Catechin inhibits Candida albicans dimorphism by disrupting Cek1 phosphorylation and cAMP synthesis

Candida albicans is a fungal pathogen that undergoes dimorphism (transformation from a yeast form to a hyphal form), wherein, the yeast form is identified as a disseminating form that plays a critical role in the early stages of Candida disease progression, while the hyphal form is found to exert additional pathogenicity by adapting to various environmental conditions. Here, we elucidated the effects of catechin on C. albicans hyphal formation. Flow cytometry analysis showed catechin inhibited FCS-induced hyphal formation. Moreover, hypha-specific gene expression in MAP kinase cascade and cAMP pathway was decreased ascribable to catechin. Furthermore, through Western blotting and cAMP synthesis analysis, we found catechin obstructs Cek1 phosphorylation in MAP kinase cascade and suppresses cAMP synthesis. These results suggest that catechin possesses anti-dimorphism activity by interfering with in vitro signal transduction. Similarly, this highlights the possible application of catechin in clinical therapy for the management and prevention of candidosis.     

Functional analysis of genes involved in beta-1, 2-Mannosylation of glycans and putative cell wall proteins in Candida albicans during hyphae transition

Structural studies of cell wall components of the pathogenic yeast Candida albicans revealed a family of genes involved in beta-mannosyltransferase associated with the addition of beta-mannose to the acid-labile fraction of cell wall phosphopeptidomannan. Despite the importance of beta-1, 2-oligomannosides in virulence, the gene expression pattern of this family during hyphae transition have not been identified in C. albicans. We investigated using RT-qPCR whether genes encoding beta-1, 2-mannosylation of glycans and putative cell wall proteins are expressed during hyphae induction. Here, we identify four out of nine putative genes encoding beta-1, 2-mannosylation of glycans, BMT2, BMT4, BMT5, and BMT6 were downregulated during 30, 60, and 180 min of hyphae transition. We also observed that expression of Candida putative GPI-anchored protein coding genes PGA6, PGA19, PGA54, PGA56, PGA58, PGA59, and PGA13 were upregulated after 60 min of hyphae transition, while the levels of expression of these genes showed moderately changes during 15 and 180 min of hyphae transition. In either case, the involvement of these genes in hyphal growth makes them putative targets for new antifungal drugs aimed at inhibiting hyphae formation in C. albicans.     

Effect of Branched α‐Oligomannoside Structures on Induction of Anti‐Candida Humoral Immune Response

Several studies have established the potential efficacy of humoral immunity, primarily mannan‐specific antibodies, in host protection against major fungal pathogen Candida albicans. In this study, we analysed humoral immune response induced by immunization with BSA‐based conjugates bearing synthetic α‐1,6‐branched oligomannosides (pentamannosides (M5) or hexamannosides (M6)) mimicking antigenic sequences of Candida cell wall mannan. We analysed the ability of antibodies prepared by immunization to recognize relevant antigenic determinants in mannan polysaccharide structure and in C. albicans yeast and hyphal morphoforms. M6‐BSA conjugate induced markedly higher levels of mannan‐specific IgG compared with M5‐BSA conjugate. In contrast to M5‐BSA conjugate, M6‐BSA conjugate induced immunoglobulin isotype class switch from IgM to IgG, as revealed also from ELISPOT analysis. Immunization‐induced antibodies showed higher reactivity with hyphal form of C. albicans cells. The reduced immunogenicity of M5‐BSA conjugate seems to be related to branching point location at terminal non‐reducing end in comparison with M6‐BSA oligomannoside with branching point at non‐terminal location. Candidacidal activity assay revealed different capacity of sera prepared by immunization with M5‐BSA and M6‐BSA conjugates to improve candidacidal activity of polymorphonuclear leucocytes. Limited capacity of α‐1,6‐branched oligomannoside – BSA conjugates to induce antibodies significantly enhancing candidacidal activity of polymorphonuclear leucocytes – was presumably related to absence of antibodies with strong reactivity to corresponding antigenic determinants in natural cell wall mannan and with reduced ability to activate complement. The study documented markedly structure‐dependent immunogenicity and limited capacity of branched α‐mannooligosides conjugates to induce production of potentially protective antibodies.     

