Benjaminiella Poitrasii Research Articles

Taguchi Design of the Experimental approach to Increase the Biomass and Cell Wall Chitosan Contents of Zygomycetous Dimorphic Fungus Benjaminiella Poitrasii

Taguchi Design of the Experimental approach to Increase the Biomass and Cell Wall Chitosan Contents of Zygomycetous Dimorphic Fungus Benjaminiella Poitrasii

Isolation and Characterization of Chitosans from Different Fungi with Special Emphasis on Zygomycetous Dimorphic Fungus Benjaminiella poitrasii: Evaluation of Its Chitosan Nanoparticles for the Inhibition of Human Pathogenic Fungi

The cell wall chitosan was extracted from fungi belonging to different taxonomic classes, namely, Benjaminiella poitrasii (Zygomycetes, dimorphic), Hanseniaspora guilliermondii, Issatchenkia orientalis, Pichia membranifaciens, and Saccharomyces cerevisiae (Ascomycetes, yeasts), and Agaricus bisporus and Pleurotus sajor-caju (Basidiomycetes). The maximum yield of chitosan was 60.89 ± 2.30 mg/g of dry mycelial biomass of B. poitrasii. The degree of deacetylation (DDA) of chitosan extracted from different fungi, as observed with 1H NMR, was in the range of 70-93%. B. poitrasii chitosan exhibited the highest DDA (92.78%). The characteristic absorption bands were observed at 3450, 1650, 1420, 1320, and 1035 cm-1 by FTIR. Compared to chitosan from marine sources (molecular weight, MW, 585 kDa), fungal chitosans showed lower MW (6.21-46.33 kDa). Further, to improve the efficacy of B. poitrasii chitosan (Bp), nanoparticles (Np) were synthesized using the ionic gelation method and characterized by dynamic light scattering (DLS). For yeast and hyphal chitosan nanoparticles (BpYCNp and BpHCNp), the average particle size was <200 nm with polydispersity index of 0.341 ± 0.03 and 0.388 ± 0.002, respectively, and the zeta potential values were 21.64 ± 0.34 and 24.48 ± 1.58 mV, respectively. The B. poitrasii chitosans and their nanoparticles were further evaluated for antifungal activity against human pathogenic Candida albicans ATCC 10231, Candida glabrata NCYC 388, Candida tropicalis ATCC 750, Cryptococcus neoformans ATCC 34664, and Aspergillus niger ATCC 10578. BpHCNps showed lower MIC90 values (0.025-0.4 mg/mL) than the chitosan polymer against the tested human pathogens. The study suggested that nanoformulation of fungal chitosan, which has low molecular weight and high % DDA, is desirable for antifungal applications against human pathogens. Moreover, chitosans as well as their nanoparticles were found to be hemocompatible and are therefore safe for healthcare applications.

NADP-dependent glutamate dehydrogenases in a dimorphic zygomycete Benjaminiella poitrasii: Purification, characterization and their evaluation as an antifungal drug target

BackgroundIt has been reported that the genes coding for NADP-dependent glutamate dehydrogenases (NADP-GDHs) showed a cause-effect relationship with Yeast-Hypha (YH) reversible transition in a zygomycete Benjaminiella poitrasii. As YH transition is significant in human pathogenic fungi for their survival and proliferation in the host, the NADP-GDHs can be explored as antifungal drug targets. MethodsThe yeast-form specific BpNADPGDH I and hyphal-form specific BpNADPGDH II of B. poitrasii were purified by heterologous expression in E. coli BL-21 cells and characterized. The structural analogs of L-glutamate, dimethyl esters of isophthalic acid (DMIP) and its derivatives were designed, synthesized and screened for inhibition of NADP-GDH activity as well as YH transition in B. poitrasii, and also in human pathogenic Candida albicans strains. ResultsThe BpNADPGDH I and BpNADPGDH II were found to be homo-hexameric proteins with native molecular mass of 282 kDa and 298 kDa, respectively and subunit molecular weights of 47 kDa and 49 kDa, respectively. Besides the distinct kinetic properties, BpNADPGDH I and BpNADPGDH II were found to be regulated by cAMP-dependent- and Calmodulin (CaM) dependent- protein kinases, respectively. The DMIP compounds showed a more pronounced effect on H-form specific BpNADPGDH II and inhibited YH transition as well as growth in B. poitrasii and C. albicans strains. ConclusionThe present study will be useful to design and develop antifungal drugs against dimorphic human pathogens using glutamate dehydrogenase as a target. SignificanceGlutamate dehydrogenases can be explored as a target against human pathogenic fungi.

