Adherence of Candida albicans and Candida dubliniensis to buccal and vaginal cells

Twenty-seven Candida albicans strains and 26 Candida dubliniensis strains, isolated from HIV patients, were tested for their adherence to buccal and vaginal epithelial cells. Both species showed important levels of adhesion to buccal and vaginal epithelial cells, although C. albicans showed the highest levels of adhesion. These results suggest that both Candida species are well adapted, in terms of adhesion capability, to the oral and vaginal environment.     

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。     

都柏林念珠菌的鉴定及与白念珠菌3种表型鉴别方法的研究

目的 从临床分离的念珠菌中进一步鉴定都柏林念珠菌,并评价3种表型鉴别白念珠菌和都柏林念珠菌的方法.方法 对17株临床分离并初步鉴定的白念珠菌和1株ATCC白念珠菌标准株,采用PHR1同源序列PCR法检测,鉴定出其中的都柏林念珠菌;分别采用45℃生长试验、YEPD(1%酵母浸膏,2%蛋白胨,2%葡萄糖)液基39℃芽管生成试验、Staib琼脂(鸟食琼脂)厚壁孢子形成试验对两种菌的表型特点进行比较.结果 17株临床分离的白念珠菌中有3株鉴定为都柏林念珠菌;45℃时,两种菌在改良沙堡弱琼脂上均无明显生长,YEPD液基中仅有1株白念珠菌生长良好;YEPD液基39℃培养2种菌均无芽管生成;Staib琼脂培养72h,3株都柏林念珠菌中有2株可形成厚壁孢子,而白念珠菌则无,与PHR1同源序列检测结果基本一致.结论 PHR1同源序列检测是鉴别都柏林念珠菌与白念珠菌的可靠方法,Staib琼脂厚壁孢子形成试验有助于鉴别两菌,45℃生长试验和YEPD液基39℃芽管生成试验则不能有效鉴别两菌.     

The closely related species Candida albicans and Candida dubliniensis can mate

Because Candida dubliniensis is closely related to Candida albicans, we tested whether it underwent white-opaque switching and mating and whether white-opaque switching depended on MTL homozygosity and mating depended on switching, as they do in C. albicans. We also tested whether C. dubliniensis could mate with C. albicans. Sequencing revealed that the MTLalpha locus of C. dubliniensis was highly similar to that of C. albicans. Hybridization with the MTLa1, MTLa2, MTLalpha1, and MTLalpha2 open reading frames of C. albicans further revealed that, as in C. albicans, natural strains of C. dubliniensis exist as a/alpha, a/a, and alpha/alpha, but the proportion of MTL homozygotes is 33%, 10 times the frequency of natural C. albicans strains. C. dubliniensis underwent white-opaque switching, and, as in C. albicans, the switching was dependent on MTL homozygosis. C. dubliniensis a/a and alpha/alpha cells also mated, and, as in C. albicans, mating was dependent on a switch from white to opaque. However, white-opaque switching occurred at unusually high frequencies, opaque cell growth was frequently aberrant, and white-opaque switching in many strains was camouflaged by an additional switching system. Mating of C. dubliniensis was far less frequent in suspension cultures, due to the absence of mating-dependent clumping. Mating did occur, however, at higher frequencies on agar or on the skin of newborn mice. The increases in MTL homozygosity, the increase in switching frequencies, the decrease in the quality of switching, and the decrease in mating efficiency all reflected a general deterioration in the regulation of developmental processes, very probably due to the very high frequency of recombination and genomic reorganization characteristic of C. dubliniensis. Finally, interspecies mating readily occurred between opaque C. dubliniensis and C. albicans strains of opposite mating type in suspension, on agar, and on mouse skin. Remarkably, the efficiency of interspecies mating was higher than intraspecies C. dubliniensis mating, and interspecies karyogamy occurred readily with apparently the same sequence of nuclear migration, fusion, and division steps observed during intraspecies C. albicans and C. dubliniensis mating and Saccharomyces cerevisiae mating.     

