Caracterização do efeito antifúngico de peptídeos presentes na peçonha de escorpiões em linhagens de Cryptococcus neoformans

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2014.O crescente número de indivíduos imunocomprometidos levou ao aumentou da incidência de micoses sistêmicas. As infecções causadas pelo fungo Cryptococcus. neoformans são a maior causa de morte relacionada a fungos em pacientes com AIDS. Este cenário se agrava com o advento de linhagens resistentes a antifúngicos disponíveis bem como a alta toxicidade desses tratamentos aos pacientes. Dessa forma, a busca por novos fármacos mais eficientes e menos tóxicos é de grande urgência. Nesse sentido, o estudo de peptídeos antimicrobianos (AMPs) é promissor, uma vez que essas moléculas possuem como um dos principais alvos a membrana citoplasmática de patógenos de forma que esses organismos estão menos propensos a desenvolver resistência. AMPs são moléculas multifuncionais, apresentando tanto atividade microbicida como imunomodulatória. São peptídeos evolutivamente conservados em diversos grupos de seres vivos e produzidos em diversos tecidos. Nos animais peçonhentos, a produção de diversos AMPs tem sido descrita nas glândulas de peçonha, sendo portanto uma interessante fonte para buscas por novas moléculas microbicidas. Neste trabalho foi caracterizado o efeito antifúngico tanto de AMPs inéditos e AMPs de já descritos na literatura presentes na peçonha de escorpiões contra duas linhagens de C. neofomans. Dos 11 peptídeos testados, sete inibiram o crescimento de C. neoformans das linhagens H99 e B3501, porém nenhum peptídeo mostrou-se mais potente que a anfotericina B. A linhagem B3501 foi mais suscetível à ação de todos os peptídeos com atividade antifúngica comparada à linhagem H99. Observou-se também que a carga residual positiva dos AMPs identificados na peçonha de escorpião pode ser um importante parâmetro para a inibição do crescimento do fungo. Foi mostrado que as células fúngicas com a cápsula mais desenvolvida são mais suscetíveis à atividade inibitória dos AMPs, assim como células melanizadas também apresentam maior suscetibilidade em comparação ás células não melanizadas. É possível que a cápsula e a melanina exerçam um importante papel na atração eletrostática do peptídeo, resultando em uma maior suscetibilidade do fungo. Foi demonstrado também por meio de microscopia eletrônica de varredura que um dos peptídeos, denominado Peptídeo 6, foi capaz de causar danos na morfologia da célula. ______________________________________________________________________________ ABSTRACTThe rising number of immunocompromised individuals is leading to an increase in the incidence of systemic mycoses. Infections caused by Cryptococcus neoformans are the major cause of death related to fungi in patients with AIDS. This scenario is aggravated by the advent of resistant strains to available antifungal agents and the high toxicity of these treatments to patients. Thus, the search for new more efficient and less toxic drugs is of great urgency. In this sense, the study of antimicrobial peptides (AMPs) is promising, since the main target of this molecules is the cytoplasmic membrane of pathogens, so these organisms are less likely to develop resistance. AMPs are multifunctional molecules with both microbicidal activity and immunomodulatory properties. These peptides are evolutionarily conserved in diverse groups of organisms and are expressed in various tissues. In venomous animals, the expression of several AMPs have been described in the venom glands and is therefore an interesting source in the search for new microbicide molecules. In this work, the effect of antifungal AMPs present in the venom of scorpions was characterized against two strains of C. neoformans. Seven peptides inhibited the growth of C. neoformans H99 and C. neoformans B3501, although, amphotericin B was more potent than any of the tested peptides. B3501 strain was more susceptible to the inhibitory activity of the tested AMPs than H99 strain. It was observed that the residual positive charge of the scorpion’s AMPs could be an important feature in their ability to induce fungal growth inhibition. It was also shown that yeast cells with larger capsule are more susceptible to the inhibitory activity of the tested AMPs. Fungal melanized cells also showed higher susceptibility compared to non-melanized cells. It is possible that the capsule and melanin play a role in electrostatic attraction of cationic peptide, enhancing the fungal susceptibility to these compounds. Scanning Electron Microscopy analysis revealed that one peptide, nominated Peptide 6, causes damage to cell morphology

Bases moleculares da resposta de Cryptococcus neoformans a peptídeos antimicrobianos derivados da peçonha de escorpião

