Activity of scorpion venom-derived antifungal peptides against planktonic cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms

The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 μM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals

Early immune response against Fonsecaea pedrosoi requires Dectin-2-mediated Th17 activity, whereas Th1 response, aided by Treg cells, is crucial for fungal clearance in later stage of experimental chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic worldwide subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. It is difficult to treat patients with the disease, mainly because of its recalcitrant nature. The correct activation of host immune response is critical to avoid fungal persistence in the tissue and disease chronification. CD4+ T cells are crucial for the development of protective immunity to F. pedrosoi infection. Here, we investigated T helper cell response dynamics during experimental CBM. Following footpad injection with F. pedrosoi hyphae and conidia, T cells were skewed towards a Th17 and Th1 phenotype. The Th17 population was the main Th cell subset found in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells, which leads to a reduction of Treg cells in the draining lymph node, resulted in decline in fungal burden after 14 days of infection. However, fungal cells were not cleared in the later stages of the disease, prolonging CBM clinical features in those animals. IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Similarly, in dectin-2 KO animals, Th17 contraction in the course of experimental CBM was accompanied by fungal burden decrease in the first 14 days of infection, although it did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi propagules and uncovered their contribution to disease resolution. The Th17 population proved to be important in eliminating fungal cells in the early stages of infection. The Th1 population, in turn, closely assisted by Treg cells, proved to be relevant not only in the elimination of fungal cells at the beginning of infection but also essential for their complete elimination in later stages of the disease in a mouse experimental model of CBM

Nutritional Conditions Modulate C. neoformans Extracellular Vesicles’ Capacity to Elicit Host Immune Response

Cryptococcus neoformans is a human pathogenic fungus that mainly afflicts immunocompromised patients. One of its virulence strategies is the production of extracellular vesicles (EVs), containing cargo with immunomodulatory properties. We evaluated EV’s characteristics produced by capsular and acapsular strains of C. neoformans (B3501 and ΔCap67, respectively) growing in nutritionally poor or rich media and co-cultures with bone marrow-derived macrophages or dendritic cells from C57BL/6 mice. EVs produced under a poor nutritional condition displayed a larger hydrodynamic size, contained more virulence compounds, and induced a more robust inflammatory pattern than those produced in a rich nutritional medium, independently of strain. We treated infected mice with EVs produced in the rich medium, and the EVs inhibited more genes related to the inflammasome than untreated infected mice. These findings suggest that the EVs participate in the pathogenic processes that result in the dissemination of C. neoformans. Thus, these results highlight the versatility of EVs’ properties during infection by C. neoformans in different tissues and support ongoing efforts to harness EVs to prevent and treat cryptococcosis

Miconazole loaded chitosan-based nanoparticles for local treatment of vulvovaginal candidiasis fungal infections

Objectives: In this study, polymeric nanoparticles based on chitosan incorporating the antifungal miconazole nitrate were fabricated and testedin vivo using murine vulvovaginal candidiasis. Methods: Nanoparticles prepared by the ionotropic gelation method presented 200 to 300 nm diameter and polydispersity indexes ranging from 0.2 to 0.4. The nanoparticles were prepared to incorporate 63.9 mg/mL of miconazole nitrate to be testedin vivo. Murine vulvovaginal candidiasis was standardized using estradiol valerate before the animals were challenged by Candida albicans. Results: The treatment using chitosan nanoparticles within miconazole nitrate presented the same therapeutic efficacy as miconazole nitrate in a commercial cream formulation, however using the antifungal content about seven-fold lower. This increase in the miconazole nitrate's therapeutic efficacy is may be due to the down-regulation of interleukin 10 (IL-10) expression. Conclusions: Our data represent a proof of concept that can be exploited to achieve an alternative and promising therapy for the treatment of vulvovaginal candidiasis

New Role of P. brasiliensis α-Glucan: Differentiation of Non-conventional Dendritic Cells

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-12-10T17:06:41Z No. of bitstreams: 1 Souza,A.C.O. New Role.pdf: 3104697 bytes, checksum: 823c2a6d2f52c4147d1c02de861826fb (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-12-10T17:15:42Z (GMT) No. of bitstreams: 1 Souza,A.C.O. New Role.pdf: 3104697 bytes, checksum: 823c2a6d2f52c4147d1c02de861826fb (MD5)Made available in DSpace on 2019-12-10T17:15:42Z (GMT). No. of bitstreams: 1 Souza,A.C.O. New Role.pdf: 3104697 bytes, checksum: 823c2a6d2f52c4147d1c02de861826fb (MD5) Previous issue date: 2019Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF), Conselho Nacional de Pesquisa (CNPq), and Decanato de Pesquisa e Pós-Graduação da Universidade de Brasília (DPP/UnB) for financial support, and CAPES for graduate students’ grants.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.The cell wall has a critical role in the host immune response to fungal pathogens. In this study, we investigated the influence of two cell wall fractions of the dimorphic fungi Paracoccidioides brasiliensis (Pb) in the in vitro generation of monocyte-derived dendritic cells (MoDCs). Monocytes were purified from the peripheral blood of healthy donors and cultivated for 7 days in medium supplemented with IL-4 and GM-CSF in the presence of Pb cell wall fractions: the alkali-insoluble F1, constituted by β-1,3-glucans, chitin and proteins, and the alkali-soluble F2, mainly constituted by α-glucan. MoDCs phenotypes were evaluated regarding cell surface expression of CD1a, DC-SIGN, HLA-DR, CD80, and CD83 and production of cytokines. The α-glucan-rich cell wall fraction downregulated the differentiation of CD1a+ MoDCs, a dendritic cell subset that stimulate Th1 responses. The presence of both cell fractions inhibited DC-SIGN and HLA-DR expression, while the expression of maturation markers was differentially induced in CD1a- MoDCs. Differentiation upon F1 and F2 stimulation induced mixed profile of inflammatory cytokines. Altogether, these data demonstrate that Pb cell wall fractions differentially induce a dysregulation in DCs differentiation. Moreover, our results suggest that cell wall α-glucan promote the differentiation of CD1a- DCs, potentially favoring Th2 polarization and contributing to pathogen persistence

Lung inflammatory lesions caused by <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> infection were reduced by Polydim-I peptide treatment.

<p>Twenty-six days after the challenge, the lungs were processed, sectioned, HE stained, and examined at 40 × or 100 × magnification. (A and B) normal lungs from PBS treated control group at 40 × and 100 × magnifications, respectively; inflammatory lesions observed in the lungs of the infected group 40 × (C) and 100 × (D). Lungs from animals infected and treated with Polydim-I (2 mg/kg/mLW) 40 × (E) and 100 × (F). The area of the inflammatory lesions were calculated and plotted in a graph (G). One-way ANOVA followed by Dunn’s test was used to determine significant differences (* p<0.05).</p

Antimycobacterial Activity of a New Peptide Polydim-I Isolated from Neotropical Social Wasp <i>Polybia dimorpha</i>

<div><p><i>Mycobacterium abscessus</i> subsp. <i>massiliense</i>, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from <i>Polybia dimorpha</i> Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> cell wall and reduce 40–90% of the bacterial load both <i>in vitro</i> and <i>in vivo</i>. The presented results encourage further studies on the use of Polydim-I as one of the components for <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> treatment.</p></div