Milk fat globule-epidermal growth factor 8 (MFGE8) prevents intestinal fibrosis.

OBJECTIVE: Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN: Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS: We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvß5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.

Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

Activated intestinal muscle cells promote preadipocyte migration: a novel mechanism for creeping fat formation in Crohn's disease.

OBJECTIVE: Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn's disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. DESIGN: Tissues from normal, UC, non-strictured and strictured Crohn's disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals. RESULTS: Crohn's disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-ß1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5ß1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix. CONCLUSION: Crohn's disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

CD101, a Novel Echinocandin, Possesses Potent Antibiofilm Activity against Early and Mature Candida albicans Biofilms.

Currently available echinocandins are generally effective against Candida biofilms, but the recent emergence of resistance has underscored the importance of developing new antifungal agents that are effective against biofilms. CD101 is a long-acting novel echinocandin with distinctive pharmacokinetic properties and improved stability and safety relative to other drugs in the same class. CD101 is currently being evaluated as a once-weekly intravenous (i.v.) infusion for the treatment of candidemia and invasive candidiasis. In this study, we determined (i) the effect of CD101 against early and mature phase biofilms formed by C. albicansin vitro and (ii) the temporal effect of CD101 on the formation of biofilms using time-lapse microscopy (TLM). Early- or mature-phase biofilms were formed on silicone elastomer discs and were exposed to the test compounds for 24 h and quantified by measuring their metabolic activity. Separate batches were observed under a confocal microscope or used to capture TLM images from 0 to 16 h. Measurements of their metabolic activity showed that CD101 (0.25 or 1 µg/ml) significantly prevented adhesion-phase cells from developing into mature biofilms (P = 0.0062 or 0.0064, respectively) and eradicated preformed mature biofilms (P = 0.04 or 0.01, respectively) compared to those of untreated controls. Confocal microscopy showed significant reductions in biofilm thicknesses for both early and mature phases (P < 0.05). TLM showed that CD101 stopped the growth of adhesion- and early-phase biofilms within minutes. CD101-treated hyphae failed to grow into mature biofilms. These results suggest that CD101 may be effective in the prevention and treatment of biofilm-associated nosocomial infections.

Effect of an Emollient on the Mycobiome of Atopic Dermatitis Patients.

INTRODUCTION: Atopic dermatitis (AD) is associated with changes in skin bacterial microbiome. Emollient treatment induces change in bacterial microbiome in AD, but its effect on fungal microbiome ("mycobiome") and their inter-kingdom correlations is unknown. We used Ion-Torrent sequencing to characterize the mycobiome of AD patients in response to emollient treatment. METHODS: Skin swabs were collected from lesional and non-lesional skin of AD patients suffering from moderate AD, after informed consent and according to GCP guidelines. Genomic DNA was extracted from each swab using the MoBio PowerSoil DNA Isolation kit and used for mycobiome sequencing analyses as described in our earlier publications. Principal coordinates analyses (PCoA), diversity, abundance, and correlations analyses were conducted in R and relevant packages using non-parametric tests (P less than .05 was significant). RESULTS: Swab samples from 10 patients (7 females, 3 males; mean age, 10.5 years) were analyzed. Emollient treatment induced a significant reduction of Scoring Atopic Dermatitis (SCORAD) score (P less than .001). PCoA showed pre-treatment and post-treatment samples clustered differently at all taxa levels. Six genera were detected in only non-lesional samples, while four were detected in only lesional samples. In non-lesional samples, Shannon diversity index was significantly increased after emollient treatment (P less than equal to .04), while lesional skin exhibited non-significant decrease. Ascomycota was the most abundant phylum and Dothideomycetes was the most abundant Class in most samples. Eight fungal species were either significantly different (P less than .05) or showed a strong trend (P less than .1) between pre- and post-treatment samples of lesional and non-lesional skin. In lesional skin, Gram-negative Pseudomonas spp. correlated significantly with pathogenic fungal species (Aspergillus, Candida spp.) in pre-treatment samples; these correlations were not detected in post-treatment samples. Moreover, lesional skin exhibited significant correlations between Gram-positive bacteria (Corynebacterium kroppenstedtiian and Staphylococcus pettenkoferi) and pathogenic Candida species in pre-treatment samples, but not in post- treated samples. DISCUSSION: Emollient treatment may induce beneficial microbial changes in the mycobiome and augment host-microbe balance on skin in AD. Clinical relevance of these results need to be investigated. J Drugs Dermatol. 2018;17(10):1039-1048.

