Delicate Metabolic Control and Coordinated Stress Response Critically Determine Antifungal Tolerance of Candida albicans Biofilm Persisters

Candida infection has emerged as a critical health care burden worldwide, owing to the formation of robust biofilms against common antifungals. Recent evidence shows that multidrug-tolerant persisters critically account for biofilm recalcitrance, but their underlying biological mechanisms are poorly understood. Here, we first investigated the phenotypic characteristics of Candida biofilm persisters under consecutive harsh treatments of amphotericin B. The prolonged treatments effectively killed the majority of the cells of biofilms derived from representative strains of Candida albicans, Candida glabrata, and Candida tropicalis but failed to eradicate a small fraction of persisters. Next, we explored the tolerance mechanisms of the persisters through an investigation of the proteomic profiles of C. albicans biofilm persister fractions by liquid chromatography-tandem mass spectrometry. The C. albicans biofilm persisters displayed a specific proteomic signature, with an array of 205 differentially expressed proteins. The crucial enzymes involved in glycolysis, the tricarboxylic acid cycle, and protein synthesis were markedly downregulated, indicating that major metabolic activities are subdued in the persisters. It is noteworthy that certain metabolic pathways, such as the glyoxylate cycle, were able to be activated with significantly increased levels of isocitrate lyase and malate synthase. Moreover, a number of important proteins responsible for Candida growth, virulence, and the stress response were greatly upregulated. Interestingly, the persisters were tolerant to oxidative stress, despite highly induced intracellular superoxide. The current findings suggest that delicate metabolic control and a coordinated stress response may play a crucial role in mediating the survival and antifungal tolerance of Candida biofilm persisters.     

Inhibition of Nucleic Acid Biosynthesis Makes Little Difference to Formation of Amphotericin B-Tolerant Persisters in Candida albicans Biofilm

Candida albicans persisters constitute a small subpopulation of biofilm cells and play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Persisters are often described as dormant, multidrug-tolerant, nongrowing cells. Persister cells are difficult to isolate and study not only due to their low levels in C. albicans biofilms but also due to their transient, reversible phenotype. In this study, we tried to induce persister formation by inducing C. albicans cells into a dormant state. C. albicans cells were pretreated with 5-fluorocytosine (planktonic cells, 0.8 μg ml⁻¹; biofilm cells, 1 μg ml⁻¹) for 6 h at 37°C, which inhibits nucleic acid and protein synthesis. Biofilms and planktonic cultures of eight C. albicans strains were surveyed for persisters after amphotericin B treatment (100 μg ml⁻¹ for 24 h) and CFU assay. None of the planktonic cultures, with or without 5-fluorocytosine pretreatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, representing approximately 0.01 to 1.93% of the total population. However, the persister levels were not significantly increased in C. albicans biofilms pretreated with 5-fluorocytosine. These results suggest that inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms.     

Targeting Fibronectin To Disrupt In Vivo Candida albicans Biofilms

New drug targets are of great interest for the treatment of fungal biofilms, which are routinely resistant to antifungal therapies. We theorized that the interaction of Candida albicans with matricellular host proteins would provide a novel target. Here, we show that an inhibitory protein (FUD) targeting Candida-fibronectin interactions disrupts biofilm formation in vitro and in vivo in a rat venous catheter model. The peptide appears to act by blocking the surface adhesion of Candida, halting biofilm formation.     

白色念珠菌对临床常用抗真菌药物的耐药性分析

目的了解白色念珠菌对临床常用抗真菌药物的耐药性。方法按照美国临床实验室标准化委员会（CLSI）推荐的酵母菌纸片扩散法敏感试验指南,对临床分离的494株白色念珠菌进行抗真菌药物的敏感性测定。结果 494株白色念珠菌对两性霉素B、制霉菌素敏感性最高,分别为96.1%和93.9%,对咪康唑的敏感性最低为51.9%;念珠菌常见临床感染部位为呼吸道,其次是泌尿道。白色念珠菌对唑类抗真菌药物存在较大的耐药性。结论白色念珠菌对常用抗真菌药物的耐药性呈上升趋势,其耐药机制有待于进一步研究。     

