scholarly journals Candida albicans cell wall mannosidases, Dfg5 and Dcw1, regulate chitin synthesis

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Abhiram Maddi ◽  
Jaewon Kim ◽  
Harleen Sohi ◽  
Sujay Busarajan ◽  
Olga Glagovyak ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescence Microscopy ◽  
Signaling Pathways ◽  
Cell Separation ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Chitin Synthases ◽  
Conditional Mutant ◽  
Calcineurin Signaling

In Candida albicans chitin synthesis is important for cell wall integrity and may also have a role in emergence of drug-resistance. Our past studies showed that cell wall mannosidases, Dfg5 and Dcw1, regulate HOG MAPK signaling. In this study, we investigated how Dfg5 and Dcw1 regulate chitin synthesis by affecting HOG, PKC and Calcium-Calcineurin signaling pathways. DFG5 and DCW1 heterologous mutants (ES1 & ES195) and a conditional mutant (ES195+methionine/cysteine) were utilized. WT SC5314 served as negative control and Hog1 knock-out mutant as positive control. Fluorescence microscopy of calcofluor white (CFW) stained mutant and control strains was performed to observe chitin accumulation. Quantitative PCR analysis was performed to measure the relative expression of chitin synthases CHS1, CHS2, CHS3 and CHS8. Incubation with chitinase was done to determine cell separation using light microscopy and scanning electron microscopy (SEM) analysis. Fluorescence microscopy showed significantly increased chitin accumulation in the mutants as compared to wild type. Chitin accumulation was observed mainly at the budding sites indicating a cause for defective cell separation phenotype. Incubation with chitinase led to cell separation in the mutants. CHS2, CHS3 and CHS8 expression was observed to be significantly upregulated in the conditional mutant and HOG1 mutant as compared to the wild type. This upregulation was also observed when the cell wall integrity PKC pathway was activated. However, activation of the Calcium-calcineurin pathway downregulated chitin synthase expression in the mutants. Our data indicates that Dfg5 and Dcw1 regulate expression of chitin synthases via HOG MAPK, PKC and Calcium-calcineurin signaling pathways.

Potent Chitin Synthase Inhibitors from Plants

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.

scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

scholarly journals Candida albicans phosphate transport, facilitating nucleotide sugar biosynthesis, contributes to cell wall stability.

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Maikel Acosta-Zaldivar ◽  
Wanjun Qi ◽  
Ning-Ning Liu ◽  
Joann Diray-Arce ◽  
Louise A. Walker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Enzymatic Reaction ◽  
Phosphate Starvation ◽  
Reaction Rates ◽  
Outer Cell ◽  
Cell Wall Polysaccharides ◽  
Nucleotide Sugar ◽  
Structural Cell ◽  
Chitin Synthases

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

scholarly journals Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

2010 ◽  
Vol 9 (9) ◽  
pp. 1329-1342 ◽  
Author(s):  
Claire A. Walker ◽  
Beatriz L. Gómez ◽  
Héctor M. Mora-Montes ◽  
Kevin S. Mackenzie ◽  
Carol A. Munro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Cell Walls ◽  
Chitin Synthesis ◽  
Melanin Production ◽  
Content Type ◽  
Encoding Gene ◽  
Chitinase Genes

ABSTRACT The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.

scholarly journals A Chitin Synthase with a Myosin-Like Motor Domain Is Essential for Hyphal Growth, Appressorium Differentiation, and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola

2007 ◽  
Vol 20 (12) ◽  
pp. 1555-1567 ◽  
Author(s):  
Stefan Werner ◽  
Janyce A. Sugui ◽  
Gero Steinberg ◽  
Holger B. Deising
Keyword(s):  
Cell Wall ◽  
Filamentous Fungi ◽  
Hyphal Growth ◽  
Motor Domain ◽  
Plant Colonization ◽  
Colletotrichum Graminicola ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Cell Wall Biogenesis ◽  
Host Cell Wall