The Emerging Pathogen Candida auris: Growth Phenotype, Virulence Factors, Activity of Antifungals, and Effect of SCY-078, a Novel Glucan Synthesis Inhibitor, on Growth Morphology and Biofilm Formation.

Candidaauris, a new multidrug-resistant Candida spp. which is associated with invasive infection and high rates of mortality, has recently emerged. Here, we determined the virulence factors (germination, adherence, biofilm formation, phospholipase and proteinase production) of 16 C. auris isolates and their susceptibilities to 11 drugs belonging to different antifungal classes, including a novel orally bioavailable 1,3-ß-d-glucan synthesis inhibitor (SCY-078). We also examined the effect of SCY-078 on the growth, ultrastructure, and biofilm-forming abilities of C. auris Our data showed that while the tested strains did not germinate, they did produce phospholipase and proteinase in a strain-dependent manner and had a significantly reduced ability to adhere and form biofilms compared to that of Candida albicans (P = 0.01). C. auris isolates demonstrated reduced susceptibility to fluconazole and amphotericin B, while, in general, they were susceptible to the remaining drugs tested. SCY-078 had an MIC90 of 1 mg/liter against C. auris and caused complete inhibition of the growth of C. auris and C. albicans Scanning electron microscopy analysis showed that SCY-078 interrupted C. auris cell division, with the organism forming abnormal fused fungal cells. Additionally, SCY-078 possessed potent antibiofilm activity, wherein treated biofilms demonstrated significantly reduced metabolic activity and a significantly reduced thickness compared to the untreated control (P < 0.05 for both comparisons). Our study shows that C. auris expresses several virulence determinants (albeit to a lesser extent than C. albicans) and is resistant to fluconazole and amphotericin B. SCY-078, the new orally bioavailable antifungal, had potent antifungal/antibiofilm activity against C. auris, indicating that further evaluation of this antifungal is warranted.

Admission to the Intensive Care Unit is Associated With Changes in the Oral Mycobiome.

A prospective exploratory study was conducted to characterize the oral mycobiome at baseline and determine whether changes occur after admission to the intensive care unit (ICU). We found that ICU admission is associated with alterations in the oral mycobiome, including an overall increase in Candida albicans.

Oral mycobiome analysis of HIV-infected patients: identification of Pichia as an antagonist of opportunistic fungi.

Oral microbiota contribute to health and disease, and their disruption may influence the course of oral diseases. Here, we used pyrosequencing to characterize the oral bacteriome and mycobiome of 12 HIV-infected patients and matched 12 uninfected controls. The number of bacterial and fungal genera in individuals ranged between 8-14 and 1-9, among uninfected and HIV-infected participants, respectively. The core oral bacteriome (COB) comprised 14 genera, of which 13 were common between the two groups. In contrast, the core oral mycobiome (COM) differed between HIV-infected and uninfected individuals, with Candida being the predominant fungus in both groups. Among Candida species, C. albicans was the most common (58% in uninfected and 83% in HIV-infected participants). Furthermore, 15 and 12 bacteria-fungi pairs were correlated significantly within uninfected and HIV-infected groups, respectively. Increase in Candida colonization was associated with a concomitant decrease in the abundance of Pichia, suggesting antagonism. We found that Pichia spent medium (PSM) inhibited growth of Candida, Aspergillus and Fusarium. Moreover, Pichia cells and PSM inhibited Candida biofilms (P = .002 and .02, respectively, compared to untreated controls). The mechanism by which Pichia inhibited Candida involved nutrient limitation, and modulation of growth and virulence factors. Finally, in an experimental murine model of oral candidiasis, we demonstrated that mice treated with PSM exhibited significantly lower infection score (P = .011) and fungal burden (P = .04) compared to untreated mice. Moreover, tongues of PSM-treated mice had few hyphae and intact epithelium, while vehicle- and nystatin-treated mice exhibited extensive fungal invasion of tissue with epithelial disruption. These results showed that PSM was efficacious against oral candidiasis in vitro and in vivo. The inhibitory activity of PSM was associated with secretory protein/s. Our findings provide the first evidence of interaction among members of the oral mycobiota, and identifies a potential novel antifungal.

Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans.

The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.

Silicon phthalocyanine 4 phototoxicity in Trichophyton rubrum.

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including age-related macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 µM Pc 4 followed by 670 to 675 nm light at 2.0 J/cm(2) reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis.

Preventing fibrosis in IBD: update on immune pathways and clinical strategies.

