Fungi, algae, plants, protozoa, and bacteria are all known to form spores, especially hardy and ubiquitous propagation structures that are also often the infectious agents of diseases. Spores can ...survive for thousands of years, frozen in the permafrost (Kochkina et al., 2012), with the oldest viable spores extracted after 250 million years from salt crystals (Vreeland, Rosenzweig, & Powers, 2000). Their resistance to high levels of UV, desiccation, pressure, heat, and cold enables the survival of spores in the harshest conditions (Setlow, 2016). For example, Bacillus subtilis spores can survive and remain viable after experiencing conditions similar to those on Mars (Horneck et al., 2012). Spores are disseminated through environmental factors. Wind, water, or animal carriage allow spores to be spread ubiquitously throughout the environment. Spores will break dormancy and begin to germinate once exposed to favorable conditions. Germination is the mechanism that converts the spore from a dormant biological organism to one that grows vegetatively and is capable of either sexual or asexual reproduction. The process of germination has been well studied in plants, moss, bacteria, and many fungi (Hohe & Reski, 2005; Huang & Hull, 2017; Vesty et al., 2016). Unfortunately, information on the complex signaling involved in the regulation of germination, particularly in fungi remains lacking. This chapter will discuss germination of fungal spores covering our current understanding of the regulation, signaling, outcomes, and implications of germination of pathogenic fungal spores. Owing to the morphological similarities between the spore-hyphal and yeast-hyphal transition and their relevance for disease progression, relevant aspects of fungal dimorphism will be discussed alongside spore germination in this chapter.
Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ...ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.
•Bacterial endosymbionts protect fungal spores from phagocytes•A secreted factor blocks growth and killing by environmental amoebas•Endosymbionts improve fungal stress resistance•Endosymbiosis also allows the evasion of vertebrate immune cells and virulence in vivo
How environmental fungi evolved virulence mechanisms to opportunistically infect humans is unclear. Itabangi et al. identify a tri-kingdom interaction, whereby a bacterial endosymbiont living within a fungus causes the generation of a secreted factor that blocks the predatory activity of amoebas and drives virulence in animal models.
Abstract
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.
Lay Summary
Candida bloodstream infections cause considerable morbidity and mortality, yet slow diagnostics delay recognition, worsening patient outcomes. We develop and validate a novel molecular approach to accurately identify Candida species directly from blood culture one day faster than standard workflows.
is an invasive fungal pathogen responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which infectious spores of
cause disease, is a key developmental process ...that transforms the dormant spore state into a vegetative one. The molecular mechanisms that 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 that take place over the course of germination. This process is characterized 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
from a single-cellular to multicellular organism.
Germination 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.
Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast ...transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the
yeast transcriptomes to those from other environmental conditions, including
growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the
putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease.
Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. In this study, we captured transcriptomes of yeasts directly out of the human cerebrospinal fluid, the most concerning site of infection. By comparing the RNA transcript levels with other conditions, we gained insights into how the basic machinery involved in metabolism and environmental responses enable this fungus to cause disease at this body site. This approach was applied to clinical isolates with diverse genotypes to begin to establish a genotype-agnostic understanding of how the yeast responds to stress. Based on these results, future studies can focus on how these genes and their pathways and networks can be targeted with new therapeutics and possibly classify yeasts with bad infection outcomes.
Cryptococcus neoformans is the causative agent of cryptococcosis, a disease with poor patient outcomes that accounts 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,
growth and genomic data for 284 C. neoformans isolates to identify clinically relevant pathogen variants within a population of clinical isolates from patients with human immunodeficiency virus (HIV)-associated cryptococcosis in Malawi. Through a genome-wide association study (GWAS) approach, we identify variants associated with the fungal burden and the 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 central nervous system (CNS) is reliant upon glycolysis in an animal model and likely impacts patient mortality, as the CNS yeast burden likely modulates patient outcome. Additionally, we find that 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 the genetic variation in C. neoformans and clinically relevant phenotypes and animal model pathogenesis, thereby shedding light on specific survival mechanisms within the CNS and identifying the pathways involved in yeast persistence.
Infection outcomes for cryptococcosis, most commonly caused by C. neoformans, are influenced by host immune responses as well as by 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 outcomes, we utilize a large collection of clinical C. neoformans samples that were isolated from patients enrolled in a clinical trial across 3 hospitals in Malawi. By combining whole-genome sequence data, clinical data, and
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.
Abstract
Background
Invasive fungal infections are increasingly common and carry high rates of morbidity and mortality, and rising rates of drug resistance, notably in Candida auris. However, there ...is a notable gap between the development of bacterial identification (ID) and antimicrobial susceptibility testing (AST) diagnostics and a relative lack thereof for fungi.
Methods
For ID, we applied phylogeny-informed rRNA-based strain identification (Phirst-ID) to Candida species, using variable regions of the 18S and 28S rRNA of 11 pathogenic Candida species. We tested 66 laboratory isolates and 62 clinical blood cultures on the NanoString (Seattle,WA) RNA detection platform. For AST, we leveraged transcriptional differences between susceptible and resistant isolates upon antifungal exposure as a phenotypic measure of susceptibility, agnostic to resistance mechanism. We performed RNA-Seq on susceptible and resistant C. albicans treated with fluconazole and C. auris treated with the major antifungal classes (voriconazole, micafungin, or amphotericin) at clinical breakpoint concentrations and screened for key transcripts whose antibiotic response best distinguished susceptible from resistant isolates.
