Candida Cell Wall Research Articles

Dectin-1 Reactivity to Paramylon Derived from <i>Euglena gracilis</i> EOD-1

Euglena gracilis is a microalga that has recently attracted attention because of its bioactivities. Paramylon (PM), a major β-1,3-glucan, constitutes 70-80% of the cells of the E. gracilis EOD-1 strain. Dectin-1 is a pattern recognition receptor that recognizes β-glucan. However, it is unclear whether PM binds to dectin-1. In this study, we investigated the reactivity of EOD1PM with dectin-1 by analyzing the binding of soluble murine and human dectin-1-Fc fusion protein (m dectin-1 Fc, h dectin-1 Fc) to EOD1PM using flow cytometry and enzyme-linked immunosorbent assay (ELISA). m Dectin-1 Fc bound to EOD1PM particles when m dectin-1-Fc is added. Furthermore, the binding specificity was examined in a competitive reaction following addition of a soluble antigen. It was found that the binding of m dectin-1-Fc to EOD1PM was not inhibited by the addition of dextran or ovalbumin but by the addition of solubilized EOD1PM or Candida cell wall- solubilized β-glucan. In addition, the h dectin-1-Fc fusion protein was found to specifically bind to EOD1PM. These results suggest that dectin-1 recognizes and binds to the β-glucan structure of EOD1PM. Dectin-1 is expressed in leukocytes as a β-glucan receptor and is involved in the expression of various biological activities; therefore, the dectin-1 pathway may be involved in the biological activity of EOD1PM.

Detection of Aspergillus spp. and Candida albicans antigenemia in a sample of Iraqi patients with severe COVID-19

Introduction: Since December 2019, coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, has spread locally in Wuhan, China, and later on, a worldwide outbreak occurred. Invasive fungal infections can cause complications in critically ill immunocompromised patients of COVID-19, especially those admitted to intensive care units and who required mechanical ventilation. Candida albicans have been the most common pathogenic species, followed by other Candida spp. Mannan is a major component of the Candida cell wall and can be detected by the enzyme-linked immunosorbent assay (ELISA) in blood and other fluids. Invasive pulmonary aspergillosis is considered a life-threatening infection, especially among immunocompromised patients. COVID-19-associated pulmonary aspergillosis has emerged as an important complication among patients in the intensive care units. Galactomannan (GM) is a major cell-wall component of Aspergillus spp. and can be found in body fluids. Blood GM can be detected by the enzyme immunoassay. The aim of the current study is to assess the frequency of aspergillosis and candidiasis among COVID-19 patients in some hospitals in Baghdad by using GM and mannan biomarkers. Methods: During the period from February 2020 to May 2021, 175 COVID-19 blood samples of patients were collected and a sandwich ELISA test was performed to detect GM Ag of Aspergillus spp. and mannan Ag of Candida spp. Results: Regarding C-reactive protein (CRP), significant differences were seen among Aspergillus/COVID-19 patients (p = 0.029). Regarding sex and age group, the results indicated that of a total of 175 adult patients with positive COVID-19, more than half of the patients were males. Regarding the distribution of mannan Ag and GM Ag in COVID-19 patients, it was seen that out of the 175 patients, 167 (95.43%) Candida mannan Ag were negative and only 8 (4.57%) were positive, and 170 (79.14%) Aspergillus GM Ag were negative and only 5 (2.86%) were positive. It was also seen that 2 patients (1.14%) who had both Candida mannan and Aspergillus GM were positive and 173 (98.6%) were negative. No statistically significant difference was seen in candidiasis and aspergillosis among patients with COVID-19 regarding age group, sex, underlying chronic diseases (hypertension and diabetes mellitus), and biochemical tests. Conclusion: COVID-19 infections increased with age and were seen more in males than in females. The percentage of infection with C. albicans and Aspergillus spp. among COVID-19 patients was not significant, and this may come from the random collection of samples from patients with different stages of illness. A significant correlation was found between Aspergillus GM Ag in COVID-19 patients and the CRP test.