Farnesol-Induced Apoptosis in Candida albicans

Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.Candida albicans is the most important human fungal pathogen, causing various diseases from superficial mucosal infections to life-threatening systemic disorders (7, 27). As a polymorphic species, C. albicans is capable of switching morphologies among yeast, hyphal, and pseudohyphal forms, and the transitions are central to its pathogenesis and biofilm formation (1, 15, 30, 32). Farnesol was recently described as a quorum-sensing molecule secreted by C. albicans that is able to prevent yeast-to-hyphal conversion (12, 26). In C. albicans, farnesol is endogenously generated in the cell by enzymatic dephosphorylation of farnesyl diphosphate, a precursor for the synthesis of sterols in the sterol biosynthesis pathway (5, 13, 31).We have previously shown that in addition to inhibiting germination in C. albicans, at high concentrations farnesol also induces cell death (14). Farnesol has been shown to induce apoptosis or programmed cell death in a number of fungal species, such as Saccharomyces cerevisiae and Aspergillus nidulans (6, 34). In C. albicans, however, farnesol-induced apoptosis has not yet been described. More recently, through analyses of typical markers of apoptosis and proteomic studies, we demonstrated that farnesol triggers apoptosis in human oral squamous carcinoma cells through the classical mammalian intrinsic and extrinsic apoptotic signaling pathways (33). More interestingly, the study also demonstrated for the first time the ability of the farnesol produced by C. albicans to induce a similar apoptotic effect on the tumor cells via the same pathways (33).Apoptosis is characterized by biochemical processes that are largely conserved throughout evolution. In mammalian cells, apoptosis is a tightly regulated process for controlling the proliferation of undesired cells (3, 10). Apoptosis is triggered by various extracellular and intracellular stimuli that result in the activation of caspases, a class of cysteine proteases that play a crucial role in the process of apoptosis by conveying the apoptotic signal in a proteolytic cascade (3, 10, 35, 39). This ultimately leads to cell disassembly, mitochondrial degradation, and growth inhibition, which are major cellular responses characteristic of apoptosis (3, 10, 21, 33, 39).Mitochondria have a central role in apoptosis, and many important aspects of the apoptotic process converge in this organelle. Mitochondria are the richest source of reactive oxygen species (ROS) in the cell, and the inhibition of the mitochondrial electron transport chain, which results in the subsequent release of ROS, is an early event in apoptotic cell death (37, 38). ROS can damage almost every essential cellular component, and in order to protect against the damaging effects of ROS, fungal cells have evolved specific defense mechanisms to neutralize ROS. The primary enzymatic antioxidants involved in protection against ROS are catalase and the superoxide dismutases (9, 16, 21, 23, 28).In C. albicans, physiological concentrations of farnesol were reported to target the mitochondria, inducing oxidative stress, and a putative caspase (a mammalian caspase homologue, MCA1) has been identified in the C. albicans genome project (orf19.5995) (4, 28, 29, 37). Furthermore, Phillips et al. (28) recently reported that C. albicans undergoes programmed cell death under certain conditions. However, in these studies, activation of the Ras1-cyclic AMP pathway was determined to be involved in the process in which the deletion of RAS1 resulted in increased survival and decreased apoptosis (28, 29). In contrast, farnesol was shown to result in lower concentrations of cyclic AMP, indicating that farnesol-induced apoptosis likely involves a different pathway (31).The exact mechanism of farnesol cytotoxicity and whether it is a part of an apoptotic pathway analogous to the caspase-mediated process of apoptosis in mammalian cells remain unclear. Therefore, to elucidate the mechanisms underlying farnesol cytotoxicity and its possible involvement in an apoptotic process in C. albicans, a global two-dimensional proteomic approach combined with a detailed assessment of standard apoptotic markers in farnesol-exposed C. albicans cells was undertaken.     

Flow Cytometry-Based Analysis of Artemisinin-Resistant Plasmodium falciparum in the Ring-Stage Survival Assay

The ring-stage survival assay (RSA) is a powerful tool for phenotyping artemisinin-resistant Plasmodium falciparum but requires experienced microscopists to count viable parasites among 10,000 erythrocytes in Giemsa-stained thin blood smears. Here we describe a rapid flow cytometric assay that accurately counts viable parasites among 250,000 erythrocytes in suspension. This method performs as well as light microscopy and can be used to standardize the collection of RSA data between research groups in laboratory and field settings.     

