scholarly journals Identification of bridgin, an unconventional linker, connects the outer kinetochore to centromeric chromatin

2019 ◽  
Author(s):  
Shreyas Sridhar ◽  
Tetsuya Hori ◽  
Reiko Nakagawa ◽  
Tatsuo Fukagawa ◽  
Kaustuv Sanyal
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Centromeric Chromatin ◽  
Microtubule Binding ◽  
Human Fungal Pathogen ◽  
Functional Analyses

ABSTRACTThe microtubule-binding outer kinetochore is linked to centromeric chromatin through the inner kinetochore CENP-CMif2, CENP-TCnn1, and CENP-UAme1 pathways. These are the only known kinetochore linker proteins across eukaryotes. Linker proteins are structurally less conserved than their outer kinetochore counterparts. Here, we demonstrate the recurrent loss of most inner kinetochore CCAN, including certain linker proteins during evolution in the fungal phylum of Basidiomycota. By studying the kinetochore interactome, a previously undescribed linker protein, bridgin was identified in the basidiomycete Cryptococcus neoformans, a human fungal pathogen. In vivo and in vitro functional analyses of bridgin reveal that it binds to the outer kinetochore and centromere chromatin simultaneously to ensure accurate kinetochore-microtubule attachments. Unlike known linker proteins, bridgin is recruited by the outer kinetochore. Homologs of bridgin were identified outside fungi. These results showcase a divergent strategy, with a more ancient origin than fungi, to link the outer kinetochore to centromeric chromatin.

scholarly journals Bridgin connects the outer kinetochore to centromeric chromatin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shreyas Sridhar ◽  
Tetsuya Hori ◽  
Reiko Nakagawa ◽  
Tatsuo Fukagawa ◽  
Kaustuv Sanyal
Keyword(s):  
Cryptococcus Neoformans ◽  
Chromosome Segregation ◽  
Human Pathogen ◽  
Centromeric Chromatin ◽  
Microtubule Binding ◽  
Functional Analyses

AbstractThe microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-CMif2, CENP-TCnn1, and CENP-UAme1 linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein “bridgin” was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin.

scholarly journals Inhibition of classical and alternative modes of respiration in C. albicans leads to cell wall remodelling and increased macrophage recognition

2018 ◽  
Author(s):  
Lucian Duvenage ◽  
Louise A. Walker ◽  
Aleksandra Bojarczuk ◽  
Simon A. Johnston ◽  
Donna M. MacCallum ◽  
...  
Keyword(s):  
Cell Wall ◽  
Electron Transport ◽  
Fungal Pathogen ◽  
Fungal Infections ◽  
Oxidase Inhibitor ◽  
Nitric Oxide Donor ◽  
Mammalian Host ◽  
Human Fungal Pathogen

AbstractThe human fungal pathogen C. albicans requires respiratory function for normal growth, morphogenesis and virulence. As such the mitochondria represent an enticing target for the development of new antifungal strategies. This possibility is further bolstered by the presence of fungal specific characteristics. However, respiration in C. albicans, as is the case in many fungal organisms, is facilitated by redundant electron transport mechanisms that makes direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we make use of chemicals with low toxicity in mammals to efficiently inhibit respiration in C. albicans. We find that use of the Nitric Oxide donor, Sodium Nitroprusside (SNP), and the alternative oxidase inhibitor, SHAM, prevent respiration, lead to a loss in viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo. We propose that SNP+SHAM treatment leads to transcriptional changes that drive cell wall re-arrangement but which also prime cells to activate transition to hyphal growth. In line with this we find that pre-treatment of C. albicans with SNP+SHAM leads to an increase in virulence. Our data reveals strong links between respiration, cell wall remodelling and activation of virulence factors. Our findings also demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals which are not damaging to the mammalian host, but that we need to develop a deeper understanding of the roles of mitochondria in cellular signalling if they are to be developed successfully as a target for new antifungals.Author SummaryCurrent approaches to tackling fungal infections are limited and new targets must be identified to protect against the emergence of resistant strains. We investigate the potential of targeting mitochondria, organelles required for energy production, growth and virulence, in the yeast human fungal pathogen Candida albicans. Our findings suggest that mitochondria can be targeted using drugs that can be tolerated by humans and that this treatment enhances their recognition by immune cells. However release of C. albicans cells from mitochondrial inhibition appears to activate a stress response that increases traits associated with virulence. Our results make it clear that mitochondria are a valid target for the development of anti-fungal strategies but that we must determine the mechanisms by which they regulate stress signalling and virulence ahead of successful therapeutic advance.

