Paracoccidioides spp.: the structural characterization of extracellular matrix, expression of glucan synthesis and associated genes and adhesins during biofilm formation.

The genus Paracoccidioides includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which comprises four phylogenetic species. A key feature distinguishing planktonic growth from biofilm is the presence of a 3D extracellular matrix (ECM). Therefore, in this study, we analyzed biofilm formation in different species of Paracoccidioides yeast phase, characterized the structural elements of the matrix of P. brasiliensis (Pb18), P. lutzii (Pl01 and 8334) and P. restrepiensis (339 and 192) and evaluated the expression of glucan genes, according to the stage of biofilm evolution for P. brasiliensis. The strains were cultivated in planktonic and biofilm form for 24-144 h. The fungi biomass and metabolic activity were determined by crystal violet and tetrazolium salt reduction (XTT) tests and colony-forming unit (CFU) by plating. The biofilm structure was designed using scanning electron microscopy and confocal laser scanning microscopy techniques. The extracellular matrix of P. brasiliensis and P. lutzii biofilms was extracted by sonication, and polysaccharides, proteins, and extracellular DNA (eDNA) were quantified. The RNA was extracted with the Trizol® reagent and quantified; then, the cDNA was synthesized to analyze the enolase expression, 14-3-3, FKS1, AGS1, GEL3, and KRE6 genes by real-time PCR. All strains of Paracoccidioides studied form a biofilm with more significant metabolic activity and biomass values in 144 h. The extracellular matrix of P. brasiliensis and P. lutzii had a higher content of polysaccharides in their composition, followed by proteins and eDNA in smaller quantities. The P. brasiliensis biofilm kinetics of formation showed greater expression of genes related to glucan's synthesis and its delivery to the external environment in addition adhesins during the biofilm's adhesion, initiation, and maturation. The GEL3 and enolase genes increased in expression within 24 h and during the biofilm maturation period, there was an increase in 14-3-3, AGS1, and FKS1. Furthermore, at 144 h, there was a decrease in KRE6 expression and an increase in GEL3. This study highlights the potential for biofilm formation for three species of Paracoccidioides and the main components of the extracellular matrix that can contribute to a better understanding of biofilm organization.

Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

Amphotericin B-loaded natural latex dressing for treating Candida albicans wound infections using Galleria mellonella model.

Amphotericin B (AmB) is the gold standard for antifungal drugs. However, AmB systemic administration is restricted because of its side effects. Here, we report AmB loaded in natural rubber latex (NRL), a sustained delivery system with low toxicity, which stimulates angiogenesis, cell adhesion and accelerates wound healing. Physicochemical characterizations showed that AmB did not bind chemically to the polymeric matrix. Electronic and topographical images showed small crystalline aggregates from AmB crystals on the polymer surface. About 56.6% of AmB was released by the NRL in 120 h. However, 33.6% of this antifungal was delivered in the first 24 h due to the presence of AmB on the polymer surface. The biomaterial's excellent hemo- and cytocompatibility with erythrocytes and human dermal fibroblasts (HDF) confirmed its safety for dermal wound application. Antifungal assay against Candida albicans showed that AmB-NRL presented a dose-dependent behavior with an inhibition halo of 30.0 ± 1.0 mm. Galleria mellonella was employed as an in vivo model for C. albicans infection. Survival rates of 60% were observed following the injection of AmB (0.5 mg.mL-1) in G. mellonella larvae infected by C. albicans. Likewise, AmB-NRL (0.5 mg.mL-1) presented survival rates of 40%, inferring antifungal activity against fungus. Thus, NRL adequately acts as an AmB-sustained release matrix, which is an exciting approach, since this antifungal is toxic at high concentrations. Our findings suggest that AmB-NRL is an efficient, safe, and reasonably priced ($0.15) dressing for the treatment of cutaneous fungal infections.

Polypeptides Targeting Paracoccidioides brasiliensis Drk1.

Considering the toxicity of conventional therapeutic approaches and the importance of precise mechanistic targets, it is important to explore signaling pathways implicated in fungal pathobiology. Moreover, treatment of paracoccidioidomycosis, a systemic mycosis caused by a dimorphic fungus, requires prolonged therapeutic regimens. Among the numerous factors underpinning the establishment of Paracoccidioides spp. infection, the capacity to transition from the mycelial to the yeast form is of pivotal importance. The Drk1 protein of Paracoccidioides brasiliensis likely plays a decisive role in this morphological shift and subsequent virulence. We identified peptides with affinity for the PbDrk1 protein using the phage-display method and assessed the effects of these peptides on P. brasiliensis. The peptides were found to inhibit the phase transition of P. brasiliensis. Furthermore, a substantial proportion of these peptides prevented adhesion to pneumocytes. Although these peptides may not possess inherent antifungal properties, they can augment the effects of certain antifungal agents. Notably, the cell wall architecture of P. brasiliensis appears to be modulated by peptide intervention, resulting in a reduced abundance of glycosylated proteins and lipids. These peptides were also evaluated for their efficacy in a Galleria mellonella model and shown to contribute to enhanced larval survival rates. The role of PbDrk1, which is notably absent in mammals, should be further investigated to improve the understanding of its functional role in P. brasiliensis, which may be helpful for designing novel therapeutic modalities.

Alternative Non-Mammalian Animal and Cellular Methods for the Study of Host-Fungal Interactions.

In the study of fungal pathogenesis, alternative methods have gained prominence due to recent global legislation restricting the use of mammalian animals in research. The principle of the 3 Rs (replacement, reduction, and refinement) is integrated into regulations and guidelines governing animal experimentation in nearly all countries. This principle advocates substituting vertebrate animals with other invertebrate organisms, embryos, microorganisms, or cell cultures. This review addresses host-fungus interactions by employing three-dimensional (3D) cultures, which offer more faithful replication of the in vivo environment, and by utilizing alternative animal models to replace traditional mammals. Among these alternative models, species like Caenorhabditis elegans and Danio rerio share approximately 75% of their genes with humans. Furthermore, models such as Galleria mellonella and Tenebrio molitor demonstrate similarities in their innate immune systems as well as anatomical and physiological barriers, resembling those found in mammalian organisms.

Green Chromatographic Methods and in Vitro Pharmacological Analysis of Varronia curassavica Leaf Derivatives.

Varronia curassavica displays anti-inflammatory, antiulcerogenic, and antioxidant activities. Herein, we employed new UHPLC -UV green chromatographic methods for the analysis of in vitro antioxidant and anti-inflammatory activities of V. curassavica and its embryotoxicity in Zebrafish. Cordialin A, brickellin, and artemetin were purified from the ethanol (EtOH) extract of V. Curassavica leaves and identified using spectrometric techniques. In line with Green Analytical Chemistry principles, the proposed UHPLC methods involve the use of ethanol as organic modifier with low mobile phase consumption, and without sample pretreatment (OLE-UHPLC-UV). The application of the Agree and HPLC-EAT tools for greenness assessment yielded this pattern: HPLC-UV (reference)

Alginate-Based 3D A549 Cell Culture Model to Study Paracoccidioides Infection.

A three-dimensional (3D) lung aggregate model based on sodium alginate scaffolds was developed to study the interactions between Paracoccidioides brasiliensis (Pb) and lung epithelial cells. The suitability of the 3D aggregate as an infection model was examined using cell viability (cytotoxicity), metabolic activity, and proliferation assays. Several studies exemplify the similarity between 3D cell cultures and living organisms, which can generate complementary data due to the greater complexity observed in these designed models, compared to 2D cell cultures. A 3D cell culture system of human A549 lung cell line plus sodium alginate was used to create the scaffolds that were infected with Pb18. Our results showed low cytotoxicity, evidence of increased cell density (indicative of cell proliferation), and the maintenance of cell viability for seven days. The confocal analysis revealed viable yeast within the 3D scaffold, as demonstrated in the solid BHI Agar medium cultivation. Moreover, when ECM proteins were added to the alginate scaffolds, the number of retrieved fungi was significantly higher. Our results highlight that this 3D model may be promising for in vitro studies of host-pathogen interactions.

Biocompatible anti-aging face mask prepared with curcumin and natural rubber with antioxidant properties.

Natural rubber latex (NRL) is a biopolymer widely used in biomedical applications. In this work, we propose an innovative cosmetic face mask, combining the NRL's biological properties with curcumin (CURC), which has a high level of antioxidant activity (AA) to provide anti-aging benefits. Chemical, mechanical and morphological characterizations were performed. The CURC released by the NRL was evaluated by permeation in Franz cells. Cytotoxicity and hemolytic activity assays were performed to assess safety. The findings showed that the biological properties of CURC were preserved after loading in the NRL. About 44.2 % of CURC was released within the first six hours, and in vitro permeation showed that 9.36 % ± 0.65 was permeated over 24h. CURC-NRL was associated with a metabolic activity higher than 70 % in 3 T3 fibroblasts, cell viability ≥95 % in human dermal fibroblasts, and a hemolytic rate ≤ 2.24 % after 24 h. Furthermore, CURC-NRL maintained the mechanical characteristics (range suitable) for human skin application. We observed that CURC-NRL preserved ~20 % antioxidant activity from curcumin-free after loading in the NRL. Our results suggest that CURC-NRL has the potential to be used in the cosmetics industry, and the experimental methodology utilized in this study can be applied to different kinds of face masks.

Membranolytic Activity Profile of Nonyl 3,4-Dihydroxybenzoate: A New Anti-Biofilm Compound for the Treatment of Dermatophytosis.

The ability of dermatophytes to live in communities and resist antifungal drugs may explain treatment recurrence, especially in onychomycosis. Therefore, new molecules with reduced toxicity that target dermatophyte biofilms should be investigated. This study evaluated nonyl 3,4-dihydroxybenzoate (nonyl) susceptibility and mechanism of action on planktonic cells and biofilms of T. rubrum and T. mentagrophytes. Metabolic activities, ergosterol, and reactive oxygen species (ROS) were quantified, and the expression of genes encoding ergosterol was determined by real-time PCR. The effects on the biofilm structure were visualized using confocal electron microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). T. rubrum and T. mentagrophytes biofilms were susceptible to nonyl and resistant to fluconazole, griseofulvin (all strains), and terbinafine (two strains). The SEM results revealed that nonyl groups seriously damaged the biofilms, whereas synthetic drugs caused little or no damage and, in some cases, stimulated the development of resistance structures. Confocal microscopy showed a drastic reduction in biofilm thickness, and transmission electron microscopy results indicated that the compound promoted the derangement and formation of pores in the plasma membrane. Biochemical and molecular assays indicated that fungal membrane ergosterol is a nonyl target. These findings show that nonyl 3,4-dihydroxybenzoate is a promising antifungal compound.

Nanotechnology-Based Approaches for Voriconazole Delivery Applied to Invasive Fungal Infections.

Invasive fungal infections increase mortality and morbidity rates worldwide. The treatment of these infections is still limited due to the low bioavailability and toxicity, requiring therapeutic monitoring, especially in the most severe cases. Voriconazole is an azole widely used to treat invasive aspergillosis, other hyaline molds, many dematiaceous molds, Candida spp., including those resistant to fluconazole, and for infections caused by endemic mycoses, in addition to those that occur in the central nervous system. However, despite its broad activity, using voriconazole has limitations related to its non-linear pharmacokinetics, leading to supratherapeutic doses and increased toxicity according to individual polymorphisms during its metabolism. In this sense, nanotechnology-based drug delivery systems have successfully improved the physicochemical and biological aspects of different classes of drugs, including antifungals. In this review, we highlighted recent work that has applied nanotechnology to deliver voriconazole. These systems allowed increased permeation and deposition of voriconazole in target tissues from a controlled and sustained release in different routes of administration such as ocular, pulmonary, oral, topical, and parenteral. Thus, nanotechnology application aiming to delivery voriconazole becomes a more effective and safer therapeutic alternative in the treatment of fungal infections.

Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures.

Histoplasma capsulatum is a fungus that causes histoplasmosis. The increased evolution of microbial resistance and the adverse effects of current antifungals help new drugs to emerge. In this work, fifty-four nitrofurans and indoles were tested against the H. capsulatum EH-315 strain. Compounds with a minimum inhibitory concentration (MIC90) equal to or lower than 7.81 µg/mL were selected to evaluate their MIC90 on ATCC G217-B strain and their minimum fungicide concentration (MFC) on both strains. The quantification of membrane ergosterol, cell wall integrity, the production of reactive oxygen species, and the induction of death by necrosis-apoptosis was performed to investigate the mechanism of action of compounds 7, 11, and 32. These compounds could reduce the extracted sterol and induce necrotic cell death, similarly to itraconazole. Moreover, 7 and 11 damaged the cell wall, causing flaws in the contour (11), or changing the size and shape of the fungal cell wall (7). Furthermore, 7 and 32 induced reactive oxygen species (ROS) formation higher than 11 and control. Finally, the cytotoxicity was measured in two models of cell culture, i.e., monolayers (cells are flat) and a three-dimensional (3D) model, where they present a spheroidal conformation. Cytotoxicity assays in the 3D model showed a lower toxicity in the compounds than those performed on cell monolayers. Overall, these results suggest that derivatives of nitrofurans and indoles are promising compounds for the treatment of histoplasmosis.

Synthesis and Evaluation of the Antifungal and Toxicological Activity of Nitrofuran Derivatives.

Fungal diseases affect more than 1 billion people worldwide. The constant global changes, the advent of new pandemics, and chronic diseases favor the diffusion of fungal pathogens such as Candida, Cryptococcus, Aspergillus, Trichophyton, Histoplasma capsulatum, and Paracoccidioides brasiliensis. In this work, a series of nitrofuran derivatives were synthesized and tested against different fungal species; most of them showed inhibitory activity, fungicide, and fungistatic profile. The minimal inhibitory concentration (MIC90) values for the most potent compounds range from 0.48 µg/mL against H. capsulatum (compound 11) and P. brasiliensis (compounds 3 and 9) to 0.98 µg/mL against Trichophyton rubrum and T. mentagrophytes (compounds 8, 9, 12, 13 and 8, 12, 13, respectively), and 3.9 µg/mL against Candida and Cryptococcus neoformans strains (compounds 1 and 5, respectively). In addition, all compounds showed low toxicity when tested in vitro on lung cell lines (A549 and MRC-5) and in vivo in Caenorhabditis elegans larvae. Many of them showed high selectivity index values. Thus, these studied nitrofuran derivatives proved to be potent against different fungal species, characterized by low toxicity and high selectivity; for these reasons, they may become promising compounds for the treatment of mycoses.

Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity.

Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host´s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 × 104 cells/cm2, and then adjusted to 2 × 106 cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded via total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens.

Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro.

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3'hydroxychalcone (3'chalc) -loaded nanoemulsion (NE) (NE3'chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3'chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3'hidroxychalcone (3'chalc) range from 0.97- 7.8 µg/mL and NE3'chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3'chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3'chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3'chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.

Drk1, a Dimorphism Histidine Kinase, Contributes to Morphology, Virulence, and Stress Adaptation in Paracoccidioides brasiliensis.

P. brasiliensis is a thermally dimorphic fungus belonging to Paracoccidioides complex, causative of a systemic, endemic mycosis limited to Latin American countries. Signal transduction pathways related to important aspects as surviving, proliferation according to the biological niches are linked to the fungal pathogenicity in many species, but its elucidation in P. brasiliensis remains poorly explored. As Drk1, a hybrid histidine kinase, plays regulators functions in other dimorphic fungi species, mainly in dimorphism and virulence, here we investigated its importance in P. brasilensis. We, therefore generated the respective recombinant protein, anti-PbDrk1 polyclonal antibody and a silenced strain. The Drk1 protein shows a random distribution including cell wall location that change its pattern during osmotic stress condition; moreover the P. brasiliensis treatment with anti-PbDrk1 antibody, which does not modify the fungus's viability, resulted in decreased virulence in G. mellonella model and reduced interaction with pneumocytes. Down-regulating PbDRK1 yielded phenotypic alterations such as yeast cells with more elongated morphology, virulence attenuation in G. mellonella infection model, lower amount of chitin content, increased resistance to osmotic and cell wall stresses, and also caspofungin, and finally increased sensitivity to itraconazole. These observations highlight the importance of PbDrk1 to P. brasiliensis virulence, stress adaptation, morphology, and cell wall organization, and therefore it an interesting target that could help develop new antifungals.

The Antimicrobial Peptide MK58911-NH2 Acts on Planktonic, Biofilm, and Intramacrophage Cells of Cryptococcus neoformans.

Cryptococcosis is associated with high rates of morbidity and mortality, especially in AIDS patients. Its treatment is carried out by combining amphotericin B and azoles or flucytosine, which causes unavoidable toxicity issues in the host. Thus, the urgency in obtaining new antifungals drives the search for antimicrobial peptides (AMPs). This study aimed to extend the understanding of the mechanism of action of an AMP analog from wasp peptide toxins, MK58911-NH2, on Cryptococcus neoformans. We also evaluated if MK58911-NH2 can act on cryptococcal cells in macrophages, biofilms, and an immersion zebrafish model of infection. Finally, we investigated the structure-antifungal action and the toxicity relationship of MK58911-NH2 fragments and a derivative of this peptide (MH58911-NH2). The results demonstrated that MK58911-NH2 did not alter the fluorescence intensity of the cell wall-binding dye calcofluor white or the capsule-binding dye 18b7 antibody-fluorescein isothiocyanate (FITC) in C. neoformans but rather reduced the number and size of fungal cells. This activity reduced the fungal burden of C. neoformans in both macrophages and zebrafish embryos as well as within biofilms. Three fragments of the MK58911-NH2 peptide showed no activity against Cryptococcus and not toxicity in lung cells. The derivative peptide MH58911-NH2, in which the lysine residues of MK58911-NH2 were replaced by histidines, reduced the activity against extracellular and intracellular C. neoformans. On the other hand, it was active against biofilms and showed reduced toxicity. In summary, these results showed that peptide MK58911-NH2 could be a promising agent against cryptococcosis. This work also opens a perspective for the verification of the antifungal activity of other derivatives.

Natural rubber dressing loaded with silver sulfadiazine for the treatment of burn wounds infected with Candida spp.

Millions of people are burned worldwide every year and 265,000 of the cases are fatal. The development of burn treatment cannot consist only of the administration of a single drug. Due to the infection risk, antibiotics are used in conjunction with gels and damp bandages. In this work, an inexpensive curative based on silver sulfadiazine (SS) and natural rubber latex (NRL) was developed to treat burn wounds. It was produced by the casting method. The infrared spectrum presented no interaction between drug and biopolymer. At the same time, electronic micrographs showed that the SS crystals are inserted on the polymeric dressing surface. Mechanical properties after the drug incorporation were considered suitable for dermal application. About 32.4% of loaded SS was released in 192 h by the dressings that also inhibited the growth of Candida albicans and Candida parapsilosis at 75.0 and 37.5 µg·mL-1, respectively. The curative proved to be biocompatible when applied to fibroblast cells, in addition to enhancing cellular proliferation and, in the hemocompatibility test, no hemolytic effects were observed. The good results in mechanical, antifungal and biological assays, combined with the average bandage cost of $0.10, represent an exciting alternative for treating burn wounds.

2-Hydroxychalcone as a Potent Compound and Photosensitizer Against Dermatophyte Biofilms.

Dermatophytes, fungi that cause dermatophytosis, can invade keratinized tissues in humans and animals. The biofilm-forming ability of these fungi was described recently, and it may be correlated with the long treatment period and common recurrences of this mycosis. In this study, we evaluated the anti-dermatophytic and anti-biofilm activity of 2-hydroxychalcone (2-chalcone) in the dark and photodynamic therapy (PDT)-mediated and to determine its mechanism of action. Trichophyton rubrum and Trichophyton mentagrophytes strains were used in the study. The antifungal susceptibility test of planktonic cells, early-stage biofilms, and mature biofilms were performed using colorimetric methods. Topographies were visualized by scanning electron microscopy (SEM). Human skin keratinocyte (HaCat) monolayers were also used in the cytotoxicity assays. The mechanisms of action of 2-chalcone in the dark and under photoexcitation were investigated using confocal microscopy and the quantification of ergosterol, reactive oxygen species (ROS), and death induction by apoptosis/necrosis. All strains, in the planktonic form, were inhibited after treatment with 2-chalcone (minimum inhibitory concentration (MIC) = 7.8-15.6 mg/L), terbinafine (TRB) (MIC = 0.008-0.03 mg/L), and fluconazole (FLZ) (1-512 mg/L). Early-stage biofilm and mature biofilms were inhibited by 2-chalcone at concentrations of 15.6 mg/L and 31.2 mg/L in all tested strains. However, mature biofilms were resistant to all the antifungal drugs tested. When planktonic cells and biofilms (early-stage and mature) were treated with 2-chalcone-mediated PDT, the inhibitory concentrations were reduced by four times (2-7.8 mg/L). SEM images of biofilms treated with 2-chalcone showed cell wall collapse, resulting from a probable extravasation of cytoplasmic content. The toxicity of 2-chalcone in HaCat cells showed higher IC50 values in the dark than under photoexcitation. Further, 2-chalcone targets ergosterol in the cell and promotes the generation of ROS, resulting in cell death by apoptosis and necrosis. Overall, 2-chalcone-mediated PDT is a promising and safe drug candidate against dermatophytes, particularly in anti-biofilm treatment.

Understanding the mechanism of action of peptide (p-BthTX-I)2 derived from C-terminal region of phospholipase A2 (PLA2)-like bothropstoxin-I on Gram-positive and Gram-negative bacteria.

Based on the antimicrobial activity of bothropstoxin-I (BthTX-I) and on the premise that a C-terminal peptide of Lys49 myotoxin can reproduce the antimicrobial activity of the parent protein, we aimed to study the mechanism of action of a peptide derived from the C-terminal region of the myotoxin BthTX-I [(p-BthTX-I)2, sequence: KKYRYHLKPFCKK, disulfide-linked dimer] against Gram-positive and Gram-negative bacteria. Fluorescence quenching technique showed that the carboxyfluorescein labeled-peptide [CF-(p-BthTX-I)2] when incubated with E. coli displayed a superior penetration activity than when incubated with S. aureus. Cell death induced by the peptide (p-BthTX-I)2 showed a loss of membrane integrity in E. coli and S. aureus; however, the mechanisms of cell death were different, characterized by the presence of necrosis-like and apoptosis-like deaths, respectively. Scanning electron microscopy studies in E. coli and S. aureus showed morphological changes in the cells, with superficial deformities, appearance of wrinkles and bubbles, and formation of vesicles. Our results demonstrate that the mechanism of action of the peptide (p-BthTX-I)2 is different in Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. Knowledge of the mechanism of action of these peptides is important, since they are promising prototypes for new antimicrobial drugs.