Methods for Manipulating Cryptococcus Spores

Frerichs, Anna B.; Huang, Mu-Shun; Ortiz, Sébastien C.; Hull, Christina M.

doi:10.3390/jof8010004

Cited by 5 publications

(3 citation statements)

References 34 publications

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“…We spotted 3 μl of 10 7 cells/ml onto V8 mating medium (pH 5) and incubated the plates in the dark at room temperature (25°C) for over 30 days. For spore isolation, we followed the protocol previously reported by Frerichs et al [ 74 ] with a slight modification. Following incubation on the mating medium, spores were harvested by scraping and resuspended in 75% Percoll buffer (Sigma-Aldrich, P1644).…”

Section: Methodsmentioning

confidence: 99%

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans

Choi,

Lee

et al. 2023

PLoS Pathog

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V-ATPase, which comprises 13–14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE (regulator of H+-ATPase of vacuolar and endosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans, and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C. neoformans. The hRAVE complex played a critical role in the pathogenicity of C. neoformans, and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis.

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Section: Methodsmentioning

confidence: 99%

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans

Choi,

Lee

et al. 2023

PLoS Pathog

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“…The INIFAP-2021 isolate was cultured on a Petri dish containing potato dextrose agar (PDA) and incubated at a temperature of 28 °C for 6 days. To harvest the spores, a modified method combining the techniques of Frerichis et al [ 54 ], Gilbert et al [ 14 ], and Shen et al [ 55 ] was used. First, a sterile crystal triangle, previously immersed in a solution of 0.01% Triton X-100 in phosphate-buffered saline (PBS), was employed to gently scrape the spores attached to the glass.…”

Section: Methodsmentioning

confidence: 99%

Identification of Virulence Factors in Entomopathogenic Aspergillus flavus Isolated from Naturally Infected Rhipicephalus microplus

Arreguin-Perez,

Miranda-Miranda,

Folch-Mallol

et al. 2023

Microorganisms

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Aspergillus flavus has been found to be an effective entomopathogenic fungus for various arthropods, including ticks. In particular, natural fungal infections in cattle ticks show promise for biocontrol of the Rhipicephalus (Boophilus) microplus tick, which is a major ectoparasite affecting cattle worldwide. Our study aimed to elucidate the specific entomopathogenic virulence factors encoded in the genome of an A. flavus strain isolated from naturally infected cattle ticks. We performed morphological and biochemical phenotyping alongside complete genome sequencing, which revealed that the isolated fungus was A. flavus related to the L morphotype, capable of producing a range of gene-coded entomopathogenic virulence factors, including ribotoxin, aflatoxin, kojic acid, chitinases, killer toxin, and satratoxin. To evaluate the efficacy of this A. flavus strain against ticks, we conducted experimental bioassays using healthy engorged female ticks. A morbidity rate of 90% was observed, starting at a concentration of 105 conidia/mL. At a concentration of 107 conidia/mL, we observed a 50% mortality rate and a 21.5% inhibition of oviposition. The highest levels of hatch inhibition (30.8%) and estimated reproduction inhibition (34.64%) were achieved at a concentration of 108 conidia/mL. Furthermore, the tick larval progeny that hatched from the infected tick egg masses showed evident symptoms of Aspergillus infection after incubation.

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“…CN and CG have been long-established model organisms within the field of medical mycology [54,55]. It is often constituted as a basis for (in vitro) studies concerning antifungal drug testing, drug resistance, mechanisms behind chronic infections and radioimmunotherapy, fungal growth, and environmental interactions [56][57][58][59]. Despite the frequent use of CN/CG in preclinical research, there are still gaps in our knowledge about the mechanisms that drive cryptococcal infection and dissemination.…”

Section: In Vitro Modelsmentioning

confidence: 99%

Preclinical Models for Cryptococcosis of the CNS and Their Characterization Using In Vivo Imaging Techniques

Roosen,

Maes,

Musetta

et al. 2024

JoF

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Infections caused by Cryptococcus neoformans and Cryptococcus gattii remain a challenge to our healthcare systems as they are still difficult to treat. In order to improve treatment success, in particular for infections that have disseminated to the central nervous system, a better understanding of the disease is needed, addressing questions like how it evolves from a pulmonary to a brain disease and how novel treatment approaches can be developed and validated. This requires not only clinical research and research on the microorganisms in a laboratory environment but also preclinical models in order to study cryptococci in the host. We provide an overview of available preclinical models, with particular emphasis on models of cryptococcosis in rodents. In order to further improve the characterization of rodent models, in particular the dynamic aspects of disease manifestation, development, and ultimate treatment, preclinical in vivo imaging methods are increasingly used, mainly in research for oncological, neurological, and cardiac diseases. In vivo imaging applications for fungal infections are rather sparse. A second aspect of this review is how research on models of cryptococcosis can benefit from in vivo imaging methods that not only provide information on morphology and tissue structure but also on function, metabolism, and cellular properties in a non-invasive way.

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Methods for Manipulating Cryptococcus Spores

Cited by 5 publications

References 34 publications

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans

Identification of Virulence Factors in Entomopathogenic Aspergillus flavus Isolated from Naturally Infected Rhipicephalus microplus

Preclinical Models for Cryptococcosis of the CNS and Their Characterization Using In Vivo Imaging Techniques

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