Infection of HEp2 epithelial cells with Candida albicans: adherence and postadherence events

We have previously observed that the infection of HEp2 epithelial cells with Candida albicans results in HEp2 cell actin rearrangement, and that a culture filtrate of C. albicans (Candida metabolite) caused the same changes and reduced membrane ruffling and motility. It was found that the Candida metabolite consisted of several proteins and nonproteinaceous components. In this study we report on the identity of three of the main proteins in the Candida metabolite, namely a secretory aspartate protease (Sap), an agglutinin-like adhesion sequence (Als) and a glucan 1,3-beta-glucosidase. The effect on HEp2 cells caused by the Candida metabolite, an inhibitor of the PKC MAP kinase signal pathway - bisindolylmaleimide (BIM), or the actin polymerization inhibitor - cytochalasin D (CyD) were studied alone and in combination. Exposure of HEp2 cells to the Candida metabolite, together with the BIM or CyD, had profound effects on HEp2 cell morphology, as compared to individually treated cells, and also reduced the adherence of the organisms to HEp2 cells. Our results show that the interaction of C. albicans with HEp2 cells is, not unexpectedly, complex, and involves changes in the host cell that may be related to the effect of Candida-secreted biomolecules.     

Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation-dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding-deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk-cortactin complex in actin polymerization downstream of tyrosine kinase signaling.     

Induction of salivary antibodies to inhibit Candida albicans adherence to human epithelial cells by tonsillar immunization in rabbits

To examine the possibility of a vaccine for Candida albicans infection in the oral cavity, we induced salivary antibodies by immunization of killed-C. albicans ATCC 18804 on the palatine tonsils of rabbits. The enzyme-linked immunosorbent assay reaction of salivary antibodies was high against C. albicans serotype A. The saliva antibodies greatly inhibited C. albicans adherence to cloned epithelial cells from human gingiva. Tonsillar immunizations of C. albicans ATCC 18804 induce salivary antibodies that prevent C. albicans adherence to epithelial cells, and thus should prove useful in the prevention of oral candidiasis caused by C. albicans serotype A.     

Nutrition-dependent modulations of protein synthesis in Candida albicans during germ-tube formation or maintenance of the yeast form in N-acetyl glucosamine media

Abstract Stationary phase, yeast-form cells of Candida albicans grown in glucose-yeast extract medium were shifted to N -acetylglucosamine (GlcNAc) and/or glucose medium, and the pattern of protein synthesized under conditions of a progressive decrease in the rate of total protein synthesis was analyzed by SDS-PAGE and autoradiography.
Marked temporal modulations in the rate of synthesis of some cytoplasmic proteins were detected both in cells forming germ-tubes (at 37°C) and in yeast cells (at 28°C). The major modulated components showed molecular weights of 63, 53, 48 and 34 kDa. These products could not be qualified as heat-shock or heat-stroke proteins, because analogous modulations were observed on shifting cells from 28°C to 37°C or from 28°C to 28°C. However, no marked modulations in the synthesis of specific proteins were detected when amino acids were added to the medium fostering germ-tube formation under conditions of unimpaired overall rate of protein synthesis.
It is suggested that the modulations observed in cells incubated in GlcNAc-glucose medium could represent a response to a nutritional stress.     

Apoptosis of phagocytic cells induced by Candida albicans and production of IL-10

Macrophages co-incubated with Candida albicans strain CR1 in vitro showed early signs of apoptosis, but evolved to necrosis after 2 h. In this study, we investigated whether strain CR1 caused apoptosis or necrosis of macrophages after its inoculation into mice peritoneal cavity, and whether this correlated with the secretion of IL-10. Peritoneal macrophages from mice that received an inoculum of C. albicans CR1 showed signs of apoptosis and necrosis from 30 min to 2 h afterwards, whereas heat-killed C. albicans did not cause those effects. IL-10 production was low during the first 6 h post-infection, when macrophages predominated in the peritoneal exudate, whereas its higher production after 24 h correlated with an increase of neutrophils in the exudate. Treatment of CR1 with pepstatin (an inhibitor of proteinases) prevented the process of apoptosis and significantly reduced IL-10 production, suggesting that the increased production of IL-10 was caused by processes occurring during the initial phase of infection, such as apoptosis, necrosis and uptake of death cells.     

