Antifungal Synergy: Mechanistic Insights into the R-1-R Peptide and Bidens pilosa Extract as Potent Therapeutics against Candida spp. through Proteomics.

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.

Exploring the Potential Mechanism of Action of Piperine against Candida albicans and Targeting Its Virulence Factors.

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.

First report of environmental isolation of Cryptococcus and Cryptococcus-like yeasts from Boyacá, Colombia.

The Cryptococcus genus comprises more than 100 species, of which C. neoformans and C. gattii are the leading cause of cryptococcosis. The distribution of C. gattii and C. neoformans species complexes has been extensively studied and widely reported globally. Other species such as Naganishia albida, Papiliotrema laurentii, and Papiliotrema flavescens have been reported as pathogenic yeasts. Since there are no reports of environmental isolation in the Boyacá region (Colombia), this study aimed to isolate and characterize Cryptococcus and Cryptococcus-like yeasts from pigeon feces, Eucalyptus, and olive trees distributed in the municipalities of Tunja and Ricaute Alto. The environmental data was recovered, and the isolations obtained were identified by microscopy, biochemical test, MALDI-TOF MS, URA5-RFLP, and sequencing of the ITS and LSU loci. For the 93 pigeon dropping samples collected in Tunja, 23 yielded to C. neoformans, 3 to N. globosa, 2 N. albida and 1 to P. laurentii. Of the 1188 samples collected from olive trees, 17 (1.43%) positive samples were identified as C. gattii species complex (4), C. neoformans species complex (2), P. laurentii (3), N. albida (2), N. globosa (5) and P. flavescens (1). Likewise, specimens of C. neoformans presented molecular type VNI and molecular type VNII; for C. gattii the molecular types found were VGIII and one VGIV by URA5-RFLP but VGIII by MALDI-TOF and sequencing of the ITS and LSU. Therefore, it can be concluded that the species of Cryptococcus, Naganishia and Papiliotrema genera, are present in the environment of Boyacá, and show a predilection for climate conditions that are typical of this region.

Combining the Peptide RWQWRWQWR and an Ethanolic Extract of Bidens pilosa Enhances the Activity against Sensitive and Resistant Candida albicans and C. auris Strains.

The antifungal activity of palindromic peptide RWQWRWQWR and its derivatives was evaluated against clinical isolates of Candida albicans and C. auris. Also, Bidens pilosa ethanolic extracts of leaves and stem were evaluated. Furthermore, combinations of peptide, extract, and/or fluconazole (FLC) were evaluated. The cytotoxicity of peptides and extracts in erythrocytes and fibroblasts was determined. The original palindromic peptide, some derivative peptides, and the ethanolic extract of leaves of B. pilosa exhibited the highest activity in some of the strains evaluated. Synergy was obtained between the peptide and the FLC against C. auris 435. The combination of the extract and the original palindromic peptide against C. albicans SC5314, C. auris 435, and C. auris 537 decreased the minimal inhibitory concentrations (MICs) by a factor of between 4 and 16. These mixtures induced changes in cell morphology, such as deformations on the cell surface. The results suggest that the combination of RWQWRWQWR and B. pilosa extract is an alternative for enhancing antifungal activity and decreasing cytotoxicity and costs and should be considered to be a promising strategy for treating diseases caused by Candida spp.

Non-natural amino acids into LfcinB-derived peptides: effect in their (i) proteolytic degradation and (ii) cytotoxic activity against cancer cells.

The dimeric peptide 26[F]: (RRWQWRFKKLG)2-K-Ahx has exhibited a potent cytotoxic effect against breast cancer cell lines, with position 26 (F) being the most relevant for anti-cancer activity. In this investigation, six analogues of the 26[F] peptide were synthesized in which the 26th position was replaced by non-natural hydrophobic amino acids, finding that some modifications increased the resistance to proteolytic degradation exerted by trypsin or pepsin. Additionally, these modifications increased the cytotoxic effect against breast cancer cells and generated cell death mediated by apoptosis pathways, activating caspases 8 and 9, and did not compromise the integrity of the cytoplasmic membrane. Finally, it was found that the modified peptides have a broad spectrum of action, since they also have a cytotoxic effect against the HeLa human cervical cancer cell line. Peptide 26[F] was inoculated in mice by ip administration and the lethal dose 50 (LD50) was between 70 and 140 mg kg-1. While for the 26[1-Nal]: (RRWQWR-1-Nal-KKLG)2-K-Ahx peptide, a dose-response test was performed, and the survival rate was 100%. These results suggested that these peptides are safe in this animal model and could be considered as promissory to develop a treatment against breast cancer.

Proteomics and Genetic Approaches Elucidate the Circulation of Low Variability Staphylococcus aureus Strains on Colombian Dairy Farms.

Staphylococcus aureus is one of the most prevalent pathogens causing bovine mastitis in the world, in part because of its ease of adaptation to various hosts and the environment. This study aimed to determine the prevalence of S. aureus in Colombian dairy farms and its relationship with the causal network of subclinical mastitis. From thirteen dairy farms enrolled, 1288 quarter milk samples (QMS) and 330 teat samples were taken from cows with positive (70.1%) and negative California Mastitis Test (CMT). In addition, 126 samples from the milking parlor environment and 40 from workers (nasal) were collected. On each dairy farm, a survey was conducted, and the milking process was monitored on the day of sampling. S. aureus was identified in 176 samples, i.e., 138 QMS, 20 from teats, 8 from the milking parlor environment, and 10 from workers' nasal swabs. Isolates identified as S. aureus underwent proteomics (clustering of mass spectrum) and molecular (tuf, coa, spa Ig, clfA, and eno genes) analysis. Regarding proteomics results, isolates were distributed into three clusters, each with members from all sources and all farms. Concerning molecular analysis, the virulence-related genes clfA and eno were identified in 41.3% and 37.8% of S. aureus isolates, respectively. We provide evidence on the circulation of S. aureus strains with limited variability among animals, humans, and the environment. The parameters with the lowest compliance in the farms which may be implicated in the transmission of S. aureus are the lack of handwashing and abnormal milk handling.

Peptide-Resorcinarene Conjugates Obtained via Click Chemistry: Synthesis and Antimicrobial Activity.

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.

The structure of associations: Method insights from analyzing 28 clinical isolates of Cryptococcus neoformans.

Clinical isolates of a fungal pathogen from a single region or country often exhibit structural clonality or phylogenetic clustering at the sequence or MLST level; such population structure can persist also in larger samples. In efforts to improve causal understanding of pathogenesis at the molecular level, genome-wide association screening methods initially designed for other kingdoms have been applied to fungi. The example of a Colombian dataset of 28 clinical Cryptococcus neoformans VNI isolates indicates where the output from standard pipelines may need to be analyzed in new ways in order to efficiently extract hypotheses for experiments from fungal genotype-phenotype data.

Collections of clinical isolates of a human fungal pathogen can consist of clusters of genetically similar isolates. Such clustering complicates the screening for genetic associations with clinically relevant traits. We propose new methods, illustrating them for the fungus causing cryptococcosis.

A Landscape of the Genomic Structure of Cryptococcus neoformans in Colombian Isolates.

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.

Changes in Length and Positive Charge of Palindromic Sequence RWQWRWQWR Enhance Cytotoxic Activity against Breast Cancer Cell Lines.

Breast cancer is one of the main causes of premature death in women; current treatments have low selectivity, generating strong physical and psychological sequelae. The palindromic peptide R-1-R (RWQWRWQWR) has cytotoxic activity against different cell lines derived from cancer and selectivity against noncancerous cells. To determine if changes in the charge/length of this peptide increase its activity, six peptides were obtained by SPPS, three of them with addition of Arg at the N, C-terminal or both and three with deletion of Arg at the N, C-terminal or both. The cytotoxic and selective activities were evaluated against MCF-7, MDA-MB-231, and MCF-12 cell lines and fibroblast primary cell culture, evidencing that the RR-1-R peptide with the inclusion of Arg in the N-terminal end maintained selectivity and increased cytotoxicity against lines derived from breast cancer. The effect of this addition regarding the type of induced cell death was evaluated by flow cytometry, showing very low rates of necrosis and a significant majority of apoptotic events with activation of both Caspase 8 and Caspase 9. This work allowed us to find a modification that generates a peptide with greater cytotoxic effects and can be considered a promising molecule for other approaches to improve anticancer peptides.

In Vitro Antifungal Activity of Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II against Cryptococcus neoformans var. grubii.

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.

Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp.

Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 µg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.

Evaluation of Anti-Candida Potential of Piper nigrum Extract in Inhibiting Growth, Yeast-Hyphal Transition, Virulent Enzymes, and Biofilm Formation.

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.

Head-to-head comparison of CLSI, EUCAST, Etest and VITEK®2 results for Candida auris susceptibility testing.

The susceptibility of 31 Candida auris clinical isolates was evaluated by four methods, namely the microdilution reference method according to Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines as well as Etest and VITEK®2. Essential agreement between the two reference methods was 90%. Etest showed a better overall agreement with the reference methods (94% and 81% for CLSI and EUCAST, respectively) than VITEK®2 (70% and 72%, respectively). Discrepancies were found for fluconazole (FLC) and amphotericin B. Considering categorical agreement (CDC tentative breakpoints), the majority of isolates were considered FLC-resistant (93.6% and 80.6% by CLSI and EUCAST, respectively). Furthermore, all isolates were considered susceptible to echinocandins by all methods. Susceptibility results should be interpreted with care if the VITEK®2 system is used to guide therapeutic decisions for C. auris infections.

Deciphering the Association among Pathogenicity, Production and Polymorphisms of Capsule/Melanin in Clinical Isolates of Cryptococcus neoformans var. grubii VNI.

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.

The Combination of Iron and Copper Increases Pathogenicity and Induces Proteins Related to the Main Virulence Factors in Clinical Isolates of Cryptococcus neoformans var. grubii.

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.

Designing Chimeric Peptides: A Powerful Tool for Enhancing Antibacterial Activity.

