scholarly journals P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity

2021 ◽  
Vol 7 (10) ◽  
pp. 843
Author(s):  
Lyubomir Dimitrov Stanchev ◽  
Juliana Rizzo ◽  
Rebecca Peschel ◽  
Lilli A. Pazurek ◽  
Lasse Bredegaard ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Biochemical Characterization ◽  
Fungal Virulence ◽  
Broad Substrate Specificity ◽  
Important Regulator ◽  
Heterodimeric Complex ◽  
P Type ◽  
Lipid Flippase

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

scholarly journals Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence inCryptococcus neoformans

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Wei Huang ◽  
Guojian Liao ◽  
Gregory M. Baker ◽  
Yina Wang ◽  
Richard Lau ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Treatment Options ◽  
Membrane Lipid ◽  
Fungal Resistance ◽  
Fungal Virulence ◽  
Innate Resistance ◽  
Fungal Meningitis ◽  
Lipid Flippase

ABSTRACTCryptococcus neoformansis a human fungal pathogen and a major cause of fungal meningitis in immunocompromised individuals. Treatment options for cryptococcosis are limited. Of the two major antifungal drug classes, azoles are active againstC. neoformansbut exert a fungistatic effect, necessitating long treatment regimens and leaving open an avenue for emergence of azole resistance. Drugs of the echinocandin class, which target the glucan synthase and are fungicidal against a number of other fungal pathogens, such asCandidaspecies, are ineffective againstC. neoformans. Despite the sensitivity of the target enzyme to the drug, the reasons for the innate resistance ofC. neoformansto echinocandins remain unknown. To understand the mechanism of echinocandin resistance inC. neoformans, we screened gene disruption and gene deletion libraries for mutants sensitive to the echinocandin-class drug caspofungin and identified a mutation ofCDC50, which encodes the β-subunit of membrane lipid flippase. We found that the Cdc50 protein localized to membranes and that its absence led to plasma membrane defects and enhanced caspofungin penetration into the cell, potentially explaining the increased caspofungin sensitivity. Loss ofCDC50also led to hypersensitivity to the azole-class drug fluconazole. Interestingly, in addition to functioning in drug resistance,CDC50was also essential for fungal resistance to macrophage killing and for virulence in a murine model of cryptococcosis. Furthermore, the surface ofcdc50Δcells contained increased levels of phosphatidylserine, which has been proposed to act as a macrophage recognition signal. Together, these results reveal a previously unappreciated role of membrane lipid flippase inC. neoformansdrug resistance and virulence.IMPORTANCECryptococcus neoformansis a fungal pathogen that is the most common cause of fungal meningitis, causing over 620,000 deaths annually. The treatment options for cryptococcosis are very limited. The most commonly used drugs are either fungistatic (azoles) or highly toxic (amphotericin B). Echinocandins are the newest fungicidal drug class that works well in treating candidiasis and aspergillosis, yet they are ineffective in treating cryptococcosis. In this study, we showed that the regulatory subunit of the lipid translocase (flippase), a protein that regulates the asymmetrical orientation of membrane lipids, is required forC. neoformansresistance to caspofungin, as well as for virulence during infection. This discovery identifies lipid flippase as a potentialC. neoformansdrug target, which plays an important role in the innate resistance ofC. neoformansto echinocandins and in fungal virulence.

scholarly journals A Predicted Mannoprotein Cmp1 Regulates Fungal Virulence in Cryptococcus neoformans

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 881
Author(s):  
Lian-Tao Han ◽  
Lei Wu ◽  
Tong-Bao Liu
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Gene Expression Pattern ◽  
Fungal Virulence ◽  
Human Fungal Pathogen ◽  
Overexpression Strain ◽  
Reproduction Process ◽  
Localization Analysis ◽  
Survival Assay

The capsule of the fungal pathogen Cryptococcus neoformans consists of glucuronoxylomannan (GXM), glucuronoxylomannogalactan (GXMGal), and mannoproteins (MPs). MPs are a kind of glycoproteins with low content but high immunogenicity, which can stimulate the immune protection of the host. However, there is not much information about the role of mannoproteins in virulence of the human fungal pathogen C. neoformans. In this study, we reported the identification and functional analysis of a predicted mannoprotein Cmp1 that regulates fungal virulence in C. neoformans. Gene expression pattern analysis indicates that the CMP1 gene was ubiquitously expressed at all stages of cryptococcal development. Subcellular localization analysis indicated that Cmp1 was localized in the cytoplasm of cryptococcal cells. Disruption or overexpression of CMP1 results in impairing capsule formation in Cryptococcus, but it does not affect the melanin production and sensitivity under various stress conditions, nor does it affect the sexual reproduction process of Cryptococcus. Survival assay showed that the pathogenicity of the cmp1Δ mutant or the CMP1 overexpression strain was significantly attenuated in a murine inhalation model of cryptococcosis. In conclusion, our findings implied that the mannoprotein Cmp1 is required for the virulence of C. neoformans.

