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.

Dynamics of Mono- and Dual-Species Biofilm Formation and Interactions Between Paracoccidioides brasiliensis and Candida albicans.

The oral cavity is a highly diverse microbial environment in which microorganisms interact with each other, growing as biofilms on biotic and abiotic surfaces. Understanding the interaction among oral microbiota counterparts is pivotal for clarifying the pathogenesis of oral diseases. Candida spp. is one of the most abundant fungi in the oral mycobiome with the ability to cause severe soft tissue lesions under certain conditions. Paracoccidioides spp., the causative agent of paracoccidioidomycosis, may also colonize the oral cavity leading to soft tissue damage. It was hypothesized that both fungi can interact with each other, increasing the growth of the biofilm and its virulence, which in turn can lead to a more aggressive infectivity. Therefore, this study aimed to evaluate the dynamics of mono- and dual-species biofilm growth of Paracoccidioides brasiliensis and Candida albicans and their infectivity using the Galleria mellonella model. Biomass and fungi metabolic activity were determined by the crystal violet and the tetrazolium salt reduction tests (XTT), respectively, and the colony-forming unit (CFU) was obtained by plating. Biofilm structure was characterized by both scanning electronic- and confocal laser scanning- microscopy techniques. Survival analysis of G. mellonella was evaluated to assess infectivity. Our results showed that dual-species biofilm with P. brasiliensis plus C. albicans presented a higher biomass, higher metabolic activity and CFU than their mono-species biofilms. Furthermore, G. mellonella larvae infected with P. brasiliensis plus C. albicans presented a decrease in the survival rate compared to those infected with P. brasiliensis or C. albicans, mainly in the form of biofilms. Our data indicate that P. brasiliensis and C. albicans co-existence is likely to occur on oral mucosal biofilms, as per in vitro and in vivo analysis. These data further widen the knowledge associated with the dynamics of fungal biofilm growth that can potentially lead to the discovery of new therapeutic strategies for these infections.

Cyto-genotoxic evaluation of novel anti-tubercular copper (II) complexes containing isoniazid-based ligands.

Considering the promising previous results of Cu (II) complexes with isoniazid active ligand against Mycobacterium tuberculosis, the main causative agent of tuberculosis, novel biological assays evaluating its toxicogenic potential were performed to ensure the safe use. The genotoxicity/mutagenicity of the complexes CuCl2(INH)2.H2O (I1), Cu(NCS)2(INH)2.5H2O (I2) and Cu(NCO)2(INH)2.4H2O (I3) was evaluated by the Comet, Micronucleus-cytome and Salmonella microsome (Ames test) assays. The cell viability using resazurin assay indicated that I1, I2 e I3 had moderate to low capacity to reduce the viability of colorectal cells (Caco-2), liver cells (HepG2), lung cells (GM 07492-A and A549) and endothelial cells (HU-VE-C). On genotoxicity/mutagenicity, I1 complex did not induce sizable levels of DNA damage in HepG2 cells (Comet assay), and gene (Ames test) and chromosomal (Micronucleus-cytome assay) mutations. Already, I2 and I3 complexes were considered mutagenic in the highest concentrations used. In light of the above, these results contribute to valuable data on the safe use of Cu(II) complexes. Considering the absence of mutagenicity and cytotoxicity of I1, this complex is a potential candidate for the development of a new drug to the treatment tuberculosis, while I2 and I3 require caution in its use.

Heat Shock Protein 60, Insights to Its Importance in Histoplasma capsulatum: From Biofilm Formation to Host-Interaction.

Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins, acting as essential regulators of diverse constitutive metabolic processes. The Hsp60 of the dimorphic fungal Histoplasma capsulatum is the major surface adhesin to mammalian macrophages and studies of antibody-mediated protection against H. capsulatum have provided insight into the complexity involving Hsp60. However, nothing is known about the role of Hsp60 regarding biofilms, a mechanism of virulence exhibited by H. capsulatum. Considering this, the present study aimed to investigate the influence of the Hsp60 on biofilm features of H. capsulatum. Also, the non-conventional model Galleria mellonella was used to verify the effect of this protein during in vivo interaction. The use of invertebrate models such as G. mellonella is highly proposed for the evaluation of pathogenesis, immune response, virulence mechanisms, and antimicrobial compounds. For that purpose, we used a monoclonal antibody (7B6) against Hsp60 and characterized the biofilm of two H. capsulatum strains by metabolic activity, biomass content, and images from scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). We also evaluated the survival rate of G. mellonella infected with both strains under blockage of Hsp60. The results showed that mAb 7B6 was effective to reduce the metabolic activity and biomass of both H. capsulatum strains. Furthermore, the biofilms of cells treated with the antibody were thinner as well as presented a lower amount of cells and extracellular polymeric matrix compared to its non-treated controls. The blockage of Hsp60 before fungal infection of G. mellonella larvae also resulted in a significant increase of the larvae survival compared to controls. Our results highlight for the first time the importance of the Hsp60 protein to the establishment of the H. capsulatum biofilms and the G. mellonella larvae infection. Interestingly, the results with Hsp60 mAb 7B6 in this invertebrate model suggest a pattern of fungus-host interaction different from those previously found in a murine model, which can be due to the different features between insect and mammalian immune cells such as the absence of Fc receptors in hemocytes. However further studies are needed to support this hypothesis.

Genotoxicological safety assessment of puree-only edible films from onion bulb (Allium cepa L.) for use in food packaging-related applications.

The production of films and coatings from onion (Allium cepa L.) to be applied as packaging is attractive, due to its high nutritional and therapeutic value. Also, it can collaborate to minimize environmental impacts caused by the improper disposal of products made from plastics. However, despite it being an innovative and novel proposal, onion films for the development of edible packaging should be evaluated before being considered nontoxic and safe for human consumption. Thus, the objective of the present study was to elucidate the cytotoxic and mutagenic profile of eluates of polymer films of Allium cepa L. obtained by the casting process and to verify their safety for commercial purposes. The analysis of cellular viability demonstrated greater cytotoxicity for unwashed hydrothermally treated pulp (HTP) than for films of washed hydrothermally treated pulp (W-HTP). Regarding the mutagenic activity, the HTP and W-HTP films were not able to statistically increase the frequencies of the biomarkers for chromosome damage (micronucleus test) at the tested concentrations. However, the HTP films showed signs of mutagenicity in the Ames test (gene mutations), suggesting caution in their use. The detection of genotoxicity is highly recommended in order to avoid the risk of genotoxic exposure to mutagens and carcinogens. In conclusion, the absence of mutagenicity and cytotoxicity observed in this study is extremely relevant, because it provides support for toxicogenic properties of the Allium cepa films with promising applicability in the food industry. PRACTICAL APPLICATION: The bioplastics made from onion bulbs are multifunctional materials, which requires safety profile assessment. The results of the mutagenicity and cytotoxicity tests suggests that especially the W-HTP films are harmless, supporting at the first level of evidence, its safety potential to be used in the food industry (food films), biodegradable packaging, and biomaterials (substrates for drug delivery system).

Antibacterial, Preservative, and Mutagenic Potential of Copaifera spp. Oleoresins Against Causative Agents of Foodborne Diseases.

Foodborne diseases (FBDs) are a serious public health concern worldwide. In this scenario, preservatives based on natural products, especially plants, have attracted researchers' attention because they offer potential antimicrobial action as well as reduced health impact. The genus Copaifera spp., which is native of tropical South America and West Africa, contains several species for which pharmacological activities, including antibacterial effects, have been described. On the basis of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm activity (inhibition and eradication), preservative capacity, and Ames test, we evaluated the antibacterial, preservative, and mutagenic potential of Copaifera spp. oleoresins against the causative agents of FBDs. The Copaifera duckei, Copaifera reticulata, Copaifera paupera, and Copaifera pubiflora oleoresins displayed promising MIC/MBC values-from 12.5 to 100 µg/mL-against Staphylococcus aureus (American Type Culture Collection [ATCC] 29213), Listeria monocytogenes (ATCC 15313), and Bacillus cereus (ATCC 14579). C. duckei, C. reticulata, C. paupera, and C. pubiflora oleoresin concentrations ranging from 25 to 200 µg/mL and from 100 to 400 µg/mL inhibited biofilm formation and eradicated biofilms, respectively. The oleoresins did not exert mutagenic effects and had superior food preservative action to sodium benzoate (positive control). In conclusion, Copaifera oleoresins exhibit potential antibacterial activity and are not mutagenic, which makes them a promising source to develop novel natural food preservatives to inhibit foodborne pathogens.