Genetic control of Candida albicans biofilm development

Candida species cause frequent infections owing to their ability to form biofilms - surface-associated microbial communities - primarily on implanted medical devices. Increasingly, mechanistic studies have identified the gene products that participate directly in the development of Candida albicans biofilms, as well as the regulatory circuitry and networks that control their expression and activity. These studies have uncovered new mechanisms and signals that govern C. albicans biofilm development and associated drug resistance, thus providing biological insight and therapeutic foresight.     

Genetic analysis of Candida albicans morphological mutants

In contrast to some other strains, Candida albicans 1001 gave rise, upon UV irradiation, to mutants displaying a 'rough colony' morphology associated with a permanent alteration in morphogenesis which determined growth of the cells mostly as pseudohyphae. One of these mutants, C. albicans 1001FR, could form sectored (rough/smooth) colonies spontaneously, and with increasing frequency by treatment with mild UV doses (32-64 microJ mm-2). Rough sectors corresponded to stable 'rough-filamentous' strains which never segregated smooth strains. On the other hand, smooth sectors consisted mainly of yeast cells which could occasionally revert to a rough-filamentous phenotype. We suggest that C. albicans 1001 is heterozygous for some gene involved in the control of morphogenesis, and that the described mutants should be of help in the characterization of the genetic control of dimorphism in C. albicans.     

白念珠菌菌丝发育的遗传调控

白念珠菌(Candidaalbicans)是人体内最重要的机会型致病真菌,能以酵母、假菌丝、菌丝等多种形态存在。白念珠菌的菌丝发育与它的致病性成正相关,这一过程由胞内多种信号转导途径所调控。现对控制白念珠菌菌丝发育的主要信号转导途径进行综述。     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

Incorporation of dexamethasone by Candida albicans

The incorporation of 3H-dexamethasone into Candida albicans has been studied. The results indicate that the steroid is incorporated unchanged and primarily into the cell wall and membrane of the organism. The incorporation appears to be of a noncovalent type.     

Keratinolytic proteinase produced by Candida albicans

Candida albicans was cultivated in various media that contained human stratum corneum, human scalp hair or keratin powder (cow's hoof) as a nitrogen source. Production of a keratinolytic proteinase (KPase) was observed when C. albicans was incubated in the medium containing stratum corneum. However, there was no production of a KPase that could digest human stratum corneum in the medium containing hair or keratin powder. alpha-fibrous protein extracted from human stratum corneum was digested by the KPase. The pH optimum of the enzyme was 4.0 and enzyme activity was inhibited by pepstatin A and chymostatin. The KPase, a kind of carboxyl proteinase, may be important for C. albicans to enable it to play a pathogenic role in vivo.     

Cell-associated collagenolytic activity by Candida albicans

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

间接免疫荧光法快速鉴别白念珠菌和都柏林念珠菌的初步研究

目的 用一种新制备的单克隆抗体MAb03．2Cl-C2鉴别生物学形态相近的白念珠菌和都柏林念珠菌。方法 用小鼠体内诱导法制备抗白念珠菌芽管胞壁外膜单克隆抗体MAb03．2Cl-C2。用不完全RPMI1640培养液、L—DMEM、H—DMEM、完全1640液、小牛血清诱导白念珠菌和都柏林念珠菌芽管及菌丝形成,间接免疫荧光（IIF）方法检测都柏林念珠菌芽管或菌丝表面有无可与该单抗相结合的成分。收集临床口腔念珠菌病标本涂片,直接做IIF试验。结果 用不完全RP-MI1640培养液37℃,6h可同时最高效率地诱导白念珠菌和都柏林念珠菌芽管或菌丝形成。单抗MAb03．2Cl-C2仅与白念珠菌芽管或菌丝特异性地结合,与都柏林念珠菌的孢子和菌丝不能结合。结论 单抗MAh03．2Cl-C2可用于白念珠菌和都柏林念珠菌实验室的速鉴别。     

