Biology subjects

Perfect, J. R.

Publications and source records attributed to Perfect, J. R..

9 recordsLinked to original sources

bioRxiv2025

Temperature and genetic background drive mobilization of diverse transposable elements in a critical human fungal pathogen

Transposable elements (TEs) are key agents of genome evolution across all domains of life. These mobile genetic elements can cause mutations through transposition or by promoting structural rearrangements. Stress conditions can amplify TE mobility, either by impairing TE suppression mechanisms or through stress-induced interactions between transcription factors and TE sequences, offering a route for rapid genetic change. As such, TEs represent an important source of adaptability within populations. To investigate the interplay between environmental stress and eukaryotic TE dynamics relevant to infectious disease, we examined how heat stress and host-mimicking medium (RPMI) affect TE mobility in the global human fungal pathogen Cryptococcus neoformans, using a collection of clinical and environmental isolates. Using a selection-based screen, we captured the mobilization of seven distinct mobile element families, encompassing diverse retrotransposons and DNA transposons, whose insertions conferred antifungal resistance. This includes a novel element, CNEST, which belongs to the CACTA, Mirage, Chapaev (CMC) supergroup. Heat stress at human body temperature (37{degrees}C) significantly increased the mobilization of a subset of these TEs, leading to higher rates of acquired antifungal resistance. Whole-genome assemblies revealed that, compared to retrotransposons, DNA transposons were hypomethylated and approximately uniformly distributed throughout the genome, features that may contribute to their frequent mobilization. We further assessed TE-driven genomic changes within hosts using serial isolates from patients with recurrent cryptococcal infections and from isolates passaged through mice. While we observed evidence of TE copy number changes near chromosome ends, we found no indication of TE-mediated alterations near gene-coding regions across any of the serial isolates. Finally, TE mobility was isolate- and strain-dependent, with significant variation even among clonally related strains collected from the same patient, emphasizing the role of genetic background in shaping TE activity. Together, these findings reveal a complex and dynamic relationship between environmental stress, genetic background, TE type-specific epigenetic regulation, and TE mobility, with important implications for adaptation and acquired antifungal resistance in C. neoformans. AUTHOR SUMMARYTransposable elements (TEs) are mobile segments of DNA that can "jump" from one place to another in a genome, causing genetic changes that can impact fitness and evolution. In this study, we investigate how different environmental conditions influence TE mobility in the pathogenic yeast Cryptococcus neoformans, which is globally distributed in the environment and causes life-threatening infections. We compared genetically diverse strains and found that growth at higher temperatures, specifically human body temperature (37{degrees}C), increases the mobility of some TEs. In contrast, other TEs were more active at lower temperatures (30{degrees}C), indicating distinct pathways of temperature regulation for different TE groups. By examining C. neoformans isolates from patients with recurrent infections, we observed genetic changes by one TE group that likely occurred during infection. We also found that TE groups are distributed differently across the genome and show varying levels of DNA methylation, a modification that can suppress their activity and control TE mobility in the cell. Overall, this work shows that environmental stresses, such as increased temperature, can accelerate genetic change by promoting TE mobilization. These changes may contribute to how C. neoformans adapts during infection or in the environment.

genetics↗

bioRxiv2025

Inhibitors of trehalose-6-phosphate synthase activity in fungal pathogens compromise thermal tolerance pathways