INTRODUCTION: Intestinal fibrosis is a common and serious complication of inflammatory bowel diseases (IBD) driving stricture formation in Crohn's disease patients and leading to submucosal damage in ulcerative colitis. Recent studies provided novel insights into the role of immune and nonimmune components in the pathogenesis of intestinal fibrosis. Those new findings may accelerate the development of anti-fibrotic treatment in IBD patients. AREAS COVERED: This review is designed to cover the recent progress in mechanistic research and therapeutic developments on intestinal fibrosis in IBD patients, including new cell clusters, cytokines, proteins, microbiota, creeping fat, and anti-fibrotic therapies. EXPERT OPINION: Due to the previously existing major obstacle of missing consensus on stricture definitions and the absence of clinical trial endpoints, testing of drugs with an anti-fibrotic mechanism is just starting in stricturing Crohn's disease (CD). A biomarker to stratify CD patients at diagnosis without any complications into at-risk populations for future strictures would be highly desirable. Further investigations are needed to identify novel mechanisms of fibrogenesis in the intestine that are targetable and ideally gut specific.

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions.

Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

Development of a 96-well catheter-based microdilution method to test antifungal susceptibility of Candida biofilms.

BACKGROUND: Candida biofilms, which are often associated with device-related infections, including catheter-related bloodstream infections, are resistant to commonly used antifungal agents. Current microtitre (96-well) plate-based methods to determine the antifungal susceptibility of these biofilms do not involve clinically relevant substrates (e.g. catheters), and are not well suited for evaluating the surface topography and three-dimensional architecture of biofilms. We describe a simple, reproducible catheter-based microtitre plate method to form biofilms and evaluate their antifungal susceptibility. METHODS: Biofilms were formed by Candida species on 5 mm catheter discs placed in microtitre plates and quantified using metabolic conversion of a formazan dye (XTT). The morphology, surface topography and three-dimensional architecture of these biofilms were evaluated by fluorescence, confocal scanning laser and scanning electron microscopy, respectively. The optimized XTT method was used to evaluate the antifungal susceptibility of formed Candida biofilms to fluconazole, voriconazole, itraconazole and anidulafungin. RESULTS: Maximum XTT activity was achieved within 90 min. All tested Candida strains formed robust biofilms on catheter discs at both 24 and 48 h (P = 0.66). Biofilms exhibited typical gross morphology, surface topography and architecture, and no difference in biofilm thickness (P = 0.37). The three tested azoles were not active against the biofilms (MIC ≥ 64 mg/L), but anidulafungin possessed potent activity against them (MIC = 0.063-0.125 mg/L). CONCLUSIONS: The developed method is simple, rapid and reproducible, and requires relatively small amounts of drug. It can be used to perform both high-resolution microscopic analysis of the topography and architecture of biofilms, and evaluation of their antifungal susceptibility.

Disruption of the ECM33 gene in Candida albicans prevents biofilm formation, engineered human oral mucosa tissue damage and gingival cell necrosis/apoptosis.

In this study we demonstrated that ΔCaecm33 double mutant showed reduced biofilm formation and causes less damage to gingival mucosa tissues. This was confirmed by the reduced level of necrotic cells and Bax/Bcl2 gene expression as apoptotic markers. In contrast, parental and Caecm33 mutant strains decreased basement membrane protein production (laminin 5 and type IV collagen). We thus propose that ECM33 gene/protein represents a novel target for the prevention and treatment of infections caused by Candida.

Human intestinal myofibroblasts deposited collagen VI enhances adhesiveness for T cells - A novel mechanism for maintenance of intestinal inflammation.

OBJECTIVE: Inflammatory bowel diseases (IBD) cause chronic intestinal damage and extracellular matrix (ECM) remodeling. The ECM may play an active role in inflammation by modulating immune cell functions, including cell adhesion, but this hypothesis has not been tested in IBD. DESIGN: Primary human intestinal myofibroblast (HIMF)-derived ECM from IBD and controls, 3D decellularized colon or ECM molecule-coated scaffolds were tested for their adhesiveness for T cells. Matrisome was analysed via proteomics. Functional integrin blockade was used to investigate the underlying mechanism. Analysis of the pediatric Crohn's disease (CD) RISK inception cohort was used to explore an altered ECM gene expression as a potential predictor for a future complicated disease course. RESULTS: HIMF-derived ECM and 3D decellularized colonic ECM from IBD bound more T cells compared to control. Control HIMFs exposed to the pro-inflammatory cytokines Iinterleukin-1ß (IL-1ß) and tumor necrosis factor (TNF) increased, and to transforming growth factor-ß1 (TGF-ß1) decreased ECM adhesiveness to T cells. Matrisome analysis of the HIMF-derived ECM revealed collagen VI as a major culprit for differences in T cell adhesion. Collagen VI knockdown in HIMF reduced adhesion T cell as did the blockage of integrin αvß1. Elevated gene expression of collagen VI in biopsies of pediatric CD patients was linked to risk for future stricturing disease. CONCLUSION: HIMF-derived ECM in IBD binds a remarkably enhanced number of T cells, which is dependent on Collagen VI and integrin αvß1. Collagen VI expression is a risk factor for a future complicated CD course. Blocking immune cells retention may represent a novel approach to treatment in IBD.