Identification of fungal trehalose for the diagnosis of invasive candidiasis by mass spectrometry

The rapidity of the diagnosis of invasive candidiasis (IC) is crucial to allow the early introduction of antifungal therapy that dramatically increases the survival rate of patients. Early diagnosis is unfortunately often delayed because Candida blood culture, the gold standard diagnostic test, is positive in only 50% of cases of IC and takes several days to obtain this result. Complementary non-culture-based methods relying on the detection of Candida cell wall polysaccharides in the serum, β-glucans and mannans, by enzymatic and immunological reagents have been successfully developed to allow a more efficient patients care. We have previously demonstrated that detection of circulating glycans by mass spectrometry could provide a reliable and cost-effective early diagnosis method called MS-DS for Mass Spectrometry of Di-Saccharide. Here, by comparing patient's sera and Candida albicans strains deficient in carbohydrates synthesis, we demonstrate that trehalose derived from fungal metabolism can be specifically targeted by MS-DS to allow early diagnosis. In particular, the use of C. albicans strains deficient in the synthesis of trehalose synthesizing enzymes Tps1 and Tps2 show that MS-DS results were correlated to the metabolism of trehalose. Finally, we demonstrate that the performance of the IC diagnosis can be significantly improved by using high resolution mass spectrometry, which opens new perspectives in the management of the disease.

Effects of CEACAM1 in oral keratinocytes on HO-1 expression induced by Candida β-glucan particles.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component β-glucan particles (β-GPs). Furthermore, the effects of CEACAM1 on β-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by β-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by β-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased β-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.

Spatial mapping of immunological epitopes in the Candida cell wall using C-type lectin probes

The primary recognition event between a fungal pathogen and the immune system normally involves the engagement of a pattern recognition receptor with specific components of the cell wall. However, the cell wall is a complex three dimensional structure whose composition changes rapidly in accordance with environmental stimuli. Therefore it is important to know what is the precise nature of the primary recognition event, how many events occur to activate the immune response and how these recognition events are affected by changes in cell wall architecture, cellular morphogenesis and physiological adaptation of the pathogen to specific niches in the human body. We address this fundamental question using four soluble immune C-Type lectin receptor-probes which recognize specific mannans and β-1,3 glucan in the cell wall. We use these C-type lectin probes to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces did not correlate with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions. These studies demonstrate that mannan-epitopes within fungal cell walls are differentially distributed and dynamically expressed as the fungus adapted to microenvironments that would be encountered in vivo.

The effect of Candida cell wall beta-glucan on treatment-resistant LL/2 cancer cell line: in vitro evaluation.

Candida albicans (C. albicans) cell wall beta-glucan has been considered as a potential agent in the treatment of cancers due to its anti-tumor properties. Therefore, in the present study, we investigated the anti-cancer effects of Candida cell wall beta-glucan on Lewis lung carcinoma cell line (LL/2) cells. Beta-glucan of C. albicans cell wall was extracted. LL/2 cell line was cultured, then sphere cells and parental cells were exposed to the different concentrations of beta-glucan extracted from C. albicans (10-6000μg/ml), for 24, 48 and 72h. Cytotoxicity of beta-glucan was assayed by MTT test, then RNA extracted from cells population (treated and untreated cells), cDNA synthetized and expression level of Sox2, Oct4, C-myc, Nanog genes were also investigated using Real-time methods. At optimal concentrations of 800 and 1000μg/ml, the extracted beta-glucan showed a significant cytotoxic effect on both parental and sphere cell populations (p < 0.05). Real-time PCR analysis revealed a decreased expression of Oct4 and Sox2 genes in treatment of cells with beta-glucan compared with control group. Since the extracted beta-glucan showed an inhibitory effect on the expression of Oct4 and Sox2 genes involved in LL/2 metastasis, therefore, beta-glucan can be considered as an anti-tumor agent because of its anti-metastatic properties, however, more in vitro and in vivo studies are needed to provide further evidence on this topic in the future.

