Cell Wall Chitin Synthesis Research Articles

Inhibitory effects of Lactobacillus brevis on Aspergillus westerdijkiae and antifungal compounds identification

Ochratoxin A (OTA), a secondary metabolite produced by Aspergillus and Penicillium, seriously contaminates grapes and its products. Inhibiting the growth of OTA-producing fungi is the best approach to reduce OTA contamination in grapes. This study was to reveal the inhibition mechanism of Lactobacillus brevis 8–2B on Aspergillus westerdijkiae. Research has found that L. brevis 8–2B could inhibit the germination, growth, and OTA synthesis of A. westerdijkiae. The antifungal activity of L. brevis 8–2B is attributed to the synergistic effect of acetic acid, lactic acid, citric acid, longifolene, methyl salicylate, 3-phenyl-2-propenal, nystatin, and itraconazole. The inhibitory effect of L. brevis 8–2B on A. westerdijkiae was the synergistic effect of these active substances. Ultrastructure observation and flow cytometry results showed that the mycelia structure, cell membrane and cell wall of A. westerdijkiae were disrupted, the cells showed apoptosis, and the ergosterol content in the cells was reduced. Transcriptome analysis revealed that the expression levels of ribosomal biosynthesis genes and cell wall chitin synthesis genes in inhibited A. westerdijkiae were up-regulated, while the expression of chitin degrading enzyme genes was down-regulated to enhance its primary growth and development. In addition, L. brevis 8–2B also inhibits the expression of amino acid synthesis and glycolysis genes in A. westerdijkiae, reducing the generation of secondary metabolite precursors such as NRPS and PKS, thereby inhibiting OTA biosynthesis. On grapes, L. brevis 8–2B could inhibit the infection of A. westerdijkiae, and prolong its shelf life. This study provides new insights for the research and development of novel agent to control OTA contamination.

The Putative Cytochrome b5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans.

Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.

Fluconazole resistant Candida auris clinical isolates have increased levels of cell wall chitin and increased susceptibility to a glucosamine-6-phosphate synthase inhibitor

In 2009 Candida auris was first isolated as fungal pathogen of human disease from ear canal of a patient in Japan. In less than a decade, this pathogen has rapidly spread around the world and has now become a major health challenge that is of particular concern because many strains are resistant to multiple class of antifungal drugs. The lack of available antifungals and rapid increase of this fungal pathogen provides an incentive for the development of new and more potent anticandidal drugs and drug combinatorial treatments. Here we have explored the growth inhibitory activity against C. auris of a synthetic dipeptide glutamine analogue, L-norvalyl-N3-(4-methoxyfumaroyl)-L-2,3- diaminopropanoic acid (Nva-FMDP), that acts as an inhibitor of glucosamine-6-phosphate (GlcN-6-P) synthase - a key enzyme in the synthesis of cell wall chitin. We observed that in contrast to FLC susceptible isolates of C. auris, FLC resistant isolates had elevated cell wall chitin and were susceptible to inhibition by Nva-FMDP. The growth kinetics of C. auris in RPMI-1640 medium revealed that the growth of FLC resistant isolates were 50–60% more inhibited by Nva-FMDP (8 μ g/ml) compared to a FLC susceptible isolate. Fluconazole resistant strains displayed increased transcription of CHS1, CHS2 and CHS3, and the chitin content of the fluconazole resistant strains was reduced following the Nva-FMDP treatment. Therefore, the higher chitin content in FLC resistant C. auris isolates may make the strain more susceptible to inhibition of the antifungal activity of the Nva-FMDP peptide conjugate.

