Role of clathrin-mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen <i>Cryptococcus neoformans</i>

Bairwa, Gaurav; Caza, Mélissa; Horianopoulos, Linda C.; Hu, Guanggan; Kronstad, James W.

doi:10.1101/352534

Cited by 2 publications

(8 citation statements)

References 79 publications

(11 reference statements)

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“…The reduced eGFP/mKATE2 values at time zero in the mutants may indicate the presence of a compensatory heme uptake mechanism that is independent of clathrin-mediated endocytosis. We previously obtained evidence for more than one heme uptake mechanism in our studies of Cig1 and Chc1 ( 26 , 35 ). Notably, the heme sensor response to the external hemin in the vps45Δ hs mutant was only slightly reduced in comparison to that of the WT hs strain, suggesting a modest influence on uptake.…”

Section: Resultsmentioning

confidence: 76%

“…Heme uptake in C. neoformans relies on clathrin-mediated endocytosis and on as-yet-unknown clathrin-independent mechanisms ( 26 ). To further examine the role of endocytosis in heme uptake and establish the utility of the heme sensor in studying heme internalization, we treated cells expressing the heme sensor (WT hs ) with the endocytosis inhibitor chlorpromazine (CPZ).…”

Section: Resultsmentioning

confidence: 99%

“…Deletion of genes involved in clathrin-mediated endocytosis (i.e., CHC1 , LAS17 , RVS161 , and RVS167 ) results in defective growth on hemin as an iron source for C. neoformans ( 26 ). To specifically assess hemin uptake, we expressed the CnHS sensor in the mutants lacking CHC1 and LAS17 , mutants lacking CIG1 or CIG1 and CFO1 ( CIG1 encodes a hemophore and CFO1 encodes a high-affinity ferric oxidoreductase), or a mutant defective in VPS45 (encoding a regulator of vesicle trafficking and fusion [ 34 ]).…”

Section: Resultsmentioning

confidence: 99%

“…The mechanisms for acquisition and use of heme-iron sources are starting to be discovered in some fungi, including Aspergillus fumigatus , Candida albicans , Candida glabrata , C. neoformans , Schizosaccharomyces pombe , and Paracoccidioides brasiliensis ( 22 – 33 ). For example, studies in C. neoformans revealed important roles in heme use for endocytosis and endomembrane trafficking machinery, including the ESCRT proteins Vps22, Vps23, and Snf7, and proteins for clathrin-mediated endocytosis such as Chc1 (clathrin heavy chain 1), Las17 (a nucleation‐promoting factor for actin assembly), and Rvs161/Rvs167 (vesicle scission proteins; amphiphysins) ( 25 , 26 , 28 ). A recent study also highlighted a role for the SNARE regulator Vps45 in iron use from heme ( 34 ).…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, the CFEM hemophore proteins in C. albicans constitute a relay network that has recently been shown to mediate heme acquisition from human serum albumin ( 36 ). For C. neoformans , defects in cellular trafficking for heme uptake result in attenuated virulence, highlighting the importance of heme iron in pathogenesis ( 25 , 26 , 35 ). Many of the iron acquisition functions in C. neoformans are regulated by iron regulatory proteins such as the monothiol glutaredoxin Grx4 and the transcription factors Cir1 and HapX ( 37 – 39 ).…”

Section: Introductionmentioning

confidence: 99%

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A Cytoplasmic Heme Sensor Illuminates the Impacts of Mitochondrial and Vacuolar Functions and Oxidative Stress on Heme-Iron Homeostasis in Cryptococcus neoformans

