Doblin Sandai

Stress adaptation in a pathogenic fungus.

Candida albicans is a major fungal pathogen of humans. This yeast is carried by many individuals as a harmless commensal, but when immune defences are perturbed it causes mucosal infections (thrush). Additionally, when the immune system becomes severely compromised, C. albicans often causes life-threatening systemic infections. A battery of virulence factors and fitness attributes promote the pathogenicity of C. albicans. Fitness attributes include robust responses to local environmental stresses, the inactivation of which attenuates virulence. Stress signalling pathways in C. albicans include evolutionarily conserved modules. However, there has been rewiring of some stress regulatory circuitry such that the roles of a number of regulators in C. albicans have diverged relative to the benign model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This reflects the specific evolution of C. albicans as an opportunistic pathogen obligately associated with warm-blooded animals, compared with other yeasts that are found across diverse environmental niches. Our understanding of C. albicans stress signalling is based primarily on the in vitro responses of glucose-grown cells to individual stresses. However, in vivo this pathogen occupies complex and dynamic host niches characterised by alternative carbon sources and simultaneous exposure to combinations of stresses (rather than individual stresses). It has become apparent that changes in carbon source strongly influence stress resistance, and that some combinatorial stresses exert non-additive effects upon C. albicans. These effects, which are relevant to fungus–host interactions during disease progression, are mediated by multiple mechanisms that include signalling and chemical crosstalk, stress pathway interference and a biological transistor.

The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans

ABSTRACT Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed in C. albicans, S. cerevisiae Icl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that like S. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation. C. albicans Icl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed in C. albicans ubi4 cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes from S. cerevisiae but absent from their C. albicans homologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure, C. albicans retains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients. IMPORTANCE Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available. Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.

Inhibitors of the Glyoxylate Cycle Enzyme ICL1 in Candida albicans for Potential Use as Antifungal Agents

Candida albicans is an opportunistic pathogen that causes candidiasis in humans. In recent years, metabolic pathways in C. albicans have been explored as potential antifungal targets to treat candidiasis. The glyoxylate cycle, which enables C. albicans to survive in nutrient-limited host niches and its. Key enzymes (e.g., isocitrate lyase (ICL1), are particularly attractive antifungal targets for C. albicans. In this study, we used a new screening approach that better reflects the physiological environment that C. albicans cells experience during infection to identify potential inhibitors of ICL. Three compounds (caffeic acid (CAFF), rosmarinic acid (ROS), and apigenin (API)) were found to have antifungal activity against C. albicans when tested under glucose-depleted conditions. We further confirmed the inhibitory potential of these compounds against ICL using the ICL enzyme assay. Lastly, we assessed the bioavailability and toxicity of these compounds using Lipinskis rule-of-five and ADMET analysis.

Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 exhibit strong antifungal effects against vulvovaginal candidiasis-causing Candida glabrata isolates

This study investigates the antagonistic effects of the probiotic strains Lactobacillus rhamnosus GR‐1 and Lactobacillus reuteri RC‐14 against vulvovaginal candidiasis (VVC)‐causing Candida glabrata.

Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits human tumor vascularization in xenograft models and modulates ERK1, VEGF-A, and FGF-2 in computer model

We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06μM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.

Resistance of Candida albicans Biofilms to Drugs and the Host Immune System

Background Candida albicans is a commensal fungus that resides on mucosal surfaces and in the gastrointestinal and genitourinary tracts in humans. However, it can cause an infection when the immune system of the host is impaired or if a niche becomes available. Many C. albicans infections are due to the organism’s ability to form a biofilm on implanted medical devices. A biofilm represents an optimal medium for the growth of C. albicans as it allows cells to be enclosed by a self-produced extracellular matrix (ECM). Objectives The present work investigated certain aspects of the resistance of C. albicans biofilms to drugs and the host immune system. Results An ECM was found to provide the infrastructure for biofilm formation, prevent disaggregation, and shield encapsulated C. albicans cells from antifungal drugs and the host’s immune system. By influencing FKS1 and upregulating multiple glucan modification genes, β-1, 3-glucan, an important component of ECM, was shown to be responsible for many of the biofilm’s drug-resistant properties. On being engulfed by ECM, the fungal cell was found to switch from glycolysis to gluconeogenesis. Resembling the cellular response to starvation, this was followed by the activation of the glyoxylate cycle that allowed the use of simple molecules as energy sources. Conclusion Mature biofilms were found to be much more resistant to antifungal agents and the host immune system than free cells. The factors responsible for high resistance included the complex architecture of biofilms, ECM, increased expression of drug efflux pumps, and metabolic plasticity.