The antifungal peptide CGA-N12 inhibits cell wall synthesis of Candida tropicalis by interacting with KRE9.

CGA-N12, an antifungal peptide derived from chromogranin A, has specific antagonistic activity against Candida spp., especially against Candida tropicalis, by inducing cell apoptosis. However, the effect of CGA-N12 on the Candida cell wall is unknown. The Candida protein KRE9, which possesses β-1,6-glucanase activity, was screened by affinity chromatography after binding to CGA-N12. In this study, the effect of CGA-N12 on KRE9 and the interaction between CGA-N12 and KRE9 was studied to clarify the effect of CGA-N12 on C. tropicalis cell wall synthesis. The effect of CGA-N12 on recombinant KRE9 β-1,6-glucanase activity was investigated by analyzing the consumption of glucose. The results showed that CGA-N12 inhibited the activity of KRE9. After C. tropicalis was treated with CGA-N12, the structure of the C. tropicalis cell wall was damaged. The interaction between CGA-N12 and KRE9 was analyzed by isothermal titration calorimetry (ITC). The results showed that their interaction process was involved an endothermic reaction, and the interaction force was mainly hydrophobic with a few electrostatic forces. The results of the fluorescence resonance energy transfer (FRET) assay showed that the distance between CGA-N12 and KRE9 was 7 ∼ 10 nm during their interaction. Therefore, we concluded that the target of CGA-N12 in the C. tropicalis cell membrane is KRE9, and that CGA-N12 weakly binds to KRE9 within a 7 ∼ 10 nm distance and inhibits KRE9 activity.

8-Hydroxyquinoline-5-sulfonamides are promising antifungal candidates for the topical treatment of dermatomycosis.

The purpose of this study was to uncover insights into the mechanism of action of the 8-hydroxyquinoline derivatives PH151 and PH153. In addition, with the future perspective of developing a topical drug for the treatment of candidiasis and dermatophytosis, the antifungal activity of a nanoemulsion formulation containing the most active compound (PH151) is also presented here. Sorbitol protection assay and scanning electron microscopy indicate that the 8-hydroxyquinoline derivatives act on the cell wall of Candida sp. and dermatophytes and they inhibit the pseudohyphae formation of C. albicans. These findings demonstrate a strong effect of these compounds on C. albicans morphogenesis, which can be considered a potential mode of action for this molecule. Besides, the nanoemulsion formulation MIC values ranged from 0·5 to 4μgml-1 demonstrating the significant antifungal activity when incorporated into a pharmaceutical formulation. Taken together, the results support the potential of these molecules as promising antifungal candidates for the treatment of candidiasis and dermatophytosis. There is an emerging need to fill the pipeline with new antifungal drugs due to the limitations presented by the currently used drugs. In this study, we have described a novel formulation with a 8-hydroxyquinoline-5-sulfonamide derivative which has presented a great potency in providing a finished product. Furthermore, the derivative has shown a selective mechanism of action confirming its potential to be developed into a new drug candidate.

Modification of Candida albicans cell wall by commensal gut bacteria

The human gut is populated with a vast community of microbes, the microbiota. Fungi comprise 0.1% of the total gut microbiota. Some of these fungi exist as benign members, however others such Candida albicans can undergo a pathogenic switch causing disease. The fungal cell wall is the first target for immune system recognition. Recent studies have suggested that Candida is decorated with different cell wall epitopes within different physiological niches, due to the impact of carbon source and oxygen availability on cell wall remodelling. Here we hypothesize that resident gut bacteria also play a major role in fungal cell wall remodelling and immune recognition. Data from our lab has shown that a common bacterium from the gut, Bacteroides thetaiotaomicron (Bt), produces an extensive repertoire of degradative enzymes to breakdown the Candida cell wall. Recently, we have identified novel enzymes in Bt from the glycoside hydrolase family 130 (GH130), which specifically target β1,2-linked mannan, a unique feature of Candida mannan. We have deleted multiple fungal mannan specific loci in Bt and examined the ability of deletion strains to utilise Candida mannan as a carbon source. These data suggest that Bt contains multiple pathways to degrade the Candida cell wall. Now we are systematically dissecting the impact of mannan degradation on the physiology of the fungus. This will provide insights into how two prominent members of the gut microbiota interact with each other, how the Candida cell wall is modified in the anaerobic environment of the gut, and the importance of this in promoting immune homeostasis.