Molecular studies of NAD- and NADP-glutamate dehydrogenases decipher the conundrum of yeast-hypha dimorphism in zygomycete Benjaminiella poitrasii.

Benjaminiella poitrasii, a zygomycete, shows glucose- and temperature-dependent yeast (Y)-hypha (H) dimorphic transition. Earlier, we reported the biochemical correlation of relative proportion of NAD- and NADP-glutamate dehydrogenases (GDHs) with Y-H transition. Further, we observed the presence of one NAD-GDH and two form-specific NADP-GDH isoenzymes in B. poitrasii. However, molecular studies are necessary to elucidate the explicit role of GDHs in regulating Y-H reversible transition. Here, we report the isolation and characterization of one NAD (BpNADGDH, 2.643 kb) and two separate genes, BpNADPGDH I (Y-form specific, 1.365 kb) and BpNADPGDH II (H-form specific, 1.368 kb) coding for NADP-GDH isoenzymes in B. poitrasii. The transcriptional profiling during Y-H transition showed higher BpNADPGDH I expression in Y cells while expression of BpNADPGDH II was higher in H cells. Moreover, the yeast-form monomorphic mutant (Y-5) did not show BpNADPGDH II expression under normal dimorphism triggering conditions. Transformation with H-form specific BpNADPGDH II induced the germ tube formation in Y-5, which confirmed the cause-effect relationship between BpNADPGDH genes and morphological outcome in B. poitrasii. Interestingly, expression of H-form specific BpNADPGDH II also induced germ tube formation in human pathogenic, non-dimorphic yeast Candida glabrata, which further corroborated our findings.

Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus Benjaminiella poitrasii.

Benjaminiella poitrasii, a dimorphic non-pathogenic zygomycetous fungus, exhibits a morphological yeast (Y) to hypha (H) reversible transition in the vegetative phase, sporangiospores (S) in the asexual phase and zygospores (Z) in the sexual phase. To study the gene expression across these diverse morphological forms, suitable reference genes are required. In the present study, 13 genes viz. ACT, 18S rRNA, eEF1α, eEF-Tu,eIF-1A, Tub-α, Tub-b, Ubc, GAPDH, Try, WS-21, NADGDH and NADPGDH were evaluated for their potential as a reference, particularly for studying gene expression during the Y-H reversible transition and also for other asexual and sexual life stages of B. poitrasii. Analysis of RT-qPCR data using geNorm, normFinder and BestKeeper software revealed that genes such as Ubc, 18S rRNA and WS-21 were expressed at constant levels in each given subset of RNA samples from all the morphological phases of B. poitrasii. Therefore, these reference genes can be used to elucidate the role of morpho-genes in B. poitrasii. Further, use of the two most stably expressed genes (Ubc and WS-21) to normalize the expression of the ornithine decarboxylase gene (Bpodc) in different morphological forms of B. poitrasii, generated more reliable results, indicating that our selection of reference genes was appropriate.

A biochemical correlate of dimorphism in a zygomycete Benjaminiella poitrasii: characterization of purified NAD-dependent glutamate dehydrogenase, a target for antifungal agents

The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from a non-pathogenic dimorphic zygomycete Benjaminiella poitrasii was previously reported to be an important biochemical correlate of the transition process. The enzyme was purified to homogeneity and characterized. It is a 371kDa native molecular weight protein made up of four identical subunits. Kinetic studies showed that unlike other NAD-GDHs, it may act as an anabolic enzyme and has more affinity towards 2-oxoglutarate than L-glutamate. Chemical modifications revealed the involvement of single histidine and lysine residues in the catalytic activity of the enzyme. The phosphorylation and dephosphorylation study showed that the NAD-GDH is present in active phosphorylated form in hyphal cells of B. poitrasii. Two of the 1,2,3 triazole linked β-lactam-bile acid conjugates synthesized in the laboratory (B18, B20) were found to be potent inhibitors of purified NAD-GDH which also significantly affected Y-H transition in B. poitrasii. Furthermore, the compound B20 inhibited germ tube formation during Y-H transition in Candida albicans strains and Yarrowia lipolytica. The possible use of NAD-GDH as a target for antifungal agents is discussed.