381. Morphologic Changes Associated With Echinocandin Tolerance Enhance Immunoevasion of Candida glabrata

BackgroundActivation of the cell wall integrity pathway and enhanced cell wall chitin synthesis are compensatory mechanisms associated with the incomplete killing of Candida glabrata by echinocandins. Echinocandin-induced morphologic changes in C. glabrata have also been described, yet their correlation with cell wall composition and macrophage responses to echinocandin treated C. glabrata are not well characterized. Elucidating these relationships is needed to understand how C. glabrata is capable of resisting both echinocandin killing and host immune responses.MethodsThree echinocandin-susceptible bloodstream isolates of C. glabrata were grown in liquid RPMI with or without inhibitory concentrations of micafungin (MFG; 0.004 µg/mL) or caspofungin (CAS; 0.008 µg/mL). Cells were stained with fluorescent markers specific for cell wall chitin, mannan, and viability, then imaged utilizing high-content single-cell techniques. Phenotypic characteristics of C. glabrata cells that survive echinocandin exposure were determined by comparing the morphology and abundance cell wall components among the viable and nonviable cell subpopulations. To identify cellular characteristics associated with reduced macrophage phagocytosis, CAS or MFG treated cells were co-incubated RAW 264.7 macrophage and imaged as above. Phenotypic characteristics of the nonphagocytized yeast cells before and after co-incubation with macrophage was compared.ResultsCompared with untreated controls, growth in MFG and CAS significantly increased the proportion of cells with multiple-buds (50% ± 10% and 40% ± 18% vs. 12% ± 6%; P < 0.001) and induced cellular enlargement (biovolume; 35 ± 9 µm3 and 80 ± 58 µm3 vs. 26 ± 5 µm3; P < 0.001). Cell enlargement, reduced cell wall mannan, and increased chitin were highly correlated with survival to MFG and CAS exposure (P < 0.001). Comparison of the drug-exposed yeast cell population before and after co-incubation with macrophage found an increased proportion of viable cells and cells with a large diameter (≥7 µM) remained un-phagocytized, indicating strong phagocytic preference for small, nonviable yeast cells.Conclusion C. glabrata cells that survive echinocandins have distinct cell wall changes and are large in size. These cells tend to evade phagocytosis by macrophages, suggesting a potential mechanism by which C. glabrata may persist despite echinocandin treatment.Disclosures N. D. Beyda, Astellas: Grant Investigator and Scientific Advisor, Consulting fee and Research grant.

Functional Differentiation of Chitin Synthases inYarrowia lipolytica

In this study, we identified seven chitin synthase-encoding genes in the genome of the dimorphic yeast Yarrowia lipolytica. Three encoded chitin synthases with myosin motor-like domains at their N-termini, and we designated these CSM1 to CSM3, whereas four were identified as CHS1 to CHS4. To investigate the functions of these seven genes, we constructed and characterized their deletion mutants. The chs2Δ mutant formed chained cells in which daughter cells were connected with mother cells and had abnormally thick septa at the bud neck. The chs4Δ mutant showed remarkably reduced chitin content in its cell wall. The chs2Δ, csm1Δ, and csm2Δ mutants were found to be highly sensitive to chitin binding dyes, calcofluor white (CFW) and Congo red, whereas the chs4Δ mutant was resistant to CFW. These results suggest that Chs2 and Chs4 play major roles in septum formation and cell wall chitin synthesis respectively, whereas Csm1 and Csm2 are involved in the maintenance of cell wall architecture and/or cell wall integrity. The populations of filamentous cells, a type of cell population that are defined by the lengths of the cellular long and short axes, decreased in the chs3Δ mutant, suggesting that Chs3 is involved in cellular morphogenesis.

Chitin Synthases with a Myosin Motor-Like Domain Control the Resistance of Aspergillus fumigatus to Echinocandins

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.

The Candida albicans Sur7 Protein Is Needed for Proper Synthesis of the Fibrillar Component of the Cell Wall That Confers Strength

The Candida albicans plasma membrane plays important roles in interfacing with the environment, morphogenesis, and cell wall synthesis. The role of the Sur7 protein in cell wall structure and function was analyzed, since previous studies showed that this plasma membrane protein is needed to prevent abnormal intracellular growth of the cell wall. Sur7 localizes to stable patches in the plasma membrane, known as MCC (membrane compartment occupied by Can1), that are associated with eisosome proteins. The sur7Δ mutant cells displayed increased sensitivity to factors that exacerbate cell wall defects, such as detergent (SDS) and the chitin-binding agents calcofluor white and Congo red. The sur7Δ cells were also slightly more sensitive to inhibitors that block the synthesis of cell wall chitin (nikkomycin Z) and β-1,3-glucan (caspofungin). In contrast, Fmp45, a paralog of Sur7 that also localizes to punctate plasma membrane patches, did not have a detectable role in cell wall synthesis. Chemical analysis of cell wall composition demonstrated that sur7Δ cells contain decreased levels of β-glucan, a glucose polymer that confers rigidity on the cell wall. Consistent with this, sur7Δ cells were more sensitive to lysis, which could be partially rescued by increasing the osmolarity of the medium. Interestingly, Sur7 is present in static patches, whereas β-1,3-glucan synthase is mobile in the plasma membrane and is often associated with actin patches. Thus, Sur7 may influence β-glucan synthesis indirectly, perhaps by altering the functions of the cell signaling components that localize to the MCC and eisosome domains.

