Posttranslational Modifications of Proteins in the Pathobiology of Medically Relevant Fungi

ABSTRACTPosttranslational modifications of proteins drive a wide variety of cellular processes in eukaryotes, regulating cell growth and division as well as adaptive and developmental processes. With regard to the fungal kingdom, most information about posttranslational modifications has been generated through studies of the model yeastsSaccharomyces cerevisiaeandSchizosaccharomyces pombe, where, for example, the roles of protein phosphorylation, glycosylation, acetylation, ubiquitination, sumoylation, and neddylation have been dissected. More recently, information has begun to emerge for the medically important fungal pathogensCandida albicans,Aspergillus fumigatus, andCryptococcus neoformans, highlighting the relevance of posttranslational modifications for virulence. We review the available literature on protein modifications in fungal pathogens, focusing in particular upon the reversible peptide modifications sumoylation, ubiquitination, and neddylation.

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Toll-Like Receptor 9 Modulates Macrophage Antifungal Effector Function during Innate Recognition of Candida albicans and Saccharomyces cerevisiae

Infection and Immunity ◽

10.1128/iai.05626-11 ◽

2011 ◽

Vol 79 (12) ◽

pp. 4858-4867 ◽

Cited By ~ 33

Author(s):

Pia V. Kasperkovitz ◽

Nida S. Khan ◽

Jenny M. Tam ◽

Michael K. Mansour ◽

Peter J. Davids ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans ◽

Fungal Pathogens ◽

Toll Like Receptor ◽

Wild Type ◽

Necrosis Factor Alpha ◽

Content Type ◽

Tnf Α ◽

Tlr9 Gene

ABSTRACTPhagocytic responses are critical for effective host defense against opportunistic fungal pathogens. Macrophages sample the phagosomal content and orchestrate the innate immune response. Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA and is activated by fungal DNA. Here we demonstrate that specific triggering of TLR9 recruitment to the macrophage phagosomal membrane is a conserved feature of fungi of distinct phylogenetic origins, includingCandida albicans,Saccharomyces cerevisiae,Malassezia furfur, andCryptococcus neoformans. The capacity to trigger phagosomal TLR9 recruitment was not affected by a loss of fungal viability or cell wall integrity. TLR9 deficiency has been linked to increased resistance to murine candidiasis and to restriction of fungal growthin vivo. Macrophages lacking TLR9 demonstrate a comparable capacity for phagocytosis and normal phagosomal maturation compared to wild-type macrophages. We now show that TLR9 deficiency increases macrophage tumor necrosis factor alpha (TNF-α) production in response toC. albicansandS. cerevisiae, independent of yeast viability. The increase in TNF-α production was reversible by functional complementation of the TLR9 gene, confirming that TLR9 was responsible for negative modulation of the cytokine response. Consistently, TLR9 deficiency enhanced the macrophage effector response by increasing macrophage nitric oxide production. Moreover, microbicidal activity againstC. albicansandS. cerevisiaewas more efficient in TLR9 knockout (TLR9KO) macrophages than in wild-type macrophages. In conclusion, our data demonstrate that TLR9 is compartmentalized selectively to fungal phagosomes and negatively modulates macrophage antifungal effector functions. Our data support a model in which orchestration of antifungal innate immunity involves a complex interplay of fungal ligand combinations, host cell machinery rearrangements, and TLR cooperation and antagonism.

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Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽

10.1128/msphere.00715-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Suresh Ambati ◽

Emma C. Ellis ◽

Jianfeng Lin ◽

Xiaorong Lin ◽

Zachary A. Lewis ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Effective Dose ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Antifungal Drugs ◽

Extracellular Matrices ◽

Content Type ◽

Order Of Magnitude ◽

Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

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Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00250-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 2-9 ◽

Cited By ~ 58

Author(s):

Frans M. Klis ◽

Chris G. de Koster ◽

Stanley Brul

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Candida Albicans ◽

Cell Size ◽

Pathogenic Fungus ◽

Turgor Pressure ◽

Hyphal Growth ◽

Biological Research ◽

Content Type ◽

Starting Point

ABSTRACTBionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeastSaccharomyces cerevisiaeand the polymorphic, pathogenic fungusCandida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation ofin vivovalues. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allowsC. albicansto cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.