Binding of Elementary Bodies by the Opportunistic Fungal Pathogen Candida albicans or Soluble β-Glucan, Laminarin, Inhibits Chlamydia trachomatis Infectivity.

Microbial interactions represent an understudied facet of human health and disease. In this study, the interactions that occur between Chlamydia trachomatis and the opportunistic fungal pathogen, Candida albicans were investigated. Candida albicans is a common component of the oral and vaginal microbiota responsible for thrush and vaginal yeast infections. Normally, Candida exist in the body as yeast. However, disruptions to the microbiota create conditions that allow expanded growth of Candida, conversion to the hyphal form, and tissue invasion. Previous studies have shown that a myriad of outcomes can occur when Candida albicans interacts with pathogenic bacteria. To determine if C. trachomatis physically interacts with C. albicans, we incubated chlamydial elementary bodies (EB) in medium alone or with C. albicans yeast or hyphal forms for 1 h. Following incubation, the samples were formaldehyde-fixed and processed for immunofluorescence assays using anti-chlamydial MOMP or anti- chlamydial LPS antibodies. Replicate samples were replenished with culture medium and incubated at 35°C for 0–120 h prior to fixation for immunofluorescence analysis or collection for EB infectivity assays. Data from this study indicates that both C. trachomatis serovar E and C. muridarum EB bind to C. albicans yeast and hyphal forms. This interaction was not blocked by pre-incubation of EB with the Candida cell wall components, mannan or β-glucans, suggesting that EB interact with a Candida cell wall protein or other structure. Bound EB remained attached to C. albicans for a minimum of 5 days (120 h). Infectivity assays demonstrated that EB bound to C. albicans are infectious immediately following binding (0h). However, once bound to C. albicans, EB infectivity decreased at a faster rate than EB in medium alone. At 6h post binding, 40% of EB incubated in medium alone remained infectious compared to only 16% of EB bound to C. albicans. Likewise, pre-incubation of EB with laminarin, a soluble preparation of β-glucan, alone or in combination with other fungal cell wall components significantly decreases chlamydial infectivity in HeLa cells. These data indicate that interactions between EB and C. albicans inhibit chlamydial infectivity, possibly by physically blocking EB interactions with host cell receptors.

Stability of cationic and amphoteric derivatives of mannan from the yeast Candida albicans

Infection with Candida albicans can prove lethal in immuno-compromised patients. It is imperative to develop a vaccine against this common organism. The amphoteric derivatives of the mannan component of the Candida cell wall may present a prospective target for the development of such a vaccine; however, the radical processing by antigen-presenting cells of the immune system is not fully understood. In this work a set of tailor-made cationic and amphoteric derivatives of three different degrees of quaternization (DSQ 0.14–0.38) has been prepared by chemical modification of ultrasonically-treated mannan and three carboxymethylated mannan derivatives (DSCM 0.13–0.32). These were exposed to free-radical attack by OH, generated in situ by the Fenton reaction. Potential changes in composition, DSQ, and molar mass distribution due to free-radical degradation were monitored by elemental analysis, NMR and FTIR spectroscopies, and size exclusion chromatography. A protective effect of quaternization against OH degradation was found. Non-isothermal thermogravimetric analysis found that the thermal stability of this mannan was also improved by chemical modification.