Infections caused by fungal pathogens such as Candida and Cryptococcus are associated with high mortality rates, partly due to limitations in the current antifungal arsenal. This highlights the need for antifungal drug targets with novel mechanisms of action. The trehalose biosynthesis pathway is a promising antifungal drug target because trehalose biosynthesis is essential for virulence in Cryptococcus neoformans and Candida albicans and is also a mediator of fungal stress responses, such as thermotolerance. To exploit its untapped antifungal potentials, we screened the St. Jude 3-point pharmacophore library to identify small molecule inhibitors of the first enzyme in the trehalose biosynthesis pathway, trehalose-6-phosphate synthase (Tps1). Structure-guided optimization of a potent hit, SJ6675, yielded a water-soluble inhibitor named 4456dh. Employing biochemical, structural and cell-based assays, we demonstrate that 4456dh inhibits Tps1 enzymatic activity, suppresses trehalose synthesis and exerts a fungicidal effect. Notably, the structure of Tps1 in complex with 4456 reveals that 4456 occupies the substrate binding pocket. Importantly, 4456dh renders normally thermotolerant fungal pathogens unable to survive at elevated temperatures, which is critical as we investigate the emergence of fungi from the environment due to a warming climate. Overall, this work develops the water-soluble 4456dh as an early-stage antifungal drug that has a distinct mechanism of action compared to existing clinical antifungals. ImportanceThe rise of fungal infections in recent years is alarming due to an increase in the vulnerable immunocompromised population, global temperature increase and limited antifungal treatment options. One of the major hurdles in developing new drugs is the identification of fungal-specific antifungal drug targets due to highly conserved cellular machinery between fungi and humans. Here, we describe a small molecule inhibitor, 4456dh, of the trehalose biosynthesis pathway. This pathway is present in fungi but not humans. Trehalose plays a critical role in stress responses such as thermotolerance in fungal pathogens and is essential for their virulence. We show that treatment with 4456dh blocks production of trehalose and renders fungal cells inviable. Thus far, 4456dh is active against two fungal pathogens of critical importance suggesting a broad-spectrum activity.

microbiology↗

bioRxiv2023

Hormonal steroids bind the Neisseria gonorrhoeae multidrug resistance regulator, MtrR, to induce a multidrug binding efflux pump and stress-response sigma factor

Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae, the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, {beta}-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.

biochemistry↗

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

Amoeba predation of Cryptococcus neoformans results in pleiotropic changes to traits associated with virulence

Phagocytic amoeboid predators such as amoeba have been proposed to select for survival traits in soil microbes such as Cryptococcus neoformans that can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Several prior studies have shown that incubation of various fungal species with amoeba can enhance their virulence. However, the mechanisms by which fungi adapt to amoeba and thus change their virulence are unknown. In this study we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged periods of time and then analyzed surviving colonies phenotypically and genetically. Surviving colonies were comprised of cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of such traits associated with virulence such as capsule size, urease production and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding for the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. In addition, isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene, which encodes Pkr1 (AMP-dependent protein kinase regulator) but were less virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes, which confer increase resistance against protozoal predation. Given the myriad of potential predators in soils the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation. Author summaryCryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. Cryptococcosis is a worldwide concern due to its high mortality rate. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for animal host in its life cycle. Previous studies showed evidence that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae behave like macrophages, an important immune cell in human body, so it is considered as a training ground for pathogens to resist macrophages. However, how C. neoformans changes its virulence through interacting with amoebae is unknown. Here, we exposed C. neoformans to amoebae for a long period of time. We found that C. neoformans cells recovered from amoebae manifested numerous changes to phenotypes related to its virulence and one of the amoeba-passaged C. neoformans cells had enhanced ability to kill macrophages. We further analyzed their genome sequences and found various mutations in different cells of amoeba-passaged C. neoformans, showing that DNA mutations may be the major cause of the phenotypic changes after interacting with amoebae. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival.

microbiology↗

bioRxiv2019

Cryptococcus neoformans resist to drastic conditions by switching to viable but non-culturable cell phenotype

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce C. neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in Cryptococcus neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial masse increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. All together, these data show that we were able to generate for the first time VBNC phenotype in Cryptococcocus neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

microbiology↗

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↗

bioRxiv2017

N VITRO AND IN VIVO CHARACTERISATION OF ISOLATES OF CRYPTOCOCCUS NEOFORMANS CAUSING MENINGITIS IN HIV-INFECTED AND UNINFECTED PATIENTS IN VIETNAM

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in HIV-uninfected individuals. This disease was associated with a single genotype of Cryptococcus neoformans (Sequence Type (ST)5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans we selected 30 representative Vietnamese isolates, compared their in vitro pathogenic potential and in vivo virulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect to in vitro virulence markers including melanin production, replication at 37{degrees}C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (p<0.001). Counter-intuitively, mice infected with ST5 isolates had significantly longer survival with lower fungal burdens at day 7 than non-ST5 isolates. Notably, ST5 isolates induced significantly greater initial inflammatory responses than non-ST5 strains, measured by TNF- concentrations (p<0.001). Despite being generally less virulent in the mouse model, we hypothesize that the significant within strain variation seen in ST5 isolates in the tested phenotypes may represent an evolutionary advantage enabling adaptation to novel niches including apparently immunocompetent human hosts.

microbiology↗

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↗