scholarly journals Biochemical Characterization of the POM-1 Metallo-β-Lactamase from Pseudomonas otitidis

2014 ◽  
Vol 59 (3) ◽  
pp. 1755-1758 ◽  
Author(s):  
Luisa Borgianni ◽  
Filomena De Luca ◽  
Maria Cristina Thaller ◽  
Yunsop Chong ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Content Type ◽  
Broad Substrate Specificity ◽  
Catalytic Activities

ABSTRACTThe POM-1 metallo-β-lactamase is a subclass B3 resident enzyme produced byPseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced inEscherichia coliBL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.

scholarly journals A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity

2017 ◽  
Vol 84 (2) ◽  
Author(s):  
Tomas Buryska ◽  
Petra Babkova ◽  
Ondrej Vavra ◽  
Jiri Damborsky ◽  
Zbynek Prokop
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Biochemical Characterization ◽  
Environmental Pollutants ◽  
Haloalkane Dehalogenase ◽  
Broad Substrate Specificity ◽  
Organic Cosolvents ◽  
Marine Metagenome ◽  
Ph Range

ABSTRACTThe haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications.IMPORTANCEWe present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.

scholarly journals Biochemical Characterization and Degradation Pattern of a Novel Endo-Type Bifunctional Alginate Lyase AlyA from Marine Bacterium Isoptericola halotolerans

Marine Drugs ◽  
2018 ◽  
Vol 16 (8) ◽  
pp. 258 ◽  
Author(s):  
Benwei Zhu ◽  
Limin Ning ◽  
Yucui Jiang ◽  
Lin Ge
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Alginate Lyase ◽  
Ionization Mass ◽  
Broad Substrate Specificity ◽  
Action Mode ◽  
Degradation Pattern ◽  
Km Value ◽  
Alginate Lyases ◽  
Layer Chromatography

Alginate lyases are important tools to prepare oligosaccharides with various physiological activities by degrading alginate. Particularly, the bifunctional alginate lyase can efficiently hydrolyze the polysaccharide into oligosaccharides. Herein, we cloned and identified a novel bifunctional alginate lyase, AlyA, with a high activity and broad substrate specificity from bacterium Isoptericola halotolerans NJ-05 for oligosaccharides preparation. For further applications in industry, the enzyme has been characterized and its action mode has been also elucidated. It exhibited the highest activity (7984.82 U/mg) at pH 7.5 and 55 °C. Additionally, it possessed a broad substrate specificity, showing high activities towards not only polyM (polyβ-d-mannuronate) (7658.63 U/mg), but also polyG (poly α-l-guluronate) (8643.29 U/mg). Furthermore, the Km value of AlyA towards polyG (3.2 mM) was lower than that towards sodium alginate (5.6 mM) and polyM (6.7 mM). TLC (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) were used to study the action mode of the enzyme, showing that it can hydrolyze the substrates in an endolytic manner to release a series of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide. This study provided extended insights into the substrate recognition and degrading pattern of the alginate lyases, with a broad substrate specificity.

scholarly journals A fungal arrestin protein contributes to cell cycle progression and pathogenesis

2019 ◽  
Author(s):  
Calla L. Telzrow ◽  
Connie B. Nichols ◽  
Natalia Castro-Lopez ◽  
Floyd L. Wormley ◽  
J. Andrew Alspaugh
Keyword(s):  
Cell Cycle ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Cell Cycle Progression ◽  
Lipid Synthesis ◽  
Adaptor Protein ◽  
Cellular Responses ◽  
Fungal Virulence ◽  
Human Fungal Pathogen ◽  
Mutant Phenotypes

