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Antifungal and antiproliferative protein from Cicer arietinum: a bioactive compound against emerging pathogens.
ABSTRACT: The emergence of epidemic fungal pathogenic resistance to current antifungal drugs has increased the interest in developing alternative antibiotics from natural sources. Cicer arietinum is well known for its medicinal properties. The aim of this work was to isolate antimicrobial proteins from Cicer arietinum. An antifungal protein, C-25, was isolated from Cicer arietinum and purified by gel filtration. C-25 protein was tested using agar diffusion method against human pathogenic fungi of ATCC strains and against clinical isolates of Candida krusei, Candida tropicalis, and Candida parapsilosis, and MIC values determined were varied from 1.56 to 12.5 ?g/mL. The SEM study demonstrated that C-25 induces the bleb-like surface changes, irregular cell surface, and cell wall disruption of the fungi at different time intervals. Cytotoxic activity was studied on oral cancer cells and normal cells. It also inhibits the growth of fungal strains which are resistant to fluconazole. It reduced the cell proliferation of human oral carcinoma cells at the concentration of 37.5 ?g/mL (IC50) and no toxic effect was found on normal human peripheral blood mononuclear cells even at higher concentration of 600 ?g/mL. It can be concluded that C-25 can be considered as an effective antimycotic as well as antiproliferative agent against human oral cancer cells.
Project description:Compared to bacteria, the role of fungi within the intestinal microbiota is poorly understood. In this study we investigated whether the presence of a "healthy" fungal community in the gut is important for modulating immune function. Prolonged oral treatment of mice with antifungal drugs resulted in increased disease severity in acute and chronic models of colitis, and also exacerbated the development of allergic airway disease. Microbiota profiling revealed restructuring of fungal and bacterial communities. Specifically, representation of Candida spp. was reduced, while Aspergillus, Wallemia, and Epicoccum spp. were increased. Oral supplementation with a mixture of three fungi found to expand during antifungal treatment (Aspergillus amstelodami, Epicoccum nigrum, and Wallemia sebi) was sufficient to recapitulate the exacerbating effects of antifungal drugs on allergic airway disease. Taken together, these results indicate that disruption of commensal fungal populations can influence local and peripheral immune responses and enhance relevant disease states.
Project description:Candida is the most common fungal pathogen of humans worldwide and has become a major clinical problem because of the growing number of immunocompromised patients, who are susceptible to infection. Moreover, the number of available antifungals is limited, and antifungal-resistant Candida strains are emerging. New and effective antifungals are therefore urgently needed. Here, we discovered a small molecule with activity against Candida spp. both in vitro and in vivo. We screened a library of 50,240 small molecules for inhibitors of yeast-to-hypha transition, a major virulence attribute of Candida albicans. This screening identified 20 active compounds. Further examination of the in vitro antifungal and anti-biofilm properties of these compounds, using a range of Candida spp., led to the discovery of SM21, a highly potent antifungal molecule (minimum inhibitory concentration (MIC) 0.2-1.6 µg/ml). In vitro, SM21 was toxic to fungi but not to various human cell lines or bacterial species and was active against Candida isolates that are resistant to existing antifungal agents. Moreover, SM21 was relatively more effective against biofilms of Candida spp. than the current antifungal agents. In vivo, SM21 prevented the death of mice in a systemic candidiasis model and was also more effective than the common antifungal nystatin at reducing the extent of tongue lesions in a mouse model of oral candidiasis. Propidium iodide uptake assay showed that SM21 affected the integrity of the cell membrane. Taken together, our results indicate that SM21 has the potential to be developed as a novel antifungal agent for clinical use.
Project description:The threat of antimicrobial resistance to society is compounded by a relative lack of new clinically effective licensed therapies reaching patients over the past three decades. This has been particularly problematic within antifungal drug development, leading to a rise in fungal infection rates and associated mortality. This paper highlights the potential of an ultrashort peptide, (naphthalene-2-ly)-acetyl-diphenylalanine-dilysine-OH (NapFFKK-OH), encompassing hydrogel-forming and antifungal properties within a single peptide motif, thus overcoming formulation (e.g., solubility, drug loading) issues associated with many currently employed highly hydrophobic antifungals. A range of fungal susceptibility (colony counts) and cell cytotoxicity (MTS cell viability, LIVE/DEAD staining® with fluorescent microscopy, haemolysis) assays were employed. Scanning electron microscopy confirmed the nanofibrous architecture of our self-assembling peptide, existing as a hydrogel at concentrations of 1% w/v and above. Broad-spectrum activity was demonstrated against a range of fungi clinically relevant to infection (Aspergillus niger, Candida glabrata, Candida albicans, Candida parapsilosis and Candida dubliniensis) with greater than 4 log10 CFU/mL reduction at concentrations of 0.5% w/v and above. We hypothesise antifungal activity is due to targeting of anionic components present within fungal cell membranes resulting in membrane disruption and cell lysis. NapFFKK-OH demonstrated reduced toxicity against mammalian cells (NCTC 929, ARPE-19) suggesting increased selectivity for fungal cells. However, further studies relating to safety for systemic administration is required, given the challenges toxicity has presented in the wider context of antimicrobial peptide drug development. Overall this study highlights the promise of NapFFKK-OH hydrogels, particularly as a topical formulation for the treatment of fungal infections relating to the skin and eyes, or as a hydrogel coating for the prevention of biomaterial related infection.
Project description: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.
