Análise do alvo predito da plumieridina em Cryptococcus neoformans
Cryptococcosis is a fungal infection caused by yeast from Cryptococcus spp. The infection starts when desiccated cells or spores are inhaled and reach the lungs. If the disease is not properly treated, the infection can progress, reach the central nervous system and result in meningococcal mening...
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| Formato: | Dissertação |
| Idioma: | pt_BR |
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Universidade Federal do Rio Grande do Norte
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| Endereço do item: | https://repositorio.ufrn.br/jspui/handle/123456789/29588 |
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| Resumo: | Cryptococcosis is a fungal infection caused by yeast from Cryptococcus spp. The infection
starts when desiccated cells or spores are inhaled and reach the lungs. If the disease is not
properly treated, the infection can progress, reach the central nervous system and result in
meningococcal meningitis and even death. Cryptococcosis treatment is carried out in three
stages and uses three drugs: fluconazole, amphotericin B and 5-flucytosine. Although effective,
the use of these drugs can result in the emergence of fungal resistance, a toxic effect for patients,
and drugs as flucytosine are not commercialized worldwide. Thus, it is proposed to investigate
the mode of action of the antifungal compound plumieridine as well as the identification of its
molecular target in C. neoformans. For this, a series of experiments were carried out in vitro
and in silico. Initially, chromatographic fractions of the aqueous extract of Allamanda
polyantha seeds were subjected to antimicrobial activity tests. The fraction with antifungal
activity was subjected to nuclear magnetic resonance analysis of carbon and hydrogen in order
to identify compounds present in the sample. Antifungal activity, evaluated through antifungal
tests, was 0.250 mg/mL and the major component in the fraction was plumieridine. Through
virtual screening based on ligand’s similarity, chitinase was identified as the molecular target
of plumieridine. Three-dimensional models of chitinases from C. neoformans were created and,
through molecular docking, interaction with residues from the active site was observed.
Chitinolytic activity inhibition assays showed that the activity is significantly reduced in the
secreted fraction and soluble cell fraction. However, chitinolytic activity is little reduced by the
presence of plumieridine in the insoluble cell fraction, where higher concentrations of the
compound are needed. Although plumieridine is able to inhibit chitinolytic activity, the
compound does not appear to be related to the transcriptional levels of chitinases of C.
neoformans, reducing only the transcriptional levels of the CHI22 gene. It was observed that
crude extracts containing chitinases from mouse macrophages, Bacillus subitilis and Tenebrio
molitor are also inhibited in the presence of plumieridine. The treatment with plumieridine still
alters the distribution pattern of the chitooligomers in the cell wall: from a polarized pattern to
a diffuse pattern through the wall. The results validate virtual screening prediction and show
that the inhibition of chitinolytic activity by plumieridine results in incomplete cell division
and, consequently, antifungal activity. Finally, the results indicate that plumieridine inhibits
chitinase and causes death of C. neoformans, however, the inhibition also occurs in other
members of the GH18 family, indicating that this is a potential inhibitor of the GH18 family. |
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