Crosstalk between calcineurin and the cell wall integrity pathways prevents chitin overexpression in Candida albicans

Echinocandins such as caspofungin are front line antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by up-regulation of chitin synthesis, thereby sustaining cell wall integrity. When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a sub-population of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable super high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway.

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Fluconazole and Lipopeptide Surfactin Interplay During Candida albicans Plasma Membrane and Cell Wall Remodeling Increases Fungal Immune System Exposure

Pharmaceutics ◽

10.3390/pharmaceutics12040314 ◽

2020 ◽

Vol 12 (4) ◽

pp. 314 ◽

Cited By ~ 3

Author(s):

Jakub Suchodolski ◽

Daria Derkacz ◽

Jakub Muraszko ◽

Jarosław J. Panek ◽

Aneta Jezierska ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Plasma Membrane ◽

Immune System ◽

Antifungal Drugs ◽

Stable Complex ◽

Host Immune System ◽

Chitin Synthesis ◽

Fungal Cell ◽

In Silico Studies

Recognizing the β-glucan component of the Candida albicans cell wall is a necessary step involved in host immune system recognition. Compounds that result in exposed β-glucan recognizable to the immune system could be valuable antifungal drugs. Antifungal development is especially important because fungi are becoming increasingly drug resistant. This study demonstrates that lipopeptide, surfactin, unmasks β-glucan when the C. albicans cells lack ergosterol. This observation also holds when ergosterol is depleted by fluconazole. Surfactin does not enhance the effects of local chitin accumulation in the presence of fluconazole. Expression of the CHS3 gene, encoding a gene product resulting in 80% of cellular chitin, is downregulated. C. albicans exposure to fluconazole changes the composition and structure of the fungal plasma membrane. At the same time, the fungal cell wall is altered and remodeled in a way that makes the fungi susceptible to surfactin. In silico studies show that surfactin can form a complex with β-glucan. Surfactin forms a less stable complex with chitin, which in combination with lowering chitin synthesis, could be a second anti-fungal mechanism of action of this lipopeptide.

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Identification of New Antifungal Agents Targeting Chitin Synthesis by a Chemical-Genetic Method

Molecules ◽

10.3390/molecules24173155 ◽

2019 ◽

Vol 24 (17) ◽

pp. 3155

Author(s):

Li ◽

Sun ◽

Zhu ◽

Bian ◽

Wang ◽

...

Keyword(s):

Cell Wall ◽

Antifungal Agents ◽

Antifungal Drugs ◽

Chitin Synthase ◽

Yeast Cells ◽

Mass Spectrometry Analysis ◽

Chitin Synthesis ◽

Glucan Synthase ◽

Genetic Method ◽

Chemical Genetic

Fungal infection is a leading cause of mortality in immunocompromised population; thus, it is urgent to develop new and safe antifungal agents. Different from human cells, fungi have a cell wall, which is composed mainly of polysaccharide glucan and chitin. The unique cell wall structure is an ideal target for antifungal drugs. In this research, a chemical-genetic method was used to isolate antifungal agents that target chitin synthesis in yeast cells. From a compound library, we isolated two benzothiazole compounds that showed greater toxicity to yeast mutants lacking glucan synthase Fks1 compared to wild-type yeast cells and mutants lacking chitin synthase Chs3. Both of them inhibited the activity of chitin synthase in vitro and reduced chitin level in yeast cells. Besides, these compounds showed clear synergistic antifungal effect with a glucan synthase inhibitors caspofungin. Furthermore, these compounds inhibited the growth of Saccharomyces cerevisiae and opportunistic pathogen Candida albicans. Surprisingly, the genome-wide mass-spectrometry analysis showed decreased protein level of chitin synthases in cells treated with one of these drugs, and this decrease was not a result of downregulation of gene transcription. Therefore, we successfully identified two new antifungal agents that inhibit chitin synthesis using a chemical-genetic method.

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Hyperactive TORC1 sensitizes yeast cells to endoplasmic reticulum stress by compromising cell wall integrity

10.1101/605824 ◽

2019 ◽

Author(s):

Khadija Ahmed ◽

David E. Carter ◽

Patrick Lajoie

Keyword(s):

Endoplasmic Reticulum ◽

Cell Wall ◽

Endoplasmic Reticulum Stress ◽

Er Stress ◽

Fungal Infections ◽

Pathogenic Fungi ◽

Stress Sensitivity ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Yeast Cells