Biosynthesis of chemical compounds by Candida albicans and Candida glabrata

BackgroundIn the last three decades the species of Candida have been of great interest due to the high mortality rates that they cause in immunocompromised and hospitalized patients. These species are opportunistic pathogens and they have inhabited other environments long before colonizing human cells. Among these environments we find wastewater from mines, and water from aquifers and soils that contain high concentrations of precious metals as well as toxic and base metals.AimsThe aim of this study was to assess whether Candida albicans and Candida glabrata are able to maintain homeostasis in the presence of zinc, copper, cobalt or silver.MethodsTo achieve the objective, each of the Candida species was exposed to every single metal individually in a salt solution. Subsequently the treated cells were lysed to evaluate the compounds formed by means of Scanning Electron Microscopy-Energy Dispersive X-ray spectroscopy (SEM-EDS).ResultsWhen analyzing the compounds that both C. albicans and C. glabrata formed in the presence of each of the metals, we found that they had synthesized silver sulfide (Ag₂S), cobalt sulfate (CoSO₄), zinc phosphate (Zn₃(PO₄)₂), or copper oxide (CuO).ConclusionsOur results indicate that both C. albicans and C. glabrata have enzymatic and non-enzymatic mechanisms that allow them to achieve homeostasis in a different specific manner for each of the single metals to which they were exposed. To our knowledge, this is the first work reporting that C. albicans and C. glabrata can reduce different metals, with the subsequent formation of sulfides, sulfates, phosphates and oxides. This ability, developed over time by these Candida species, is probably a kind of biochemical mechanism in order to survive and colonize many different environments, from water or soil to humans. For this reason, C. albicans and C. glabrata make up an excellent model of study, both from a medical and biotechnical point of view.     

用ELISA一步法检测白色念珠菌

用白色念珠菌临床分离株免疫家兔并经纯化获得特异性抗体,建立了检测临床标本中白色念珠菌抗原的ＥＬＩＳＡ一步法,此法的敏感性为９９％,特异性为９８３％,与其它念珠菌和临床常见细菌均无交叉反应,整个反应只需１小时即可完成,可望成为一种取代培养法的快速诊断方法     

Characteristics of biofilm formation by Candida albicans

A variety of manifestations of Candida albicans infections are associated with the formation of biofilms on the surface of biomaterials. Cells in biofilms display phenotypic traits that are dramatically different from their free-floating planktonic counterparts, such as increased resistance to anti-microbial agents and protection form host defenses. Here, we describe the characteristics of C. albicans biofilm development using a 96 well microtitre plate model, microscopic observations and a colorimetric method based on the use of a modified tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide, XTT) to monitor metabolic activities of cells within the biofilm. C. albicans biofilm formation was characterized by initial adherence of yeast cells (0-2 h), followed by germination and micro-colony formation (2-4 h), filamentation (4-6 h), monolayer development (6-8 h), proliferation (8-24 h) and maturation (24-48 h). The XTT-reduction assay showed a linear relationship between cellular density of the biofilm and metabolic activity. Serum and saliva pre-conditioning films increased the initial attachment of C. albicans, but had minimal effect on subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize C. albicans biofilms. Mature C. albicans biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. albicans biofilms displayed a complex three dimensional structure which demonstrated spatial heterogeneity and a typical architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. albicans cells against clinically used fluconazole and amphotericin B as compared to their planktonic counterparts.     

Inhibition of endothelium-dependent relaxation by Candida albicans

This study examines the effects of Candida albicans on acethylcholine-induced, endothelium-dependent relaxation of thoracic aorta of rabbits, precontracted by phenylephrine (10(-7) M). Isolated vessel rings were incubated with C. albicans, Saccharomyces cerevisiae, or their mannans, and endothelium-dependent relaxation was measured by the induction of acethylcholine. Endothelium-dependent relaxation remained unaffected after 3 hours by either C. albicans or S. cerevisiae, or their mannans. After 24 hours, however, incubation with C. albicans had completely abolished relaxation, whereas relaxation was decreased by mannan of C. albicans and continued unaffected by S. cerevisiae. In contrast, no change was registered with a 24 hours incubation of C. Albicans in a sodium nitroprusside-induced, endothelium-independent, vascular smooth muscle relaxation. Microscopical investigation of the morphological structure of vessel walls revealed penetration of C. albicans on the intimal surface after 3 hours incubation and infiltration of the yeast through the vessel wall after 24 hours. No changes in vessel morphology occurred after 3 or 24 hours with S. cerevisiae or the mannan of C. albicans. These results show the ability of C. albicans to inhibit endothelium-dependent, but not endothelium-independent, relaxation of vascular smooth muscle and may have important implications for functional damage to endothelial cells and the regulation of vessel tone and blood flow.     