Stress-Induced Phenotypic Switching in Candida albicans

Candida albicans is both a common commensal and an opportunistic pathogen, being a prevalent cause of mucosal and systemic infections in humans. Phenotypic switching between white and opaque forms is a reversible transition that influences virulence, mating behavior, and biofilm formation. In this work, we show that a wide range of factors induces high rates of switching from white to opaque. These factors include different forms of environmental stimuli such as genotoxic and oxidative stress, as well as intrinsic factors such as mutations in DNA repair genes. We propose that these factors increase switching to the opaque phase via a common mechanism—inhibition of cell growth. To confirm this hypothesis, growth rates were artificially manipulated by varying expression of the CLB4 cyclin gene; slowing cell growth by depleting CLB4 resulted in a concomitant increase in white-opaque switching. Furthermore, two clinical isolates of C. albicans, {"type":"entrez-protein","attrs":{"text":"P37005","term_id":"729917","term_text":"P37005"}}P37005 and L26, were found to naturally exhibit both slow growth and high rates of white-opaque switching. Notably, suppression of the slow growth phenotype suppressed hyperswitching in the {"type":"entrez-protein","attrs":{"text":"P37005","term_id":"729917","term_text":"P37005"}}P37005 isolate. Based on the sensitivity of the switch to levels of the master regulator Wor1, we propose a model for how changes in cellular growth modulate white-opaque switching frequencies.     

A rapid screening test to distinguish between Candida albicans and Candida dubliniensis using NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy combined with a statistical classification strategy (SCS) successfully distinguished between Candida albicans and Candida dubliniensis. 96% of the isolates from an independent test set were identified correctly. This proves that this rapid approach is a valuable method for the identification and chemotaxonomic characterisation of closely related taxa. Most discriminatory regions were correlated with metabolite profiles, indicating biochemical differences between the two species.     

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.     

Candida dubliniensis and Candida albicans display surface variations consistent with observed intergeneric coaggregation

Adherence of yeasts to other microorganisms and epithelial cell surfaces is important in their colonization. Comparative studies based on the coaggregation of Candida dubliniensis versus Candida albicans with Fusobacterium nucleatum and other oral bacteria suggested differences in the surfaces of these yeasts. Transmission electron microscopy was used to test the hypothesis that there are morphologic variations in the cell surface of these two species. C. dubliniensis type strain CD36 and C. albicans ATCC 18804 were grown on Sabouraud's dextrose agar at various growth temperatures. In some experiments suspensions of yeast cells were treated with dithiothreitol. Fixation for transmission electron microscopy was accomplished using dimethylsulfoxide and alcian blue added to 3% paraformaldehyde and 1% glutaraldahyde in cacodylate buffer. The cell wall of both species was predominantly electron lucent and was visibly differentiated into several layers. A thin electron dense outer layer was seen with clearly visible fibrillar structures, closely associated to the cytoplasmic membrane. The length of the fibrils of the C. albicans cells grown at 37 degrees C was approximately two times greater than those of the cells grown at 25 degrees C. The fibrils of the 37 degrees C-grown cells were thin, distinct and tightly packed whereas those of the 25 degrees C-grown cells appeared blunt, loosely spaced and aggregated. C. dubliniensis demonstrated short, blunt fibrils appearing similar to those of the 25 degrees C-grown C. albicans cells. C. dubliniensis showed no difference in the density, length and arrangement of fibrils between the 25 degrees C and 37 degrees C growth temperatures. The shortest and most aggregated fibrils seen were of the 45 degrees C-grown C. albicans cells. Dithiothreitoltreated 37 degrees C-grown C. albicans cells revealed a distorted and partially destroyed fibrillar layer. In this investigation C. dubliniensis, unlike C. albicans, displayed an outer fibrillar layer that did not vary with variations in growth temperature. In addition, the fibrils on the C. dubliniensis cells were similar to those of the 25 degrees C-grown C. albicans in that they were considerably shorter and less dense than those of the 37 degrees C-grown C. albicans cells. It can be postulated, that C. dubliniensis exhibits constant cell surface characteristics consistent with hydrophobicity and that this property may give this species an ecological advantage. Therefore, C. dubliniensis may compete well in oral environments via enhanced attachment to oral microbes and other surfaces, perhaps even more efficiently than C. albicans.     