Flocculation of dimorphic yeast Benjaminiella poitrasii is altered by modulation of NAD-glutamate dehydrogenase

A strategy to control flocculation is investigated using dimorphic yeast, Benjaminiella poitrasii as a model. Parent form of this yeast (Y) exhibited faster flocculation (11.1 min) than the monomorphic yeast form mutant Y-5 (12.6 min). Atomic force microscopy revealed higher surface roughness of Y (439.34 rms) than Y-5 (52 rms). Also, the former had a zeta potential of −65.97 ± 3.45 as against −50.21 ± 2.49 for the latter. Flocculation of both Y and Y-5 could be altered by supplementing either substrates or inhibitor of NAD-glutamate dehydrogenase (NAD-GDH) in the growth media. The rate of flocculation was promoted by α-ketoglutarate or isophthalic acid and decelerated by glutamate with a statistically significant inverse correlation to corresponding NAD-GDH levels. These interesting findings open up new possibilities of using NAD-GDH modulating agents to control flocculation in fermentations for easier downstream processing.

Efficient synthesis of antifungal pyrimidines via palladium catalyzed Suzuki/Sonogashira cross-coupling reaction from Biginelli 3,4-dihydropyrimidin-2(1 H)-ones

An efficient regioselective approach to the synthesis of tetrasubstituted pyrimidines was developed by sequential functionalization of easily available Biginelli 3,4-dihydropyrimidine-2(1 H)-ones via dehydrogenation, chlorination followed by palladium catalyzed C–C Suzuki/Sonogashira coupling reaction. All the synthesized compounds were evaluated in vitro for their antifungal activities against Candida albicans, Cryptococcus neoformans, Benjaminiella poitrasii, Yarrowia lipolytica, and Fusarium oxysporum, and antibacterial activities against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus.

Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure.

The effects of changes in external osmotic pressure on chitin synthase activity of a dimorphic fungus, Benjaminiella poitrasii, have been investigated. Mycelial and yeast cells incubated in medium of low osmolality (distilled water, 0 mOsm) for 10 min had 2-3-fold higher specific activities of native chitin synthase in mixed membrane preparations than cells that had been subjected to a high osmolality medium (1.2 M sorbitol in distilled water, 1612 mOsm). Cells suspended in media of different osmolalities for 10 min were also affected in the extent of germ tube formation. Germ tube formation was highest in cells incubated in low osmolality medium. The addition of protein phosphatase inhibitors (cyclosporin A, 1.2 micrograms/ml; cantharidin, 20 microM) abolished the effect of hypo-osmotic stress on chitin synthase activation of yeast mixed membrane preparations. The presence of protein kinase inhibitors (genistein, 40 micrograms/ml; H-7, 100 microM) and a Ca2+ channel blocker (verapamil, 50 microM) reduced chitin synthase activity to 50-60% of that observed in cells under hypo-osmotic shock. These inhibitors also inhibited germ tube formation. This suggests that chitin synthase activity and yeast hyphal morphogenesis are both subject to regulation by osmotic pressure, phosphorylation and calcium.

Morphology-associated expression of NADP-dependent glutamate dehydrogenases during yeast-mycelium transition of a dimorphic fungus Benjaminiella poitrasii.