Biofilm formation by Candida albicans and Streptococcus mutans in the presence of farnesol: a quantitative evaluation

The aim of this study was to evaluate the effect of the QS molecule farnesol on single and mixed species biofilms formed by Candida albicans and Streptococcus mutans. The anti-biofilm effect of farnesol was assessed through total biomass quantification, counting of colony forming units (CFUs) and evaluation of metabolic activity. Biofilms were also analyzed by scanning electron microscopy (SEM). It was observed that farnesol reduced the formation of single and mixed biofilms, with significant reductions of 37% to 90% and 64% to 96%, respectively, for total biomass and metabolic activity. Regarding cell viability, farnesol treatment promoted significant log reductions in the number of CFUs, ie 1.3–4.2 log₁₀ and 0.67–5.32 log₁₀, respectively, for single and mixed species biofilms. SEM images confirmed these results, showing decreases in the number of cells in all biofilms. In conclusion, these findings highlight the role of farnesol as an alternative agent with the potential to reduce the formation of pathogenic biofilms.     

Glutathione levels during thermal induction of the yeast-to-mycelial transition in Candida albicans

Glutathione (GSH) levels were directly monitored by reverse phase HPLC during the thermal yeast-to-mycelial induction of Candida albicans. The GSH levels decreased approximately 100-fold within 120 min which corresponded to the time of maximal yeast-to-mold conversion. The yeast to mold conversion was inhibited by 1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one (CDDP), a thiol-specific alkylator, which prevented the decline in GSH levels. These results are discussed with respect to the potential involvement of intracellular GSH levels in regulation of the yeast-to-mold dimorphism in Candida albicans.     

Nitric oxide-dependent killing of Candida albicans by murine peritoneal cells during an experimental infection

Abstract The phagocytic and candidacidal activities of the peritoneal cells of Candida albicans -infected mice were studied 20 days following experimental infection. Both activities were enhanced during infection. The production of nitric oxide (NO) by the peritoneal cells of infected mice was determined, and an increase in the nitrite concentration in supernatants of peritoneal cell cultures was detected. The period of NO production by the peritoneal cells coincided partially with the period of enhanced C. albicans killing. The inhibition of NO synthesis by N -monomethyl- l -arginine was concomitant with inhibition of candidacidal activity. We conclude that NO systhesis is the primary candidacidal mechanism of the murine peritoneal cells activated by C. albicans infection.     

白色念珠菌CaBEM1基因的克隆及其在酿酒酵母丝状生长中的作用

白色念珠菌在不同的生长条件下能发生显著的形态变化 ,这种变化由多种调控因子与信号转导途径所调控。酿酒酵母的G1期细胞周期蛋白Cln1和Cln2参与其形态发生 ,cln1/cln1、cln2 /cln2双缺失株不能形成菌丝。把白色念珠菌基因组文库导入cln1/cln1、cln2 /cln2缺失株 ,筛选能校正菌丝形成缺陷的基因 ,分离得到白色念珠菌中的CaBEM 1基因。从核苷酸序列推导 ,CaBEM1编码一种 6 32个氨基酸的蛋白质 ,氨基酸序列分析表明在其N端有 2个SH3结构域 ,中部有 1个PX结构域 ,C端有 1个PB1结构域 ;CaBem1的氨基酸序列与酿酒酵母的Bem1同源性达 38% ,与裂殖酵母的Scd2同源性达 32 %。在酿酒酵母的缺失株中异源表达CaBEM1,能够部分校正它们在氮源缺乏条件下的菌丝形成缺陷。这种菌丝形成的校正作用绕过MAPK途径和cAMP/PKA途径 ,表明CaBem1在菌丝形成中的作用可能位于这两条信号转导途径的下游     

Differential profiles of soluble proteins during the initiation of morphogenesis in Candida albicans

