Project description:Fungal infections in humans are increasing worldwide and are currently mostly treated with a relative limited set of antifungals. Resistance to antifungals is increasing, for example, in Aspergillus fumigatus and Candida auris, and expected to increase for many medically relevant fungal species in the near future. We have developed and patented a set of cathelicidin-inspired antimicrobial peptides termed 'PepBiotics'. These peptides were initially selected for their bactericidal activity against clinically relevant Pseudomonas aeruginosa and Staphylococcus aureus isolates derived from patients with cystic fibrosis and are active against a wide range of bacteria (ESKAPE pathogens). We now report results from studies that were designed to investigate the antifungal activity of PepBiotics against a set of medically relevant species encompassing species of Aspergillus, Candida, Cryptococcus, Fusarium, Malassezia, and Talaromyces. We characterized a subset of PepBiotics and show that these peptides strongly affected metabolic activity and/or growth of a set of medically relevant fungal species, including azole-resistant A. fumigatus isolates. PepBiotics showed a strong inhibitory activity against a large variety of filamentous fungi and yeasts species at low concentrations (≤1 μM) and were fungicidal for at least a subset of these fungal species. Interestingly, the concentration of PepBiotics required to interfere with growth or metabolic activity varied between different fungal species or even between isolates of the same fungal species. This study shows that PepBiotics display strong potential for use as novel antifungal compounds to fight a large variety of clinically relevant fungal species.
Project description: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 μg/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 μg/mL (IC50) and no toxic effect was found on normal human peripheral blood mononuclear cells even at higher concentration of 600 μg/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:The use of antifungal drugs in the therapy of fungal diseases can lead to the development of antifungal resistance. Resistance has been described for virtually all antifungal agents in diverse pathogens, including Candida and Aspergillus species. The majority of resistance mechanisms have also been elucidated at the molecular level in these pathogens. Drug resistance genes and genome mutations have been identified. Therapeutic choices are limited for the control of fungal diseases, and it is tempting to combine several drugs to achieve better therapeutic efficacy. In the recent years, several novel resistance patterns have been observed, including antifungal resistance originating from environmental sources in Aspergillus fumigatus and the emergence of simultaneous resistance to different antifungal classes (multidrug resistance) in different Candida species. This review will summarize these current trends.
Project description:The emergence of novel pathogens, exemplified recently by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need for rapidly deployable and adaptable diagnostic assays to assess their impact on human health and guide public health responses in future pandemics. In this study, we developed an automated multiplex microscopy assay coupled with machine learning-based analysis for antibody detection. To achieve multiplexing and simultaneous detection of multiple viral antigens, we devised a barcoding strategy utilizing a panel of HeLa-based cell lines. Each cell line expressed a distinct viral antigen, along with a fluorescent protein exhibiting a unique subcellular localization pattern for cell classification. Our robust, cell segmentation and classification algorithm, combined with automated image acquisition, ensured compatibility with a high-throughput approach. As a proof of concept, we successfully applied this approach for quantitation of immunoreactivity against different variants of SARS-CoV-2 spike and nucleocapsid proteins in sera of patients or vaccinees, as well as for the study of selective reactivity of monoclonal antibodies. Importantly, our system can be rapidly adapted to accommodate other SARS-CoV-2 variants as well as any antigen of a newly emerging pathogen, thereby representing an important resource in the context of pandemic preparedness.
Project description:Antimicrobial peptides are prominent components of the plant immune system acting against a wide variety of pathogens. Legume plants from the inverted repeat lacking clade (IRLC) have evolved a unique gene family encoding nodule-specific cysteine-rich NCR peptides acting in the symbiotic cells of root nodules, where they convert their bacterial endosymbionts into non-cultivable, polyploid nitrogen-fixing cells. NCRs are usually 30-50 amino acids long peptides having a characteristic pattern of 4 or 6 cysteines and highly divergent amino acid composition. While the function of NCRs is largely unknown, antimicrobial activity has been demonstrated for a few cationic Medicago truncatula NCR peptides against bacterial and fungal pathogens. The advantages of these plant peptides are their broad antimicrobial spectrum, fast killing modes of actions, multiple bacterial targets, and low propensity to develop resistance to them and no or low cytotoxicity to human cells. In the IRLC legumes, the number of NCR genes varies from a few to several hundred and it is possible that altogether hundreds of thousands of different NCR peptides exist. Due to the need for new antimicrobial agents, we investigated the antimicrobial potential of 104 synthetic NCR peptides from M. truncatula, M. sativa, Pisum sativum, Galega orientalis and Cicer arietinum against eight human pathogens, including ESKAPE bacteria. 50 NCRs showed antimicrobial activity with differences in the antimicrobial spectrum and effectivity. The most active peptides eliminated bacteria at concentrations from 0.8 to 3.1 μM. High isoelectric point and positive net charge were important but not the only determinants of their antimicrobial activity. Testing the activity of shorter peptide derivatives against Acinetobacter baumannii and Candida albicans led to identification of regions responsible for the antimicrobial activity and provided insight into their potential modes of action. This work provides highly potent lead molecules without hemolytic activity on human blood cells for novel antimicrobial drugs to fight against pathogens.
