Induction of germ-tube formation by Candida albicans in amino acid liquid synthetic medium at 25°C

Candida albicans (3153A) was found to exhibit extensive germ-tube and mycelial development at 25°C when transferred from amino acid synthetic medium at pH 6 to medium of pH 7. Significant germ-tube formation was detectable after approximately 8 h and in all experimental treatments, the peaks of maximal germination occurred at approximately 40–44 h. Such a transition was not only dependent on the initial pH of the medium but also on the glucose concentration and inoculum size. The optimum initial glucose concentration and inoculum size for maximal germ-tube development was 1.25% and 2×10⁶ cells ml^–1 respectively and above or below these values the extent of germ-tube formation was greatly reduced.     

Regulation of chitin synthesis during germ-tube formation in Candida albicans

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (K _i =1.2M) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.Non-Standard Abbreviations GlcNac N-acetyl glucosamine - UDP-GlcNac UDP-N-acetyl glucosamine - PMSF phenylmethylsulphonylfluoride     

A new minimal synthetic medium for germ-tube production in Candida albicans

A new minimal synthetic medium, with low amount of glucose, without aminoacids, vitamins and neutral pH, which induces germ-tubes production in Candida albicans, is reported in this work. The results indicate a perfect agreement between the germ-tube test performed with the standard method in human or animal serum and this test performed in minimal synthetic medium. In this medium the germ-tube test for the presumptive identification of Candida albicans can be performed with the same formality, time and reproducibility as those in human or animal serum. This constitutes an interesting finding because it is easy to prepare, to store and is highly reproducible.     

The first clinical isolates of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in Slovakia

Candida dubliniensis, yeast closely related to Candida albicans, is a new pathogen associated mainly with infections of immunocompromised hosts. In this study, we report the first isolation of three isolates of C. dubliniensis in Slovakia. The first selection of both C. albicans and C. dubliniensis from the other Candida species was done on the basis of specific green color of primoculture grown on CHROMagar Candida. The presumptive identification was completed by supplemental tests: germ-tube formation, production of chlamydospores, ability or inability to grow at 42 and 45,°C and by commercial set API 20C AUX. Parallely, the discrimination between both species was performed by PCR assay using primers specific for Candida dubliniensis     

N-Acetyl-d-Glucosamine Induces Germination in Candida albicans through a Mechanism Sensitive to Inhibitors of cAMP-Dependent Protein Kinase

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-d-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.     

紫草素对白色念珠菌的抑制作用机制

【目的】研究紫草素抑制白色念珠菌的作用机制。【方法】通过微量稀释法测定紫草素对白色念珠菌的最低抑菌浓度(MIC)和最低杀菌浓度(MFC);紫外分光光度法测定紫草素对白色念珠菌细胞膜渗透性的影响;扫描电镜观察紫草素对菌体形态的影响;激光共聚焦显微镜测定紫草素对白色念珠菌细胞内钙离子浓度的影响;卵黄平板培养基法检测紫草素对白色念珠菌的细胞膜磷脂酶活性的影响;RT-PCR检测紫草素对白色念珠菌PLB1和PLB2基因表达量的影响。【结果】紫草素对白色念珠菌有较强的抑制作用,其对白色念珠菌的MIC和MFC分别为16μg/m L和32μg/mL。紫草素能破坏白色念珠菌细胞膜的完整性,使细胞膜的通透性增加,导致细胞内DNA和RNA等大分子物质的泄漏和细胞内钙离子的流失。其中MIC的紫草素作用菌体16 h后,上清液中的DNA和RNA等大分子含量与对照组相比增加了117.32%(P0.01);细胞内的[Ca~(2+)]降低了72.02%(P0.01)。扫描电镜结果也证明了紫草素对白色念珠菌细胞膜的破坏作用。紫草素也能抑制白色念珠菌分泌磷脂酶,且呈浓度剂量依赖。其中,与对照组相比,MIC的紫草素能使白色念珠菌分泌磷脂酶的量下降56.3%(P0.01)。RT-PCR结果显示,紫草素能抑制编码磷脂酶B的基因PLB1和PLB2的表达量,其中1/2 MIC的紫草素作用白色念珠菌16 h后,与对照组相比,PLB1和PLB2基因的相对表达量分别降低了56.4%和61.4%(P0.01)。【结论】紫草素对白色念珠菌有较强的抑杀作用,其作用机制是通过破坏白色念珠菌细胞膜的完整性,增加菌体细胞膜的通透性,导致细胞内DNA和RNA等大分子的泄漏和细胞内[Ca~(2+)]的流失,最终引起菌体的死亡。而紫草素对白色念珠菌磷脂酶分泌的抑制作用,致使其不能及时维护和修复由紫草素造成的细胞膜的破坏和损伤,也是导致菌体死亡的原因。     