scholarly journals Mixed Infections and In Vivo Evolution in the Human Fungal Pathogen Cryptococcus neoformans

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Marie Desnos-Ollivier ◽  
Sweta Patel ◽  
Adam R. Spaulding ◽  
Caroline Charlier ◽  
Dea Garcia-Hermoso ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Molecular Diversity ◽  
Therapeutic Strategies ◽  
Mixed Infections ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Life Threatening

ABSTRACTKoch’s postulates are criteria establishing a causal relationship between a microbe and a disease that lead to the assumption that diseases are caused by a single strain or its evolved forms.Cryptococcus neoformansis a life-threatening human fungal pathogen responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis. To assess the molecular diversity of clinical isolates and gain knowledge ofC. neoformansbiology in the host, we analyzed clinical cultures collected during the prospective CryptoA/D study. Using molecular analysis of unpurified isolates, we demonstrated that mixed infections in humans are more common than previously thought, occurring in almost 20% of patients diagnosed with cryptococcosis. These mixed infections are composed of different mating types, serotypes, and/or genotypes. We also identified genetically related haploid and diploid strains in the same patients. Experimental infections and quantitative PCR show that these ploidy changes can result from endoreplication (duplication of DNA content) and that shuttling between haploid and diploid states can occur, suggestingin vivoevolution. Thus, the concept of one strain/one infection does not hold true forC. neoformansand may apply to other environmentally acquired fungal pathogens. Furthermore, the possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens.IMPORTANCECryptococcus  neoformansis a life-threatening human fungal pathogen that is present in the environment and is responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis in HIV-infected patients. To assess the molecular diversity of clinical isolates and gain knowledge ofC. neoformansbiology in the host, we analyzed clinical cultures collected during a prospective study on cryptococcosis. Using molecular analysis of unpurified isolates, we uncovered an unexpectedly high frequency (almost 20%) of mixed infections. We further demonstrated that these mixed infections could result from infestation by multiple strains acquired from the environment. We also made the serendipitous discovery ofin vivoevolution leading to endoreplication of the yeasts within the host. Thus, the concept of one strain causing one infection does not hold true forC. neoformansand potentially for other environmentally acquired fungal pathogens. The possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens.

scholarly journals Trojan Horse Transit Contributes to Blood-Brain Barrier Crossing of a Eukaryotic Pathogen

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Felipe H. Santiago-Tirado ◽  
Michael D. Onken ◽  
John A. Cooper ◽  
Robyn S. Klein ◽  
Tamara L. Doering
Keyword(s):  
Experimental Evidence ◽  
Cryptococcus Neoformans ◽  
Blood Brain Barrier ◽  
Fungal Pathogen ◽  
Trojan Horse ◽  
Brain Barrier ◽  
Barrier Crossing ◽  
Blood Brain ◽  
The Brain

