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Previous reports have demonstrated that the peptide derived from LfcinB, R-1-R, exhibits anti-Candida activity, which is enhanced when combined with an extract from the Bidens pilosa plant. However, the mechanism of action remains unexplored. In this research, a proteomic study was carried out, followed by a bioinformatic analysis and biological assays in both the SC5314 strain and a fluconazole-resistant isolate of Candida albicans after incubation with R-1-R. The proteomic data revealed that treatment with R-1-R led to the up-regulation of most differentially expressed proteins compared to the controls in both strains. These proteins are primarily involved in membrane and cell wall biosynthesis, membrane transport, oxidative stress response, the mitochondrial respiratory chain, and DNA damage response. Additionally, proteomic analysis of the C. albicans parental strain SC5314 treated with R-1-R combined with an ethanolic extract of B. pilosa was performed. The differentially expressed proteins following this combined treatment were involved in similar functional processes as those treated with the R-1-R peptide alone but were mostly down-regulated (data are available through ProteomeXchange with identifier PXD053558). Biological assays validated the proteomic results, evidencing cell surface damage, reactive oxygen species generation, and decreased mitochondrial membrane potential. These findings provide insights into the complex antifungal mechanisms of the R-1-R peptide and its combination with the B. pilosa extract, potentially informing future studies on natural product derivatives. [ABSTRACT FROM AUTHOR]

Plant-derived compounds have proven to be a source of inspiration for new drugs. In this study, piperine isolated from the fruits of Piper nigrum showed anti-Candida activity. Furthermore, the mechanisms of action of piperine and its impact on virulence factors in Candida albicans, which have not been comprehensively understood, were also assessed. Initially, piperine suppressed the hyphal transition in both liquid and solid media, hindered biofilm formation, and resulted in observable cell distortions in scanning electron microscope (SEM) samples, for both fluconazole-sensitive and fluconazole-resistant C. albicans strains. Additionally, the morphogenetic switches triggered by piperine were found to rely on the activity of mutant C. albicans strains. Secondly, piperine treatment increased cell membrane permeability and disrupted mitochondrial membrane potential, as evidenced by propidium iodine and Rhodamine 123 staining, respectively. Moreover, it induced the accumulation of intracellular reactive oxygen species in C. albicans. Synergy was obtained between the piperine and the fluconazole against the fluconazole-sensitive strain. Interestingly, there were no hemolytic effects of piperine, and it resulted in reduced cytotoxicity on fibroblast cells at low concentrations. The results suggest that piperine could have a dual mode of action inhibiting virulence factors and modulating cellular processes, leading to cell death in C. albicans. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is one of the top ten threats to public health, as reported by the World Health Organization (WHO). One of the causes of the growing AMR problem is the lack of new therapies and/or treatment agents; consequently, many infectious diseases could become uncontrollable. The need to discover new antimicrobial agents that are alternatives to the existing ones and that allow mitigating this problem has increased, due to the rapid and global expansion of AMR. Within this context, both antimicrobial peptides (AMPs) and cyclic macromolecules, such as resorcinarenes, have been proposed as alternatives to combat AMR. Resorcinarenes present multiple copies of antibacterial compounds in their structure. These conjugate molecules have exhibited antifungal and antibacterial properties and have also been used in anti-inflammatory, antineoplastic, and cardiovascular therapies, as well as being useful in drug and gene delivery systems. In this study, it was proposed to obtain conjugates that contain four copies of AMP sequences over a resorcinarene core. Specifically, obtaining (peptide)4-resorcinarene conjugates derived from LfcinB (20–25): RRWQWR and BF (32–34): RLLR was explored. First, the synthesis routes that allowed obtaining: (a) alkynyl-resorcinarenes and (b) peptides functionalized with the azide group were established. These precursors were used to generate (c) (peptide)4-resorcinarene conjugates by azide-alkyne cycloaddition CuAAC, a kind of click chemistry. Finally, the conjugates' biological activity was evaluated: antimicrobial activity against reference strains and clinical isolates of bacteria and fungi, and the cytotoxic activity over erythrocytes, fibroblast, MCF-7, and HeLa cell lines. Our results allowed establishing a new synthetic route, based on click chemistry, for obtaining macromolecules derived from resorcinarenes functionalized with peptides. Moreover, it was possible to identify promising antimicrobial chimeric molecules that may lead to advances in the development of new therapeutic agents. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans species complexes are recognized as environmental fungi responsible for lethal meningoencephalitis in immunocompromised individuals. Despite the vast knowledge about the epidemiology and genetic diversity of this fungus in different regions of the world, more studies are necessary to comprehend the genomic profiles across South America, including Colombia, considered to be the second country with the highest number of Cryptococcosis. Here, we sequenced and analyzed the genomic architecture of 29 Colombian C. neoformans isolates and evaluated the phylogenetic relationship of these strains with publicly available C. neoformans genomes. The phylogenomic analysis showed that 97% of the isolates belonged to the VNI molecular type and the presence of sub-lineages and sub-clades. We evidenced a karyotype without changes, a low number of genes with copy number variations, and a moderate number of single-nucleotide polymorphisms (SNPs). Additionally, a difference in the number of SNPs between the sub-lineages/sub-clades was observed; some were involved in crucial fungi biological processes. Our study demonstrated the intraspecific divergence of C. neoformans in Colombia. These findings provide evidence that Colombian C. neoformans isolates do not probably require significant structural changes as adaptation mechanisms to the host. To the best of our knowledge, this is the first study to report the whole genome sequence of Colombian C. neoformans isolates. [ABSTRACT FROM AUTHOR]