Lower filamentation rates of Candida dubliniensis contribute to its lower virulence in comparison with Candida albicans

Candida albicans and C. dubliniensis are very closely related yeast species. In this study, we have conducted a thorough comparison of the ability of the two species to produce hyphae and their virulence in two infection models. Under all induction conditions tested C. albicans consistently produced hyphae more efficiently than C. dubliniensis. In the oral reconstituted human epithelial model, C. dubliniensis isolates grew exclusively in the yeast form, while the C. albicans strains produced abundant hyphae that invaded and caused significant damage to the epithelial tissue. In the oral-intragastric infant mouse infection model, C. dubliniensis strains were more rapidly cleared from the gastrointestinal tract than C. albicans. Immunosuppression of Candida-infected mice caused dissemination to internal organs by both species, but C. albicans was found to be far more effective at dissemination than C. dubliniensis. These data suggest that a major reason for the comparatively low virulence of C. dubliniensis is its lower capacity to produce hyphae.     

Extensive chromosome rearrangements distinguish the karyotype of the hypovirulent species Candida dubliniensis from the virulent Candida albicans

Candida dubliniensis and Candida albicans, the most common human fungal pathogen, have most of the same genes and high sequence similarity, but C. dubliniensis is less virulent. C. albicans causes both mucosal and hematogenously disseminated disease, C. dubliniensis mostly mucosal infections. Pulse-field electrophoresis, genomic restriction enzyme digests, Southern blotting, and the emerging sequence from the Wellcome Trust Sanger Institute were used to determine the karyotype of C. dubliniensis type strain CD36. Three chromosomes have two intact homologues. A translocation in the rDNA repeat on chromosome R exchanges telomere-proximal regions of R and chromosome 5. Translocations involving the remaining chromosomes occur at the Major Repeat Sequence. CD36 lacks an MRS on chromosome R but has one on 3. Of six other C. dubliniensis strains, no two had the same electrophoretic karyotype. Despite extensive chromosome rearrangements, karyotypic differences between C. dubliniensis and C. albicans are unlikely to affect gene expression. Karyotypic instability may account for the diminished pathogenicity of C. dubliniensis.     

Purification and germination of Candida albicans and Candida dubliniensis chlamydospores cultured in liquid media

Candida albicans and Candida dubliniensis are the only Candida sp. that have been observed to produce chlamydospores. The function of these large, thick-walled cells is currently unknown. In this report, we describe the production and purification of chlamydospores from these species in defined liquid media. Staining with the fluorescent dye FUN-1 indicated that chlamydospores are metabolically active cells, but that metabolic activity is undetectable in chlamydospores that are >30 days old. However, 5–15-day-old chlamydospores could be induced to produce daughter chlamydospores, blastospores, pseudohyphae and true hyphae depending on the incubation conditions used. Chlamydospores that were preinduced to germinate were also observed to escape from murine macrophages following phagocytosis, suggesting that these structures may be viable in vivo . Mycelium-attached and purified chlamydospores rapidly lost their viability in water and when subjected to dry stress, suggesting that they are unlikely to act as long-term storage structures. Instead, our data suggest that chlamydospores represent an alternative specialized form of growth by C. albicans and C. dubliniensis .     

The Usefulness of DNA Sequencing After Extraction by Whatman FTA Filter Matrix Technology and Phenotypic Tests for Differentiation of Candida albicans and Candida dubliniensis

Since C. dubliniensis is similar to C. albicans phenotypically, it can be misidentified as C. albicans. We aimed to investigate the prevalence of C. dubliniensis among isolates previously identified as C. albicans in our stocks and to compare the phenotypic methods and DNA sequencing of D1/D2 region on the ribosomal large subunit (rLSU) gene. A total of 850 isolates included in this study. Phenotypic identification was performed based on germ tube formation, chlamydospore production, colony colors on chromogenic agar, inability of growth at 45 °C and growth on hypertonic Sabouraud dextrose agar. Eighty isolates compatible with C. dubliniensis by at least one phenotypic test were included in the sequence analysis. Nested PCR amplification of D1/D2 region of the rLSU gene was performed after the fungal DNA extraction by Whatman FTA filter paper technology. The sequencing analysis of PCR products carried out by an automated capillary gel electrophoresis device. The rate of C. dubliniensis was 2.35 % (n = 20) among isolates previously described as C. albicans. Consequently, none of the phenotypic tests provided satisfactory performance alone in our study, and molecular methods required special equipment and high cost. Thus, at least two phenotypic methods can be used for identification of C. dubliniensis, and molecular methods can be used for confirmation.     

Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO₂ induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO₂, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.     

Utility of random amplified polymorphic DNA in the discrimination between Candida albicans and Candida dubliniensis

Candida dubliniensisis a recently described species closely related to Candida albicans. Since the discrimination between both species by conventional mycological methods is not easy, many researchers have been trying DNA-related techniques in order to identify C. dubliniensis correctly. In this study, we propose the use of the random amplification of polymorphic DNA (RAPD) with a commercialized short primer which discriminates between both species. This oligonucleotide, AB1-12, allowed also separating C. albicans isolates into four different genotypes. These genotypes were different from the unique genotype observed in C. dubliniensis.     

The presence of fluconazole-resistant Candida dubliniensis strains among Candida albicans isolates from immunocompromised or otherwise debilitated HIV-negative Turkish patients

The newly described species Candida dubliniensis phenotipically resembles Candida albicans in many respects and so it could be easily misidentified. The present study aimed at determining the frequency at which this new Candida species was not recognized in the authors' university hospital clinical laboratory and to assess antifungal susceptibility. In this study, six identification methods based on significant phenotypic characteristics each proposed as reliable tests applicable in mycology laboratories for the differentiation of the two species were performed together to assess the clinical strains that were initially identified as C. albicans. Only the isolates which have had the parallel results in all methods were assessed as C. dubliniensis. One hundred and twenty-nine C. albicans strains isolated from deep mycosis suspected patients were further examined. Three of 129 C. albicans (2 from oral cavity, 1 from sputum) were reidentified as C. dubliniensis. One of the strains isolated from oral cavity and that from the sputum were obtained at two months intervals from the same patient with acute myeloid leukemia, while the other oral cavity strain was obtained from a patient who had previously been irradiated for a laryngeal malignancy. Isolates were all susceptible in vitro to amphotericin B, with the MIC range 0.125 to 0.5 &mgr;g/ml, resistant to fluconazole, with MICs >/=64 &mgr;g/ml, and resistant to ketoconazole, with MICs >/=16 &mgr;g/ml, dose-dependent to itraconazole with a MIC range 0.25-0.5 &mgr;g/ml, and susceptible to flucytosine, with a MIC range 1-4 &mgr;g/ml.     

Differential Interaction of the Two Related Fungal Species Candida albicans and Candida dubliniensis with Human Neutrophils

Candida albicans, the most common facultative human pathogenic fungus is of major medical importance, whereas the closely related species Candida dubliniensis is less virulent and rarely causes life-threatening, systemic infections. Little is known, however, about the reasons for this difference in pathogenicity, and especially on the interactions of C. dubliniensis with the human immune system. Because innate immunity and, in particular, neutrophil granulocytes play a major role in host antifungal defense, we studied the responses of human neutrophils to clinical isolates of both C. albicans and C. dubliniensis. C. dubliniensis was found to support neutrophil migration and fungal cell uptake to a greater extent in comparison with C. albicans, whereas inducing less neutrophil damage and extracellular trap formation. The production of antimicrobial reactive oxygen species, myeloperoxidase, and lactoferrin, as well as the inflammatory chemokine IL-8 by neutrophils was increased when stimulated with C. dubliniensis as compared with C. albicans. However, most of the analyzed macrophage-derived inflammatory and regulatory cytokines and chemokines, such as IL-1α, IL-1β, IL-1ra, TNF-α, IL-10, G-CSF, and GM-CSF, were less induced by C. dubliniensis. Similarly, the amounts of the antifungal immunity-related IL-17A produced by PBMCs was significantly lower when challenged with C. dubliniensis than with C. albicans. These data indicate that C. dubliniensis triggers stronger early neutrophil responses than C. albicans, thus providing insight into the differential virulence of these two closely related fungal species, and suggest that this is, in part, due to their differential capacity to form hyphae.     