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2019.Nas últimas décadas, tem se observado um aumento na incidência de infecções fúngicas, enquanto o número de classes de antifúngicos disponíveis para a terapia continua o mesmo. A resistência de algumas espécies de fungos aos fármacos usados atualmente na clínica, associada à alta toxicidade impõe a busca por novas moléculas, mais eficientes e menos tóxicas, para o desenvolvimento de novas terapias para o tratamento de micoses. Peptídeos antimicrobianos (AMPs) são moléculas de defesa evolutivamente conservadas, multifuncionais produzidas por diversos organismos e são potenciais candidatos para o desenvolvimento de novos fármacos. Uma fonte interessante desses AMPs é a peçonha de escorpiões. Em trabalhos prévios, nosso grupo descreveu a atividade antifúngica de diversos AMPs da peçonha de escorpião contra Cryptococcus neoformans e Candida spp. Este trabalho visa melhor caracterizar a atividade antifúngica e elucidar o mecanismo de ação de peptídeos de peçonha de escorpião, em especial do peptídeo ToAP2. Os peptídeos ToAP1 e ToAP2 foram testados em linhagens de Cryptococcus spp com diferentes tamanhos basais de cápsula, sendo observado que a cápsula não protege o fungo contra a atividade do peptídeo. Testes utilizando células submetidas a tratamentos prévios para indução da cápsula ou melanização demonstraram que a modulação desses dois atributos de virulência de C. neoformans aumentam a susceptibilidade do fungo aos peptídeos. Observou-se que o peptídeo ToAP2 é capaz de permeabilizar a membrana plasmática das leveduras de C. neoformans e destruir organelas membranosas dentro da célula. Além disso, o tratamento com esse peptídeo causou um aparente aumento na espessura das fibras dos polissacarídeos da cápsula. Também foi avaliada a atividade dos dois peptídeos contra biofilmes de C. neoformans e o peptídeo ToAP2 inibiu fortemente a formação do biofilme, interferindo possivelmente na adesão das células. Para melhor elucidar o mecanismo de ação do peptídeo ToAP2, foi realizada uma varredura de uma biblioteca de mutantes de C. neoformans para identificar processos moleculares importantes para tolerância do fungo a esse AMP. Foram identificados 42 mutantes mais sensíveis ao peptídeo que a linhagem selvagem. A maioria dos genes cuja deleção leva a um aumento na suscetibilidade do fungo está envolvida com o tráfego de membrana. Também observou-se um envolvimento de genes relacionados à biossíntese de ergosterol e esfingolipídeos, à síntese e manutenção da parede celular e ao transporte de membrana na tolerância desse fungo ao AMP. Por fim, uma análise do perfil transcritômico do fungo tratado com o peptídeo ToAP2, anfotericina B e com a combinação dessas duas moléculas é apresentada nesta tese. Nossos dados sugerem que a membrana é o principal alvo desse peptídeo e que a permeabilização da bicamada lipídica possa ser o seu principal mecanismo de ação. Os resultados aqui obtidos reforçam o potencial dessas moléculas no desenvolvimento de novas terapias antifúngicas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).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 currently available antifungals imposes 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 of the innate immune response of diverse organisms. An interesting and potential source of AMPs is scorpion venom. Our group has previously described several scorpion venom AMPs with promising antifungal activity against Cryptococcus neoformans and Candida spp. This work aims to further characterize the antifungal activity and to elucidate the mechanism of action of those AMPs, in particular peptide ToAP2. The scorpion peptides ToAP1 and ToAP2 were tested against several Cryptococcus spp strains contrasting in terms of basal capsule thickness. It was observed that the capsule does not protect the fungi from the peptide antifungal activity. Tests using cells with induced capsule and melanin showed that modulation of these two virulence factors of C. neoformans increases the susceptibility of the yeast to peptides. ToAP2 permeabilizes the membrane of C. neoformans and destroys the membrane-bound organelles inside the cell. ToAP2-treated cells showed an apparent increase in polysaccharide fiber density. Also, ToAP1 and ToAP2 were tested against C. neoformans biofilms. ToAP2 strongly inhibited biofilm formation, possibly interfering with fungal initial adhesion. We performed a genetic screen to identify important molecular processes that mediate tolerance of C. neoformans to the AMPs in order to further elucidate ToAP2 mechanism of action. We identified 42 mutants which were more sensitive to the peptide than the wild-type strain. The largest group of genes whose deletion increase the yeast susceptibility to the peptide are involved in membrane trafficking. Ergosterol and sphingolipid biosynthesis pathway, cell wall synthesis and maintenance and transporters are also necessary for fugal tolerance to the peptide. Finally, an initial analysis of the transcriptomic profile of the yeast treated with ToAP2, amphotericin B and the combination of both antifungals is presented in this work. Our data indicate that the fungal membrane is the main target of ToAP2 and permeabilization of the lipid bilayer might be its main mechanism of action. Our results reinforce the potential of those AMPs as promising candidates for the development of new antifungal drugs

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

Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance

Antimicrobial peptides (AMPs) are natural antibiotics produced by various organisms such as mammals, arthropods, plants, and bacteria. In addition to antimicrobial activity, AMPs can induce chemokine production, accelerate angiogenesis, and wound healing and modulate apoptosis in multicellular organisms. Originally, their antimicrobial mechanism of action was thought to consist solely of an increase in pathogen cell membrane permeability, but it has already been shown that several AMPs do not modulate membrane permeability in the minimal lethal concentration. Instead, they exert their effects by inhibiting processes such as protein and cell wall synthesis, as well as enzyme activity, among others. Although resistance to these molecules is uncommon several pathogens developed different strategies to overcome AMPs killing such as surface modification, expression of efflux pumps, and secretion of proteases among others. This review describes the various mechanisms of action of AMPs and how pathogens evolve resistance to them

A hidden battle in the dirt: Soil amoebae interactions with Paracoccidioides spp.

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits