Publicação
Involvement of the gene CaRLM1 in Candida albicans virulence
| Resumo: | The cell wall is an essential structure that maintains the viability of fungal cells, conferring their typical morphology and protection. As the most external cellular structure of pathogenic microorganisms, it also carries important antigenic determinants and mediates adhesion to the host tissues, being crucial to initiate colonization and to cause disease. Saccharomyces cerevisiae and Candida albicans respond to cell wall perturbations by activation of the cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway (also known as the PKC pathway). In S cerevisiae one of the transcription factors of this signalling cascade is the MADS-box protein Rlm1, of which an orthologue was identified in C. albicans based solely in sequence homology. In this thesis we aimed at studying the molecular diversity and functional characterization of the RLM1 gene as well as its involvement in C. albicans cell wall maintenance and virulence. Candida albicans Rlm1 is a transcription factor that presents a great variability at its C-terminus, conferred by the CAI microsatellite. One hundred twenty-three C. albicans isolates were genotyped with CAI microsatellite (CAA/G)n and 35 alleles were found with repeat units varying from 11 to 49. Interestingly the strains with higher number of (CAA/G) repetitions displayed higher tolerance to cell wall stress agents. These observations suggested that CAI repetitive region confers a high genetic variability to the RLM1 gene, which is reflected in different strain susceptibilities to different stress conditions, conferring a higher plasticity to C. albicans isolates. To determine if C. albicans RLM1 is involved in the CWI pathway, as described for S. cerevisiae, a rlm1Δ/rlm1Δ mutant was constructed, using the SAT1-flipping strategy to avoid the use of auxotrophic markers, and its functional characterization was performed. The wild-type (WT), mutant (rlm1Δ/rlm1Δ) and complemented (rlm1Δ/rlm1Δ+RLM1) strains were tested with several cell wall stress agents in parallel with S.cerevisiae rlm1Δ mutant and WT strains. Candida albicans rlm1Δ/rlm1Δ mutant displayed phenotypes associated to cell wall deficiency such as, hypersensitivity to Congo red, caspofungin and calcofluor white. Upon osmotic stabilization with 1M sorbitol, the caspofungin phenotype was reverted, suggesting cell wall weakening in the mutant. Quantification of cell wall components showed a two-fold increase in chitin and mannans in the C. albicans rlm1Δ/rlm1Δ mutant in comparison with the WT strain. The S. cerevisiae rlm1Δ mutant displayed several phenotypic differences in comparison with the rlm1Δ/rlm1Δ mutant of C. albicans: insensitivity to Congo red and caspofungin, more resistance to calcofluor white, and higher sensitivity to SDS. In agreement with a transcription factor function, we found evidence indicating nuclear localization of the Rlm1-GFP fusion protein. Microarray analysis showed that the absence of a functional C. albicans RLM1 significantly increased transcription of genes involved in cell adhesion, like ECE1, ALS1, ALS3, HWP1, RBT1, and decreased transcription of genes involved in the catabolism of carbohydrates, DAK2, GLK4, NHT1 and TPS1. The increased transcription of genes involved in cell adhesion correlated well with adhesion and biofilm assays. These results and the homology with other MADS-box Rlm1 transcription factors strongly suggest that C. albicans RLM1, like the S. cerevisiae orthologue, is involved in cell wall remodeling. Furthermore, the increase of cell adhesion binding proteins involved in biofilm formation was confirmed in the rlm1Δ/rlm1Δ mutant in comparison with the WT strain, suggesting that C. albicans Rlm1 acts as a negative biofilm regulator. The involvement of the Rlm1 transcription factor of the human fungal pathogen C. albicans in virulence was evaluated in a murine model of disseminated candidiasis. Mice infected with rlm1Δ/rlm1Δ mutant cells presented a higher survival time than mice infected with the WT and complemented strains, both presenting higher fungal burden and invasive micelial growth through kidneys in hystopathological analysis. Additionally, in the murine macrophage-like cell line J744A, the TNF-α was lower in response to rlm1Δ/rlm1Δ mutant and the cellular toxicity, measured as extracellular lactate dehydrogenase activity, caused by this mutant was significantly lower in comparison with the WT and complemented strains. Finally, qRT-PCR determination showed that the expression of the cell wall-related genes, CRH11 and PHR2, was clearly higher in rlm1Δ/rlm1Δ mutant in relation to WT strain, in mRNA of kidney samples after 7 days post-infection. Since these proteins are part important of the cell wall and in vivo the yeast cells are under constant cell wall stress by the immune system, it is conceivable that the mutant presents a higher expression of these genes, which may compensate the weakened cell wall. Overall, these results showed that the transcription factor Rlm1 is involved in the stability of the cell wall in the interaction with the host, being important for the virulence of C. albicans and invasion of the kidneys during hematogenously disseminated candidiasis. Altogether, the objective proposted in this thesis were achieved, demonstrating that RLM1 gene is an important transcription factor involved in C. albicans cell wall remodelling, such that the mutant is practically avirulent. Additionally, we also think that this yeast developed a way of increasing the genetic variability of this important gene (through CAI repetitive region), confering a high plasticity to C. albicans isolates. |
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| Autores principais: | Silva, Yolanda Bedsabé Delgado |
