Candida albicans ABG1 gene is involved in endocytosis

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.     

热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2的功能评价

【目的】热带假丝酵母是发酵法生产二元酸的重要工业菌株,具有较高的ω-氧化活性。脂肪醛脱氢酶在ω-氧化途径中起重要作用,催化脂肪醛生成脂肪酸,但其具体催化功能及对细胞生理影响还未被系统研究。本文通过删除脂肪醛脱氢酶基因CtAld1和CtAld2鉴定了其在ω-氧化途径中的功能。【方法】通过基因组信息挖掘获得热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2序列,在此基础上,通过同源重组敲除CtAld1和CtAld2基因。考察突变株的生长和胞内脂肪醛脱氢酶活性变化,并评价CtAld1和CtAld2基因敲除对细胞二元酸合成能力的影响。【结果】分别获得了热带假丝酵母突变株XZX-1(ΔCtAld1/ΔCtAld1)、XZX-2(ΔCtAld2/ΔCtAld2)和XZX-12(ΔCtAld1/ΔCtAld1,ΔCtAld2/ΔCtAld2)。在以十二烷为唯一碳源的培养基中,敲除CtAld2基因显著抑制细胞的生长,胞内脂肪醛脱氢酶活性降低为出发菌株的30%;敲除CtAld1基因尽管会使细胞损失一部分醛脱氢酶活性,但能够一定程度地提升细胞在十二烷中的生长性能。敲除CtAld1或CtAld2会降低菌株二元酸产量,组合敲除CtAld1和CtAld2严重削弱菌株十二碳二元酸的合成能力。【结论】CtAld2对热带假丝酵母细胞的生长和十二碳二元酸的合成具有重要作用,缺失CtAld1或CtAld2基因降低细胞的二元酸合成能力。CtAld1和CtAld2可作为热带假丝酵母ω-氧化途径代谢工程改造的潜在靶点。     

Etoposide and Camptothecin Reduce Growth,Viability, the Generation of Petite Mutants,and Recognize the Active Site of DNA Topoisomerase I and II Enzymes in Candida glabrata

Candidemia, one of the most common invasive fungal infections in hospitalized patients, can lead to death and huge financial losses. Candida albicans is the main causative agent of this disorder and Candida glabrata occupies the second or third place, for which new therapeutic alternatives must be found. The objective of the present study was to evaluate the inhibitory effect of etoposide and camptothecin (inhibitors of deoxyribonucleic acid (DNA) topoisomerase) on the C. glabrata CBS138 strain. Etoposide and camptothecin showed better or similar MIC (minimum inhibitory concentration) (5 and 2.5 μg/mL, respectively), with respect to fluconazole (8 μg/mL) and itraconazole (4 μg/mL). They also suppressed colony formation during the 12-h test. On the other hand, petite colonies were less formed by exposing C. glabrata to etoposide or camptothecin (indicating low toxicity), with respect fluconazole and itraconazole. Such colonies are phenotypically observed as limited growth in medium containing a non-fermentable carbon source, and are genotypically characterized by a partial or total loss of mitochondrial DNA (mtDNA) fragments. Using PCR techniques and cell staining with 4′,6-diamidino-2-phenylindole (DAPI), loss of mtDNA was detected only in yeast cells treated with fluconazole. Additionally, molecular docking studies with etoposide and camptothecin showed recognition in the active site of the Topo I and II enzymes from C. glabrata. Since etoposide and camptothecin showed good inhibitory activity and low toxicity on C. glabrata; they should certainly be of interest for the treatment of C. glabrata infections and the design and development of new antifungal compounds derived from these drugs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00942-6.     

Kinetic resolution of ibuprofen catalyzed by Candida rugosa lipase in ionic liquids

Candida rugosa lipase-catalyzed esterification of ibuprofen with 1-propanol was conducted in seven ionic liquids and the results were compared with those in isooctane. Although the enzyme showed comparable or higher activity in some ionic liquids compared to that in isooctane, only in the case of [BMIM]PF6 was the enantioselectivity (E = 24.1) almost twice that (E = 13.0) of isooctane. In another six ionic liquids the enzyme enantioselectivity was much poorer (E = 1.1-6.4). At the same conversion of 30%, E of [BMIM]PF6 is more than triple that of isooctane. The lipase stability in [BMIM]PF6 was improved by 25% of that in isooctane. It was concluded that [BMIM]PF6 could be applied to substitute the conventional organic solvent (isooctane) in the esterification of ibuprofen.     