Genotypic subgrouping of clinical isolates of Candida albicans and Candida dubliniensis by 25S intron analysis

AIMS: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates. METHODS: Twenty-one invasive and 18 noninvasive isolates were examined by PCR amplification of a transposable intron region in the 25S rRNA gene. Isolates were genotyped following analysis of the size of resulting DNA amplicons. The isolates could be subdivided into four genotypes (A-D). RESULTS: There was no significant difference between the frequency and genotype distribution of the invasive and noninvasive Candida isolates. IMPACT OF THE STUDY: Therapeutic prophylaxis against candidal infections remains an area of controversy. Any diagnostic markers that reflect the potential of isolates to become invasive should be fully explored, so that more focused antifungal intervention should be targeted at these patients with these potential invasive markers. This study demonstrated that analysis of the transposable intron region in the 25S rRNA gene may be useful in helping to differentiate C. albicans from C. dubliniensis isolates, without the need for sequence analysis, which may not be readily available at primary diagnostic laboratories. However, employment of this genotypic assay is not a suitable locus to determine invasiveness and other more reliable markers of invasiveness should be sought.     

Phenotypic resistance to amphotericin B in Candida albicans: relationship to glucan metabolism

The phenotypic resistance to amphotericin methyl ester (AME) of stationary phase cultures of Candida albicans was decreased by alkaline pH values and by treatment with 2-mercaptoethanol or glucanase preparations, and was increased by acid pH values, increased aeration, treatment with N-ethylmaleimide, or the presence of inhibitors of protein synthesis such as trichodermin. The effects of such treatments on endogenous glucanase activity and on the incorporation of glucose residues into the 'glucan fraction' of the organism were studied. The changes in the endogenous levels of lytic activities on laminarin [as a measure of the total (1 leads to 3)-beta-D-glucanase] and on p-nitrophenyl-beta-D-glucoside [reflecting the exo-(1 leads to 3)-beta-D-glucanase] were followed in C. albicans cells under a variety of conditions. Treatments which increased AME sensitivity stimulated both total and exo-(1 leads to 3)-beta-D-glucanase activities, while treatments which promoted resistance decreased the levels of both (1 leads to 3)-beta-D-glucanases. Changes in the 'glucan fraction' were followed by incubating suspensions of organisms in the presence of trace amounts of [U-14C]glucose. The rate of incorporation of radioactivity fell during the first 2-3 d of stationary phase culture and then rose to high values by 7-8 d; AME resistance increased throughout this period. The rate of incorporation was markedly stimulated by prior treatment of the organisms with 2-mercaptoethanol or glucanase and inhibited by trichodermin or treatment with N-ethylmaleimide. The addition in the concentration range 0.3-3 mM of the glucose analogues beta-D-allose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose or 5-thio-D-glucose to cultures 24 h after inoculation prevented any further increase in AME resistance for the next 2-3 d and resulted in a decrease in the level of resistance established at the time of addition. Radioactivity from 14C- or 3H-labelled analogues added, 24 h after inoculation, to stationary phase cultures was incorporated into the 'glucan fraction' of the organisms. The incorporation of glucose residues into the 'glucan fraction' is controlled by the activity of glucanases in producing glucose acceptor sites. The results reported confirm that there is a correlation between glucan metabolism, glucanase activity and resistance to AME, in that any factor leading to increased glucanase action also results in decreased resistance and vice versa, while incorporation of certain glucose analogues into the 'glucan fraction' delays the further increase in resistance.     

Lack of Candida africana and Candida dubliniensis in vaginal Candida albicans isolates in Turkey using HWP1 gene polymorphisms

Candida africana differs from the common strains of C. albicans and C. dubliniensis morphologically, physiologically, genetically, and, in particular, clinically. This fungal pathogen is primarily recovered from genital specimens, especially in vaginal specimens. In this investigation, we reexamined 195 vaginal C. albicans isolates for the presence of C. africana and C. dubliniensis by using hyphal wall protein 1 (HWP1) gene polymorphisms. All study isolates were confirmed to be C. albicans, and none were verified as either C. africana or C. dubliniensis. In conclusion, the HWP1 gene polymorphisms offer a useful tool in the discrimination of C. africana, C. albicans, and C. dubliniensis. Further studies may highlight the pathogenesis and importance of this yeast in vulvovaginal candidiasis.