Differential and enrichment media for selective culture and recognition of yeast species from clinical material

An enrichment medium was developed and evaluated for isolation of fluconazole-resistant minority yeast species in a hospital setting. The enrichment medium was made by adding fluconazole (10 µg/ml) to yeast nitrogen base/glucose broth. Under laboratory conditions the broth permitted detection of 20 of 20Candida krusei isolates and 20 of 20Candida glabrata isolates in mixtures withCandida albicans even when theCandida albicans cells outnumbered those of the other species by 1000:1. Results of culture on the enrichment medium were compared with those obtained on routine agar media and on a yeast differential agar which facilitates detection of mixed yeast species by their colony colours. Only oneCandida glabrata isolate was detected on the enrichment broth but not found on routine culture of 68 yeast-positive clinical specimens. However, bacterial over-growth in some broths may have retarded the appearance of other yeast isolates. On the yeast differential agar, 20 clinical specimens were found to contain mixtures of yeast species compared with only 2 on routine culture.     

The role of diclofenac sodium in the dimorphic transition in Candida albicans

Diclofenac sodium is a non-steroidal anti-inflammatory drug that inhibits filamentation in Candida albicans. Here we examined the effect of diclofenac sodium on hypha formation in C. albicans. The C. albicans cells were treated with various concentrations of diclofenac sodium (50, 100, 200 and 500 μg/ml) and incubated at 37 °C for 2 h. The characteristics of hypha formation were then assessed microscopically in both liquid and solid media. The results indicated that the effect of diclofenac sodium was dependent on the concentration of this compound, and preincubation with 500 μg/ml diclofenac sodium completely inhibited hypha formation in both liquid and solid media.RT-qPCR analysis of RNA extracted from C. albicans indicated that the levels of expression of agglutinin-like sequence 3 (ALS3), RAS1, EFG1 mRNA, which are regulated by the cAMP-EFG1 pathway in C. albicans and three hypha-specific genes (ALS1, ECE1 and HWP1), were decreased in diclofenac sodium treated cells compared to the levels in controls. Our results also demonstrated that diclofenac sodium possesses potent anti yeast-hypha transition activity in vitro and it could be useful in combined therapy with conventional antifungal agents in the management of treatment of Candida albicans infections.     

Use of specialised isolation media for recognition and identification ofCandida dubliniensis isolates from HIV-infected patients

During a study of oral rinses of 130 HIV-infected individuals, both typical and atypicalCandida albicans colonies were isolated from ten patients on a yeast differential medium. TypicalCandida albicans colonies were light green; atypical colonies were dark green. Both types of colonies were germ tube-positive and produced chlamydospores. However, DNA fingerprinting of the atypical isolates with the Ca3Candida albicans-specific probe showed that they belonged to the recently described speciesCandida dubliniensis.Candida dubliniensis colonies could also be differentiated fromCandida albicans colonies on isolation plates by the absence of fluorescence of colonies on methyl blue-Sabouraud agar under Wood's light. Among other phenotypic characteristics, only the absence of intracellular -glucosidase activity reliably distinguishedCandida albicans fromCandida dubliniensis.Candida dubliniensis may be underreported in clinical samples because most currently used isolation and identification methods fail to recognize this yeast.     

Rapid Identification of Yeasts Commonly Found in Positive Blood Cultures by Amplification of the Internal Transcribed Spacer Regions 1 and 2

A multiplex PCR method using one universal and eight species-specific primers was developed to rapidly identify eight yeast species found in positive blood cultures. The species-specific primers were designed from the internal transcribed spacer regions 1 and 2 of the rRNA gene, whereas the universal primer was located at the 26S rRNA gene. The eight species were Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans. The PCR products (116 to 630 bp) were different in length and could be effectively separated and recognized by polyacrylamide gel electrophoresis. By testing 234 positive blood cultures (237 isolates), 234 (98.7%) isolates of the above eight species were correctly identified by the multiplex PCR. The present method is simple to perform and can be completed within 6 h.     

Memory IL‐22‐producing CD4+ T cells specific for Candida albicans are present in humans

Co‐expression of IL‐22 and IL‐17 has been identified and demonstrated to be involved in the immunopathogenesis of some autoimmune diseases as well as the defense against pathogenic infections in animal studies. However, the properties of IL‐22‐producing cells in humans remain largely unclear. In the present study, we showed that IL‐22 could be induced from human PBMC following various polyclonal stimulations. The majority of IL‐22‐producing cells in PBMC were CD4⁺ T cells with a memory cell phenotype. In addition, we found that a subset of IL‐22⁺ T cells produced IL‐22 alone, whereas other IL‐22⁺ T cells co‐expressed cytokines typical of Th1, Th2 and Th17 cells. Importantly, stimulation of PBMC from healthy adults with heat‐inactivated Candida albicans (C. albicans) yeast or hyphae resulted in IL‐22 production by central and effector memory CD4⁺ T cells. Moreover, CD4⁺CCR6⁺ but not CD4⁺CCR6^? T cells produced IL‐22 when stimulated with either C. albicans or PMA and ionomycin. In addition, PBMC from the individuals infected with C. albicans produced a significantly higher amount of IL‐22 compared with healthy controls following stimulation with C. albicans. These data demonstrate that IL‐22‐producing T cells in humans may play an important role in the defense against fungal infections such as C. albicans.     