N-Acetyl-d-Glucosamine Induces Germination in Candida albicans through a Mechanism Sensitive to Inhibitors of cAMP-Dependent Protein Kinase

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-d-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.     

In vitro phagocytosis of several candida berkhout species by murine leukocytes

In vitro phagocytosis of thirteen Candida berkhout species by rat leukocytes was studied to assess a possible correlation between pathogenicity and phagocytosisYeast-leukocyte suspensions were mixed up for 3 h and phagocytic index, germ-tube formation and leukocyte candidacidal activity were evaluated. Highest values for phagocytosis were reached in all cases at the end of the first hour. Leukocyte candidacidal activity was absent. Only C. albicans produced germ-tubes.The various phagocytosis indices were determined depending on the Candida species assayed. Under these conditions, the more pathogenic species presented the lower indices of phagocytosis.It is determined that the in vitro phagocytic index may bear a close relationship with the pathogenicity of the Candida berkhout.     

Relationship between cyclic adenosine 3′:5′-monophosphate and germination inCandida albicans

Germination of yeast-like cells ofCandida albicans is preceded by a significant decrease in intracellular levels of cyclic AMP during the early stage of germ-tube induction. These levels increased thereafter as germ-tube formation proceeded. The intracellular concentration of cyclic AMP was measured with a cyclic AMP radioimmunoassay and with a competitive assay method using a cyclic AMP-binding protein. Under inducing conditions, germtube formation was inhibited by the addition of cyclic AMP or compounds that are known to elevate the intracellular cyclic nucleotide concentration.     

Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

Background  

Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts.     

In vitro efficacy of the lipopeptide biosurfactant surfactin-C15 and its complexes with divalent counterions to inhibit Candida albicans biofilm and hyphal formation

Abstract

Surfactin is a type of cyclic lipopeptide biosurfactant implicated in a wide range of applications. Although its antimicrobial activity has been characterized, its effect on Candida albicans physiology remains to be elucidated. The present study evaluated the influence of surfactin-C₁₅ (SF) and its complexes with divalent counterions on C. albicans biofilm formation and preformed biofilms. The SF and metal(II)-SF complexes inhibited biofilm formation and reduced the metabolic activity of mature biofilms in a concentration-dependent manner. The same concentrations of the compounds studied dislodged preexisting biofilms grown on polystyrene plates. Moreover, SF and its metal(II) complexes reduced the mRNA expression of hypha-specific genes HWP1, ALS1, ALS3, ECE1 and SAP4 without exhibiting significant growth inhibition. Further research showed that the compounds tested reduced cellular surface hydrophobicity (CSH). These results suggest that SF and metal(II)-SF complexes could be used as anti-biofilm agents against C. albicans hypha-related infections in clinical practice.     

Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols

doi: 10.1111/j.1741‐2358.2011.00485.x
Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols Objective: The aim of this study was to investigate Candida albicans biofilm formation on denture liners and to analyse the efficacy of cleaning protocols. Material and methods: Specimens were prepared from four silicone‐based soft denture liners. After artificial ageing and surface free energy determination, specimens were incubated with saliva (2 h) and Candida albicans ATCC 10231 for either short‐ (2.5 h) or long‐term (24 h) biofilm formation. Adherent cells were determined either after incubation of specimens with Candida albicans or after treatment with different denture cleaning protocols. Statistical analysis was performed using one‐way anova and the Games–Howell test (α = 0.05). Results: For both short‐ and long‐term biofilm formation, similar amounts of Candida albicans cells were found on the surface of the different liners (p = 0.295 and 0.178, respectively). For both short‐ and long‐term biofilm formation, the highest cleaning efficacy was observed for sodium hypochlorite (NaOCl; p < 0.01). The efficacy of the chemical denture cleaner in removing long‐term Candida albicans biofilms was significantly lower than the efficacy of removal by brushing (p < 0.001). Conclusion: Different silicone‐based soft denture liners yield similar Candida albicans biofilm formation on their surface. The highest efficacy for the removal of Candida albicans biofilms was identified for NaOCl. Chemical denture cleaners appear to have rather low efficacy to remove mature Candida albicans biofilms.     

白色念珠菌生物被膜研究进展

难治性真菌感染的临床分析发现,病灶感染病原常以生物被膜的形态存在。生物被膜的形成可帮助真菌躲避宿主细胞免疫系统清除和药物的攻击,所造成的持续性感染严重威胁人类健康,因此,认识研究真菌生物被膜及其耐药机理对于防治临床真菌感染有着重大意义。白色念珠菌是一种临床感染常见的条件性致病菌,也是目前真菌生物被膜研究的主要研究模型。白色念珠菌生物被膜主要由多糖、蛋白质和DNA构成,其形成由微生物间的群体感应调控,并受到环境中营养成分及其附着物表面性质影响。研究发现,胞外基质的屏障作用、耐药基因的表达等机制与生物被膜耐药性的产生密切相关。本文就白色念珠菌生物被膜的形成过程、结构组成、形成的影响因素、现有研究模型、耐药机制和治疗策略等几个方面介绍近年来的研究进展。     

Inhibition on <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm formation using divalent cation chelators (EDTA)

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37°C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37°C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependant manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.     

Adhesive Properties and Hydrolytic Enzymes of Oral <Emphasis Type="Italic">Candida albicans</Emphasis> Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.     

Isolation of genes from Candida albicans by complementation in Saccharomyces cerevisiae

Summary A genomic library of the asexual pathogenic yeast Candida albicans was constructed in the S. cerevisiae vector YEp13. The library contains a representation of the entire genome with a probability of 99%. The expression of the genes of C. albicans in S. cerevisiae was examined and two mutations his3-1 and trp1-289 of S. cerevisiae were complemented by the cloned genes of C. albicans. The hybridization data indicates that the plasmids complementing the mutations of S. cerevisiae contain sequences from C. albicans.     

Mutations on CaENO1 in Candida albicans inhibit cell growth in the presence of glucose

Summary Enolase (2-phospho-D-glycerate hydrolase) is an enzymatic component of the glycolytic pathway and is conserved through evolution. The TR-CaENO1/Caeno1 stain, of which the expression of CaENO1 is under control of the tetracycline-regulatable (TR) expression system, is utilized for elucidating the functions of CaENO1 in Candida albicans. As expected, there was no detectable CaENO1 mRNA when the TR-CaENO1/Caeno1 cells grew on media containing doxycycline repressing the expression of TR-CaENO1.The TR-CaENO1/Caeno1 cells were arrested in media containing doxycycline in the presence of glucose but not in non-fermentable carbon sources, such as glycerol. Furthermore, the TR-CaENO1/Caeno1 cells were also arrested in media containing 4% serum. In this study, we have showed that CaENO1 is required for the cell growth of C. albicans in the presence of glucose. Our findings may help us to design new and more effective antifungal agents for preventing and treating bloodstream fungal infections by blocking the function(s) of enolases.