ABSTRACT The blood-brain barrier (BBB) protects the central nervous system (CNS) by restricting the passage of molecules and microorganisms. Despite this barrier, however, the fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that is estimated to kill over 600,000 people annually. Cryptococcal infection begins in the lung, and experimental evidence suggests that host phagocytes play a role in subsequent dissemination, although this role remains ill defined. Additionally, the disparate experimental approaches that have been used to probe various potential routes of BBB transit make it impossible to assess their relative contributions, confounding any integrated understanding of cryptococcal brain entry. Here we used an in vitro model BBB to show that a “Trojan horse” mechanism contributes significantly to fungal barrier crossing and that host factors regulate this process independently of free fungal transit. We also, for the first time, directly imaged C. neoformans-containing phagocytes crossing the BBB, showing that they do so via transendothelial pores. Finally, we found that Trojan horse crossing enables CNS entry of fungal mutants that cannot otherwise traverse the BBB, and we demonstrate additional intercellular interactions that may contribute to brain entry. Our work elucidates the mechanism of cryptococcal brain invasion and offers approaches to study other neuropathogens. IMPORTANCE The fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that kills hundreds of thousands of people each year. One route that has been proposed for this brain entry is a Trojan horse mechanism, whereby the fungus crosses the blood-brain barrier (BBB) as a passenger inside host phagocytes. Although indirect experimental evidence supports this intriguing mechanism, it has never been directly visualized. Here we directly image Trojan horse transit and show that it is regulated independently of free fungal entry, contributes to cryptococcal BBB crossing, and allows mutant fungi that cannot enter alone to invade the brain. IMPORTANCE The fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that kills hundreds of thousands of people each year. One route that has been proposed for this brain entry is a Trojan horse mechanism, whereby the fungus crosses the blood-brain barrier (BBB) as a passenger inside host phagocytes. Although indirect experimental evidence supports this intriguing mechanism, it has never been directly visualized. Here we directly image Trojan horse transit and show that it is regulated independently of free fungal entry, contributes to cryptococcal BBB crossing, and allows mutant fungi that cannot enter alone to invade the brain.

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

2020 ◽  
Vol 138 ◽  
pp. 103365
Author(s):  
Garrick W.K. Spencer ◽  
Sheena M.H. Chua ◽  
Paige E. Erpf ◽  
Maha S.I. Wizrah ◽  
Taylor G. Dyba ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Fluorescent Protein ◽  
Human Fungal Pathogen

scholarly journals New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ren-Yi Lu ◽  
Ting-Jun-Hong Ni ◽  
Jing Wu ◽  
Lan Yan ◽  
Quan-Zhen Lv ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Pathogenic Fungi ◽  
Control Group ◽  
Survival Times ◽  
Content Type ◽  
Fungal Burden ◽  
Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

scholarly journals Structural analysis of fungal pathogenicity-related casein kinase α subunit, Cka1, in the human fungal pathogen Cryptococcus neoformans

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Belinda X. Ong ◽  
Youngki Yoo ◽  
Myeong Gil Han ◽  
Jun Bae Park ◽  
Myung Kyung Choi ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Kinase Assay ◽  
Α Subunit ◽  
Dynamic Architecture ◽  
Human Fungal Pathogen ◽  
Life Threatening ◽  
The Difference ◽  
Anti Fungal

Abstract CK2α is a constitutively active and highly conserved serine/threonine protein kinase that is involved in the regulation of key cellular metabolic pathways and associated with a variety of tumours and cancers. The most well-known CK2α inhibitor is the human clinical trial candidate CX-4945, which has recently shown to exhibit not only anti-cancer, but also anti-fungal properties. This prompted us to work on the CK2α orthologue, Cka1, from the pathogenic fungus Cryptococcus neoformans, which causes life-threatening systemic cryptococcosis and meningoencephalitis mainly in immunocompromised individuals. At present, treatment of cryptococcosis remains a challenge due to limited anti-cryptococcal therapeutic strategies. Hence, expanding therapeutic options for the treatment of the disease is highly clinically relevant. Herein, we report the structures of Cka1-AMPPNP-Mg2+ (2.40 Å) and Cka1-CX-4945 (2.09 Å). Structural comparisons of Cka1-AMPPNP-Mg2+ with other orthologues revealed the dynamic architecture of the N-lobe across species. This may explain for the difference in binding affinities and deviations in protein-inhibitor interactions between Cka1-CX-4945 and human CK2α-CX-4945. Supporting it, in vitro kinase assay demonstrated that CX-4945 inhibited human CK2α much more efficiently than Cka1. Our results provide structural insights into the design of more selective inhibitors against Cka1.

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