The Role of Candida dubliniensis in Oral Candidiasis in Human Immunodeficiency Virus-Infected Individuals

There is an increasing interest in non-albicans Candida species because of the increasing number of fungal infections they cause. Most of these infections can be found in immunocompromised individuals, especially in those infected with human immunodeficiency virus (HIV). Candida dubliniensis is a recently identified yeast, mostly isolated in HIV-positive individuals with oral candidiasis. Candida dubliniensis is a germ tube- and chlamydospore-form yeast. Thus, it shares diagnostic characteristics with Candida albicans. Probably, Candida dubliniensis has been present in the community for a long time and has been misidentified as Candida albicans. Significant phenotypic characteristics of Candida dubliniensis (difference in the carbohydrate assimilation profile, difference in colony color on CHROMagar Candida, and positive tetrazolium test, etc.) have been found, but none of them seem to be sufficient alone for the definitive identification of the species. Recently, PCR tests were developed to discriminate Candida albicans from Candida dubliniensis. However, these prove difficult in the context of routine mycologi-cal diagnostics. Moreover, an increased resistance to antifungal drugs has been described. This shows the importance of identification of Candida dubliniensis. To elucidate the current insight into Candida dubliniensis, the phenotypic and genotypic characteristics as well as the prevalence and the antifungal drug susceptibilities of this species are discussed from a clinical standpoint.     

Mixed oropharyngeal candidiasis due toCandida albicans and non-albicans Candida strains in HIV-infected patients

In order to determine the clinical significance of mixed oropharyngeal candidiasis (Candida albicans plus a non-albicans strain ofCandida) in patients infected with HIV-1, a retrospective chart review was done in 12 HIV-1-infected patients with a clinical episode of oropharyngeal candidiasis, in whom a mixed culture ofCandida albicans (found to be fluconazole-sensitive) plus a non-albicans species ofCandida was obtained from their oral cavities. This group was compared with 26 HIV-positive patients (control group) with oropharyngeal candidiasis due toCandida albicans (found to be fluconazole-sensitive). Antifungal susceptibility testing was performed by a broth microdilution test with RPMI-2% glucose. A fungal strain was considered fluconazole-sensitive if its MIC was < 0.5 g/ml. Both the study and control groups had similar clinical and demographic characteristics. All the patients were severely immunocompromised, with a mean CD4+ lymphocyte count of 63/mm³ (95% Cl 41–84) and 80/mm³ (95% Cl 25–135) in the study and control groups, respectively. In the study group, seven patients hadCandida albicans andCandida krusei in their oral cavity, four hadCandida albicans andCandida glabrata, and one hadCandida albicans andCandida tropicalis. Antifungal therapy consisted of ketoconazole (5 patients in the study group, 14 in the control group) or fluconazole (7 patients in the study group, 12 in the control group); no statistically significant difference in clinical outcome was observed. Fungal strain persistence after therapy was frequently observed in both groups. It is concluded that non-albicans strains ofCandida, less sensitive to azole drugs than theirCandida albicans counterparts, are not clinically relevant in episodes of mixed oropharyngeal candidiasis in HIV-1-infected patients.     

Antimicrobial effects of liquid anesthetic isoflurane on Candida albicans

Candida albicans is a dimorphic fungus that can grow in yeast morphology or hyphal form depending on the surrounding environment. This ubiquitous fungus is present in skin and mucus membranes as a potential pathogen that under opportunistic conditions causes a series of systemic and superficial infections known as candidiasis, moniliasis or simply candidiasis. There has been a steady increase in the prevalence of candidiasis that is expressed in more virulent forms of infection. Although candidiasis is commonly manifested as mucocutaneous disease, life-threatening systemic invasion by this fungus can occur in every part of the body. The severity of candidal infections is associated with its morphological shift such that the hyphal morphology of the fungus is most invasive. Of importance, aberrant multiplication of Candida yeast is also associated with the pathogenesis of certain mucosal diseases. In this study, we assessed the anti-candidal activity of the volatile anesthetic isoflurane in liquid form in comparison with the anti-fungal agent amphotericin B in an in vitro culture system. Exposure of C. albicans to isoflurane (0.3% volume/volume and above) inhibited multiplication of yeast as well as formation of hyphae. These data suggest development of potential topical application of isoflurane for controlling a series of cutaneous and genital infections associated with this fungus. Elucidiation of the mechanism by which isoflurane effects fungal growth could offer therapeutic potential for certain systemic fungal infections.     