Gene disruption and characterization of a class V chitin synthase in Botrytis cinerea

Cell-wall chitin biosynthesis in the phytopathogenic fungus Botrytis cinerea involves 7 classes of chitin synthases. In this study, we disrupted a gene encoding a chitin synthase with a myosin-like motor domain (BcchsV) through Agrobacterium tumefaciens mediated transformation. The resulting mutant was not significantly affected in either growth characteristics or pathogenicity on tomato leaves. Surprisingly, the BcchsV mutant exhibited a 31% (m/m) increase in its chitin content compared with the wild-type strain. In addition, the BcchsV mutant showed increased sensitivity to Calcofluor White and slightly enhanced tolerance to cell-wall disturbing substances and osmosis regulators, including SDS, sorbitol, and NaCl. These results suggest that Bcchs does not play an essential role in the synthesis of cell-wall chitin in B. cinerea. However, disruption of this gene provoked a compensatory mechanism regulating the cellular response to cell-wall damage.

Effect of plagiochin E, an antifungal macrocyclic bis(bibenzyl), on cell wall chitin synthesis in Candida albicans

To investigate the effect of plagiochin E (PLE), an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L, on cell wall chitin synthesis in Candida albicans. The effect of PLE on chitin synthesis in Candida albicans was investigated at the cellular and molecular levels. First, the ultrastructural changes were observed under transmission electron microscopy (TEM). Second, the effects of PLE on chitin synthetase (Chs) activities in vitro were assayed using 6-O-dansyl-N-acetylglucosamine as a fluorescent substrate, and its effect on chitin synthesis in situ was assayed by spheroplast regeneration. Finally, real-time RT-PCR was performed to assay its effect on the expression of Chs genes (CHS). Observation under TEM showed that the structure of the cell wall in Candida albicans was seriously damaged, which suggested that the antifungal activity of PLE was associated with its effect on the cell wall. Enzymatic assays and spheroplast regeneration showed that PLE inhibited chitin synthesis in vitro and in situ. The results of the PCR showed that PLE significantly downregulated the expression of CHS1, and upregulated the expression of CHS2 and CHS3. Because different Chs is regulated at different stages of transcription and post-translation, the downregulation of CHS1 would decrease the level of Chs1 and inhibit its activity, and the inhibitory effects of PLE on Chs2 and Chs3 would be at the post-translational level or by the inhibition on the enzyme-active center. These results indicate that the antifungal activity of PLE would be attributed to its inhibitory effect on cell wall chitin synthesis in Candida albicans.

2,3-Butanedione monoxime increases sensitivity to Nikkomycin Z in the budding yeast Saccharomyces cerevisiae.

Nikkomycin Z (NZ) is a competitive inhibitor of chitin synthase III in the yeast Saccharomyces cerevisiae. Myosin type II-deficient yeast strains (myo1) display a dramatic reduction in growth when chitin synthase III activity is inhibited by NZ, supporting the contention that actomyosin motility plays an important role in maintaining cell wall integrity. A proposed inhibitor of cortical actin polymerization in vitro, 2,3-butanedione monoxime (BDM), also inhibits growth of wild-type yeast strains at a concentration of 20 mM. In this study, we assayed for potential in vivo interplay between BDM-sensitive cell functions and cell wall chitin synthesis by testing for increased sensitivity to NZ during co-treatment with BDM at sub-inhibitory concentrations. Our results show that BDM can increase the sensitivity of yeast cells to Nikkomycin Z.

Characterization of Yeast Yea4p, a Uridine Diphosphate-N-acetylglucosamine Transporter Localized in the Endoplasmic Reticulum and Required for Chitin Synthesis

Chitin is an essential cell wall component, synthesis of which is regulated throughout the cell cycle in the yeast Saccharomyces cerevisiae. We cloned an S. cerevisiae gene, YEA4, whose product is homologous to the Kluyveromyces lactis uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) transporter. An epitope-tagged Yea4p localized mainly in the 10,000 x g pellet (P2), suggesting endoplasmic reticulum (ER) localization. Membrane vesicles from the P2 fraction showed an 8-fold higher UDP-GlcNAc transport activity in cells harboring a multicopy YEA4 plasmid than in cells harboring vector alone. The activity distribution is identical with the protein distribution in P2, whether the gene is overexpressed or not, suggesting its native localization in P2. Immunolocalization of epitope-tagged Yea4p further revealed ER localization. The increase in transport activity due to the YEA4 overexpression is specific for UDP-GlcNAc, but not for UDP-galactose and GDP-mannose. Deltayea4-disrupted cells showed a reduced rate of UDP-GlcNAc transport, contained less chitin, and were larger and rounder in shape than the wild type cells. Our results indicate that YEA4 encodes an ER-localized UDP-GlcNAc transporter that is required for cell wall chitin synthesis in S. cerevisiae.