Project description:Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection.
Project description:BACKGROUND:Fungal infections affect various parts of the body and can be difficult to treat. Aspergillus infection causes a spectrum of diverse diseases particularly in lung according to host immunity. The two major entities are invasive pulmonary aspergillosis and chronic pulmonary aspergillosis. Candida infections can be superficial or invasive. Superficial infections often affect the skin or mucous membranes. However, invasive fungal infections are often life-threatening. Advances in nanotechnology have opened new horizons in nanomedicine, allowing the synthesis of nanoparticles that can be assembled into complex architectures. Novel studies and technologies are devoted to understanding the mechanisms of disease for the design of new drugs. OBJECTIVES:In the present study, the antifungal activity of biogenic selenium nanoparticles (Se NPs) against Aspergillus fumigatus and Candida albicans was investigated. MATERIALS AND METHODS:Se-reducing bacteria previously identified as Bacillus sp. MSh-1 were used for the intracellular biosynthesis of elemental Se NPs. The shape, size, and purity of the extracted NPs were determined with various instrumental techniques. The nanoparticles antifungal characterization mainly derives from the following pathways: (i) to generate sustained flux of nano-ions from the compounds that deposited on special substrates or imbedded in colloidal or semisolid matrices. (ii) To transport active those ions to sensitive targets on plasma membrane of fungi. RESULTS:The results of energy-dispersive X-ray demonstrated that the purified NPs consisted of only Se. In addition, transmission electron micrographs showed that 120- to 140-nm spherical Se NPs were the most common. An antifungal assay was performed with a standard Clinical and Laboratory Standards Institute broth microdilution method. Minimum inhibitory concentration (MIC) measurements of the antifungal activity of the Se NPs against C. albicans (70 ?g/mL) and A. fumigatus (100 ?g/mL) showed that yeast cells were more sensitive than mold cells. CONCLUSIONS:The MICs against A. fumigatus (100 ?g/mL) and C. albicans (70 ?g/mL) showed that biogenic Se NPs are useful antifungal agents.
Project description:The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against Aspergillus fumigatus were tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates (n = 96) with a MIC range of 0.016 to 0.25 ?g/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 ?g/ml. PC1244 was a strong tight-binding inhibitor of recombinant A. fumigatus CYP51A and CYP51B (sterol 14?-demethylase) enzymes and strongly inhibited ergosterol synthesis in A. fumigatus with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 ?g/ml), especially Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae PC1244 also proved to be quickly absorbed into both A. fumigatus hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 ?g/ml) which indicated that it was 8-fold more potent than voriconazole. In vivo, once-daily intranasal administration of PC1244 (3.2 to 80 ?g/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible A. fumigatus substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed in vivo effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of A. fumigatus infection in the lungs of humans.
Project description:Development of new antimicrobial agents, capable of combating resistant and multidrug-resistant fungal and bacterial clinical strains, is necessary. This study presents the synthesis and antimicrobial screening of 42 2-substituted-1,4-benzenediols, being 10 novel compounds. In total, 23 compounds showed activity against fungi and/or bacteria. Benzenediol compounds 2, 5, 6, 8, 11, and 12 demonstrated broad spectrum antimicrobial actions, including resistant and multidrug-resistant species of dermatophytes (Trichophyton mentagrophytes), Candida spp. and the ESKAPE panel of bacteria. Minimum inhibitory concentrations of these compounds for fungi and bacterial strains ranged from 25 to 50?µg/ml and 8-128?µg/ml, respectively. The antifungal mechanism of action is related to the fungal cell wall of dermatophytes and membrane disruption to dermatophytes and yeasts, in the presence of compound 8. Specific structural changes, such as widespread thinning along the hyphae and yeast lysis, were observed by scanning electron microscopy. The effects of compound 8 on cell viability are dose-dependent; however they did not cause genotoxicity and mutagenicity in human leukocyte cells nor haemolysis. Moreover, the compounds were identified as nonirritant by the ex-vivo Hen's egg test-chorioallantoic membrane (HET-CAM). The furan-1,4-benzenediol compound 5 showed in vivo efficacy to combat S. aureus infection using embryonated chicken eggs. Therefore, the compounds 8, and 5 are promising as hits for the development of new antimicrobial drugs with reduced toxicity.
Project description:Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear.The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model.Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2?g/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load.Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent.The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development.
Project description:Oral candidiasis is closely associated with changes in oral fungal biodiversity and is caused primarily by Candida albicans. However, the widespread use of empiric and prophylactic antifungal drugs has caused a shift in fungal biodiversity towards other Candida or yeast species. Recently, next-generation sequencing (NGS) has provided an improvement over conventional culture techniques, allowing rapid comprehensive analysis of oral fungal biodiversity. In this study, we used NGS to examine the oral fungal biodiversity of 27 patients with pseudomembranous oral candidiasis (POC) and 66 healthy controls. The total number of fungal species in patients with POC and healthy controls was 67 and 86, respectively. The copy number of total PCR products and the proportion of non-C. albicans, especially C. dubliniensis, in patients with POC, were higher than those in healthy controls. The detection patterns in patients with POC were similar to those in controls after antifungal treatment. Interestingly, the number of fungal species and the copy number of total PCR products in healthy controls increased with aging. These results suggest that high fungal biodiversity and aging might be involved in the pathogenesis of oral candidiasis. We therefore conclude that NGS is a useful technique for investigating oral candida infections.