Quercetin Sensitizes Fluconazole-Resistant Candida albicans To Induce Apoptotic Cell Death by Modulating Quorum Sensing

Quorum sensing (QS) regulates group behaviors of Candida albicans such as biofilm, hyphal growth, and virulence factors. The sesquiterpene alcohol farnesol, a QS molecule produced by C. albicans, is known to regulate the expression of virulence weapons of this fungus. Fluconazole (FCZ) is a broad-spectrum antifungal drug that is used for the treatment of C. albicans infections. While FCZ can be cytotoxic at high concentrations, our results show that at much lower concentrations, quercetin (QC), a dietary flavonoid isolated from an edible lichen (Usnea longissima), can be implemented as a sensitizing agent for FCZ-resistant C. albicans NBC099, enhancing the efficacy of FCZ. QC enhanced FCZ-mediated cell killing of NBC099 and also induced cell death. These experiments indicated that the combined application of both drugs was FCZ dose dependent rather than QC dose dependent. In addition, we found that QC strongly suppressed the production of virulence weapons—biofilm formation, hyphal development, phospholipase, proteinase, esterase, and hemolytic activity. Treatment with QC also increased FCZ-mediated cell death in NBC099 biofilms. Interestingly, we also found that QC enhances the anticandidal activity of FCZ by inducing apoptotic cell death. We have also established that this sensitization is reliant on the farnesol response generated by QC. Molecular docking studies also support this conclusion and suggest that QC can form hydrogen bonds with Gln969, Thr1105, Ser1108, Arg1109, Asn1110, and Gly1061 in the ATP binding pocket of adenylate cyclase. Thus, this QS-mediated combined sensitizer (QC)-anticandidal agent (FCZ) strategy may be a novel way to enhance the efficacy of FCZ-based therapy of C. albicans infections.     

白假丝酵母菌致食物中毒的病原学鉴定及药敏试验结果的报告

目的明确白假丝酵母菌的鉴定过程,探寻治疗白假丝酵母菌致食物中毒的药物。方法收集16例食物中毒者吐、泻物标本及剩余饭菜,酱油。白假丝酵母菌的鉴定用快速微生物鉴定系统(API)20C Aux V3.0;药敏试验用FUNGUS-11药敏最低抑菌浓度(MIC)测定试验条。结果检样呕吐物、水样便、剩菜、酱油中检出白假丝酵母菌,食物中毒主要是白假丝酵母菌引起的,白假丝酵母菌对两性霉素B和制霉菌素敏感。结论综合流行病学调查、临床症状、实验室鉴定结果,确定该次食物中毒由白假丝酵母菌引起;首选治疗药物为两性霉素B和制霉菌素。     

Sonography of Candida albicans cystitis

Candida albicans infections are opportunistic and range from asymptomatic infections to life-threatening ones. Noted on sonography in three proven cases, Candida albicans cystitis produced a mildly thickened bladder wall and a discrete, well-defined, dense fluid-fluid interface within the bladder, debris that contained the long threadlike pseudomycelia of the fungus. This fluid-fluid interface shifted with changes in position. The sonographic appearance, although not pathognomonic, was similar in all three cases. Sonography was used to monitor the progress of therapy. In all three cases, conservative treatment successfully cleared the vesical debris without significant sequelae or the formation of concretions analogous to bezoars and the bladder returned to normal except for minimal residual wall thickening.     

生物膜中白念珠菌的耐药性研究

白念珠菌（Canida albicans）是人类最常见的条件致病真菌,健康者带菌率以口腔最高,约80％。当机体免疫力下降或使用免疫抑制剂时,白念珠菌可导致严重的感染性疾病,在美国医院感染中列第4位,其病死率为30％～70％。研究发现,在难治性白念珠菌感染的病灶中大多存在生物膜。自然界中99％微生物以生物膜的形式存在,65％人类感染性疾病与生物膜相关。     

Reduced biocide susceptibility in Candida albicans biofilms

Candida biofilm formation is common during infection and environmental growth. We tested the impacts of three biocides (ethanol [EtOH], H₂O₂, and sodium dodecyl sulfate) on Candida albicans, C. parapsilosis, and C. glabrata biofilms. Higher concentrations of the biocides were required for efficacy against biofilms than for efficacy against planktonic controls. A combination study with two biocides (EtOH and H₂O₂) and fluconazole demonstrated that the combination had enhanced efficacy.     