Results
From lab culture, Candida Phirst-ID distinguished 11 common pathogenic Candida species, including C. auris, with 100% accuracy. From clinical blood cultures, we correctly identified all 57 monomicrobial Candida species, without misidentifying 3 non-Candida isolates. For AST, in C. albicans,the response of ERG genes to fluconazole exposure cleanly distinguished 8 susceptible from 8 resistant isolates. RNA-seq data from C. auris exposed to amphotericin B, micafungin, and voriconazole suggest differential regulation in several key transcripts between susceptible and resistant isolates, and we are investigating their diagnostic utility in NanoString assays.
Transcriptional profiling provides accurate AST in yeast.
(a) RNA-Seq data showing showing transcriptional response in susceptible (left) but not resistant (right) isolates of C. albicans isolates upon fluconazole exposure. Transcripts with false discovery rate (FDR) < 0.1 in susceptible isolates are shown in red in both plots; no transcripts reach statistical significance in resistant isolates. (b) Heatmaps of normalized NanoString data for 10 fluconazole-responsive transcripts from 12 isolates of C. albicans, arranged by increasing MIC, with AST classification below. (c) One-dimensional projections of NanoString data clearly distinguish C. albicans isolates by fluconazole susceptibility.
Conclusion
Our Candida species ID assay is a simple, robust assay that can provide accurate results within hours directly from blood culture bottles which we are extending into a pan-fungal assay tested on clinical specimens. The transcriptional signature of select genes in fluconazole exposed C. albicans distinguishes isolates’ susceptibility and we are working to identify transcripts with such behavior in C. auris.
Disclosures
All Authors: No reported disclosures
Abstract
Talaromycosis, a severe and invasive fungal infection caused by Talaromyces marneffei, is difficult to treat and impacts those living in endemic regions of Southeast Asia, India, and China. ...While 30% of infections result in mortality, our understanding of the genetic basis of pathogenesis for this fungus is limited. To address this, we apply population genomics and genome-wide association study approaches to a cohort of 336 T. marneffei isolates collected from patients who enrolled in the Itraconazole vs Amphotericin B for Talaromycosis trial in Vietnam. We find that isolates from northern and southern Vietnam form two distinct geographical clades, with isolates from southern Vietnam associated with increased disease severity. Leveraging longitudinal isolates, we identify multiple instances of disease relapse linked to unrelated strains, highlighting the potential for multistrain infections. In more frequent cases of persistent talaromycosis caused by the same strain, we identify variants arising over the course of patient infections that impact genes predicted to function in the regulation of gene expression and secondary metabolite production. By combining genetic variant data with patient metadata for all 336 isolates, we identify pathogen variants significantly associated with multiple clinical phenotypes. In addition, we identify genes and genomic regions under selection across both clades, highlighting loci undergoing rapid evolution, potentially in response to external pressures. With this combination of approaches, we identify links between pathogen genetics and patient outcomes and identify genomic regions that are altered during T. marneffei infection, providing an initial view of how pathogen genetics affects disease outcomes.
Sephton-Clark et al. take population genomic approaches to assess relatedness of longitudinal Talaromycosis cases and signals of selection in two distinct clades identified across Vietnam. They observe isolates from the Southern region associate with more severe disease outcomes, and they and identify multiple genes implicated in clinical phenotypes through GWAS analysis.
Abstract
S2.5 Rare yeasts, September 21, 2022, 3:00 PM - 4:30 PM
Candida auris (C. auris) is a recently emerged human fungal pathogen of growing concern due to its ability to acquire extensive ...multidrug resistance (MDR) to all four antifungal drug classes. The unprecedented extent of MDR in C. auris, suggests accelerated resistance evolution, novel mechanisms of resistance, and/or potential fitness compensation. Despite being the first fungus to be officially considered an urgent antimicrobial resistance threat by the CDC (US), insights into the resistance mechanisms and evolutionary dynamics of C. auris are still scarce.
By using high-throughput in vitro experimental evolution with various antifungal drugs, we have obtained a library of resistant strains from four different clades. Through both genome and targeted sequencing, we have discovered novel mutations, especially for polyene resistance, which indicate new mechanisms of resistance and fitness compensation. For the validation of mutations, we have optimized a recyclable CRISPR/Cas9 tool for C. auris based on the C. albicans HIS-FLP system.
By mapping drug susceptibility responses of evolved strains across a library of several antifungals and repurposed drugs, we have discovered trends of cross-resistance and collateral sensitivity. Both phenomena have been extensively studied in tumors and bacteria but remain unexplored in fungi. In the light of these observations, we explore novel treatment schemes that prevent antifungal drug resistance development in C. auris and other pathogenic fungi.
A pet cockatoo was the suspected source of
recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence ...analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI-SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.
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