Candida albicans is a dimorphic fungus that can grow either as yeast or as mycelia. The mycelial form may be required for tissue penetration and therefore may have a role in pathogenesis. The protein profiles of the cell-free S100 fraction from budding yeast cells and germ tube-forming cells (an early stage of the transition between yeast and mycelia) were evaluated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yeast growth or germ tube formation was induced in carbon-starved cells at 37° C by either glucose, galactose or N-acetylglucosamine at pH 4.5 or pH 6.7. More than 400 constitutively synthesised polypeptides were identified on 2-D PAGE by silver staining. A few polypeptides which seem to reflect the release from carbon starvation were detected, but no polypeptides unique to either morphology were observed. Fractionation of S100 preparations by polyethylenimine or heparin-agarose affinity chromatography, which have been used to detect DNA-binding proteins, revealed several proteins that were synthesised on the resumption of cell growth or in response to pH difference. Heparin-agarose also bound novel polypeptides in the size range 130–200 kDa that were preferentially synthesised in germ tube-forming cells. These results suggest that any protein factors that might exert a regulatory role early in germ tube formation are of low abundance, and that a minor group of soluble proteins involved in C. albicans morphogenesis may be differentially synthesised. Received: 11 March 1996 / Accepted: 10 July 1996     

CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响

目的 研究CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响.方法 显微镜下观察,计算菌丝形成率,比较CRK1基因缺失菌（Δcrk1菌）及标准菌SC5314形成菌丝的能力;建立肠黏膜模型,计算黏附率,评价CRK1基因缺失对白念珠菌黏附的影响;MTT法及结晶紫法（CV）评价CRK1基因缺失对白念珠菌生物被膜形成的影响.结果 与SC5314相比,Δcrk1菌分别在10％胎牛血清和RPMI-1640培养条件下形成菌丝能力均较弱,两者之间有统计学差异;Δcrk1菌在60、90、120 min时对肠黏膜的黏附数明显少于标准菌SC5314,两者之间有统计学差异;通过MTT法、结晶紫法两种方法证实了,在经48 h培养后,Δcrk1菌与其标准菌SC5314相比,形成生物膜的能力弱,两者之间差异有统计学差异.结论 CRK 1基因缺失影响白念珠菌菌丝二态性的转化,进而影响黏附力和生物被膜的形成.     

Influence of aeration of Candida albicans during culturing on their surface aggregation in the presence of adhering Streptococcus gordonii

Candida albicans surfaces are extremely sensitive to changes in growth conditions. In this study, adhesion to glass of aerated and non-aerated C. albicans ATCC 10261 in the presence and absence of adhering Streptococcus gordonii NCTC 7869 was determined in a parallel plate flow chamber. In addition, the influence of aeration on the yeast cell surface hydrophobicity, surface charge, and elemental cell surface composition was measured. S. gordonii adhering at the glass surface caused a reduction in the initial deposition rate of C. albicans, regardless of aeration. In a stationary end-point, only adhesion of non-aerated C. albicans was suppressed by the adhering S. gordonii. Non-aerated yeasts had a higher O/C elemental surface concentration ratio, indicative of cell surface polysaccharides, than aerated yeasts, at the expense of nitrogen-rich cell surface proteins. Both yeasts were essentially uncharged, but the nitrogen-rich cell surface of aerated yeasts had a slightly higher water contact angle than non-aerated yeasts. Summarizing, this study suggests that highly localized, hydrophobic cell surface proteins on C. albicans are a prerequisite for their interaction with adhering streptococci.     

乳杆菌代谢物对白色念珠菌影响的体外试验研究

目的从阴道微生态平衡角度出发,观察乳杆菌(DM9811)代谢物对白色念珠菌的抑杀效果,并行组份分析,探索CV(candidal vaginitis,CV)的生态疗法.方法取CV患者阴道分泌物30例,分离培养,鉴定,保留白色念珠菌菌株.1.以提取液(以下简称提取液)Ⅰ和Ⅱ分别代替蒸馏水配制培养基SⅠ和SⅡ,将保留的白色念珠菌株传代5次后,配制菌液,接种于S、SⅠ和SⅡ中,培养后活菌计数.2.对提取液Ⅰ和Ⅱ倍比稀释,并代替蒸馏水配制多个培养基SⅠ和SⅡ,测定SⅠ和SⅡ中没有菌落生长的最低有效浓度(MIC).3.气相色谱测定提取液中挥发酸类成分并定量.4.选择提取液最低浓度加1的方法配制培养基,比较提取液和乙酸水溶液对白色念珠菌的抑制效果.结果1.白色念珠菌生长24 h后计活菌数,将SⅠ与S,SⅡ与S中活菌数相比较,非参数配对T检验P＜0.01.2.提取液Ⅰ最低有效浓度为1:4,提取液Ⅱ最低有效浓度为1:2.3.气相色谱测定提取液Ⅱ中挥发酸类主要成分为乙酸,浓度为0.235 9 S/100ml.4.提取液和乙酸对白色念珠菌的生长都有抑制作用,但提取液的抑制作用明显强于乙酸.结论乳酸杆菌DM9811代谢物对白色念珠菌有明显的抑菌和杀菌作用,可作为一种新型的生态制剂进行开发,用于治疗念珠菌阴道炎.     