Benjaminiella poitrasii, a dimorphic zygomycetous fungus possesses three glutamate dehydrogenases, one requiring NAD while the other two use NADP as a coenzyme. In the activity staining after electrophoresis on native polyacrylamide gel NAD- dependent glutamate dehydrogenase revealed the presence of one enzyme that was expressed in both, yeast- and mycelium-form cells. While in case of NADP- dependent glutamate dehydrogenase two distinct activity bands that were differentially expressed in yeast- and mycelium-form cells were seen. Interestingly, during yeast-mycelium transition and reverse, quantitative changes in form-specific native NADP-dependent glutamate dehydrogenase activities were seen. The biochemical data on temperature and pH optima, thermostability, and kinetic properties confirmed the presence of two NADP-dependent proteins in B. poitrasii, parent strain. The monomorphic mutant (Y-5, yeast form) showed NADP- glutamate dehydrogenase similar to parent yeast-form enzyme. For the first time the significance of differential expression of these enzymes during morphological transition in B. poitrasii has been suggested.

The zygomycetous fungus, Benjaminiella poitrasii contains a large family of differentially regulated chitin synthase genes

Benjaminiella poitrasii is a zygomycetous, non-pathogenic dimorphic fungus. Chitin synthases are the membrane bound enzymes involved in the synthesis of chitin and are key enzymes in the cell wall metabolism. Multiplicity of these enzymes is a common occurrence. Here, we identify eight distinct CHS genes in B. poitrasii as confirmed through DNA sequence and Southern analysis. These genes are related to other fungal CHS genes. BpCHS1-4 are class I–III chitin synthases while BpCHS5-8 are class IV–V chitin synthases. These eight genes are differentially expressed during morphogenesis and under different growth conditions. Two of these genes viz. BpCHS2 and BpCHS3 appear to be specific to the mycelial growth form. These are the first B. poitrasii sequences to be reported. Based on CHS gene sequences, B. poitrasii chitin synthase genes place it with other zygomycetes on a fungal phylogenetic tree.

Isolation and regeneration of protoplasts from the yeast and mycelial form of the dimorphic zygomycete Benjaminiella poitrasii: Role of chitin metabolism for morphogenesis during regeneration

Experimental parameters for isolation and regeneration of protoplasts from the mycelial and yeast form cells of the dimorphic zygomycete Benjamininiella poitrasii are reported. Using a chitosanase containing preparation from Streptomyces sp. MCl we obtained protoplasts after 5 h incubation with a yield of 2+/-0.3 x 10(6) ml(-1) and 3+/-0.4 x 10(7) ml(-1) for the mycelial and yeast form, respectively. During regeneration under conditions triggering dimorphism the two morphological forms were observed after 36 h. Initially, for 10-12 h only an irregular mass was formed as a result of deregulated cell wall synthesis. Among the tested inhibitors influencing cell wall metabolism, chitin metabolism inhibitors showed distinctive effects on the regeneration of protoplasts suggesting that the respective enzymes significantly contribute to determining the morphogenesis of the dimorphic fungus B. poitrasii.

Dimorphism in Benjaminiella poitrasii: involvement of intracellular endochitinase and N-acetylglucosaminidase activities in the yeast-mycelium transition.

The chitinase and N-acetylglucosaminidase activities in cell-wall-bound and free fractions in the dimorphic fungus Benjaminiella poitrasii were studied as a function of morphological (unicellular yeast-mycelium) transition. The specific activities of chitinases of cell-wall-free, particularly in the membrane fraction, were significantly different in the yeast and mycelial forms. During the yeast-mycelium transition, the N-acetylglucosaminidase activity isolated in a membrane preparation increased steadily. The activity of the yeast cells (0.83 +/- 0.17 nkat/mg protein) increased 17-fold to 14.2 +/- 1.7 nkat/mg protein in 1-d-old mycelial cells. The endochitinase activity increased 12-fold between 6 and 12 h and thereafter practically remained unchanged up to 24 h. A reverse trend in the chitinolytic activities was observed during the mycelium-yeast transition. Isoelectrofocussing (pH range 3.5-10) of mixed membrane fraction free of particulate fraction of parent and morphological (Y-5, yeast-form) mutant cells separated endochitinase and N-acetylglucosaminidase activity into two pH ranges, viz. 4.3-5.7 and 6.1-7.7, respectively. The predominant N-acetylglucosaminidase activity observed at pH 6.9 and 7.1 for the parent strain membrane fraction was undetected in the mutant preparation. The results suggested that the membrane-bound (either tightly or loosely) chitinolytic enzymes, particularly, N-acetylglucosaminidase, significantly contributed to the morphological changes in B. poitrasii.