Project description:Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2-4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides.
Project description:The identification of dystrophin and the causative role of mutations in this gene in Duchenne and Becker muscular dystrophies (D/BMD) was expected to lead to timely development of effective therapies. Despite over 20 years of research, corticosteroids remain the only available pharmacological treatment for DMD, although significant benefits and extended life have resulted from advances in the clinical care and management of DMD individuals. Effective treatment of DMD will require dystrophin restitution in skeletal, cardiac, and smooth muscles and nonmuscle tissues; however, modulation of muscle loss and regeneration has the potential to play an important role in altering the natural history of DMD, particularly in combination with other treatments. Emerging biological, molecular, and small molecule therapeutics are showing promise in ameliorating this devastating disease, and it is anticipated that regulatory environments will need to display some flexibility in order to accommodate the new treatment paradigms.
Project description:The development of chemotherapies against eukaryotic pathogens is especially challenging because of both the evolutionary conservation of drug targets between host and parasite, and the evolution of strain-dependent drug resistance. There is a strong need for new nontoxic drugs with broad-spectrum activity against trypanosome parasites such as Leishmania and Trypanosoma. A relatively untested approach is to target macromolecular interactions in parasites rather than small molecular interactions, under the hypothesis that the features specifying macromolecular interactions diverge more rapidly through coevolution. We computed tRNA Class-Informative Features in humans and independently in eight distinct clades of trypanosomes, identifying parasite-specific informative features, including base pairs and base mis-pairs, that are broadly conserved over approximately 250 million years of trypanosome evolution. Validating these observations, we demonstrated biochemically that tRNA:aminoacyl-tRNA synthetase (aaRS) interactions are a promising target for anti-trypanosomal drug discovery. From a marine natural products extract library, we identified several fractions with inhibitory activity toward Leishmania major alanyl-tRNA synthetase (AlaRS) but no activity against the human homolog. These marine natural products extracts showed cross-reactivity towards Trypanosoma cruzi AlaRS indicating the broad-spectrum potential of our network predictions. We also identified Leishmania major threonyl-tRNA synthetase (ThrRS) inhibitors from the same library. We discuss why chemotherapies targeting multiple aaRSs should be less prone to the evolution of resistance than monotherapeutic or synergistic combination chemotherapies targeting only one aaRS.
Project description:Current agrochemicals used in crop farming mainly consist of synthetic compounds with harmful effects on the environment and human health. Crop-associated fungal endophytes, which play many ecological roles including defense against pathogens, represent a promising source for bioactive and ecologically safer molecules in agrochemical discovery. The methanolic extract of the endophyte Menisporopsis sp. LCM 1078 was evaluated in vitro against the plant pathogens Boeremia exigua, Calonectria variabilis, Colletotrichum theobromicola, Colletotrichum tropicale, and Mycena cytricolor. Bioassay-guided isolation using chromatographic techniques followed by detailed chemical characterization by NMR and mass spectrometry led to the identification of menisporopsin A, which showed inhibitory activity in a dose-dependent manner against the five fungal pathogens including an endophytic strain (Colletotrichum tropicale), with MIC values in the range of 0.63-10.0 μg/mL showing a potency equivalent to the broadly employed agrochemical mancozeb.
Project description:Unscientific use of synthetic fungicides in plant disease management has environmental ramifications, such as disease resurgence and serious health problems due to their carcinogenicity. This has prompted the identification and development of eco-friendly greener alternatives. Eclipta alba extract was evaluated for its antifungal activity in in vitro and in vivo against sorghum fungal pathogens Fusarium thapsinum, Alternaria alternata, Epicoccum sorghinum, and Curvularia lunata. The column purified methanolic extract of E. alba exhibited good antifungal activity against the target pathogens. The MIC was observed at 80 mg/mL for all tested pathogenic fungi, whereas MFC was 80 mg/mL for E. sorghinum, 100 mg/mL for F. thapsinum, A. alternata, and C. lunata. In vitro germination percentage was significantly high in seeds treated with E. alba extract (98%) over untreated control (91%). Significant disease protection of 95% was observed in greenhouse and 66% disease protection was noticed in field experiments. The efficacy of E. alba extract in field conditions was improved with the use of E. alba extract formulation. The profile of phytochemicals in E. alba methanol fractions was obtained by ultra-performance liquid chromatography (UPLC) mass spectroscopy. The [M-H]- at m/z 313.3, m/z 797.9, and m/z 269.0 revealed the presence of wedelolactone, eclalbasaponin II, and apigenin, respectively. The H-nuclear magnetic resonance spectroscopy (¹H-NMR) chemical shift value supported the findings of the mass spectrometry. The results highlighted the possible use of E. alba methanolic extract as alternative to chemical fungicide in sorghum disease management.