Central venous catheter-associated Nocardia bacteremia in cancer patients.

Central venous catheters, often needed by cancer patients, can be the source of Nocardia bacteremia. We evaluated the clinical characteristics and outcomes of 17 cancer patients with Nocardia bacteremia. For 10 patients, the bacteremia was associated with the catheter; for the other 7, it was a disseminated infection. N. nova complex was the leading cause of bacteremia. Nocardia promoted heavy biofilm formation on the surface of central venous catheter segments tested in an in vitro biofilm model. Trimethoprim- and minocycline-based lock solutions had potent in vitro activity against biofilm growth. Patients with Nocardia central venous catheter-associated bloodstream infections responded well to catheter removal and antimicrobial drug therapy, whereas those with disseminated bacteremia had poor prognoses.

Gentian violet exhibits activity against biofilms formed by oral Candida isolates obtained from HIV-infected patients.

The effect of gentian violet against Candida albicans and non-Candida albicans biofilms formed on polymethylmethacrylate strips was evaluated using a dry weight assay and confocal laser scanning microscopy. The ability of gentian violet to inhibit Candida albicans germination was also assessed. Gentian violet activity against Candida biofilms was demonstrated by a reduction in dry weight, disruption of biofilm architecture, and reduced biofilm thickness. Additionally, gentian violet inhibited Candida germination in a concentration-dependent manner.

Lipidomics of Candida albicans biofilms reveals phase-dependent production of phospholipid molecular classes and role for lipid rafts in biofilm formation.

Candida albicans-associated bloodstream infections are linked to the ability of this yeast to form biofilms. In this study, we used lipidomics to compare the lipid profiles of C. albicans biofilms and planktonic cells, in early and mature developmental phases. Our results showed that significant differences exist in lipid composition in both developmental phases. Biofilms contained higher levels of phospholipid and sphingolipids than planktonic cells (nmol per g biomass, P<0.05 for all comparisons). In the early phase, levels of lipid in most classes were significantly higher in biofilms compared to planktonic cells (P≤0.05). The ratio of phosphatidylcholine to phosphatidylethanolamine was lower in biofilms compared to planktonic cells in both early (1.17 vs 2.52, P≤0.001) and late (2.34 vs 3.81, P≤0.001) developmental phases. The unsaturation index of phospholipids decreased with time, with this effect being particularly strong for biofilms. Inhibition of the biosynthetic pathway for sphingolipid [mannosyl diinositolphosphoryl ceramide, M(IP)2C] by myriocin or aureobasidin A, and disruption of the gene encoding inositolphosphotransferase (Ipt1p), abrogated the ability of C. albicans to form biofilms. The differences in lipid profiles between biofilms and planktonic Candida cells may have important implications for the biology and antifungal resistance of biofilms.

Disruption of sphingolipid biosynthetic gene IPT1 reduces Candida albicans adhesion and prevents activation of human gingival epithelial cell innate immune defense.

We demonstrated the effect of a Candida albicans sphingolipid biosynthetic gene, IPT1, on the interaction between gingival epithelial and Candida cells using monolayer cultures and engineered human oral mucosa tissue (EHOM). Disrupting the IPT1 gene greatly reduced Candida adhesion to gingival epithelial cells, compared to the wild-type and revertant strains. The yeasts adhesion to epithelial cells may activate toll-like receptors (TLRs). Cell response against Candida infection was thus investigated by evaluating TLR expression and antimicrobial peptide (AMP) production. The wild-type and revertant strains both activated TLR2, TLR4, TLR6, and TLR9 gene expression in the epithelial cells, whereas the Δipt1 mutant Candida strain had no effect on this expression. This finding was supported by an increased AMP expression (human ß-defensin HBD-2 and HBD-3) in the EHOM tissue infected with the wild-type and revertant Candida strains, and a decreased expression in the Δipt1 mutant-infected model. HBD protein secretion confirmed the absence of any effect by the Δipt1 on epithelial cell innate defense. This is the first study to demonstrate that a disruption of the IPT1 gene affects Candida-host interaction, thus preventing TLR activation and ß-defensin expression.