Cell wall mannan of Candida krusei mediates dendritic cell apoptosis and orchestrates Th17 polarization via TLR-2/MyD88-dependent pathway

Dendritic cells (DCs) abundantly express diverse receptors to recognize mannans in the outer surface of Candida cell wall, and these interactions dictate the host immune responses that determine disease outcomes. C. krusei prevalence in candidiasis worldwide has increased since this pathogen has developed multidrug resistance. However, little is known how the immune system responds to C. krusei. Particularly, the molecular mechanisms of the interplay between C. krusei mannan and DCs remain to be elucidated. We investigated how C. krusei mannan affected DC responses in comparison to C. albicans, C. tropicalis and C. glabrata mannan. Our results showed that only C. krusei mannan induced massive cytokine responses in DCs, and led to apoptosis. Although C. krusei mannan-activated DCs underwent apoptosis, they were still capable of initiating Th17 response. C. krusei mannan-mediated DC apoptosis was obligated to the TLR2 and MyD88 pathway. These pathways also controlled Th1/Th17 switching possibly by virtue of the production of the polarizing cytokines IL-12 and IL-6 by the C. krusei mannan activated-DCs. Our study suggests that TLR2 and MyD88 pathway in DCs are dominant for C. krusei mannan recognition, which differs from the previous reports showing a crucial role of C-type lectin receptors in Candida mannan sensing.

The Role of Candida albicans Transcription Factor RLM1 in Response to Carbon Adaptation.

Candida albicans is the main causative agent of candidiasis and one of the most frequent causes of nosocomial infections worldwide. In order to establish an infection, this pathogen supports effective stress responses to counter host defenses and adapts to changes in the availability of important nutrients, such as alternative carbon sources. These stress responses have clear implications on the composition and structure of Candida cell wall. Therefore, we studied the impact of lactate, a physiologically relevant carbon source, on the activity of C. albicans RLM1 transcriptional factor. RLM1 is involved in the cell wall integrity pathway and plays an important role in regulating the flow of carbohydrates into cell wall biosynthesis pathways. The role of C. albicans RLM1 in response to lactate adaptation was assessed in respect to several virulence factors, such as the ability to grow under cell wall damaging agents, filament, adhere or form biofilm, as well as to immune recognition. The data showed that growth of C. albicans cells in the presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle on both the yeast and the host cells. In addition, we found that adaptation of C. albicans cells to lactate reduces their internalization by immune cells and consequent % of killing, which could be correlated with a lower exposure of the cell wall β-glucans. In addition, absence of RLM1 has a minor impact on internalization, compared with the wild-type and complemented strains, but it reduces the higher efficiency of lactate grown cells at damaging phagocytic cells and induces a high amount of IL-10, rendering these cells more tolerable to the immune system. The data suggests that RLM1 mediates cell wall remodeling during carbon adaptation, impacting their interaction with immune cells.

Differential dendritic cell responses to cell wall mannan of Candida albicans, Candida parapsilosis, and Candida dubliniensis.

Mannan (mannosylated glycoproteins) in the outermost layer of the Candida cell wall may be the first molecules that interact with host dendritic cells (DCs) and activate immune responses that determine disease outcomes. However, little is known about how different mannan structures of common oral Candida species affect DC activation. The effects of heat-inactivated (HI) yeast cells and soluble mannan of Candida albicans, Candida parapsilosis, and Candida dubliniensis on bone marrow-derived DC (BMDC) responses were compared. HI Candida and the mannan exhibited different effects on BMDC activation and functions, which could be due to other carbohydrate compositions in the yeast cell wall. Among Candida mannan, the C. albicans mannan was the weakest stimulus and induced only interferon (IFN)-γ production. This suggests the possibility that C. albicans mannan may skew T helper (Th) responses from protective Th17 toward Th1. In contrast, C. parapsilosis mannan caused strong BMDC activation and high production of several proinflammatory cytokines which possibly promote hyperinflammation. Meanwhile, C. dubliniensis mannan induced moderate BMDC responses, which may correlate with its lower pathogenicity. Therefore, mannan of each Candida species play distinct roles in DC responses and may be involved in the immunopathogenesis and disease severity of oral candidiasis as well as other Candida infection.