限磷对产甘油假丝酵母甘油合成与胞内磷积累的影响

研究了磷酸盐限量对产甘油假丝酵母甘油合成与胞内磷积累的影响。结果表明, 当酵母细胞从适磷或富磷培养基转接入低磷培养基时, 发酵过程中胞内积累的磷逐渐减少; 而当菌体从低磷培养基转接入适磷或富磷培养基时, 发酵过程中胞内聚磷酸盐的积累量迅速增加。当细胞在第14小时和第38小时从适磷培养基转接入低磷培养基时甘油得率分别高达60.9%和61.4%, 而甘油产率则分别为2.03 g/(L·h)和2.23 g/(L·h)。这些现象说明限制发酵培养基中的磷浓度是产甘油假丝酵母高产甘油的必要条件, 并为其反复分批发酵法生产甘油提供了重要依据。     

REMI介导电转化产甘油假丝酵母

为了分离耐高渗和甘油代谢相关基因,以Zeocin为选择标记,利用REMI技术电转化产甘油假丝酵母Candida glycerinogenes。考察了7种限制性内切酶对转化的影响,选择Hind III进一步优化了转化的几个条件。结果表明,在OD₆₀₀≈1.3 时收集细胞,在1.5 kV 电压下,感受态细胞浓度为2.0×10⁹个细胞/mL,100U Hind III时,能获得129个转化子/μg DNA的较高转化率,58% 的转化子稳定,表明REMI技术适合于产甘油假丝酵母的转化。     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

Inhibition of Candida rugosa lipase by saponins, flavonoids and alkaloids

Lipase inhibitors have generated a great interest because they could help in the prevention or the therapy of lipase-related diseases. Therefore, the aim of the work was to evaluate by HPLC, and using Candida rugosa lipase as model, the inhibitory effect of several saponins: β-aescin, digitonin, glycyrrhizic acid (GA) and Quillaja saponin (QS); flavonoids: 3-hydroxyflavone, 5-hydroxyflavone, (±)-catechin and kaempferol; and alkaloids: aspidospermine, papaverine, physostigmine, pilocarpine, raubasine, rescinnamine, reserpine and trigonelline.

The inhibition produced by most of these compounds is described here for the first time. Saponins appeared very active, being β-aescin and digitonin the most active compounds (IC₅₀ = 0.8–2.4 × 10⁻⁵ M). The inhibitory activity of flavonoids was lower than that of saponins (except GA), and (±)-catechin and kaempferol were the most active. Alkaloids was the most heterogeneous group assayed, varying from rescinnamine, with an IC₁₆ similar to that of digitonin, to papaverine and others which showed almost no inhibition.

In conclusion, β-aescin, digitonin, kaempferol or (±)-catechin, strong lipase inhibitors with a low toxicity and present herbal drugs used for lipase-related diseases such as acne or ulcer, are promising candidates for the prevention or the treatment of these diseases.     

Simple and reliable detection of slime production of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">spp</Emphasis>. directly from blood culture bottles: Comparison of visual tube method and transmission electron microscopy

Early detection of slime production may be useful for clinical decision because of its suggestive property for potential pathogenic capacity of a Candida strain especially in patients with a prosthetic device. In this study we aimed to compare the visual tube method (VTM) with transmission electron microscopy (TEM) in order to confirm the reliability of the former method. In order to demonstrate the reproducibility of the tube method and to determine the correct timing for the test, Candida isolates directly obtained from blood culture (DBC) bottles and their two subsequent subcultures were used. The results of this study showed that VTM is a simple and reliable method which can be used in every clinical mycology laboratory, provided that the test is applied on DBC isolates; as the ability of slime production is decreased or lost even after the first subculturing. We suggest that this simple method can be used and may have some contributions to the ongoing studies on the controversial issue concerning removal of biomaterials in candidemic patients.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.