ABSTRACTArrestins, a structurally specialized and functionally diverse group of proteins, are central regulators of adaptive cellular responses in eukaryotes. Previous studies on fungal arrestins have demonstrated their capacity to modulate diverse cellular processes through their adaptor functions, facilitating the localization and function of other proteins. However, the mechanisms by which arrestin-regulated processes are involved in fungal virulence remain unexplored. We have identified a small family of four arrestins - Ali1, Ali2, Ali3, and Ali4 - in the human fungal pathogen Cryptococcus neoformans. Using complementary microscopy, proteomic, and reverse genetic techniques, we have defined a role for Ali1 as a novel contributor to cytokinesis, a fundamental cell cycle-associated process. We observed that Ali1 strongly interacts with proteins involved in lipid synthesis, and that ali1Δ mutant phenotypes are rescued by supplementation with lipid precursors that are used to build cellular membranes. From these data, we hypothesize that Ali1 contributes to cytokinesis by serving as an adaptor protein, facilitating the localization of enzymes that modify the plasma membrane during cell division, specifically the fatty acid synthases, Fas1 and Fas2. Finally, we assessed the contributions of the C. neoformans arrestin family to virulence, to better understand the mechanisms by which arrestin-regulated adaptive cellular responses influence fungal infection. We observed that the C. neoformans arrestin family contributes to virulence, and that the individual arrestin proteins likely fulfill distinct functions that are important for disease progression.IMPORTANCETo survive in unpredictable conditions, all organisms must adapt to stressors by regulating adaptive cellular responses. Arrestin proteins are conserved regulators of adaptive cellular responses in eukaryotes. Studies that have been limited to mammals and model fungi have demonstrated that disruption of arrestin-regulated pathways is detrimental for viability. The human fungal pathogen Cryptococcus neoformans causes more than 180,000 infection-related deaths annually, especially among immunocompromised patients. In addition to being genetically-tractable, C. neoformans has a small arrestin family of four members, lending itself to a comprehensive characterization of its arrestin family. This study serves as a functional analysis of arrestins in a pathogen, particularly in the context of fungal fitness and virulence. We investigate the functions of one arrestin protein, Ali1, and define its novel contributions to cytokinesis. We additionally explore the virulence contributions of the C. neoformans arrestin family and find that they contribute to disease establishment and progression.

scholarly journals A Fungal Arrestin Protein Contributes to Cell Cycle Progression and Pathogenesis

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Calla L. Telzrow ◽  
Connie B. Nichols ◽  
Natalia Castro-Lopez ◽  
Floyd L. Wormley ◽  
J. Andrew Alspaugh
Keyword(s):  
Cell Cycle ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Reverse Genetics ◽  
Lipid Synthesis ◽  
Adaptor Protein ◽  
Cellular Responses ◽  
Fungal Virulence ◽  
Content Type ◽  
Human Fungal Pathogen

ABSTRACT Arrestins, a structurally specialized and functionally diverse group of proteins, are central regulators of adaptive cellular responses in eukaryotes. Previous studies on fungal arrestins have demonstrated their capacity to modulate diverse cellular processes through their adaptor functions, facilitating the localization and function of other proteins. However, the mechanisms by which arrestin-regulated processes are involved in fungal virulence remain unexplored. We have identified a small family of four arrestins, Ali1, Ali2, Ali3, and Ali4, in the human fungal pathogen Cryptococcus neoformans. Using complementary microscopy, proteomic, and reverse genetics techniques, we have defined a role for Ali1 as a novel contributor to cytokinesis, a fundamental cell cycle-associated process. We observed that Ali1 strongly interacts with proteins involved in lipid synthesis, and that ali1Δ mutant phenotypes are rescued by supplementation with lipid precursors that are used to build cellular membranes. From these data, we hypothesize that Ali1 contributes to cytokinesis by serving as an adaptor protein, facilitating the localization of enzymes that modify the plasma membrane during cell division, specifically the fatty acid synthases Fas1 and Fas2. Finally, we assessed the contributions of the C. neoformans arrestin family to virulence to better understand the mechanisms by which arrestin-regulated adaptive cellular responses influence fungal infection. We observed that the C. neoformans arrestin family contributes to virulence, and that the individual arrestin proteins likely fulfill distinct functions that are important for disease progression. IMPORTANCE To survive under unpredictable conditions, all organisms must adapt to stressors by regulating adaptive cellular responses. Arrestin proteins are conserved regulators of adaptive cellular responses in eukaryotes. Studies that have been limited to mammals and model fungi have demonstrated that the disruption of arrestin-regulated pathways is detrimental for viability. The human fungal pathogen Cryptococcus neoformans causes more than 180,000 infection-related deaths annually, especially among immunocompromised patients. In addition to being genetically tractable, C. neoformans has a small arrestin family of four members, lending itself to a comprehensive characterization of its arrestin family. This study serves as a functional analysis of arrestins in a pathogen, particularly in the context of fungal fitness and virulence. We investigate the functions of one arrestin protein, Ali1, and define its novel contributions to cytokinesis. We additionally explore the virulence contributions of the C. neoformans arrestin family and find that they contribute to disease establishment and progression.

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