Mannan and phosphomannan from Kuraishia capsulata yeast

Linear mannan and branched phosphomannan were identified as exopolysaccharides produced by Kuraishia capsulata yeast. Their structures were determined using nuclear magnetic resonance spectroscopy. The repeating unit of mannan was found to be a trisaccharide →6)-α-Manp-(1→2)-α-Manp-(1→2)-α-Manp-(1→, while the phosphomannan was shown to be built of β-Manp-(1→2)-α-Manp-(1 disaccharide blocks linked by phosphodiester bonds via C-1 and C-6 of the reducing unit. The production of both polysaccharides was shown to depend on the phosphate concentration in the culture medium. In the absence of phosphate, only mannan was obtained, while an excess of KH2PO4 led to the exclusive production of phosphomannan. Chemical depolymerisation of phosphomannan led to the formation of disaccharide β-Manp-(1→2)-(6-P)-Manp, representing the repeating unit of the hydrolysed polysaccharide. The treatment of the disaccharide with alkaline phosphatase resulted in the formation of disaccharide β-Manp-(1→2)-Manp. The latest products can be transformed into glycosyl donors applicable further in the synthesis of oligosaccharides related to Candida cell wall polysaccharides.

Quantitative Analysis of Candida Cell Wall Components by Flow Cytometry with Triple-Fluorescence Staining

This work was supported by the European Commission within the FP7 Framework Programme [Fungitect-Grant No 602125]. We also thank Thomas Sauer, Vienna Biocenter Campus (VBC), Austria, for technical support at the FACS facility of the MFPL, Karl Kuchler, MFPL-Department of Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria and Ernst Thuer, Centre for Genomic Regulation, Barcelona, Spain, for advice on statistical approaches. Neil Gow acknowledges the support of the Wellcome Trust and the MRC Centre for Medical Mycology

AFM combined to ATR-FTIR reveals Candida cell wall changes under caspofungin treatment.

Fungal pathogens from Candida genus are responsible for severe life-threatening infections and the antifungal arsenal is still limited. Caspofungin, an antifungal drug used for human therapy, acts as a blocking agent of the cell wall synthesis by inhibiting the β-1,3-glucan-synthase encoded by FKS genes. Despite its efficiency, the number of genetic mutants that are resistant to caspofungin is increasing. An important challenge to improve antifungal therapy is to understand cellular phenomenon that are associated with drug resistance. Here we used atomic force microscopy (AFM) combined to Fourier transform infrared spectroscopy in attenuated total reflection mode (ATR-FTIR) to decipher the effect of low and high drug concentration on the morphology, mechanics and cell wall composition of two Candida strains, one susceptible and one resistant to caspofungin. Our results confirm that caspofungin induces a dramatic cell wall remodelling via activation of stress responses, even at high drug concentration. Additionally, we highlighted unexpected changes related to drug resistance, suggesting that caspofungin resistance associated with FKS gene mutations comes from a combination of effects: (i) an overall remodelling of yeast cell wall composition; and (ii) cell wall stiffening through chitin synthesis. This work demonstrates that AFM combined to ATR-FTIR is a valuable approach to understand at the molecular scale the biological mechanisms associated with drug resistance.

Morphological and physiological changes induced by contact-dependent interaction between Candida albicans and Fusobacterium nucleatum.