ABSTRACTThe disruption of protein folding homeostasis in the endoplasmic reticulum (ER) results in an accumulation of toxic misfolded proteins and activates a network of signaling events collectively known as the unfolded protein response (UPR). While UPR activation upon ER stress is well characterized, how other signaling pathways integrate into the ER proteostasis network is unclear. Here, we sought to investigate how the target of rapamycin complex 1 (TORC1) signaling cascade acts in parallel with the UPR to regulate ER stress sensitivity. Using S. cerevisiae, we found that TORC1 signaling is attenuated during ER stress and constitutive activation of TORC1 increases sensitivity to ER stressors such as tunicamycin and inositol deprivation. This phenotype is independent of the UPR. Transcriptome analysis revealed that TORC1 hyperactivation results in cell wall remodelling. Conversely, hyperactive TORC1 sensitizes cells to cell wall stressors, including the antifungal caspofungin. Elucidating the crosstalk between the UPR, cell wall integrity, and TORC1 signaling may uncover new paradigms through which the response to protein misfolding is regulated, and thus have crucial implications for the development of novel therapeutics against pathogenic fungal infections.IMPORTANCEThe prevalence of pathogenic fungal infections, coupled with the emergence of new fungal pathogens, has brought these diseases to the forefront of global health problems. While antifungal treatments have advanced over the last decade, patient outcomes have not substantially improved. These shortcomings are largely attributed to the evolutionary similarity between fungi and humans, which limits the scope of drug development. As such, there is a pressing need to understand the unique cellular mechanisms that govern fungal viability. Given that Saccharomyces cerevisiae is evolutionarily related to a number of pathogenic fungi, and in particular to the Candida species, most genes from S. cerevisiae are highly conserved in pathogenic fungal strains. Here we show that hyperactivation of TORC1 signaling sensitizes S. cerevisiae cells to both endoplasmic reticulum stress and cell wall stressors by compromising cell wall integrity. Therefore, targeting TORC1 signaling and endoplasmic reticulum stress pathways may be useful in developing novel targets for antifungal drugs.

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Endoplasmic Reticulum α-Glycosidases of Candida albicans Are Required for N Glycosylation, Cell Wall Integrity, and Normal Host-Fungus Interaction

Eukaryotic Cell ◽

10.1128/ec.00350-07 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2184-2193 ◽

Cited By ~ 83

Author(s):

Héctor M. Mora-Montes ◽

Steven Bates ◽

Mihai G. Netea ◽

Diana F. Díaz-Jiménez ◽

Everardo López-Romero ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Wall ◽

Candida Albicans ◽

Systemic Infection ◽

Cell Wall Integrity ◽

Yeast Cells ◽

Chain Elongation ◽

Cell Wall Integrity Pathway ◽

Oligosaccharide Processing ◽

Host Fungus

ABSTRACT The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc3Man9GlcNAc2 N-glycan is processed by α-glucosidases I and II and α1,2-mannosidase to generate Man8GlcNAc2. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for α-glucosidase I, α-glucosidase II catalytic subunit, and α1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated β-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.

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Interface of Candida albicans Biofilm Matrix-Associated Drug Resistance and Cell Wall Integrity Regulation

Eukaryotic Cell ◽

10.1128/ec.05126-11 ◽

2011 ◽

Vol 10 (12) ◽

pp. 1660-1669 ◽

Cited By ~ 94

Author(s):

Jeniel E. Nett ◽

Hiram Sanchez ◽

Michael T. Cain ◽

Kelly M. Ross ◽

David R. Andes

Keyword(s):

Drug Resistance ◽

Cell Wall ◽

Candida Albicans ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Candidate Gene Approach ◽

Content Type ◽

Resistant Phenotype ◽

Cell Wall Integrity Pathway ◽

Biofilm Matrix

ABSTRACTCandida albicansfrequently infects medical devices by growing as a biofilm, i.e., a community of adherent organisms entrenched in an extracellular matrix. During biofilm growth,Candidaspp. acquire the ability to resist high concentrations of antifungal drugs. One recently recognized biofilm resistance mechanism involves drug sequestration by matrix β-1,3 glucan. Using a candidate gene approach, we investigated potentialC. albicansβ-1,3-glucan regulators, based on their homology toSaccharomyces cerevisiae, includingSMI1and protein kinase C (PKC) pathway components. We identified a role for theSMI1in biofilm matrix glucan production and development of the associated drug resistance phenotype. This pathway appears to act through transcription factor Rlmp and glucan synthase Fks1p. The phenotypes of these mutant biofilms mimicked those of thesmi1Δ/smi1Δ biofilm, and overexpression ofFKS1in thesmi1Δ/smi1Δ mutant restored the biofilm resistant phenotype. However, control of this pathway is distinct from that of the upstream PKC pathway because thepkc1Δ/pkc1Δ,bck1Δ/bck1Δ,mkk2Δ/mkk2Δ, andmkc1Δ/mkc1Δ biofilms retained the resistant phenotype of the parent strain. In addition, resistance to cell-perturbing agents and gene expression data do not support a significant role for the cell wall integrity pathway during the biofilm formation. Here we show that Smi1p functions in conjunction with Rlm1p and Fks1p to produce drug-sequestering biofilm β-glucan. Our work provides new insight into how theC. albicansbiofilm matrix production and drug resistance pathways intersect with the planktonic cell wall integrity pathway. This novel connection helps explain how pathogens in a multicellular biofilm community are protected from anti-infective therapy.

