of the emerging human fungal pathogen Candida auris

BackgroundCandida auris was first described in 2009, and has since caused nosocomial outbreaks, invasive infections and fungaemia across 11 countries in five continents. An outbreak of C. auris occurred in a specialised cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak reported worldwide, involving a total of 72 patients.\n\nMethodsTo understand the epidemiology of C. auris infection within this hospital, we sequenced the genomes of outbreak isolates using Oxford Nanopore Technologies and Illumina in order to type antifungal resistance alleles and to explore the outbreak within its local and global context.\n\nFindingsPhylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin. The outbreak showed similar diversity to that of the entire clade and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations are novel for this pathogen.\n\nInterpretationMultiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission as well as implementing outbreak control measures.\n\nFundingAntimicrobial Research Collaborative, Imperial College London

epidemiology↗

bioRxiv2017

Genome analysis reveals evolutionary mechanisms of adaptation in systemic dimorphic fungi

Dimorphic fungal pathogens cause a significant human disease burden and unlike most fungal pathogens affect immunocompetent hosts. To examine the origin of virulence of these fungal pathogens, we compared genomes of classic systemic, opportunistic, and non-pathogenic species, including Emmonsia and two basal branching, non-pathogenic species in the Ajellomycetaceae, Helicocarpus griseus and Polytolypa hystricis. We found that gene families related to plant degradation, secondary metabolites synthesis, and amino acid and lipid metabolism are retained in H. griseus and P. hystricis. While genes involved in the virulence of dimorphic pathogenic fungi are conserved in saprophytes, changes in the copy number of proteases, kinases and transcription factors in systemic dimorphic relative to non-dimorphic species may have aided the evolution of specialized gene regulatory programs to rapidly adapt to higher temperatures and new nutritional environments. Notably, both of the basal branching, non-pathogenic species appear homothallic, with both mating type locus idiomorphs fused at a single locus, whereas all related pathogenic species are heterothallic. These differences revealed that independent changes in nutrient acquisition capacity have occurred in the Onygenaceae and Ajellomycetaceae, and underlie how the dimorphic pathogens have adapted to the human host and decreased their capacity for growth in environmental niches.

genomics↗

bioRxiv2017

Identification of environmental and genetic factors important for Cryptococcus neoformans titan cell formation using new in vitro inducing conditions

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 {micro}m cells and large titan cells (> 10 {micro}m and up to 100 {micro}m). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 {micro}m), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.\n\nAuthor SummaryCryptococcus neoformans is a yeast that is capable of morphological change upon interaction with the host. Particularly, in the lungs of infected mice, a subpopulation of yeast enlarges, producing cells up to 100 {micro}m in cell body diameter - referred to as titan cells. Along with their large size, the titan cells have other unique characteristics such as thickened cell wall, dense capsule, polyploidization, large vacuole with peripheral nucleus and cellular organelles. The generation of a large number of such cells outside the lungs of mice has been described but was not reproducible nor standardized. Here we report standardized, reproducible, robust conditions for generation of titan cells and explored the environmental and genetic factors underlying the genesis of these cells. We showed that titan cells were generated upon stresses such as change in the incubation medium, nutrient deprivation, hypoxia and low pH. Using collections of well characterized reference strains and clinical isolates, we validated with our model that the cAMP/PKA/Rim101 pathway is a major genetic determinant of titan cell formation. This study opens the way for a more comprehensive picture of the ontology of morphological changes in Cryptococcus neoformans and its impact on pathobiology of this deadly pathogen.

microbiology↗

bioRxiv2017

Epigenetic maintenance of DNA methylation after evolutionary loss of the de novo methyltransferase

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles, yet has been lost many times in diverse eukaryotic lineages. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase, Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 MYA. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 MY through a process analogous to Darwinian evolution of the genome.

molecular biology↗

bioRxiv2017

Population Genomics Of Cryptococcus neoformans var. grubii Reveals New Biogeographic Relationships And Finely Maps Hybridization

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically HIV/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterized the major subdivisions, their relative diversity and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype A/A) and of hybrids with C. neoformans var. neoformans (serotype A/D) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil revealed that the previously African VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions demonstrating the highly dispersive nature of this pathogen.\n\nAuthor SummaryCryptococcus neoformans var. grubii is a human fungal pathogen of immunocompromised individuals that has global clinical impact, causing half a million deaths per year. Substantial genetic substructure exists for this pathogen, with two lineages found globally (VNI, VNII) whereas a third has appeared confined to sub-Saharan Africa (VNB). Here, we utilized genome sequencing of a large set of global isolates to examine the genetic diversity, hybridization, and biogeography of these lineages. We found that while the three major lineages are well separated, recombination between the lineages has occurred, notably resulting in hybrid isolates with segmented ancestry across the genome. In addition, we showed that isolates from South America are placed within the VNB lineage, formerly thought to be confined to Africa, and that there is phylogenetic separation between these geographies that substantially expands the diversity of these lineages. Our findings provide a new framework for further studies of the dynamics of natural populations of C. neoformans var. grubii.

genomics↗