Fungal spore germination into yeast or mycelium: possible implications of dimorphism in evolution and human pathogenesis

The ability of dimorphism in fungi is conventionally regarded as a reversible change between the two vegetative forms, yeast and mycelium, in response to environmental change. A zygomycetous isolate, Benjaminiella poitrasii, exhibited yeast-mycelium transition in response to the change in temperature (37-28 degrees C) and decrease in glucose concentration. For the first time the presence of dimorphic response during asexual and sexual spore germination is reported under the dimorphism-triggering conditions in B. poitrasii. The zygospores germinated into budding yeast when subjected to yeast-form supporting conditions. The mycelium-form favoring conditions gave rise to true mycelium. Similarly, the asexual spores displayed a dimorphic response during germination. Our observations suggest that dimorphism is an intrinsic ability present in the vegetative, asexual, and sexual forms of the fungus. As dimorphic fungi are intermediate to the unicellular yeast and the filamentous forms, understanding of the dimorphic character could be useful to trace the evolutionary relationships among taxonomically different fungi. Moreover, the implications of spore germination during the onset of pathogenesis and in drug development for human health care are discussed.

Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure

The effects of changes in external osmotic pressure on chitin synthase activity of a dimorphic fungus, Benjaminiella poitrasii, have been investigated. Mycelial and yeast cells incubated in medium of low osmolality (distilled water, 0 mOsm) for 10 min had 2–3-fold higher specific activities of native chitin synthase in mixed membrane preparations than cells that had been subjected to a high osmolality medium (1.2 M sorbitol in distilled water, 1612 mOsm). Cells suspended in media of different osmolalities for 10 min were also affected in the extent of germ tube formation. Germ tube formation was highest in cells incubated in low osmolality medium. The addition of protein phosphatase inhibitors (cyclosporin A, 1.2 μg/ml; cantharidin, 20 μM) abolished the effect of hypo-osmotic stress on chitin synthase activation of yeast mixed membrane preparations. The presence of protein kinase inhibitors (genistein, 40 μg/ml; H-7, 100 μM) and a Ca 2+ channel blocker (verapamil, 50 μM) reduced chitin synthase activity to 50–60% of that observed in cells under hypo-osmotic shock. These inhibitors also inhibited germ tube formation. This suggests that chitin synthase activity and yeast hyphal morphogenesis are both subject to regulation by osmotic pressure, phosphorylation and calcium.

Dimorphism in Benjaminiella poitrasii: cell wall chemistry of parent and two stable yeast mutants.

In the dimorphic zygomycetous fungus Benjaminiella poitrasii, the cell wall compositions of mycelial phase (M), yeast phase (Y) and its yeast form mutants (Y-2 and Y-5) were studied. Chitosan was abundant in M-phase (26.6%) whereas lesser amounts were present in Y-phase (17.3%) and in mutants Y-2 (19.6%) or Y-5 (17.3%). Although chitin was present as a smaller fraction of the total glucosaminoglycan in each of different cell wall preparations, it was almost 3 times more prevalent in M-phase than the Y-phase cells. Cross-linking studies among the various cell wall components in B. poitrasii, suggest linkages among mannans and proteins and glucans and glucosaminoglycans.

Dimorphism of Benjaminiella poitrasii: Isolation and biochemical studies of morphological mutants

The yeast-mycelium dimorphism of the genus Benjaminiella poitrasii has been investigated. To understand the mechanism of dimorphism two stable yeast-phase mutants (Y-1 & Y-2) and one slow growing mycelial mutant (M-1) of B. poitrasii were isolated after NTG treatment of parent strain spores and studied for their biochemical characteristics. Effects of (i) kind and concentration of carbon source, (ii) presence of complex organic nitrogen and (iii) C:N ratio in the growth medium on the morphology of parent and mutant strains were carried out at 28 degrees C under shaking conditions. Ethanol induced morphological change and its reversal were studied in all the strains in order to elucidate the possible mechanism of morphogenesis.