miR-146a is a critical target associated with multiple biological pathways of skin aging.

Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm-associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm. Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed. Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer. Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells' health.

Cryptococcus neoformans Δsgl1 Vaccination Requires Either CD4+ or CD8+ T Cells for Complete Host Protection.

Cryptococcus neoformans is a fungal pathogen causing life-threatening meningoencephalitis in susceptible individuals. Fungal vaccine development has been hampered by the fact that cryptococcosis occurs during immunodeficiency. We previously reported that a C. neoformans mutant (Δsgl1) accumulating sterylglucosides (SGs) is avirulent and provides complete protection to WT challenge, even under CD4+ T cell depletion, an immunodeficient condition commonly associated with cryptococcosis. We found high levels of SGs in the lungs post-immunization with Δsgl1 that decreased upon fungal clearance. Th1 cytokines increased whereas Th2 cytokines concurrently decreased, coinciding with a large recruitment of leukocytes to the lungs. Depletion of B or CD8+ T cells did not affect either Δsgl1 clearance or protection from WT challenge. Although CD4+ T cell depletion affected clearance, mice were still protected indicating that clearance of the mutant was not necessary for host protection. Protection was lost only when both CD4+ and CD8+ T cells were depleted, highlighting a previously unexplored role of fungal-derived SGs as an immunoadjuvant for host protection against cryptococcosis.

Semisynthesis of Functional Glycosylphosphatidylinositol-Anchored Proteins.

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell- and tissue-specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one-pot intein-mediated ligation (OPL) to obtain GPI-anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP119 were prepared. Glypiation did not alter the structure of eGFP and MSP119 proteins in solution, but it induced a strong pro-inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections.

Age Associated Decrease of MT-1 Melatonin Receptor in Human Dermal Skin Fibroblasts Impairs Protection Against UV-Induced DNA Damage.

The human body follows a physiological rhythm in response to the day/night cycle which is synchronized with the circadian rhythm through internal clocks. Most cells in the human body, including skin cells, express autonomous clocks and the genes responsible for running those clocks. Melatonin, a ubiquitous small molecular weight hormone, is critical in regulating the sleep cycle and other functions in the body. Melatonin is present in the skin and, in this study, we showed that it has the ability to dose-dependently stimulate PER1 clock gene expression in normal human dermal fibroblasts and normal human epidermal keratinocytes. Then we further evaluated the role of MT-1 melatonin receptor in mediating melatonin actions on human skin using fibroblasts derived from young and old subjects. Using immunocytochemistry, Western blotting and RT-PCR, we confirmed the expression of MT-1 receptor in human skin fibroblasts and demonstrated a dramatic age-dependent decrease in its level in mature fibroblasts. We used siRNA technology to transiently knockdown MT-1 receptor in fibroblasts. In these MT-1 knockdown cells, UV-dependent oxidative stress (H2O2 production) was enhanced and DNA damage was also increased, suggesting a critical role of MT-1 receptor in protecting skin cells from UV-induced DNA damage. These studies demonstrate that the melatonin pathway plays a pivotal role in skin aging and damage. Moreover, its correlation with skin circadian rhythm may offer new approaches for decelerating skin aging by modulating the expression of melatonin receptors in human skin.

Sphingolipid biosynthetic pathway is crucial for growth, biofilm formation and membrane integrity of Scedosporium boydii.

Aim: Glycosphingolipids are conserved lipids displaying a variety of functions in fungal cells, such as determination of cell polarity and virulence. They have been considered as potent targets for new antifungal drugs. The present work aimed to test two inhibitors, myriocin and DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol, in Scedosporium boydii, a pathogenic fungus which causes a wide range of disease. Materials & methods: Mass spectrometry, microscopy and cell biology approaches showed that treatment with both inhibitors led to defects in fungal growth and membrane integrity, and caused an increased susceptibility to the current antifungal agents. Conclusion: These data demonstrate the antifungal potential of drugs inhibiting sphingolipid biosynthesis, as well as the usefulness of sphingolipids as promising targets for the development of new therapeutic options.

Cryptococcus neoformans Glucuronoxylomannan and Sterylglucoside Are Required for Host Protection in an Animal Vaccination Model.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis.IMPORTANCE The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis.

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis.

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.

Plasma membrane lipids and their role in fungal virulence.

There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies.

Transmembrane transporter expression regulated by the glucosylceramide pathway in Cryptococcus neoformans.

BACKGROUND: The sphingolipid glucosylceramide (GlcCer) and factors involved in the fungal GlcCer pathways were shown earlier to be an integral part of fungal virulence, especially in fungal replication at 37 °C, in neutral/alkaline pH and 5 % CO2 environments (e.g. alveolar spaces). Two mutants, ∆gcs 1 lacking glucosylceramide synthase 1 gene (GCS1) which catalyzes the formation of sphingolipid GlcCer from the C9-methyl ceramide and ∆smt1 lacking sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position nine of the sphingosine backbone of ceramide, of this pathway were attenuated in virulence and have a growth defect at the above-mentioned conditions. These mutants with either no or structurally modified GlcCer located on the cell-membrane have reduced membrane rigidity, which may have altered not only the physical location of membrane proteins but also their expression, as the pathogen's mode of adaptation to changing need. Importantly, pathogens are known to adapt themselves to the changing host environments by altering their patterns of gene expression. RESULTS: By transcriptional analysis of gene expression, we identified six genes whose expression was changed from their wild-type counterpart grown in the same conditions, i.e. they became either down regulated or up regulated in these two mutants. The microarray data was validated by real-time PCR, which confirmed their fold change in gene expression. All the six genes we identified, viz siderochrome-iron transporter (CNAG_02083), monosaccharide transporter (CNAG_05340), glucose transporter (CNAG_03772), membrane protein (CNAG_03912), membrane transport protein (CNAG_00539), and sugar transporter (CNAG_06963), are membrane-localized and have significantly altered gene expression levels. Therefore, we hypothesize that these genes function either independently or in tandem with a structurally modified cell wall/plasma membrane resulting from the modifications of the GlcCer pathway and thus possibly disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology. CONCLUSION: Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.