Chimeric peptides containing short sequences derived from bovine Lactoferricin (LfcinB) and Buforin II (BFII) were synthetized using solid-phase peptide synthesis (SPPS) and characterized via reversed-phase liquid chromatography and mass spectrometry. The chimeras were obtained with high purity, demonstrating their synthetic viability. The chimeras' antibacterial activity against Gram-positive and Gram-negative strains was evaluated. Our results showed that all the chimeras exhibited greater antibacterial activity against the evaluated strains than the individual sequences, suggesting that chemical binding of short sequences derived from AMPs significantly increased the antibacterial activity. For each strain, the chimera with the best antibacterial activity exerted a bacteriostatic and/or bactericidal effect, which was dependent on the concentration. It was found that: (i) the antibacterial activity of a chimera is mainly influenced by the linked sequences, the palindromic motif RLLRRLLR being the most relevant one; (ii) the inclusion of a spacer between the short sequences did not significantly affect the chimera's synthesis process; however, it enhanced its antibacterial activity against Gram-negative and Gram-positive strains; on the other hand, (iii) the replacement of Arg with Lys in the LfcinB or BFII sequences improved the chimeras' synthesis process without significantly affecting their antibacterial activity. These results illustrate the great importance of the synthesis of chimeric peptides for the generation of promising antibacterial peptides.

Galleria mellonella as a Novelty in vivo Model of Host-Pathogen Interaction for Malassezia furfur CBS 1878 and Malassezia pachydermatis CBS 1879.

Malassezia furfur and Malassezia pachydermatis are lipophilic and lipid dependent yeasts, associated with the skin microbiota in humans and domestic animals, respectively. Although they are commensals, under specific conditions they become pathogens, causing skin conditions, such as pityriasis versicolor, dandruff/seborrheic dermatitis, folliculitis in humans, and dermatitis and otitis in dogs. Additionally, these species are associated with fungemia in immunocompromised patients and low-weight neonates in intensive care units with intravenous catheters or with parenteral nutrition and that are under-treatment of broad-spectrum antibiotics. The host-pathogen interaction mechanism in these yeasts is still unclear; for this reason, it is necessary to implement suitable new host systems, such as Galleria mellonella. This infection model has been widely used to assess virulence, host-pathogen interaction, and antimicrobial activity in bacteria and fungi. Some advantages of the G. mellonella model are: (1) the immune response has phagocytic cells and antimicrobial peptides that are similar to those in the innate immune response of human beings; (2) no ethical implications; (3) low cost; and (4) easy to handle and inoculate. This study aims to establish G. mellonella as an in vivo infection model for M. furfur and M. pachydermatis. To achieve this objective, first, G. mellonella larvae were first inoculated with different inoculum concentrations of these two Malassezia species, 1.5 × 106 CFU/mL, 1.5 × 107 CFU/mL, 1.5 × 108 CFU/mL, and 11.5 × 109 CFU/mL, and incubated at 33 and 37°C. Then, for 15 days, the mortality and melanization were evaluated daily. Finally, the characterization of hemocytes and fungal burden assessment were as carried out. It was found that at 33 and 37°C both M. furfur and M. pachydermatis successfully established a systemic infection in G. mellonella. M. pachydermatis proved to be slightly more virulent than M. furfur at a temperature of 37°C. The results suggest that larvae mortality and melanization is dependent on the specie of Malassezia, the inoculum concentration and the temperature. According to the findings, G. mellonella can be used as an in vivo model of infection to conduct easy and reliable approaches to boost our knowledge of the Malassezia genus.

Development and Validation of an in-House Library of Colombian Candida auris Strains with MALDI-TOF MS to Improve Yeast Identification.

: 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.

Selective cytotoxic effect against the MDA-MB-468 breast cancer cell line of the antibacterial palindromic peptide derived from bovine lactoferricin.

The cytotoxic effect against the breast cancer cell line MDA-MB-468 of the palindromic peptide LfcinB (21-25)Pal: 1RWQWRWQWR9 and its analogous peptides, obtained via alanine scanning, was evaluated. The results indicate that the palindromic peptide exhibited a concentration-dependent cytotoxic effect against this cell line. The cytotoxic effect of the palindromic peptide was fast and selective and was sustained for up to 48 h of treatment. MDA-MB-468 cells treated with the palindromic peptide exhibited severe cellular damage, acquiring rounded forms and shrinkage, a behavior typical of apoptotic events. The analogous peptides exhibited fewer cytotoxic effects than the original palindromic peptide, suggesting that the substitution of any amino acid with alanine diminishes the cytotoxic effect. The Arg and Trp residues proved to be the most relevant for the cytotoxic effect; the analogous peptides with substitutions of Trp with Ala did not induce a change in cellular morphology, while analogous peptides with substitutions of Arg or Gln with Ala induced cellular damage. Also, neither the palindromic peptide nor its analogues exerted a significant cytotoxic effect on normal fibroblasts, indicating that the peptides had a selective cytotoxic effect on cancerous cells. The peptide LfcinB (21-25)Pal, and its analogues exhibited antibacterial activity against E. coli and S. aureus strains and a selective cytotoxic effect against the breast cancer cell line MDA-MB-468.