Publicação
Candida glabrata biofilms: mechanisms of antifungal resistance and matrix role
| Resumo: | Candidiasis are the most common fungal diseases, especially among immunosuppressed, hospitalized and patients with prolonged contact to medical devices. Generally, Candida albicans is the most encountered Candida spp., however, in the last decades, non-Candida albicans Candida (NCAC) spp. have been increasing their responsibility in the cases of candidiasis. In this group, one of the most important species is Candida glabrata. This species presents azole resistance, like Candida krusei and a quick capacity to develop resistance to echinocandins, similar to Candida parapsilosis. Although not having capacity to produce certain important enzymes (e.g. hydrolases), C. glabrata has an essential feature that explains part of its recalcitrance to antifungals: the capacity to form biofilms. The present thesis has the general purpose of increasing the current knowledge on the different and less explored biofilms of C. glabrata, their mechanisms of resistance to antifungal drugs, and ultimately contributing to the path into a more consistent optimization and patient response to the present therapies. Thus, to begin, a method to study the fluconazole's diffusion through the biofilm matrices was developed and then the effectiveness of voriconazole in C. glabrata biofilms was determined. It was concluded that voriconazole had higher activity than fluconazole and its diffusion was also deeper. Some changes were noticed in the ERG3, ERG6 and ERG11 genes expression. Additionally, an association of ascorbic acid (immunostimulator and β-glucan degradator) and fluconazole was applied to biofilms of C. glabrata. Biofilms C. glabrata were weakened, but not entirely degraded, and the number of cells was higher, due to the assimilation of the glucose derived from the β- glucan hydrolysis. In the context of polyenes, it was assessed the capacity of C. glabrata to form biofilms in the presence of clinical concentrations of amphotericin B. This capacity was confirmed, demonstrating the resistance of this species, even when using drugs with a good antifungal activity. In another approach, the comparison between two amphotericin B formulations – deoxycholate and liposomal – was performed in C. glabrata biofilms, and other Candida spp.. Both formulations have a good biomass reduction capacity, but the liposomal has a better activity against biofilm cells. The concomitant use of a polyene and an azole was showed to be mostly antagonist, since when applying amphotericin B and posaconazole, the effect in the number of cells, biomass reduction and Fractional inhibitory concentration index (FICI) calculation was not better than using each drug alone. Caspofungin and micafungin showed to induce great changes in C. glabrata biofilm matrices. These echinocandins were evaluated in terms of planktonic and biofilm cell susceptibility, showing to be better in the first case than in the latter. The biomass reduction was very good in both cases, but the variations regarding the quantity of proteins were variable. The amount of β-glucans generally decreased after the echinocandins’ contact, in opposition with the other drug’s response. Regarding transcriptomics, firstly a set of genes responsible for the production of matrix components was selected and their expression was assessed, on C. glabrata biofilm cells. In a general manner, all genes were overexpressed, but the ones related to the production of β-1,3- glucans revealed a higher expression. It was concluded that the cells invest more in the replacement of these biopolymers than the others and that they might have a greater impact in the biofilm cells lower susceptibility to antifungal drugs. Next, a C. glabrata mutant knocked-out on the MNN2 gene was studied in the biofilm form. The biofilm cells were evaluated in terms of drug susceptibility, cell wall, and the biofilm composition was also determined. There were no visible differences among the cell walls of the strains, but the mutant showed to have a great biomass reduction, after a drug stress. The quantity of mannans of the biofilm cells in the mutant decreased. The biofilm cell walls composition showed an increase of the quantity of β-1,3-glucans in the C. glabrata Dmnn2, when compared to the reference strain, possibly compensating the reduction of mannans. This increase in β-1,3-glucans could explain the reduction in the biofilm susceptibility. The in vivo approach permitted an exploration on the drug and immune response of an infection related solely to biofilm cells of C. glabrata and then treated with two echinocandins. The results showed that, generally, the immune response in C. glabrata is very distinctive from C. albicans. The fungal burden was higher in the liver than in the kidneys. Neither caspofungin nor micafungin was effective in fully eradicating C. glabrata biofilm cells and it was noticed a predominant mononuclear immune response in the spleen, liver and kidneys of the infected mice. In summary, the present thesis compiles important data that increase the knowledge on the mechanisms of antifungal resistance of C. glabrata biofilms, contributing to the path into a more sustained and defined understanding on how the biofilm matrices and the biofilm cells contribute to more severe infections. Moreover, it is underlined the importance on how these biofilm-derivedinfections need to be addressed differently from the the planktonic ones, replacing the current protocols. |
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| Autores principais: | Rodrigues, Célia Fortuna |