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Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal

mSphere ◽

10.1128/msphere.00610-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Irsa Shoukat ◽

Corey Frazer ◽

John S. Allingham

Keyword(s):

Candida Albicans ◽

Mitotic Spindle ◽

Fungal Pathogens ◽

Spindle Assembly ◽

Spindle Pole ◽

Content Type ◽

Bipolar Spindle ◽

Spindle Elongation ◽

Simultaneous Loss ◽

Bipolar Spindles

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

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Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance inAspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02593-17 ◽

2018 ◽

Vol 62 (6) ◽

Cited By ~ 9

Author(s):

Fabio Gsaller ◽

Takanori Furukawa ◽

Paul D. Carr ◽

Bharat Rash ◽

Christoph Jöchl ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Fungal Pathogens ◽

Regulatory Protein ◽

Transcriptional Regulators ◽

Primary Factor ◽

Ph Sensing ◽

Content Type ◽

Ph Dependent ◽

Mechanistic Basis

ABSTRACTThe antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is licensed for the treatment of fungal diseases; however, it is rarely used as a monotherapeutic to treatAspergillusinfection. Despite being potent against other fungal pathogens, 5FC has limited activity againstAspergillus fumigatuswhen standardin vitroassays are used to determine susceptibility. However, in modifiedin vitroassays where the pH is set to pH 5, the activity of 5FC increases significantly. Here we provide evidence thatfcyB, a gene that encodes a purine-cytosine permease orthologous to known 5FC importers, is downregulated at pH 7 and is the primary factor responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional regulators that are responsible for the repression offcyBand, consequently, mediators of 5FC resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We propose that the activity of 5FC might be enhanced by the perturbation of factors that repressfcyBexpression, such as PacC or other components of the pH-sensing machinery.

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Candida albicans Msi3p, a homolog of the Saccharomyces cerevisiae Sse1p of the Hsp70 family, is involved in cell growth and fluconazole tolerance

FEMS Yeast Research ◽

10.1111/j.1567-1364.2012.00822.x ◽

2012 ◽

Vol 12 (6) ◽

pp. 728-737 ◽

Cited By ~ 8

Author(s):

Jun-ichi Nagao ◽

Tamaki Cho ◽

Jun Uno ◽

Keigo Ueno ◽

Rieko Imayoshi ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans ◽

Cell Growth ◽

Hsp70 Family

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Aspergillus fumigatus Intrinsic Fluconazole Resistance Is Due to the Naturally Occurring T301I Substitution in Cyp51Ap

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00905-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5420-5426 ◽

Cited By ~ 26

Author(s):

Florencia Leonardelli ◽

Daiana Macedo ◽

Catiana Dudiuk ◽

Matias S. Cabeza ◽

Soledad Gamarra ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Parental Strain ◽

Amino Acid Residues ◽

Intrinsic Resistance ◽

Single Nucleotide ◽

Fluconazole Resistance ◽

Content Type ◽

Natural Polymorphism

ABSTRACTAspergillus fumigatusintrinsic fluconazole resistance has been demonstrated to be linked to theCYP51Agene, although the precise molecular mechanism has not been elucidated yet. Comparisons betweenA. fumigatusCyp51Ap andCandida albicansErg11p sequences showed differences in amino acid residues already associated with fluconazole resistance inC. albicans. The aim of this study was to analyze the role of the natural polymorphism I301 inAspergillus fumigatusCyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue inA. fumigatusCyp51Ap was replaced with a threonine (analogue to T315 atCandida albicansfluconazole-susceptible Erg11p) by changing one single nucleotide in theCYP51Agene. Also, aCYP51Aknockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 μg/ml) than the parental strain (MIC, 640 μg/ml), while no changes in MIC were observed for other azole- and non-azole-based drugs. These data strongly implicate theA. fumigatusCyp51Ap I301 residue in the intrinsic resistance to fluconazole.