Studies on the Mode of Action of Polyoxins

Polyoxins A-M, which belong to the class of peptidyl-pyrimidine nucleoside antibiotics, are produced by Streptomyces cacaoi var. asoensis. Polyoxins, except C and I, are active against various phytopathogenic fungi. Polyoxins A, B and D are shown to inhibit selectively the incorporation of 14C-glucosamine into cell wall chitin by washed mycelia of Alternaria kikuchiana, Cochliobolus miyabeanus and Pyricularia oryzae at levels which are comparable to their minimal inhibitory concentrations for the fungal frowth. At the same time, each active polyoxin causes an unusually increased accumulation of UDP-14C-N-acetylglucosamine (UDP-GlcNAc). Polyoxins A-M, except C and I, markedly inhibit chitin synthetase preparation from P. oryzae in competition with the substrate, UDP-GlcNAc. Michaelis constant, Km, for UDP-GlcNAc is 3.3×10-3M and inhibitor constant, Ki, for the active polyoxins is found to be in the range of 3.3×10-5-3.4×10-6M. At higher concentrations such as 3mM, polyoxin C also competitively inhibits the enzyme, and the Ki is close to the Km. It is concluded that polyoxins selectively inhibit the synthesis of cell wall chitin and that the primary effect is the competitive inhibition of chitin synthetase. To correlate the polyoxin structure to the enzyme inhibition, the values of binding affinity, -ΔGbind*, for polyoxins and various N-aminoacyl derivatives of polyoxin C are calculated from the Ki values, and the values of partial binding affinity, -Δg*, for structural moieties which constitute the polyoxin J molecule are estimated from the -ΔGbind values. The results indicate that the carbamoylpolyoxamic acid moiety of polyoxins serves to stabilize the complexing of polyoxin with the enzyme. The same kinetic study using nucleosides, nucleotides and nucleotide sugars suggests the presence of a specific binding site on the enzyme for the uridine moiety of UDP-GlcNAc, where the pyrimidine nucleoside moiety of polyoxins also binds. From the pKi-pH plots for the derivatives of polyoxin C and other polyoxins, it is found that the ionized amino group at C-2″ position plays a very important role for the binding of polyoxins to chitin synthetase.* -ΔG=-RTlnKi-ΔGbind=-ΔG+2.4-ΔGbind=-(Δg′+Δg″+Δg′′′+…+Δgn) (kcal/mol, 25°C)

Mechanism of polyoxin resistance

In polyoxin-resistant strains of Alternaria kikuchiana, the synthesis of cell-wall chitin was scarcely inhibited by polyoxin B, although chitin synthetase preparations of them showed a level of polyoxin sensitivity similar to those of sensitive strains. A decrease of14C-labelled polyoxin uptake was correlated with an increased level of polyoxin resistance in various strains. The amount of14C-polyoxin A or3H-polyoxin A accumulated in a highly resistant strain was less than 10% of that in a sensitive strain. These results indicate that resistant strains have a reduced capacity to take up polyoxin A or B.

Feedback Inhibition of l -Glutamine d -Fructose 6-Phosphate Amidotransferase by Uridine Diphosphate N -Acetylglucosamine in Neurospora crassa

The enzyme, l-glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) of Neurospora crassa, which catalyzes the formation of glucosamine 6-phosphate was shown to be subject to feedback inhibition by uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc). The conclusion is based on the following observations. UDP-GlcNAc, the direct precursor of chitin, did not accumulate in the cell even when its utilization for the synthesis of cell wall chitin was interrupted by the antibiotic polyoxin D, a competitive inhibitor of the chitin synthetase (EC 2.4.1.16). Furthermore, the cellular level of UDP-GlcNAc rose in a short period of time when the amidotransferase was bypassed in vivo by the addition of glucosamine to the growing medium of the fungus. The amidotransferase was purified from N. crassa approximately 85-fold. Kinetic studies showed that UDP-GlcNAc was a potent and specific inhibitor of the amidotransferase, and that it did not alter the Michaelis constant for either l-glutamine or d-fructose 6-phosphate, suggesting that the inhibitor binds at a site on the enzyme distinct from the active site.