Role of Sterylglucosidase 1 (Sgl1) on the pathogenicity of Cryptococcus neoformans: potential applications for vaccine development.

Cryptococcosis caused by Cryptococcus neoformans and Cryptococcus gattii affects a large population and is a cause of significant morbidity and mortality. Despite its public health burden, there are currently no vaccines against cryptococcosis and new strategies against such infections are needed. In this study, we demonstrate that C. neoformans has the biochemical ability to metabolize sterylglucosides (SGs), a class of immunomodulatory glycolipids. Genetic manipulations that eliminate cryptococccal sterylglucosidase lead to the accumulation of SGs and generate a mutant strain (Δsgl1) that is non-pathogenic in the mouse models of cryptococcosis. Interestingly, this mutant strain acts as a vaccine strain and protects mice against cryptococcosis following infection with C. neoformans or C. gattii. The immunity induced by the Δsgl1 strain is not CD4(+) T-cells dependent. Immunocompromised mice, which lack CD4(+) T-cells, are able to control the infection by Δsgl1 and acquire immunity against the challenge by wild-type C. neoformans following vaccination with the Δsgl1 strain. These findings are particularly important in the context of HIV/AIDS immune deficiency and suggest that the Δsgl1 strain might provide a potential vaccination strategy against cryptococcosis.

Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids.

UNLABELLED: Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE: Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

A novel small molecule methyltransferase is important for virulence in Candida albicans.

Candida albicans is an opportunistic pathogen capable of causing life-threatening infections in immunocompromised individuals. Despite its significant health impact, our understanding of C. albicans pathogenicity is limited, particularly at the molecular level. One of the largely understudied enzyme families in C. albicans are small molecule AdoMet-dependent methyltransferases (smMTases), which are important for maintenance of cellular homeostasis by clearing toxic chemicals, generating novel cellular intermediates, and regulating intra- and interspecies interactions. In this study, we demonstrated that C. albicans Crg1 (CaCrg1) is a bona fide smMTase that interacts with the toxin in vitro and in vivo. We report that CaCrg1 is important for virulence-related processes such as adhesion, hyphal elongation, and membrane trafficking. Biochemical and genetic analyses showed that CaCrg1 plays a role in the complex sphingolipid pathway: it binds to exogenous short-chain ceramides in vitro and interacts genetically with genes of glucosylceramide pathway, and the deletion of CaCRG1 leads to significant changes in the abundance of phytoceramides. Finally we found that this novel lipid-related smMTase is required for virulence in the waxmoth Galleria mellonella, a model of infection.

The role of procalcitonin in neonatal intensive care unit patients with candidemia.

Candidemia is a major infectious complication in neonatal patients. The isolation of yeasts from blood is still the "gold standard" for its diagnosis, but other laboratory markers (i.e., circulating antigens) have been studied with varying specificities and sensitivities. The aim of this study was to evaluate the role of procalcitonin for the diagnosis of candidemia in neonatal patients at high risk. To verify if the use of different commercial methods can highlight dissimilar results of sensitivity and/or specificity, the determination of procalcitonin serum levels was estimated by two systems. Overall, 90 patients from a Neonatal Intensive Care Units were enrolled, of whom six developed Candida bloodstream infection. Four of six infants with candidemia had slight increase of procalcitonin values (0.5-1 ng/mL). Only one baby showed very high levels but he had fungal and bacterial sepsis at the same time, while no elevation was observed in the sixth patient. No statistically significant difference was observed between two different methods at the time of monitoring (p>0.643). Both methods showed a sensitivity of 83.3 % at diagnosis, while the specificity was 73.8 and 63.1 % by methods A and B, respectively. In the light of the low sensibility and specificity of this assay, we can assume that the determination of procalcitonin would not seem to play a significant role in the diagnosis of fungal infection in neonatal patients.

Pseudomonas aeruginosa inhibits the growth of Cryptococcus species.

Pseudomonas aeruginosa is a ubiquitous and opportunistic bacterium that inhibits the growth of different microorganisms, including Gram-positive bacteria and fungi such as Candida spp. and Aspergillus fumigatus. In this study, we investigated the interaction between P. aeruginosa and Cryptococcus spp. We found that P. aeruginosa PA14 and, to a lesser extent, PAO1 significantly inhibited the growth of Cryptococcus spp. The inhibition of growth was observed on solid medium by the visualization of a zone of inhibition of yeast growth and in liquid culture by viable cell counting. Interestingly, such inhibition was only observed when P. aeruginosa and Cryptococcus were co-cultured. Minimal inhibition was observed when cell-cell contact was prevented using a separation membrane, suggesting that cell contact is required for inhibition. Using mutant strains of Pseudomonas quinoline signaling, we showed that P. aeruginosa inhibited the growth of Cryptococcus spp. by producing antifungal molecules pyocyanin, a redox-active phenazine, and 2-heptyl-3,4-dihydroxyquinoline (PQS), an extracellular quorum-sensing signal. Because both P. aeruginosa and Cryptococcus neoformans are commonly found in lung infections of immunocompromised patients, this study may have important implication for the interaction of these microbes in both an ecological and a clinical point of view.