Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis

Stone, Neil; Rhodes, Johanna; Fisher, Matthew C.; Mfinanga, Sayoki; Kivuyo, Sokoine; Rugemalila, Joan; Segal, Ella Shtifman; Needleman, Leor; Molloy, Síle F.; Kwon‐Chung, June; Harrison, Thomas S.; Hope, William; Berman, Judith; Bicanic, Tihana

doi:10.1172/jci124516

Cited by 134 publications

(156 citation statements)

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“…Cryptococcus neoformans displays fluconazole hetero-resistance: transient resistance in a subpopulation of cells mediated by aneuploidy of chromosomes that carry genes involved in fluconazole detoxification, and this impacts on disease outcomes (55–58). Gerstein et al previously demonstrated that in vivo- derived titans provide daughters with a survival advantage compared to in vivo- derived typical cells.…”

Section: Discussionmentioning

confidence: 99%

Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

Zhou

Zafar

Sephton-Clark

et al. 2020

Preprint

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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 μm titan cells, 5-7 μm yeast cells and 2-4 μm 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 μm) 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.

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Section: Discussionmentioning

confidence: 99%

Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

Zhou

Zafar

Sephton-Clark

et al. 2020

Preprint

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“…above discussed, the antifungal of choice are different, as fluconazole is indicate for Cryptococcus sp. while yeasts of the genus Rhodotorula are resistant to this antifungal class [43]. It is important to note that yeasts belonging to Rhodotorula genus have a high level of fluconazole resistance, a greater tolerance for itraconazole and susceptibility for amphotericin B [44, 45].…”

Section: Discussionmentioning

confidence: 99%

Approaches by Rhodotorula mucilaginosa from a chronic kidney disease patient for elucidating the pathogenicity profile by this emergent species

Jarros¹,

Veiga²,

Corrêa³

et al. 2019

Preprint

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26Background: Traditionally known as a common contaminant, Rhodotorula mucilaginosa is 27 among the leading causes of invasive fungal infections by non-candida yeasts. They affect 28 mainly immunocompromised individuals, often mimicking the cryptococcosis infection, 29 despite invasive infections by Rhodotorula are still not well explained. Thus, here we aimed to 30 characterize microbiologically clinical isolates of R. mucilaginosa isolated from colonization 31 of a patient with chronic renal disease (CKD), as well as to evaluate their phylogeny, antifungal 32 susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible 33 infection. 34 Methodology/Principal Findings: For this study, two isolates of R. mucilaginosa from oral 35 colonization of a CKD patient were isolated, identified and characterized by classical 36 (genotypic and phenotypic) methods. Susceptibility to conventional antifungals was evaluated, 37 followed by biofilm production, measured by different techniques (total biomass, metabolic 38 activity, colony forming units and extracellular matrix quantification). Finally, the 39 pathogenicity of yeast was evaluated by infection of Tenebrio molitor larvae. 40All isolates were resistant to azole and sensitive to polyenes and they were able to adhere and 41 form biofilm on the abiotic surface of polystyrene. In general, similar profiles among isolates 42 were observed over the observed periods (2, 24, 48 and 72 hours). Regarding extracellular 43 matrix components of biofilms at different maturation ages, R. mucilaginosa was able to 44 produce eDNA, eRNA, proteins, and polysaccharides that varied according to time and the 45 strain. The death curve in vivo model showed a large reduction in the survival percentage of 46 the larvae was observed in the first 24 hours, with only 40% survival at the end of the 47 Conclusions/Significance: We infer that colonization of chronic renal patients by R. 49 mucilaginosa offers a high risk of serious infection. And also emphasize that the correct 50 identification of yeast is the main means for an efficient treatment. 51 52 53 3 Author Summary 54The genus Rhodotorula is known to be a common contaminant, however, it has been 55 increasing in the last years, reports of different forms infections by this yeast, reaching mainly 56 individuals with secondary diseases or with low immunity. However, very little is known about 57 the mechanism that triggers the disease. Thus, this study aims to characterize microbiologically 58 clinical isolates of R. mucilaginosa isolated from a patient with chronic renal disease, as well 59 as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order 60 to infer the potential to become a possible infection. It was possible to characterize in general 61 the clinical isolates, to determine that they are resistant to an important class of the antifungal 62 agents which are the azoles. In addition, they are able to adhere and to form biofilm on abiotic 63 surfaces, t...

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“…These microevolutionary changes have been documented in AIDS patients with chronic and recurrent cryptococcal infections (12)(13)(14)(15)(16), as well as in murine models of infection (13,16). Resulting phenotypes include resistance to azole drugs (12,14) and differences in capsule structure, melanization and thermotolerance (12,13,17,18), all of which are factors that increase persistence of Cryptococcus in the host. In addition, up to 20% of cryptococcal cells form giant polyploid cells (titan cells) during infection that are resistant to phagocytosis and have enhanced survival and dissemination (19,20).…”

Section: Introductionmentioning

confidence: 99%

“…In response, these fungi can undergo rapid genomic adaptations that enhance survival and enable drug resistance (11). Previously reported alterations arising during cryptococcal infection include base substitutions, insertions, deletions, target gene copy-number variation, and chromosomal duplications (12)(13)(14). These microevolutionary changes have been documented in AIDS patients with chronic and recurrent cryptococcal infections (12)(13)(14)(15)(16), as well as in murine models of infection (13,16).…”

Section: Introductionmentioning

confidence: 99%

“…Previously reported alterations arising during cryptococcal infection include base substitutions, insertions, deletions, target gene copy-number variation, and chromosomal duplications (12)(13)(14). These microevolutionary changes have been documented in AIDS patients with chronic and recurrent cryptococcal infections (12)(13)(14)(15)(16), as well as in murine models of infection (13,16). Resulting phenotypes include resistance to azole drugs (12,14) and differences in capsule structure, melanization and thermotolerance (12,13,17,18), all of which are factors that increase persistence of Cryptococcus in the host.…”

Section: Introductionmentioning

confidence: 99%

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Transposon mobilization in the human fungal pathogenCryptococcus deneoformansis mutagenic during infection and promotes drug resistancein vitro

Gusa

Williams

Cho

et al. 2020

Preprint

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ABSTRACTWhen transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeast-like fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNAi pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.SIGNIFICANCE STATEMENTThe incidence of infections due to fungal pathogens has dramatically increased in the past few decades with similar increases in human populations with weakened or suppressed immune systems. Understanding the mechanisms by which organisms rapidly adapt during human infection to enhance virulence and evolve drug resistance is important for developing effective treatments. We find that transposon mobilization in the human pathogen Cryptococcus causes genomic mutations in a murine model of infection and promotes resistance to antifungal drugs in vitro. Thermotolerance is a key virulence determinant for pathogenic fungi during the environment-to-host transition, and we demonstrate that a temperature increase is sufficient to trigger transposon mobilization in vitro. The link between temperature stress and transposon-associated mutations may significantly impact adaptation to the host during infection, including the acquisition of drug resistance.

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Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis

Cited by 134 publications

References 53 publications

Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

Approaches by Rhodotorula mucilaginosa from a chronic kidney disease patient for elucidating the pathogenicity profile by this emergent species

Transposon mobilization in the human fungal pathogenCryptococcus deneoformansis mutagenic during infection and promotes drug resistancein vitro

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