Candida albicans and Fusobacterium nucleatum are well-studied oral commensal microbes with pathogenic potential that are involved in various oral polymicrobial infectious diseases. Recently, we demonstrated that F. nucleatum ATCC 23726 coaggregates with C. albicans SN152, a process mainly mediated by fusobacterial membrane protein RadD and Candida cell wall protein Flo9. The aim of this study was to investigate the potential biological impact of this inter-kingdom interaction. We found that F. nucleatum ATCC 23726 inhibits growth and hyphal morphogenesis of C. albicans SN152 in a contact-dependent manner. Further analysis revealed that the inhibition of Candida hyphal morphogenesis is mediated via RadD and Flo9 protein pair. Using a murine macrophage cell line, we showed that the F. nucleatum-induced inhibition of Candida hyphal morphogenesis promotes C. albicans survival and negatively impacts the macrophage-killing capability of C. albicans. Furthermore, the yeast form of C. albicans repressed F. nucleatum-induced MCP-1 and TNFα production in macrophages. Our study suggests that the interaction between C. albicans and F. nucleatum leads to a mutual attenuation of virulence, which may function to promote a long-term commensal lifestyle within the oral cavity. This finding has significant implications for our understanding of inter-kingdom interaction and may impact clinical treatment strategies.

The GCA1 gene encodes a glycosidase-like protein in the cell wall of Candida albicans.

Candida albicans Gca1p is a putative glucoamylase enzyme which contains 946 amino acids, 11 putative sites for N-glycosylation and 9 for O-glycosylation. Gca1p was identified in β-mercaptoethanol extracts from isolated cell walls of strain C. albicans SC5314 and it is involved in carbohydrate metabolism. The significance and the role of this protein within the cell wall structure were studied in the corresponding mutants. The homozygous mutant showed that GCA1 was not an essential gene for cell viability. Subsequent phenotypic analysis performed in the mutants obtained did not show significant difference in the behavior of mutant when compared with the wild strain SC5314. Zymoliase, Calcofluor White, Congo red, SDS, caffeine or inorganic compounds did not affect the integrity of the cell wall. No differences were observed when hyphal formation assays were carried out. However, an enzyme assay in the presence of substrate p-nitrophenyl-α-D-glucopyranoside enabled us to detect a significant decrease in glycosidase activity in the mutants compared with the parental strain, revealing the function of Gca1.

Nanoscopic Cell Wall Architecture of an Immunogenic Ligand in Candida Albicans during Antifungal Drug Treatment

Candida albicans is a common cause of high-mortality disseminated fungal infections. Morphotype switching between yeasts and filamentous hyphae is important for virulence. Innate immune cells use C-type lectins to recognize Candida cell wall polysaccharides. In particular, Dectin-1 recognizes fungal cell wall beta-glucan, leading to phagocytosis and pro-inflammatory responses. However, C. albicans partially evades innate immune recognition by restricting glucan surface exposure. Treatment with the antifungal caspofungin increases surface exposure of beta-glucan and leukocyte phagocytosis. Our objective was to better understand the nanoscale structure of glucan exposures that C. albicans presents to leukocytes. We probed exposed glucan with AF647-soluble Dectin-1 ectodomain and used direct Stochastic Optical Reconstruction Microscopy (dSTORM) to quantify glucan exposure on C. albicans yeast and hyphal cell walls, both in the resting state and after treatment with caspofungin. Strikingly, the vast majority of glucan exposure sites were consistent with single receptor binding sites in all cases. In resting yeasts, the multiple-probe exposure sites were few and small. After caspofungin treatment, multi-probe exposures were larger and surface density of glucan exposures was increased. Untreated C. albicans hyphae had a higher glucan exposure surface density than yeasts. Lateral hyphal cell walls imaged by dSTORM underwent little quantitative or qualitative change in glucan exposure geometry after drug treatment, but confocal imaging revealed higher glucan exposure after caspofungin treatment at the actively growing and remodeling cell walls of hyphal tips. Similarly, in budding yeasts, we found consistently higher glucan exposure in the actively remodeling cell wall of daughter cells. These findings enable future mechanistic investigation of the contribution of glucan nanopatterning to C. albicans evasion of innate immune recognition via Dectin-1 and the use of nanopatterning of glucan for therapeutic interventions in fungal infectious disease.