Genetic control of susceptibility to infection with Candida albicans in mice

Candida albicans is an opportunistic pathogen that causes acute disseminated infections in immunocompromised hosts, representing an important cause of morbidity and mortality in these patients. To study the genetic control of susceptibility to disseminated C. albicans in mice, we phenotyped a group of 23 phylogenetically distant inbred strains for susceptibility to infection as measured by extent of fungal replication in the kidney 48 hours following infection. Susceptibility was strongly associated with the loss-of-function mutant complement component 5 (C5/Hc) allele, which is known to be inherited by approximately 40% of inbred strains. Our survey identified 2 discordant strains, AKR/J (C5-deficient, resistant) and SM/J (C5-sufficient, susceptible), suggesting that additional genetic effects may control response to systemic candidiasis in these strains. Haplotype association mapping in the 23 strains using high density SNP maps revealed several putative loci regulating the extent of C. albicans replication, amongst which the most significant were C5 (P value = 2.43×10⁻¹¹) and a novel effect on distal chromosome 11 (P value = 7.63×10⁻⁹). Compared to other C5-deficient strains, infected AKR/J strain displays a reduced fungal burden in the brain, heart and kidney, and increased survival, concomitant with uniquely high levels of serum IFNγ. C5-independent genetic effects were further investigated by linkage analysis in an [A/JxAKR/J]F2 cross (n = 158) where the mutant Hc allele is fixed. These studies identified a chromosome 11 locus (Carg4, Candida albicans resistance gene 4; LOD = 4.59), and a chromosome 8 locus (Carg3; LOD = 3.95), both initially detected by haplotype association mapping. Alleles at both loci were inherited in a co-dominant manner. Our results verify the important effect of C5-deficiency in inbred mouse strains, and further identify two novel loci, Carg3 and Carg4, which regulate resistance to C. albicans infection in a C5-independent manner.     

Flocculation phenomenon of Candida albicans by lysozyme

Young colonies of Sabouraud's glucose agar room temperature culture ofCandida species from human isolation were suspended in distilled water. The suspension was mixed with a solution of lysozyme and incubated in a 37° C water bath. Within 3–5 hours, various species ofCandida cells showed flocculation to varying degrees which occurred at varying periods of onset. Among sevenCandida species,Candida albicans andCandida stellatoidea showed the strongest flocculation, earliest onset and most solution clarity than did any other species.Candida stellatoidea was indistinguishable fromCandida albicans in its degree of flocculation, and in the clarity of solution.Candida species may be arranged in the following order according to their decreasing positivity in flocculation:

1. Candida albicans
2. Candida stellatoidea
3. Candida tropicalis
4. Candida krusei
5. Candida pseudotropicalis
6. Candida parapsilosis
7. Candida guilliermondii
8. Saccharomyces species may be placed afterCandida guilliermondii.

It seems possible to separate theCandida species into 3 groups by the rate of flocculation, and clarity of solution. Group I.Candida albicans andCandida stellatoidea. Group II.Candida tropicalis, C. krusei andCandida pseudotropicalis. Group III.Candida parapsilosis andCandida guilliermondii. Saccharomyces specimens (S. cerevisiae and others) were placed after group III.     

Bioassay for siderophore utilization by Candida albicans

A convenient plate assay which is sensitive to medium pH has been developed to evaluate potential siderophores of Candida albicans. Adding a siderophore to a filter paper disk on chemically defined Lee's agar (final pH 7.2) seeded with the test strain reversed the growth inhibitory effects of the supplemented (25-100 micrograms/ml) iron chelator ethylenediaminedi(o-hydroxyphenylacetic acid), to provide a zone of growth stimulation. This bioassay has been used to demonstrate the structure-activity relationships of ferrichrome and several water-soluble hydroxamate peptide building blocks of this natural siderophore. Of all compounds so evaluated, ferrichrome exhibited the best activity.     

Genetic structure of typical and atypical populations of Candida albicans from Africa

Atypical isolates of the pathogenic yeast Candida albicans have been reported with increasing frequency. To investigate the origin of a set of atypical isolates and their relationship to typical isolates, we employed a combination of molecular phylogenetic and population genetic analyses using rDNA sequencing, PCR fingerprinting, and analysis of co-dominant DNA nucleotide polymorphisms to characterize the population structure of one typical and two atypical populations of C. albicans from Angola and Madagascar. The extent of clonality and recombination was assessed in each population. The analyses revealed that the structure of all three populations of C. albicans was predominantly clonal but, as in previous studies, there was also evidence for recombination. Allele frequencies differed significantly between the typical and the atypical populations, suggesting very low levels of gene flow between them. However, allele frequencies were quite similar in the two atypical C. albicans populations, suggesting that they are closely related. Phylogenetic analysis of partial sequences encoding the nuclear 26S rDNA demonstrated that all three populations belong to a single monophyletic group, which includes the type strain of C. albicans.