| Ano: | 2012 |
| 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 cell wall is an essential structure that maintains the viability of fungal cells, conferring their typical morphology and protection. As the most external cellular structure of pathogenic microorganisms, it also carries important antigenic determinants and mediates adhesion to the host tissues, being crucial to initiate colonization and to cause disease. Saccharomyces cerevisiae and Candida albicans respond to cell wall perturbations by activation of the cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway (also known as the PKC pathway). In S cerevisiae one of the transcription factors of this signalling cascade is the MADS-box protein Rlm1, of which an orthologue was identified in C. albicans based solely in sequence homology. In this thesis we aimed at studying the molecular diversity and functional characterization of the RLM1 gene as well as its involvement in C. albicans cell wall maintenance and virulence. Candida albicans Rlm1 is a transcription factor that presents a great variability at its C-terminus, conferred by the CAI microsatellite. One hundred twenty-three C. albicans isolates were genotyped with CAI microsatellite (CAA/G)n and 35 alleles were found with repeat units varying from 11 to 49. Interestingly the strains with higher number of (CAA/G) repetitions displayed higher tolerance to cell wall stress agents. These observations suggested that CAI repetitive region confers a high genetic variability to the RLM1 gene, which is reflected in different strain susceptibilities to different stress conditions, conferring a higher plasticity to C. albicans isolates. To determine if C. albicans RLM1 is involved in the CWI pathway, as described for S. cerevisiae, a rlm1Δ/rlm1Δ mutant was constructed, using the SAT1-flipping strategy to avoid the use of auxotrophic markers, and its functional characterization was performed. The wild-type (WT), mutant (rlm1Δ/rlm1Δ) and complemented (rlm1Δ/rlm1Δ+RLM1) strains were tested with several cell wall stress agents in parallel with S.cerevisiae rlm1Δ mutant and WT strains. Candida albicans rlm1Δ/rlm1Δ mutant displayed phenotypes associated to cell wall deficiency such as, hypersensitivity to Congo red, caspofungin and calcofluor white. Upon osmotic stabilization with 1M sorbitol, the caspofungin phenotype was reverted, suggesting cell wall weakening in the mutant. Quantification of cell wall components showed a two-fold increase in chitin and mannans in the C. albicans rlm1Δ/rlm1Δ mutant in comparison with the WT strain. The S. cerevisiae rlm1Δ mutant displayed several phenotypic differences in comparison with the rlm1Δ/rlm1Δ mutant of C. albicans: insensitivity to Congo red and caspofungin, more resistance to calcofluor white, and higher sensitivity to SDS. In agreement with a transcription factor function, we found evidence indicating nuclear localization of the Rlm1-GFP fusion protein. Microarray analysis showed that the absence of a functional C. albicans RLM1 significantly increased transcription of genes involved in cell adhesion, like ECE1, ALS1, ALS3, HWP1, RBT1, and decreased transcription of genes involved in the catabolism of carbohydrates, DAK2, GLK4, NHT1 and TPS1. The increased transcription of genes involved in cell adhesion correlated well with adhesion and biofilm assays. These results and the homology with other MADS-box Rlm1 transcription factors strongly suggest that C. albicans RLM1, like the S. cerevisiae orthologue, is involved in cell wall remodeling. Furthermore, the increase of cell adhesion binding proteins involved in biofilm formation was confirmed in the rlm1Δ/rlm1Δ mutant in comparison with the WT strain, suggesting that C. albicans Rlm1 acts as a negative biofilm regulator. The involvement of the Rlm1 transcription factor of the human fungal pathogen C. albicans in virulence was evaluated in a murine model of disseminated candidiasis. Mice infected with rlm1Δ/rlm1Δ mutant cells presented a higher survival time than mice infected with the WT and complemented strains, both presenting higher fungal burden and invasive micelial growth through kidneys in hystopathological analysis. Additionally, in the murine macrophage-like cell line J744A, the TNF-α was lower in response to rlm1Δ/rlm1Δ mutant and the cellular toxicity, measured as extracellular lactate dehydrogenase activity, caused by this mutant was significantly lower in comparison with the WT and complemented strains. Finally, qRT-PCR determination showed that the expression of the cell wall-related genes, CRH11 and PHR2, was clearly higher in rlm1Δ/rlm1Δ mutant in relation to WT strain, in mRNA of kidney samples after 7 days post-infection. Since these proteins are part important of the cell wall and in vivo the yeast cells are under constant cell wall stress by the immune system, it is conceivable that the mutant presents a higher expression of these genes, which may compensate the weakened cell wall. Overall, these results showed that the transcription factor Rlm1 is involved in the stability of the cell wall in the interaction with the host, being important for the virulence of C. albicans and invasion of the kidneys during hematogenously disseminated candidiasis. Altogether, the objective proposted in this thesis were achieved, demonstrating that RLM1 gene is an important transcription factor involved in C. albicans cell wall remodelling, such that the mutant is practically avirulent. Additionally, we also think that this yeast developed a way of increasing the genetic variability of this important gene (through CAI repetitive region), confering a high plasticity to C. albicans isolates. |
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