Bairwa

Sánchez‐León

et al. 2020

mBio

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Pathogens must compete with hosts to acquire sufficient iron for proliferation during pathogenesis. The pathogenic fungus Cryptococcus neoformans is capable of acquiring iron from heme, the most abundant source in vertebrate hosts, although the mechanisms of heme sensing and acquisition are not entirely understood. In this study, we adopted a chromosomally encoded heme sensor developed for Saccharomyces cerevisiae to examine cytosolic heme levels in C. neoformans using fluorescence microscopy, fluorimetry, and flow cytometry. We validated the responsiveness of the sensor upon treatment with exogenous hemin, during proliferation in macrophages, and in strains defective for endocytosis. We then used the sensor to show that vacuolar and mitochondrial dysregulation and oxidative stress reduced the labile heme pool in the cytosol. Importantly, the sensor provided a tool to further demonstrate that the drugs artemisinin and metformin have heme-related activities and the potential to be repurposed for antifungal therapy. Overall, this study provides insights into heme sensing by C. neoformans and establishes a powerful tool to further investigate mechanisms of heme-iron acquisition in the context of fungal pathogenesis. IMPORTANCE Invasive fungal diseases are increasing in frequency, and new drug targets and antifungal drugs are needed to bolster therapy. The mechanisms by which pathogens obtain critical nutrients such as iron from heme during host colonization represent a promising target for therapy. In this study, we employed a fluorescent heme sensor to investigate heme homeostasis in Cryptococcus neoformans. We demonstrated that endocytosis is a key aspect of heme acquisition and that vacuolar and mitochondrial functions are important in regulating the pool of available heme in cells. Stress generated by oxidative conditions impacts the heme pool, as do the drugs artemisinin and metformin; these drugs have heme-related activities and are in clinical use for malaria and diabetes, respectively. Overall, our study provides insights into mechanisms of fungal heme acquisition and demonstrates the utility of the heme sensor for drug characterization in support of new therapies for fungal diseases.

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Cytoplasmic Heme Sensor Illuminates the Impacts of Mitochondrial and Vacuolar Functions and Oxidative Stress on Heme-Iron Homeostasis in Cryptococcus neoformans

Bairwa

Sánchez‐León

et al. 2020

mBio

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Comparative miRNA Transcriptomics of Mouse and Macaque Reveals MYOC is An Inhibitor for C. neoformans Invasion into Brain

Li¹,

Han²,

et al. 2022

Preprint

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Cryptococcal meningitis is a major risk for immune compromised patients mainly caused by the human opportunistic pathogen Cryptococcus neoformans, yet mechanisms of the brain or CNS dissemination remain to elucidate, which is the deadest process for the disease. Meanwhile, illustrations of clinically relevant responses in cryptococcosis were limited, as the low availabilities of clinical samples. In this study, macaque and mouse infection models were employed and miRNA-mRNA transcriptomes were performed and combined, which revealed cytoskeleton was a centric pathway regulated in both two organisms. Notably, myocilin, a novel modulator encoded by MYOC, was identified during cryptococcal pneumonia. An enhanced fungal burden was achieved in the brains of MYOC transgenic mice. Importantly, C. neoformans dissemination to the brain was inhibited by cytoskeleton inhibitor, which ruined “Trojan Horse” by dampening migration and phagocytosis of macrophages, but toxic to hosts. Taking together, this study provides new pathways and modulators during C. neoformans infections and reveals fundamental roles of cytoskeleton and MYOC in blocking fungal CNS dissemination that facilitate the development of novel drug targets for therapies of meningoencephalitis caused by C. neoformans and other pathogenic microorganisms.Author summaryFungal CNS dissemination is vital and lethal, which is the most important step for both fungal pathogenesis and host defense. Many milestones have been made to illustrate processes of invasion, such as the “Trojan Horse” derived by macrophages. However, blocking this “road” is still on the way. In our work, to mimic real responses in humans, macaque and mouse were included, miRNA-Seq and mRNA-Seq were combined, and we identified the core-pathway (cytoskeleton) during C. neoformans pathogenesis, which plays important roles in migration and phagocytosis for macrophages, key factors for macrophage “Trojan horse”, and cytoskeleton was also ruined in HIV/AIDS patients. Fungal brain dissemination was weakened by cytoskeleton inhibitor in mice. Meanwhile, we identified a novel modulator for macrophage “Trojan Horse”, MYOC, involved with C. neoformans brain dissemination. Consistently, we assume macrophages are the key executor cells for fungal CNS dissemination, and we propose the processes can be shut down by cytoskeleton or MYOC inhibitors. Our research would not only help to understand the high prevalence of cryptococcal meningitis in HIV/AIDS but also provides putative ways to block fungal cells invading the brain.

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Role of clathrin-mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans

Cited by 2 publications

References 79 publications

A Cytoplasmic Heme Sensor Illuminates the Impacts of Mitochondrial and Vacuolar Functions and Oxidative Stress on Heme-Iron Homeostasis in Cryptococcus neoformans

A Cytoplasmic Heme Sensor Illuminates the Impacts of Mitochondrial and Vacuolar Functions and Oxidative Stress on Heme-Iron Homeostasis in Cryptococcus neoformans

Comparative miRNA Transcriptomics of Mouse and Macaque Reveals MYOC is An Inhibitor for C. neoformans Invasion into Brain

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