Systemic Infection Following Intravenous Inoculation of Mice with Candida albicans int1 Mutant Strains

The Candida albicans gene INT1 is associated with epithelial adhesion, hyphal formation, and virulence. C. albicans strains carrying two, one, or no functional INT1 alleles were used to assess the association between mortality and C. albicans persistence in the liver and kidney of intravenously inoculated mice. Mice were injected with 10⁵C. albicans CAF2 (parent strain, INT1/INT1), C. albicans CAG3 (homozygous disruptant, Int1/int1), or C. albicans CAG5 (heterozygous reintegrant, int1/int1 + INT1). Mortality was monitored and mice were sacrificed on Days 1, 7, 14, and 21 for quantitative analysis of kidney and liver microbes, with histologic analysis of these tissues as well. Mortality was highest for mice injected with the wild-type strain CAF2 (INT1/INT1) and lowest for mice injected with the homozygous disruptant CAG3 (int/int1). Yeast were readily cleared from the liver of all mice injected with any of the three C. albicans strains. Although the mutant strains CAG3 and CAG5 are defective for hyphal formation in vitro, there was histological evidence of abundant hyphal formation in the renal pelvis of mice injected with these strains. Compared to the wild-type strain, mutant strains were associated with reduced mortality but increased C. albicans persistence in the kidney. Thus, the absolute ability to form hyphae in the kidney did not appear to modulate either C. albicans-induced mortality or the course of progressive infection in the kidney. In addition, reduced virulence was paradoxically associated with increased, not decreased, persistence of C. albicans in the kidney.     

Identification,characterization and sequence of Candida albicans repetitive DNAs Rel-1 and Rel-2

Two moderately repetitive DNA elements, Rel-1 and Rel-2, were identified in a screen for clones that hybridized to a Candida albicans minichromosome. Rel-1, a 223-bp sequence, is C. albicans-specific. The 2789-bp Rel-2 sequence hybridizes weakly to C. stellatoidia DNA but not to DNA from several other yeast species. Genomic Southern-blot analysis indicated that Rel-1 and Rel-2 are often closely associated in the genome, suggesting that they may be subsequences of a larger repetitive element. Small subrepeats are located in the nucleotide sequence of both clones. Hybridization demonstrated that Rel-2 contains both repetitive and unique DNA sequences. The repetitive DNA is present on most, and perhaps all, C. albicans chromosomes. The unique sequence maps to chromosome 7; however, in some strains, it is also present on additional chromosomes.     

Differentiation of <Emphasis Type="Italic">Candida albicans</Emphasis> from non-<Emphasis Type="Italic">albicans</Emphasis> yeast directly from blood cultures by Gram stain morphology

Clusters of pseudohyphae are commonly seen on Gram stain of blood cultures from patients with Candida albicans fungemia. Whether this morphologic feature is useful for differentiating C. albicans from other yeasts has not previously been systematically evaluated. Yeast morphology on Gram stain of blood cultures from consecutive patients with fungemia detected by the Bactec automated blood culture system was prospectively assessed and correlated with the final culture-based yeast identification. The distribution of yeast in 60 consecutive patients with fungemia included Candida spp. (C. albicans 43%, Candida glabrata 28%, Candida parapsilosis 8%, Candida krusei 7%, Candida tropicalis 5%, Candida dubliniensis 2%, and Candida lusitaniae 2%), Rhodotorula mucilaginosa 2%, and Cryptococcus neoformans 3%. Upon analysis of the first positive blood culture bottle per patient, the presence of clustered pseudohyphae on Gram stain had a sensitivity, specificity, positive predictive value, and negative predictive value of 85, 97, 96, and 89%, respectively, for C. albicans. The sensitivity and specificity of aerobic vs Myco/F blood culture bottles were 96 and 95% vs 25 and 100%, p < 0.001, respectively. Inter-rater agreement for ten separate observations among five reviewers was 100%. The presence of pseudohyphae clusters by Gram stain of blood cultures is useful in distinguishing C. albicans from non-albicans yeast. Additional studies are necessary to determine the clinical impact of these findings and their validity with other blood culture systems.     

Rab14 Regulates Maturation of Macrophage Phagosomes Containing the Fungal Pathogen Candida albicans and Outcome of the Host-Pathogen Interaction

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogen Candida albicans undergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated by C. albicans hyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment of C. albicans cells. Phagosomes containing live C. albicans cells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing by C. albicans. Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.     

Activity of coumarin against Candida albicans biofilms

Objective

The aim of this study was to investigate the antibiofilm activity of coumarin against Candida albicans.