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High Pressure Freezing/Freeze Substituion: Comparison of Chemical Fixation Versus Cryoimmobilization of Candida Albicans Cultured in Cellulose Tubing

Microscopy and Microanalysis ◽

10.1017/s143192760001549x ◽

1999 ◽

Vol 5 (S2) ◽

pp. 434-435

Author(s):

S. Erlandsen ◽

A Holzer ◽

M. Gavin ◽

C. Frethem ◽

C. Wells

Keyword(s):

High Pressure ◽

Cell Wall ◽

Candida Albicans ◽

Freeze Substitution ◽

Host Cells ◽

Yeast Cells ◽

Outer Cell ◽

High Pressure Freezing ◽

Outer Cell Wall

In the interactions of Candida albicans with host cells, the cell wall of the yeast may play important roles in the adhesion of yeast cells to tissues. The outer cell wall of yeast (e.g. Saccharomyces cerevisiae, C. albicans) has been shown to consist of a dense network of radially projecting fibrils composed of mannoproteins that are known as fimbriae and which previously have required cryopreservation either by jet propane freezing or by plunge freezeing for their visualization. High pressure freezing provides an advantage over jet or plunge freezing in terms of the higher consistancey in the quality of freezing, and the minimization of formation of ice I with this method. Hohenberg et al reported a method utilizing cellulose capillary tubes to cryoimmobilize suspensions of microoganisms by high pressure freezing (HPF) and freeze substitution (FS), and herein, we describe an adaptation of this method by culturing microorganisms within the tubing to increase cell density prior to high pressure freezing and freeze substution.

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Control of β-glucan exposure by the endo-1,3-glucanase Eng1 in Candida albicans modulates virulence

PLoS Pathogens ◽

10.1371/journal.ppat.1010192 ◽

2022 ◽

Vol 18 (1) ◽

pp. e1010192

Author(s):

Mengli Yang ◽

Norma V. Solis ◽

Michaela Marshall ◽

Rachel Garleb ◽

Tingting Zhou ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Opportunistic Pathogen ◽

Antifungal Drugs ◽

Yeast Cells ◽

Immune Recognition ◽

Dependent Manner ◽

Wild Type ◽

Fungal Cell ◽

Disseminated Candidiasis

Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes β-glucan in the fungal cell wall. C. albicans β-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how β-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates β-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated β-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated β-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.

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Elevated Chitin Content Reduces the Susceptibility of Candida Species to Caspofungin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01486-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 146-154 ◽

Cited By ~ 98

Author(s):

Louise A. Walker ◽

Neil A. R. Gow ◽

Carol A. Munro

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Point Mutations ◽

Antifungal Drugs ◽

Cell Wall Polysaccharide ◽

Chitin Synthesis ◽

Fungal Cell ◽

Content Type ◽

Chitin Content ◽

Compensatory Increase

ABSTRACTThe echinocandin antifungal drugs inhibit synthesis of the major fungal cell wall polysaccharide β(1,3)-glucan. Echinocandins have good efficacy againstCandida albicansbut reduced activity against otherCandidaspecies, in particularCandida parapsilosisandCandida guilliermondii. Treatment ofCandida albicanswith a sub-MIC level of caspofungin has been reported to cause a compensatory increase in chitin content and to select for sporadic echinocandin-resistantFKS1point mutants that also have elevated cell wall chitin. Here we show that elevated chitin in response to caspofungin is a common response in variousCandidaspecies. Activation of chitin synthesis was observed in isolates ofC. albicans,Candida tropicalis,C. parapsilosis, andC. guilliermondiiand in some isolates ofCandida kruseiin response to caspofungin treatment. However,Candida glabrataisolates demonstrated no exposure-induced change in chitin content. Furthermore, isolates ofC. albicans,C. krusei,C. parapsilosis, andC. guilliermondiiwhich were stimulated to have higher chitin levels via activation of the calcineurin and protein kinase C (PKC) signaling pathways had reduced susceptibility to caspofungin. Isolates containing point mutations in theFKS1gene generally had higher chitin levels and did not demonstrate a further compensatory increase in chitin content in response to caspofungin treatment. These results highlight the potential of increased chitin synthesis as a potential mechanism of tolerance to caspofungin for the major pathogenicCandidaspecies.