| Assunto: | Candida glabrata biofilm matrix antifungal resistance biofilme matriz antifúngico resistência |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | português |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Candidiasis are the most common fungal diseases, especially among immunosuppressed, hospitalized and patients with prolonged contact to medical devices. Generally, Candida albicans is the most encountered Candida spp., however, in the last decades, non-Candida albicans Candida (NCAC) spp. have been increasing their responsibility in the cases of candidiasis. In this group, one of the most important species is Candida glabrata. This species presents azole resistance, like Candida krusei and a quick capacity to develop resistance to echinocandins, similar to Candida parapsilosis. Although not having capacity to produce certain important enzymes (e.g. hydrolases), C. glabrata has an essential feature that explains part of its recalcitrance to antifungals: the capacity to form biofilms. The present thesis has the general purpose of increasing the current knowledge on the different and less explored biofilms of C. glabrata, their mechanisms of resistance to antifungal drugs, and ultimately contributing to the path into a more consistent optimization and patient response to the present therapies. Thus, to begin, a method to study the fluconazole's diffusion through the biofilm matrices was developed and then the effectiveness of voriconazole in C. glabrata biofilms was determined. It was concluded that voriconazole had higher activity than fluconazole and its diffusion was also deeper. Some changes were noticed in the ERG3, ERG6 and ERG11 genes expression. Additionally, an association of ascorbic acid (immunostimulator and β-glucan degradator) and fluconazole was applied to biofilms of C. glabrata. Biofilms C. glabrata were weakened, but not entirely degraded, and the number of cells was higher, due to the assimilation of the glucose derived from the β- glucan hydrolysis. In the context of polyenes, it was assessed the capacity of C. glabrata to form biofilms in the presence of clinical concentrations of amphotericin B. This capacity was confirmed, demonstrating the resistance of this species, even when using drugs with a good antifungal activity. In another approach, the comparison between two amphotericin B formulations – deoxycholate and liposomal – was performed in C. glabrata biofilms, and other Candida spp.. Both formulations have a good biomass reduction capacity, but the liposomal has a better activity against biofilm cells. The concomitant use of a polyene and an azole was showed to be mostly antagonist, since when applying amphotericin B and posaconazole, the effect in the number of cells, biomass reduction and Fractional inhibitory concentration index (FICI) calculation was not better than using each drug alone. Caspofungin and micafungin showed to induce great changes in C. glabrata biofilm matrices. These echinocandins were evaluated in terms of planktonic and biofilm cell susceptibility, showing to be better in the first case than in the latter. The biomass reduction was very good in both cases, but the variations regarding the quantity of proteins were variable. The amount of β-glucans generally decreased after the echinocandins’ contact, in opposition with the other drug’s response. Regarding transcriptomics, firstly a set of genes responsible for the production of matrix components was selected and their expression was assessed, on C. glabrata biofilm cells. In a general manner, all genes were overexpressed, but the ones related to the production of β-1,3- glucans revealed a higher expression. It was concluded that the cells invest more in the replacement of these biopolymers than the others and that they might have a greater impact in the biofilm cells lower susceptibility to antifungal drugs. Next, a C. glabrata mutant knocked-out on the MNN2 gene was studied in the biofilm form. The biofilm cells were evaluated in terms of drug susceptibility, cell wall, and the biofilm composition was also determined. There were no visible differences among the cell walls of the strains, but the mutant showed to have a great biomass reduction, after a drug stress. The quantity of mannans of the biofilm cells in the mutant decreased. The biofilm cell walls composition showed an increase of the quantity of β-1,3-glucans in the C. glabrata Dmnn2, when compared to the reference strain, possibly compensating the reduction of mannans. This increase in β-1,3-glucans could explain the reduction in the biofilm susceptibility. The in vivo approach permitted an exploration on the drug and immune response of an infection related solely to biofilm cells of C. glabrata and then treated with two echinocandins. The results showed that, generally, the immune response in C. glabrata is very distinctive from C. albicans. The fungal burden was higher in the liver than in the kidneys. Neither caspofungin nor micafungin was effective in fully eradicating C. glabrata biofilm cells and it was noticed a predominant mononuclear immune response in the spleen, liver and kidneys of the infected mice. In summary, the present thesis compiles important data that increase the knowledge on the mechanisms of antifungal resistance of C. glabrata biofilms, contributing to the path into a more sustained and defined understanding on how the biofilm matrices and the biofilm cells contribute to more severe infections. Moreover, it is underlined the importance on how these biofilm-derivedinfections need to be addressed differently from the the planktonic ones, replacing the current protocols. |
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