Effect of 2, 4-di-tert-butylphenol on growth and biofilm formation by an opportunistic fungus Candida albicans

Candida albicans, an opportunistic pathogen, has been known to form hypoxic biofilms on medical devices which in turn confers resistance towards antifungals, resulting in subsequent therapeutic failures. Inclusion of anti-biofilm agents in the control of infections is a topic of current interest in developing potential anti-infectives. The in vitro anti-fungal and anti-biofilm efficacy of 2,4-di-tert-butyl phenol [DTBP] was evaluated in this study, which revealed the potential fungicidal action of DTBP at higher concentrations where fluconazole failed to act completely. DTBP also inhibited the production of hemolysins, phospholipases and secreted aspartyl proteinase which are the crucial virulence factors required for the invasion of C. albicans. Various anti-biofilm assays and morphological observations revealed the efficacy of DTBP in both inhibiting and disrupting biofilms of C. albicans. Inhibition of hyphal development, a key process that aids in initial adhesion of C. albicans, was observed, and this could be a mechanism for the anti-biofilm activity of DTBP.     

Biofilm formation by Candida albicans mutants for genes coding fungal proteins exhibiting the eight-cysteine-containing CFEM domain

Several features and functions of a Candida albicans gene, PGA10 (also designated as RBT51), coding for a putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as CFEM (Common in several Fungal Extracellular Membrane proteins), are described. The ORF of the gene (ORF19.5674) encoded a protein of 250 amino acids, with a predicted molecular mass of 25.17 kDa. The product of the PGA10 gene also exhibited some features reminiscent of a class II-type hydrophobin. Deletion of PGA10 resulted in a cascade of pleiotropic effects, mostly affecting cell-surface-related properties. Thus, the null pga10Delta mutant displayed an increased sensitivity to cell-wall-perturbing agents and formed fragile biofilms that appeared partially split and weakly attached to the substratum. The biofilm-forming ability of several C. albicans mutants with single, double and triple deletions of genes encoding other protein species also containing the CFEM domain (RBT5 and WAP1/CSA1) was determined. These mutants also exhibited an abnormal ability to form biofilms. Overall, the evidence presented here suggests that fungal proteins containing the CFEM domain (Pga10p/Rbt51p, Rbt5p and Wap1p/Csa1p) may play a key role in the formation, development and/or maintenance of the biofilm structure in C. albicans.     

Antifungal drugs effect adherence of Candida albicans to acrylic surfaces by changing the zeta-potential of fungal cells

The effect of sub-inhibitory concentrations of antifungal drugs on the adherence of Candida albicans to acrylic surfaces was investigated. Among five antifungals tested, azalomycin F and aculeacin A significantly enhanced the adherence. The zeta-potential of fungal cells was affected by antifungal drugs, whereas no significant change in cell surface hydrophobicity was observed. The relationship obtained between the change in the adherence and that in zeta-potential suggests that the enhanced adherence was caused by decreased electric repulsive forces.     

汉防己甲素及其联合氟康唑对白念珠菌细胞周期影响的初步研究

目的 探讨汉防己甲素联合氟康唑对白念珠菌细胞周期的影响.方法 将白念珠菌CA-1菌悬液与汉防己甲素和（或）氟康唑共培养12h,应用流式细胞仪测定空白对照组、汉防己甲素组、氟康唑组及汉防己甲素联合氟康唑组DNA含量.比较细胞周期各期DNA含量变化并计算增殖抑制率PI％,分析汉防己甲素及其联合氟康唑对白念珠菌细胞周期的影响.结果 汉防己甲素、氟康唑组与对照组S期DNA含量相比,分别能增加17.25％ （P =0.018）与6.54％ （P ＞0.05）,其联合运用效果更明显,S期DNA含量可增加31.52％ （P =0.002）.这说明汉防己甲素能将白念珠菌细胞阻滞在S期.结论 汉防己甲素能抑制白念珠菌细胞DNA合成,阻断白念珠菌细胞周期进程,抑制细胞分裂,其与氟康唑联用时阻滞作用更显著.