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The Regulatory Function of the Molecular Chaperone Hsp90 in the Cell Wall Integrity of Pathogenic Fungi

Current Proteomics ◽

10.2174/1570164615666180820155807 ◽

2018 ◽

Vol 16 (1) ◽

pp. 44-53

Author(s):

Marina Campos Rocha ◽

Camilla Alves Santos ◽

Iran Malavazi

Keyword(s):

Cell Wall ◽

Antifungal Therapy ◽

Molecular Chaperone ◽

Regulatory Protein ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Regulatory Function ◽

Loss Of Function ◽

Hsp90 Inhibition

Different signaling cascades including the Cell Wall Integrity (CWI), the High Osmolarity Glycerol (HOG) and the Ca2+/calcineurin pathways control the cell wall biosynthesis and remodeling in fungi. Pathogenic fungi, such as Aspergillus fumigatus and Candida albicans, greatly rely on these signaling circuits to cope with different sources of stress, including the cell wall stress evoked by antifungal drugs and the host’s response during infection. Hsp90 has been proposed as an important regulatory protein and an attractive target for antifungal therapy since it stabilizes major effector proteins that act in the CWI, HOG and Ca2+/calcineurin pathways. Data from the human pathogen C. albicans have provided solid evidence that loss-of-function of Hsp90 impairs the evolution of resistance to azoles and echinocandin drugs. In A. fumigatus, Hsp90 is also required for cell wall integrity maintenance, reinforcing a coordinated function of the CWI pathway and this essential molecular chaperone. In this review, we focus on the current information about how Hsp90 impacts the aforementioned signaling pathways and consequently the homeostasis and maintenance of the cell wall, highlighting this cellular event as a key mechanism underlying antifungal therapy based on Hsp90 inhibition.

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Potent Chitin Synthase Inhibitors from Plants

Current Bioactive Compounds ◽

10.2174/1573407213666180719145831 ◽

2020 ◽

Vol 16 (1) ◽

pp. 58-63

Author(s):

Amrutha Vijayakumar ◽

Ajith Madhavan ◽

Chinchu Bose ◽

Pandurangan Nanjan ◽

Sindhu S. Kokkal ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Caenorhabditis Elegans ◽

Aspergillus Niger ◽

Small Molecules ◽

Tip Growth ◽

Chitin Synthase ◽

Chitin Synthesis ◽

Hyphal Tip Growth ◽

Hyphal Tip

Background: Chitin is the main component of fungal, protozoan and helminth cell wall. They help to maintain the structural and functional characteristics of these organisms. The chitin wall is dynamic and is repaired, rearranged and synthesized as the cells develop. Active synthesis can be noticed during cytokinesis, laying of primary septum, maintenance of lateral cell wall integrity and hyphal tip growth. Chitin synthesis involves coordinated action of two enzymes namely, chitin synthase (that lays new cell wall) and chitinase (that removes the older ones). Since chitin synthase is conserved in different eukaryotic microorganisms that can be a ‘soft target’ for inhibition with small molecules. When chitin synthase is inhibited, it leads to the loss of viability of cells owing to the self- disruption of the cell wall by existing chitinase. Methods: In the described study, small molecules from plant sources were screened for their ability to interfere with hyphal tip growth, by employing Hyphal Tip Burst assay (HTB). Aspergillus niger was used as the model organism. The specific role of these small molecules in interfering with chitin synthesis was established with an in-vitro method. The enzyme required was isolated from Aspergillus niger and its activity was deduced through a novel method involving non-radioactively labelled substrate. The activity of the potential lead molecules were also checked against Candida albicans and Caenorhabditis elegans. The latter was adopted as a surrogate for the pathogenic helminths as it shares similarity with regard to cell wall structure and biochemistry. Moreover, it is widely studied and the methodologies are well established. Results: Out of the 11 compounds and extracts screened, 8 were found to be prospective. They were also found to be effective against Candida albicans and Caenorhabditis elegans. Conclusion: Purified Methyl Ethyl Ketone (MEK) Fraction1 (F1) of Coconut (Cocos nucifera) Shell Extract (COSE) was found to be more effective against Candida albicans with an IC50 value of 3.04 μg/mL and on L4 stage of Caenorhabditis elegans with an IC50 of 77.8 μg/mL.

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