Aplicação dos íntrons do grupo I do mitogenoma de Cryptococcus neoformans e Cryptococcus gattii para identificação de espécies e sua associação com susceptibilidade a antifúngicos
The species complexes of Cryptococcus neoformans and Cryptococcus gattii are composed of pathogenic fungi that kill over 112,000 people annually worldwide, having meningoencephalitis as the main symptom of cryptococcosis. Characteristics such as virulence and antifungal susceptibility can vary wi...
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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/handle/123456789/54835 |
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| Resumo: | The species complexes of Cryptococcus neoformans and Cryptococcus gattii are
composed of pathogenic fungi that kill over 112,000 people annually worldwide, having
meningoencephalitis as the main symptom of cryptococcosis. Characteristics such as virulence
and antifungal susceptibility can vary within each species according to fungal genotype: C.
neoformans is divided into molecular types VNI, VNII, VNIII, and VNIV, and C. gattii into
VGI, VGII, VGIII, and VGIV. Therefore, specific molecular markers are necessary for
differential diagnosis. Autocatalytic group I introns can be a potential target for distinguishing
between molecular types of these yeasts, as they have polymorphisms regarding their presence
and base pair sequences. Furthermore, since the auto-splicing of these elements is vital for the
fungal cell, they are considered important therapeutic targets since they are absent from the
human genome. Therefore, this study aimed to evaluate the presence of group I introns in the
mitochondrial cob and cox1 genes of Cryptococcus fungal isolates, search for ORFs with
endonuclease genes in intronic sequences, investigate the potential use of these genetic
elements as molecular markers for differentiation of genotypes and/or species, and analyze their
relationship with antifungal susceptibility andin addition to study their origin, distribution, and
evolution through phylogenetic analyses. The data obtained from intron amplification of cob
and cox1 genes showed that the C. neoformans complex has, on average, fewer introns in its
mitogenome, and there is a great polymorphism of presence and size of these elements among
and within genotypes, making it impossible to use a single intronic marker to differentiate
genotype and/or species in the C. neoformans and C. gattii complexes. However, differentiation
between species is possible with combinations of PCRs of mtLSU and cox1 introns for C.
neoformans species and mtLSU and cob introns for C. gattii. Approximately 80.5% of the
sequenced introns had homing endonucleases, and phylogenetic analyses showed that introns
occupying the same insertion site form monophyletic clades and probably have a common
ancestor that invaded this site before the species diverged. There was only one case of
heterologous invasion, probably originating from horizontal transfer between a VGIV genotype
isolate and other fungi. Regarding susceptibility assays to antifungal agents, it was observed
that the minimum inhibitory concentration (MIC) values of fluconazole, 5-flucytosine, and
pentamidine were statistically associated with species complexes, with C. gattii having higher
MICs for fluconazole and C. neoformans having higher MICs for 5-flucytosine and
pentamidine. The presence of introns was related to susceptibility to amphotericin B, for which
a higher number of introns was associated with lower MIC values, and to 5-flucytosine and
pentamidine, for which the influence of the presence of introns varied between complexes:
regarding 5-flucytosine, the presence of introns was related to lower susceptibility in C.
neoformans and higher in C. gattii, and the opposite scenario was observed for pentamidine,
for which introns were associated with higher susceptibility in C. neoformans and lower in C.
gattii. |
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