Publicação
Clinical Candida species co-infection and associated virulence
| Resumo: | The Candida genus consists of approximately 200 species of fungi and collectively represents a highly heterogenic group. Clinically, the most important specie is Candida albicans, an opportunistic fungal pathogen of humans that frequently causes superficial infections of oral and vaginal mucosal surfaces of debilitated individuals. This microorganism is, however, also commonly encountered as a commensal in healthy individuals where it is a component of the normal microflora. Nowadays, non-Candida albicans Candida (NCAC) species such as Candida glabrata, Candida parapsilosis and Candida tropicalis are also becoming frequently identified as potential human pathogens. Thus, the principal aim of this thesis was to obtain significant insight into the virulence mechanisms of Candida species, with special relevance to those colonising the vaginal tissue, as well as to evaluate their resistance to new antifungal agents. The treatment of human infections caused by Candida (candidosis) is difficult, especially due to the eukaryotic nature of fungal cells. Furthermore, several Candida species exhibit both intrinsic and acquired resistance to common antifungal agents and biofilms produced by Candida are also less susceptible. Thus, the first goal of this thesis was to perform a screening of the antifungal potential of natural plant extracts (Castanea sativa, Filipendula ulmaria, Rosa micrantha and Cistus ladanifer) and four phenolic compounds (gallic acid, catechin, luteolin and quercetin), identified from these plants, against C. albicans, C. glabrata, C. parapsilosis and C. tropicalis. The minimum inhibitory concentration (MIC) of plant extracts and phenolic compounds was determined according to standard methods. The antifungal potential of the phenolic compounds was also tested against Candida biofilms, and was assessed by quantification of colony forming units (CFUs). Overall, all plant extracts, as well as the phenolic compounds, especially gallic acid, revealed promising antifungal activity against Candida species. However, only gallic acid and quercetin demonstrated a slight effect against Candida species biofilms. Although the majority of the studies regarding Candida infection of human mucosal surfaces often include only single species, often candidosis is associated with mixed colonisation. The second aim of this research was to examine the interactions and expression of virulence factors by C. albicans and C. glabrata using a reconstituted human vaginal epithelium (RHVE). The pathogenesis of C. albicans and C. glabrata single and co-infections were investigated using peptide nucleic probe fluorescent in situ hybridization (PNA FISH), confocal laser scanning microscopy (CLSM) and a novel qRT-PCR protocol for Candida quantification in the tissues. RHVE damage was evaluated by measuring lactate dehydrogenase activity. Candida virulence gene (HWP1, ALS, EPA, PLB, PLD and SAP) expression was evaluated by qRT-PCR. It was shown that although C. albicans was a higher coloniser and invader of vaginal tissue, than C. glabrata, the invasiveness of C. glabrata strains was enhanced in the presence of C. albicans. Additionally, the results suggest an important role of HWP1, PLD1 and ALS3 virulence factors in C. albicans and C. glabrata pathogenicity. It is known that several environmental factors may be altered in vivo, to facilitate the conversion of Candida from a harmless commensal to a pathogenic organism. In the vaginal environment, hormonal changes are amongst such influencing factors. Therefore, the effect of progesterone (hormone) in C. albicans biofilm formation and RHVE colonisation and invasion was examined, using the same techniques described above. It was found that progesterone decreased the capacity of C. albicans to form biofilms and it was shown that C. albicans was a higher RHVE coloniser in the absence of progesterone. Gene expression by C. albicans infecting the vaginal epithelium suggests an important role of BCR1 and HWP1 virulence factor in C. albicans pathogenicity. In summary, this work emphasised the importance of studying new natural products as potential antifungal agents and also provided more insight into the mechanisms of vaginal infections caused by Candida species, in mono and mixed cultures, as well as in the presence of progesterone. |
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| Autores principais: | Alves, Carlos |
| Assunto: | Engenharia e Tecnologia::Engenharia Médica |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The Candida genus consists of approximately 200 species of fungi and collectively represents a highly heterogenic group. Clinically, the most important specie is Candida albicans, an opportunistic fungal pathogen of humans that frequently causes superficial infections of oral and vaginal mucosal surfaces of debilitated individuals. This microorganism is, however, also commonly encountered as a commensal in healthy individuals where it is a component of the normal microflora. Nowadays, non-Candida albicans Candida (NCAC) species such as Candida glabrata, Candida parapsilosis and Candida tropicalis are also becoming frequently identified as potential human pathogens. Thus, the principal aim of this thesis was to obtain significant insight into the virulence mechanisms of Candida species, with special relevance to those colonising the vaginal tissue, as well as to evaluate their resistance to new antifungal agents. The treatment of human infections caused by Candida (candidosis) is difficult, especially due to the eukaryotic nature of fungal cells. Furthermore, several Candida species exhibit both intrinsic and acquired resistance to common antifungal agents and biofilms produced by Candida are also less susceptible. Thus, the first goal of this thesis was to perform a screening of the antifungal potential of natural plant extracts (Castanea sativa, Filipendula ulmaria, Rosa micrantha and Cistus ladanifer) and four phenolic compounds (gallic acid, catechin, luteolin and quercetin), identified from these plants, against C. albicans, C. glabrata, C. parapsilosis and C. tropicalis. The minimum inhibitory concentration (MIC) of plant extracts and phenolic compounds was determined according to standard methods. The antifungal potential of the phenolic compounds was also tested against Candida biofilms, and was assessed by quantification of colony forming units (CFUs). Overall, all plant extracts, as well as the phenolic compounds, especially gallic acid, revealed promising antifungal activity against Candida species. However, only gallic acid and quercetin demonstrated a slight effect against Candida species biofilms. Although the majority of the studies regarding Candida infection of human mucosal surfaces often include only single species, often candidosis is associated with mixed colonisation. The second aim of this research was to examine the interactions and expression of virulence factors by C. albicans and C. glabrata using a reconstituted human vaginal epithelium (RHVE). The pathogenesis of C. albicans and C. glabrata single and co-infections were investigated using peptide nucleic probe fluorescent in situ hybridization (PNA FISH), confocal laser scanning microscopy (CLSM) and a novel qRT-PCR protocol for Candida quantification in the tissues. RHVE damage was evaluated by measuring lactate dehydrogenase activity. Candida virulence gene (HWP1, ALS, EPA, PLB, PLD and SAP) expression was evaluated by qRT-PCR. It was shown that although C. albicans was a higher coloniser and invader of vaginal tissue, than C. glabrata, the invasiveness of C. glabrata strains was enhanced in the presence of C. albicans. Additionally, the results suggest an important role of HWP1, PLD1 and ALS3 virulence factors in C. albicans and C. glabrata pathogenicity. It is known that several environmental factors may be altered in vivo, to facilitate the conversion of Candida from a harmless commensal to a pathogenic organism. In the vaginal environment, hormonal changes are amongst such influencing factors. Therefore, the effect of progesterone (hormone) in C. albicans biofilm formation and RHVE colonisation and invasion was examined, using the same techniques described above. It was found that progesterone decreased the capacity of C. albicans to form biofilms and it was shown that C. albicans was a higher RHVE coloniser in the absence of progesterone. Gene expression by C. albicans infecting the vaginal epithelium suggests an important role of BCR1 and HWP1 virulence factor in C. albicans pathogenicity. In summary, this work emphasised the importance of studying new natural products as potential antifungal agents and also provided more insight into the mechanisms of vaginal infections caused by Candida species, in mono and mixed cultures, as well as in the presence of progesterone. |
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