Propranolol inhibits hyphal development in Candida albicans

Propranolol was used to investigate the role of phosphatidic acid (PA) and diacylglycerol in the dimorphic transition in Candida albicans. Propranolol was able to inhibit the appearance of germ tubes without decreasing growth rate. Data suggest that inhibition of morphogenesis may be due to binding by propranolol of PA derived from PLD1 hydrolysis of phosphatidylcholine.     

介绍两种鉴定白色念珠菌的方法

应用含０．０１％阿尼林蓝ＷＳ染料的ＹＭ固体培基（ＹＭＡＢ）和以对－硝基苯酚－Ｎ－乙酰－β－Ｄ－氨基半乳糖苷（ＮＧＬ）为底物鉴定白色念珠菌（白念菌），同时进行芽管试验。生长于ＹＭＡＢ上的白念菌温育１２～１８小时后在伍德灯下可观察到黄绿色荧光。ＮＧＬ酶法温育９０分钟后，白念菌酶类可分解底物呈现明显黄色。两种方法均正确鉴定了５５例白念菌中的５４例（９８．２％）。阿尼林蓝法中，１株类星形念珠菌也出现荧光，特异性９２．８％，敏感性及预示值均高于芽管法。ＮＧＬ酶法中，７株近平滑念珠菌呈阳性，特异性５０％，但反应快速。将两种方法并用，可使正确鉴定率达１００％，且两种方法简便、价廉，易于推广。     

Susceptibility of Candida albicans to Miconazole

A total of 439 clinical isolates of Candida albicans were tested for susceptibility to miconazole by the agar dilution technique. When tests were read at 48 and 24 h, 56 and 84%, respectively, of the strains were completely inhibited by 4.0 mug of miconazole per ml, the estimated upper limit of probable clinical susceptibility.     

含光滑念珠菌与白色念珠菌白带的比较分析

目的分析女性含白色念珠菌和光滑念珠菌白带的性状及镜下特征,以便认识并区分这两类念珠菌所引起的妇科感染。方法连续选取2000例做白带检查的妇科门诊患者,询问患者的典型症状并观察单纯含念珠菌妇科涂片的镜下特征,用显色培养基对白带内的真菌进行鉴定,比较分析白色念珠菌组与光滑念珠菌组的差异。结果共检出476例含念珠菌标本,其中白色念珠菌393例,光滑念珠菌60例,其他念珠菌23例。白色念珠菌与光滑念珠菌阴道感染患者均表现出较高的性交痛症状。与白色念珠菌比较,光滑念珠菌组白带无豆渣样改变,患者少见外阴瘙痒;高倍视野下白细胞数量较少,上皮细胞、乳酸杆菌及真菌孢子数量较多,清洁度较好。结论含光滑念珠菌白带与白色念珠菌白带比较具有较为明显的差异,光滑念珠菌感染后患者临床症状及白带镜下特征均无白色念珠菌典型。     

In-vitro resistance to imidazole antifungals in Candida albicans

Three strains of Candida albicans from patients with chronic mucocutaneous candidosis (CMC) who relapsed during prolonged treatment with ketoconazole were compared with three other Can. albicans strains by means of an agar incorporation MIC method, a broth microdilution method for IC30 determination, and a hyphal elongation assessment method. The MICs of ketoconazole, tioconazole and miconazole were difficult to interpret, but were much higher for the three CMC strains if low inoculum concentrations and short incubation times were used. The IC30S of the three imidazoles were much higher for the CMC strains than for the others. The imidazoles all caused minimal inhibition of hyphal elongation by the three CMC strains at concentrations up to 50 mg/l. Concentrations of 0.5 mg/l caused marked inhibition of the other strains.