Chitin synthesis contributes to cell wall biogenesis and is essential for invasion of solid substrata and pathogenicity of filamentous fungi. In contrast to yeasts, filamentous fungi contain up to 10 chitin synthases (CHS), which might reflect overlapping functions and indicate their complex lifestyle. Previous studies have shown that a class VI CHS of the maize anthracnose fungus Colletotrichum graminicola is essential for cell wall synthesis of conidia and vegetative hyphae. Here, we report on cloning and characterization of three additional CHS genes, CgChsI, CgChsIII, and CgChsV, encoding class I, III, and V CHS, respectively. All CHS genes are expressed during vegetative and pathogenic development. ΔCgChsI and ΔCgChsIII mutants did not differ significantly from the wild-type isolate with respect to hyphal growth and pathogenicity. In contrast, null mutants in the CgChsV gene, which encodes a CHS with an N-terminal myosin-like motor domain, are strongly impaired in vegetative growth and pathogenicity. Even in osmotically stabilized media, vegetative hyphae of ΔCgChsV mutants exhibited large balloon-like swellings, appressorial walls appeared to disintegrate during maturation, and infection cells were nonfunctional. Surprisingly, ΔCgChsV mutants were able to form dome-shaped hyphopodia that exerted force and showed host cell wall penetration rates comparable with the wild type. However, infection hyphae that formed within the plant cells exhibited severe swellings and were not able to proceed with plant colonization efficiently. Consequently, ΔCgChsV mutants did not develop macroscopically visible anthracnose disease symptoms and, thus, were nonpathogenic.

scholarly journals Deletions of the Endocytic Components VPS28 and VPS32 in Candida albicans Lead to Echinocandin and Azole Hypersensitivity

2006 ◽  
Vol 50 (10) ◽  
pp. 3492-3495 ◽  
Author(s):  
Muriel Cornet ◽  
Claude Gaillardin ◽  
Mathias L. Richard
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Signaling Pathways ◽  
Antifungal Agents ◽  
Cell Wall Disruption

ABSTRACT Vps28p and Vps32p act in both the endocytic and the pH signaling pathways in yeasts and are required for Candida albicans virulence. Here, we show that deletions of VPS28 and VPS32 increase the susceptibility of C. albicans to cell wall disruption agents, echinocandin and azole antifungal agents.

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

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 314 ◽  
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.

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae

1983 ◽  
Vol 3 (5) ◽  
pp. 922-930
Author(s):  
R L Roberts ◽  
B Bowers ◽  
M L Slater ◽  
E Cabib
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Wall ◽  
Permissive Temperature ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Specific Staining ◽  
Septal Region ◽  
Wall Components ◽  
Generalized Distribution

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

scholarly journals The effect of farnesol on amino acid incorporation by a wild-type and cell-wall variant strain ofCandida albicans

2005 ◽  
Vol 51 (8) ◽  
pp. 715-718 ◽  
Author(s):  
Phyllis C Braun
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Amino Acid ◽  
Quorum Sensing ◽  
Cell Surface ◽  
Wild Type ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Ofcandida Albicans ◽  
Density Growth

Farnesol, a quorum sensing (QS) signal, is produced by Candida albicans during high density growth and has been found to inhibit morphogenesis. This QS auto-inducing signal was discovered to increase amino acid incorporation by C. albicans when concentrations of farnesol increased to 10 µg/mL in yeast nitrogen broth. Farnesol concentrations greater than 10 µg/mL abolished the enhanced incorporation, and the magnitude of the increased incorporation was dependent on cell-surface hydrophobicity.Key words: Candida albicans, farnesol, amino acid incorporation.

scholarly journals The Candida albicans Exocyst Subunit Sec6 Contributes to Cell Wall Integrity and Is a Determinant of Hyphal Branching

2015 ◽  
Vol 14 (7) ◽  
pp. 684-697 ◽  
Author(s):  
Alba A. Chavez-Dozal ◽  
Stella M. Bernardo ◽  
Hallie S. Rane ◽  
Gloria Herrera ◽  
Vibhati Kulkarny ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Mutant Strain ◽  
Secretory Protein ◽  
Hyphal Branching ◽  
Content Type ◽  
Polarized Secretion ◽  
Late Endosomes ◽  
Conditional Mutant ◽  
Macrophage Killing

ABSTRACTThe yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To studySEC6function inCandida albicans, we generated a conditional mutant strain in whichSEC6was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack ofSEC6expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting thatC. albicansSec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role ofSEC6inC. albicansvirulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis ofC. albicans.

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