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.

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.

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 Structural mechanism of Myb–MuvB assembly

2018 ◽  
Vol 115 (40) ◽  
pp. 10016-10021 ◽  
Author(s):  
Keelan Z. Guiley ◽  
Audra N. Iness ◽  
Siddharth Saini ◽  
Sarvind Tripathi ◽  
Joseph S. Lipsick ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Biochemical Analysis ◽  
Adaptor Protein ◽  
Cancer Cell Proliferation ◽  
Cycle Progression ◽  
Structural Mechanism ◽  
Transcriptional Regulatory ◽  
Regulatory Complex ◽  
Cycle Dependent

The MuvB transcriptional regulatory complex, which controls cell-cycle-dependent gene expression, cooperates with B-Myb to activate genes required for the G2 and M phases of the cell cycle. We have identified the domain in B-Myb that is essential for the assembly of the Myb–MuvB (MMB) complex. We determined a crystal structure that reveals how this B-Myb domain binds MuvB through the adaptor protein LIN52 and the scaffold protein LIN9. The structure and biochemical analysis provide an understanding of how oncogenic B-Myb is recruited to regulate genes required for cell-cycle progression, and the MMB interface presents a potential therapeutic target to inhibit cancer cell proliferation.

scholarly journals Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans

2020 ◽  
Vol 140 ◽  
pp. 103368 ◽  
Author(s):  
Fabiana Freire M. Oliveira ◽  
Hugo Costa Paes ◽  
Luísa Defranco F. Peconick ◽  
Fernanda L. Fonseca ◽  
Clara Luna Freitas Marina ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Membrane Composition ◽  
Human Fungal Pathogen

scholarly journals The Role of Oxidoreductase-Like Protein Olp1 in Sexual Reproduction and Virulence of Cryptococcus neoformans

2020 ◽  
Vol 8 (11) ◽  
pp. 1730
Author(s):  
Qi-Kun Yu ◽  
Lian-Tao Han ◽  
Yu-Juan Wu ◽  
Tong-Bao Liu
Keyword(s):  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Expression Patterns ◽  
Fungal Virulence ◽  
Growth Defects ◽  
Human Fungal Pathogen ◽  
Yeast Extract Peptone Dextrose ◽  
Virulence Attenuation ◽  
Protein Encoding ◽  
Fungal Mating

Cryptococcus neoformans is a basidiomycete human fungal pathogen causing lethal meningoencephalitis, mainly in immunocompromised patients. Oxidoreductases are a class of enzymes that catalyze redox, playing a crucial role in biochemical reactions. In this study, we identified one Cryptococcus oxidoreductase-like protein-encoding gene OLP1 and investigated its role in the sexual reproduction and virulence of C. neoformans. Gene expression patterns analysis showed that the OLP1 gene was expressed in each developmental stage of Cryptococcus, and the Olp1 protein was located in the cytoplasm of Cryptococcus cells. Although it produced normal major virulence factors such as melanin and capsule, the olp1Δ mutants showed growth defects on the yeast extract peptone dextrose (YPD) medium supplemented with lithium chloride (LiCl) and 5-fluorocytosine (5-FC). The fungal mating analysis showed that Olp1 is also essential for fungal sexual reproduction, as olp1Δ mutants show significant defects in hyphae growth and basidiospores production during bisexual reproduction. The fungal nuclei imaging showed that during the bilateral mating of olp1Δ mutants, the nuclei failed to undergo meiosis after fusion in the basidia, indicating that Olp1 is crucial for regulating meiosis during mating. Moreover, Olp1 was also found to be required for fungal virulence in C. neoformans, as the olp1Δ mutants showed significant virulence attenuation in a murine inhalation model. In conclusion, our results showed that the oxidoreductase-like protein Olp1 is required for both fungal sexual reproduction and virulence in C. neoformans.

scholarly journals Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Shannon K. Esher ◽  
Kyla S. Ost ◽  
Lukasz Kozubowski ◽  
Dong-Hoon Yang ◽  
Min Su Kim ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Posttranslational Modification ◽  
Fungal Pathogen ◽  
Content Type ◽  
Processing Enzymes ◽  
Human Fungal Pathogen ◽  
Modification Process ◽  
Substrate Proteins ◽  
Processing Steps

ABSTRACT Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus. Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.

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