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Chromatin-Mediated Regulation of Genome Plasticity in Human Fungal Pathogens

Genes ◽

10.3390/genes10110855 ◽

2019 ◽

Vol 10 (11) ◽

pp. 855 ◽

Cited By ~ 3

Author(s):

Buscaino

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Environmental Changes ◽

Structure And Function ◽

Model Systems ◽

Genome Plasticity ◽

And Function ◽

Heterochromatin Structure

Human fungal pathogens, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, are a public health problem, causing millions of infections and killing almost half a million people annually. The ability of these pathogens to colonise almost every organ in the human body and cause life-threating infections relies on their capacity to adapt and thrive in diverse hostile host-niche environments. Stress-induced genome instability is a key adaptive strategy used by human fungal pathogens as it increases genetic diversity, thereby allowing selection of genotype(s) better adapted to a new environment. Heterochromatin represses gene expression and deleterious recombination and could play a key role in modulating genome stability in response to environmental changes. However, very little is known about heterochromatin structure and function in human fungal pathogens. In this review, I use our knowledge of heterochromatin structure and function in fungal model systems as a road map to review the role of heterochromatin in regulating genome plasticity in the most common human fungal pathogens: Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans.

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Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating

Eukaryotic Cell ◽

10.1128/ec.00090-07 ◽

2007 ◽

Vol 6 (6) ◽

pp. 907-918 ◽

Cited By ~ 28

Author(s):

Dana Schaefer ◽

Pierre Côte ◽

Malcolm Whiteway ◽

Richard J. Bennett

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans ◽

Cell Growth ◽

Cell Fusion ◽

Aspartyl Protease ◽

Wild Type ◽

Pheromone Activity ◽

Important Regulator ◽

Mutant Cells ◽

Type Strains

ABSTRACT Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Δbar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in α cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Δbar1 a and α cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.

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Distinct and Redundant Roles of the Two MYST Histone Acetyltransferases Esa1 and Sas2 in Cell Growth and Morphogenesis of Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00275-12 ◽

2013 ◽

Vol 12 (3) ◽

pp. 438-449 ◽

Cited By ~ 20

Author(s):

Xiongjun Wang ◽

Peng Chang ◽

Jianping Ding ◽

Jiangye Chen

Keyword(s):

Candida Albicans ◽

Cell Growth ◽

Histone Acetylation ◽

Synergistic Effects ◽

Histone Acetyltransferases ◽

Content Type ◽

Functional Roles ◽

Oxidative Stresses ◽

Human Body Temperature ◽

Mutant Cells

ABSTRACTCandida albicansis associated with humans, as both a harmless commensal organism and a pathogen. Adaption to human body temperature is extremely important for its growth and morphogenesis.Saccharomyces cerevisiaeEsa1, a member of the MYST family HATs (histone acetyltransferases) and the catalytic subunit of the NuA4 complex, and its homologues in other eukaryotes have been shown to be essential for cell growth. To investigate the functional roles of two MYST family HATs, Esa1 and Sas2 inC. albicans, we deletedESA1andSAS2in theC. albicansgenome and performed cell growth analyses. Our results demonstrated thatC. albicansEsa1 is not essential for general growth but is essential for filamentous growth. Theesa1/esa1mutant cells exhibited sensitivity to thermal, genotoxic, and oxidative stresses but tolerance to cold, osmotic, and cell wall stresses. In contrast, thesas2/sas2mutant adapted to growth at higher temperatures and promoted filament formation at lower temperatures, resembling the phenotype of aC. albicansstrain overexpressingESA1. Cells with deletions of bothESA1andSAS2were inviable, reflecting the functional redundancy in cell growth.C. albicansEsa1 and Sas2 have distinct and synergistic effects on histone acetylation at H4K5, H4K12, and H4K16. Esa1 contributes mainly to acetylation of H4K5 and H4K12, whereas Sas2 contributes to acetylation of H4K16. Our findings suggest thatC. albicansEsa1 and Sas2 play opposite roles in cell growth and morphogenesis and contribute coordinately to histone acetylation and gene regulation.

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