Cat’s whiskers ( Orthosiphon stamineus ) tea modulates arthritis pathogenesis via the angiogenesis and inflammatory cascade

BackgroundOrthosiphon stamineus is used traditionally to treat gout, arthritis, and inflammatory related conditions. The in vitro anti-inflammatory effects of the plant have been scientifically investigated. The goal of the present study was to evaluate the potential of the 50% ethanol extract of O. stamineus (EOS) to treat rheumatoid arthritis.MethodsAnti-arthritic activity was assessed using the in vitro heat denaturation test and the (FCA)-induced arthritis model. Efficacy was assessed by measurements of paw edema and granulation, X-ray radiography, fluorescence molecular tomography (FMT), and histological evaluation. Levels of (TNF-α), interleukin-1 (IL-1), and (COX-1 and COX-2) were analyzed in vitro in lipopolysaccharide (LPS)-stimulated human macrophage (U937). TNF-α and IL-1 levels in the serum samples of arthritic rats were also measured using an ELISA kit.ResultsTreatment with EOS resulted in dose-dependent inhibition of paw edema in acute and chronic models of inflammation. It also inhibited significantly the production of TNF-α, IL-1 COX-1, and COX-2 in the LPS-stimulated U937 macrophages. EOS significantly suppressed FCA-induced paw edema as well as the serum levels of TNF-α and IL-1. X-rays of the synovial joint of the hind leg showed considerable improvement in joint integrity and recovery of tibia-talus bones from degeneration and osteoporotic lesions. Histology of proximal interphalangeal joints of EOS-treated animals showed obvious protection of cartilage and soft tissue. Finally, FMT analysis strongly supported the anti-arthritic effect of EOS. EOS had high phenolic and total flavonoid content as well as strong antioxidant activity.ConclusionsResults illustrated that the anti-arthritic properties of O. stamineus could be beneficial for prevention and management of rheumatoid arthritis and other chronic inflammatory disorders.Graphical abstractIllustration of the Anti- arthritis efficacy of Orthosiphon Stamineus standardized extract.

Growth, biofilm formation, antifungal susceptibility and oxidative stress resistance of Candida glabrata are affected by different glucose concentrations

Glucose is an important fuel source to support many living organisms. Its importance in the physiological fitness and pathogenicity of Candida glabrata, an emerging human fungal pathogen has not been extensively studied. The present study aimed to investigate the effects of glucose on the growth, biofilm formation, antifungal susceptibility and oxidative stress resistance of C. glabrata. In addition, its effect on the expression of a putative high affinity glucose sensor gene, SNF3 was also investigated. Glucose concentrations were found to exert effects on the physiological responses of C. glabrata. The growth rate of the species correlated positively to the amount of glucose. In addition, low glucose environments were found to induce C. glabrata to form biofilm and resist amphotericin B. Conversely, high glucose environments promoted oxidative stress resistance of C. glabrata. The expression of CgSNF3 was found to be significantly up-regulated in low glucose environments. The expression of SNF3 gene in clinical isolates was found to be higher compared to ATCC laboratory strains in low glucose concentrations, which may explain the better survivability of clinical isolates in the low glucose environment. These observations demonstrated the impact of glucose in directing the physiology and virulence fitness of C. glabrata through the possible modulation by SNF3 as a glucose sensor, which in turn aids the species to adapt, survive and thrive in hostile host environment.

Tualang honey improves human corneal epithelial progenitor cell migration and cellular resistance to oxidative stress in vitro

Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3±0.4%) were observed compared to the untreated population (20.5±0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro.

The Role of Isocitrate Lyase (ICL1) in the Metabolic Adaptation of Candida albicans Biofilms

Background A major characteristic of Candida biofilm cells that differentiates them from free-floating cells is their high tolerance to antifungal drugs. This high resistance is attributed to particular biofilm properties, including the accumulation of extrapolymeric substances, morphogenetic switching, and metabolic flexibility. Objectives This study evaluated the roles of metabolic processes (in particular the glyoxylate cycle) on biofilm formation, antifungal drug resistance, morphology, and cell wall components. Methods Growth, adhesion, biofilm formation, and cell wall carbohydrate composition were quantified for isogenic Candida albicans ICL1/ICL1, ICL1/icl1, and icl1/icl1 strains. The morphology and topography of these strains were compared by light microscopy and scanning electron microscopy. FKS1 (glucan synthase), ERG11 (14-α-demethylase), and CDR2 (efflux pump) mRNA levels were quantified using qRT-PCR. Results The ICL1/icl1 and icl1/icl1 strains formed similar biofilms and exhibited analogous drug-tolerance levels to the control ICL1/ICL1 strains. Furthermore, the drug sequestration ability of β-1, 3-glucan, a major carbohydrate component of the extracellular matrix, was not impaired. However, the inactivation of ICL1 did impair morphogenesis. ICL1 deletion also had a considerable effect on the expression of the FKS1, ERG11, and CDR2 genes. FKS1 and ERG11 were upregulated in ICL1/icl1 and icl1/icl1 cells throughout the biofilm developmental stages, and CDR2 was upregulated at the early phase. However, their expression was downregulated compared to the control ICL1/ICL1 strain. Conclusions We conclude that the glyoxylate cycle is not a specific determinant of biofilm drug resistance.