Cryptococcosis is associated with high rates of morbidity and mortality. The limited number of antifungal agents, their toxicity, and the difficulty of these molecules in crossing the blood–brain barrier have made the exploration of new therapeutic candidates against Cryptococcus neoformans a priority task. To optimize the antimicrobial functionality and improve the physicochemical properties of AMPs, chemical strategies include combinations of peptide fragments into one. This study aimed to evaluate the binding of the minimum activity motif of bovine lactoferricin (LfcinB) and buforin II (BFII) against C. neoformans var. grubii. The antifungal activity against these chimeras was evaluated against (i) the reference strain H99, (ii) three Colombian clinical strains, and (iii) eleven mutant strains, with the aim of evaluating the possible antifungal target. We found high activity against these strains, with a MIC between 6.25 and 12.5 µg/mL. Studies were carried out to evaluate the effect of the combination of fluconazole treatments, finding a synergistic effect. Finally, when fibroblast cells were treated with 12.5 µg/mL of the chimeras, a viability of more than 65% was found. The results obtained in this study identify these chimeras as potential antifungal molecules for future therapeutic applications against cryptococcosis. [ABSTRACT FROM AUTHOR]

Due to the increased incidence of fungal infections and the emergence of antifungal resistance mainly by Candida species, the need for safe and effective novel therapies is imperative. Consequently, plants and herbs are a powerful source to combat infections. Here, we evaluated the anti-Candida potential of an ethanolic extract from Piper nigrum. The phytochemical analysis of P. nigrum revealed bioactive compounds such as alkaloids, terpenoids, and tannis. Our results showed that P. nigrum extract suppressed the virulence factors of C. albicans strains, including hyphae formation in both liquid and solid media, reduced secretion of phospholipases/proteinases, and affected biofilm formation. Furthermore, the P. nigrum extract showed no hemolytic effect in vitro and exhibited reduced cytotoxicity on Vero cells and G. mellonella larvae at concentrations that inhibited hyphae and biofilm in C. albicans. Moreover, the extract demonstrated antifungal activity against C. auris strains. In conclusion, the P. nigrum extract affected the growth and morphogenesis of Candida (even in resistant strains), demonstrating that this plant has an anti-candida activity and represents a promising resource for discovering novel antifungal compounds. [ABSTRACT FROM AUTHOR]

Background: Cryptococcus neoformans is an opportunistic fungal pathogen that can cause meningitis in immunocompromised individuals. The objective of this work was to study the relationship between the phenotypes and genotypes of isolates of clinical origin from different cities in Colombia. Methods: Genome classification of 29 clinical isolates of C. neoformans var. grubii was performed using multilocus sequence typing (MLST), and genomic sequencing was used to genotype protein-coding genes. Pathogenicity was assessed in a larval model, and melanin production and capsule size were evaluated in vitro and in vivo. Results: Eleven MLST sequence types (STs) were found, the most frequent being ST69 (n = 9), ST2, ST93, and ST377 (each with n = 4). In the 29 isolates, different levels of pigmentation, capsule size and pathogenicity were observed. Isolates classified as highly pathogenic showed a tendency to exhibit larger increases in capsule size. In the analysis of polymorphisms, 48 non-synonymous variants located in the predicted functional domains of 39 genes were found to be associated with capsule size change, melanin, or pathogenicity. Conclusions: No clear patterns were found in the analysis of the phenotype and genotype of Cryptococcus. However, the data suggest that the increase in capsule size is a key variable for the differentiation of pathogenic isolates, regardless of the method used for its induction. [ABSTRACT FROM AUTHOR]

Background: Candida auris is characterized for having a high genetic variability among species. MALDI-TOF MS library contains spectra from only three strains of C. auris, which makes difficult the identification process and gives low scores at the species level. Our aim was to construct and validate an internal library to improve C. auris identification with Colombian clinical strains. Methods: From 30 clinical strains, 770 mass spectra were obtained for the construction of the database. The validation was performed with 300 strains to compare the identification results in the BDAL and C. auris Colombia libraries. Results: Our library allowed a complete, 100% identification of the evaluated strains and a significant improvement in the scores obtained, showing a better performance compared to the Bruker BDAL library. Conclusions: The strengthening of the database is a great opportunity to improve the scoring and C. auris identification. Library data are available via ProteomeXchange with identifier PXD016387. [ABSTRACT FROM AUTHOR]

In fungi, metals are associated with the expression of virulence factors. However, it is unclear whether the uptake of metals affects their pathogenicity. This study aimed to evaluate the effect of iron/copper in modulating pathogenicity and proteomic response in two clinical isolates of C. neoformans with high and low pathogenicity. Methods: In both isolates, the effect of 50 µM iron and 500 µM copper on pathogenicity, capsule induction, and melanin production was evaluated. We then performed a quantitative proteomic analysis of cytoplasmic extracts exposed to that combination. Finally, the effect on pathogenicity by iron and copper was evaluated in eight additional isolates. Results: In both isolates, the combination of iron and copper increased pathogenicity, capsule size, and melanin production. Regarding proteomic data, proteins with increased levels after iron and copper exposure were related to biological processes such as cell stress, vesicular traffic (Ap1, Vps35), cell wall structure (Och1, Ccr4, Gsk3), melanin biosynthesis (Hem15, Mln2), DNA repair (Chk1), protein transport (Mms2), SUMOylation (Uba2), and mitochondrial transport (Atm1). Increased pathogenicity by exposure to metal combination was also confirmed in 90% of the eight isolates. Conclusions: The combination of these metals enhances pathogenicity and increases the abundance of proteins related to the main virulence factors. [ABSTRACT FROM AUTHOR]