Biology subjects

Sephton-Clark, P. C. S.

Publications and source records attributed to Sephton-Clark, P. C. S..

4 recordsLinked to original sources

bioRxiv2022

A novel rRNA hybridization-based approach to rapid, accurate Candida identification directly from blood culture

Invasive fungal infections are increasingly common and carry high morbidity and mortality, yet fungal diagnostics lag behind bacterial diagnostics in rapidly identifying the causal pathogen. We previously devised a fluorescent hybridization-based assay to identify bacteria within hours directly from blood culture bottles without subculture, called phylogeny-informed rRNA-based strain identification (Phirst-ID). Here, we adapt this approach to unambiguously identify 11 common pathogenic Candida species, including C. auris, with 100% accuracy from laboratory culture (33 of 33 strains in a reference panel, plus 33 of 33 additional isolates tested in a validation panel). In a pilot study on 62 consecutive positive clinical blood cultures from two hospitals that showed yeast on Gram stain, Candida Phirst-ID matched the clinical laboratory result for 58 of 59 specimens represented in the 11-species reference panel, without misclassifying the 3 off-panel species. It also detected mixed Candida species in 2 of these 62 specimens, including the one discordant classification, that were not identified by standard clinical microbiology workflows; in each case the presence of both species was validated by both clinical and experimental data. Finally, in three specimens that grew both bacteria and yeast, we paired our prior bacterial probeset with this new Candida probeset to detect both pathogen types using Phirst-ID. This simple, robust assay can provide accurate Candida identification within hours directly from blood culture bottles, and the conceptual approach holds promise for pan-microbial identification in a single workflow.

microbiology↗

bioRxiv2021

Genomic variation across a clinical Cryptococcus population linked to disease outcome

Cryptococcus neoformans is the causative agent of cryptococcosis, a disease with poor patient outcomes, accounting for approximately 180,000 deaths each year. Patient outcomes may be impacted by the underlying genetics of the infecting isolate; however, our current understanding of how genetic diversity contributes to clinical outcomes is limited. Here, we leverage clinical, in vitro growth and genomic data for 284 C. neoformans isolates to identify clinically relevant pathogen variants within a population of clinical isolates from patients with HIV-associated cryptococcosis in Malawi. Through a genome-wide association study (GWAS) approach, we identify variants associated with fungal burden and growth rate. We also find both small and large-scale variation, including aneuploidy, associated with alternate growth phenotypes, which may impact the course of infection. Genes impacted by these variants are involved in transcriptional regulation, signal transduction, glycosylation, sugar transport, and glycolysis. We show that growth within the CNS is reliant upon glycolysis in an animal model, and likely impacts patient mortality, as CNS yeast burden likely modulates patient outcome. Additionally, we find genes with roles in sugar transport are enriched in regions under selection in specific lineages of this clinical population. Further, we demonstrate that genomic variants in two genes identified by GWAS impact virulence in animal models. Our approach identifies links between genetic variation in C. neoformans and clinically relevant phenotypes and animal model pathogenesis; shedding light on specific survival mechanisms within the CNS and identifying pathways involved in yeast persistence. ImportanceInfection outcomes for cryptococcosis, most commonly caused by C. neoformans, are influenced by host immune responses, as well as host and pathogen genetics. Infecting yeast isolates are genetically diverse; however, we lack a deep understanding of how this diversity impacts patient outcomes. To better understand both clinical isolate diversity and how diversity contributes to infection outcome, we utilize a large collection of clinical C. neoformans samples, isolated from patients enrolled in a clinical trial across 3 hospitals in Malawi. By combining whole-genome sequence data, clinical data, and in vitro growth data, we utilize genome-wide association approaches to examine the genetic basis of virulence. Genes with significant associations display virulence attributes in both murine and rabbit models, demonstrating that our approach can identify potential links between genetic variants and patho-biologically significant phenotypes.

genomics↗

bioRxiv2020

Characteristics of Titan cell formation vary across clinical isolates of Cryptococcus neoformans

Fungal morphology significantly impacts the host response. Filamentation and tissue penetration by Candida and Aspergillus species are essential for virulence, while growth as a yeast allows the thermal dimorphic fungi Coccidiodes, Histoplasma, and Talaromyces to reside inside phagocytes and disseminate. The basidiomycete Cryptococcus neoformans exhibits an unusual yeast-to-titan transition thought to enhance pathogenicity by increasing fungal survival in the host lung and dissemination to the central nervous system. In a common laboratory strain (H99), in vitro and in vivo titan induction yields a heterogenous population including >10 m titan cells, 5-7 m yeast cells and 2-4 m titanides. Previous reports have shown that titan cells are associated with enhanced virulence and the generation of aneuploid cells that facilitate stress adaptation and drug resistance, while small (>10 m) cells are associated with increased dissemination. However, the relationship between titan cells, small cells, and titanides remains unclear. Here, we characterize titanides and small cells in H99 and three clinical isolates and show that titanides share the lipid membrane order of their titan mothers and the G0 quiescent-like DNA staining of mating spores. In addition, we show that both titanizing and non-titanizing isolates exhibit altered capsule structure and PAMP exposure over time during in vitro culture, and generate aneuploidy in vivo. Author summaryThe human fungal pathogen Cryptococcus neoformans causes 200,000 HIV-associated deaths each year. In the lung, Cryptococcus makes an unusual yeast-to-titan morphological switch that contributes to disease development by altering immune polarization and introducing aneuploidy underlying host stress and drug resistance. Specifically, a proportion of 5 um haploid yeast endoreduplicate and swell, converting to large (> 10 um) polyploid titan cells that can then produce genetically distinct daughter cells. We recently developed an in vitro protocol for inducing large titan cells and additionally observed a novel small "titanide" cell type. Here we investigate the nature and origin of these small cells, demonstrating that they emerge during both in vitro and in vivo mouse-passaged titan induction in the well characterised lab strain H99 and are also apparent in a titanizing clinical isolate, Zc8. We show that these titanide cells share features with titan mothers (lipid order) and with spores produced during heterothalic mating. Finally, we show that the capacity of clinical isolates to produce both titan and titanide cells impacts aneuploidy and the emergence of drug resistance in vivo.

microbiology↗

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↗