Broadening the spectrum of fluorescent protein tools for use in the encapsulated human fungal pathogen Cryptococcus neoformans

Spencer, Garrick W.K.; Chua, Sheena M.H.; Erpf, Paige E.; Wizrah, Maha S.I.; Dyba, Taylor G.; Condon, Nicholas D.; Fraser, James A.

doi:10.1016/j.fgb.2020.103365

Cited by 7 publications

(6 citation statements)

References 54 publications

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“…To determine whether pre-phagocytosis had the same effect in a mammalian infection, we performed a competition assay using EGFP-expressing MECs and TC-experienced Cn expressing mRuby3 ( 20 , 21 ) (JMD225, produced from the same parental strain as JMD163). These were inoculated together into A/J mice via the intranasal route.…”

Section: Resultsmentioning

confidence: 99%

“…A nuclear-localized mRuby3 expression strain (JMD225) was constructed using CRISPR, similar to the prior construction of the green fluorescent JMD163 strain ( 19 ). mRuby3 was chosen based on its impressive fluorescence in Spencer et al ( 21 ). The mRuby3 construct was amplified from pGWKS7 (AddGene #139414) using primers JMD228 (5′-cat cta tca cAT GGT CTC CAA GGG CGA G-3′) and JMD231 (5′-tgg cgg atc cCT TGT AGA GCT CGT CCA TGC-3′) (lower case letters indicate overlap region for fusion PCR).…”

Section: Methodsmentioning

confidence: 99%

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Survival in macrophages induces enhanced virulence in Cryptococcus

Nielson,

Jezewski,

Wellington

et al. 2024

mSphere

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Cryptococcus is a ubiquitous environmental fungus and frequent colonizer of human lungs. Colonization can lead to diverse outcomes, from clearance to long-term colonization to life-threatening meningoencephalitis. Regardless of the outcome, the process starts with an encounter with phagocytes. Using the zebrafish model of this infection, we have noted that cryptococcal cells first spend time inside macrophages before they become capable of pathogenic replication and dissemination. What “licensing” process takes place during this initial encounter, and how are licensed cryptococcal cells different? To address this, we isolated cryptococcal cells after phagocytosis by cultured macrophages and found these macrophage-experienced cells to be markedly more virulent in both zebrafish and mouse models. Despite producing a thick polysaccharide capsule, they were still subject to phagocytosis by macrophages in the zebrafish. Analysis of antigenic cell wall components in these licensed cells demonstrated that components of mannose and chitin are more available for staining than they are in culture-grown cells or cells with capsule production induced in vitro . Cryptococcus is capable of exiting or transferring between macrophages in vitro , raising the likelihood that this fungus alternates between intracellular and extracellular life during growth in the lungs. Our results raise the possibility that intracellular life has its advantages over time, and phagocytosis-induced alteration in mannose and chitin exposure is one way that makes subsequent rounds of phagocytosis more beneficial to the fungus. IMPORTANCE Cryptococcosis begins in the lungs and can ultimately travel through the bloodstream to cause devastating infection in the central nervous system. In the zebrafish model, small amounts of cryptococcus inoculated into the bloodstream are initially phagocytosed and become far more capable of dissemination after they exit macrophages. Similarly, survival in the mouse lung produces cryptococcal cell types with enhanced dissemination. In this study, we have evaluated how phagocytosis changes the properties of Cryptococcus during pathogenesis. Macrophage-experienced cells (MECs) become “licensed” for enhanced virulence. They out-disseminate culture-grown cells in the fish and out-compete non-MECs in the mouse lung. Analysis of their cell surface demonstrates that MECs have increased availability of cell wall components mannose and chitin substances involved in provoking phagocytosis. These findings suggest how Cryptococcus might tune its cell surface to induce but survive repeated phagocytosis during early pathogenesis in the lung.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Survival in macrophages induces enhanced virulence in Cryptococcus

Nielson,

Jezewski,

Wellington

et al. 2024

mSphere

View full text Add to dashboard Cite

show abstract

“…To determine if pre-phagocytosis had the same effect in a mammalian infection, we performed a competition assay using EGFP-expressing MECs and TC-conditioned Cc expressing mRuby3 (20, 21) (JMD225, produced from the same parental strain as JMD163). These were inoculated together into A/J mice via the intranasal route.…”

Section: Resultsmentioning

confidence: 99%

Survival in macrophages induces an enhanced virulence inCryptococcus

Nielson,

Jezewski,

Wellington

et al. 2023

Preprint

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Cryptococcusis a ubiquitous environmental fungus and frequent colonizer of human lungs. Colonization can lead to diverse outcomes, from clearance to long-term colonization, and to life-threatening meningoencephalitis. Regardless of the outcome, the process starts with an encounter with phagocytes. Using the zebrafish model of this infection, we have noted that cryptococcal cells first spend time inside macrophages before they become capable of pathogenic replication and dissemination. What ‘licensing’ process takes place during this initial encounter, and how are licensed cryptococcal cells different? To address this, we isolated cryptococcal cells after phagocytosis by cultured macrophages and found these macrophage-experienced cells to be markedly more virulent in both zebrafish and mouse models. Despite producing a thick polysaccharide capsule, they were still subject to phagocytosis by macrophages in the zebrafish. Analysis of antigenic cell wall components in these licensed cells demonstrated that components of mannose and chitin are more available to staining than they are in culture-grown cells or cells with capsule production induced in vitro.Cryptococcusis capable of exiting or transferring between macrophages in vitro, raising the likelihood that this fungus alternates between intracellular and extracellular life during growth in the lungs. Our results raise the possibility that intracellular life has its advantages over time, and phagocytosis-induced alteration in mannose and chitin exposure is one way that makes subsequent rounds of phagocytosis more beneficial to the fungus.IMPORTANCECryptococcosis begins in the lungs and can ultimately travel through the bloodstream to cause devastating infection in the central nervous system. In the zebrafish model, small amounts of cryptococcus inoculated into the bloodstream are initially phagocytosed, and become far more capable of dissemination after they exit macrophages. Similarly, survival in the mouse lung produces cryptococcal cell types with enhanced dissemination. In this study we have evaluated how phagocytosis changes the properties ofCryptococcusduring pathogenesis. Macrophage experienced cells (MECs) become ‘licensed’ for enhanced virulence. They out-disseminate culture grown cells in the fish and out-compete non-MECs in the mouse lung. Analysis of their cell surface demonstrates that MECs have increased availability of cell wall components mannose and chitin—substances involved in provoking phagocytosis. These findings suggest howCryptococcusmight tune its cell surface to induce but survive repeated phagocytosis during early pathogenesis in the lung.

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“…Afterwards, Upadhya and collaborators ( 45 ) engineered a red-emitting derivative of the H99 congenic strain KN99 using mCherry, which allowed fluorescence-based ex vivo determinations of intracellular and extracellular lung fungal burdens during experimental murine infection. Recently, Spencer and colleagues ( 46 ) expanded the current red-green color palette of engineered fluorescent C. neoformans strains. Despite not being as fluorescently robust as the above-mentioned transformant strains, our PAB-GFP transformant also showed low background fluorescence, was successfully used to aid in analyses of C. neoformans -host interactions, and, importantly, allowed a more detailed exploration of interesting features of the biology of C. neoformans , including the recently described viable but nonculturable cell phenotype.…”

Section: Discussionmentioning

confidence: 99%