Final Report Summary - CAINFECTOME (Unravelling the infectome of Candida albicans)
Candida albicans is a major fungal pathogen and can infect virtually every organ of its human host. This ability relies on the expression of different virulence factors, although the exact mechanisms by which this fungus causes disease remain largely unknown. One of the main proposed virulence attributes is the ability of the fungus to grow both as ovoid yeast and as filamentous hyphae.
CAINFECTOME aimed to identify novel molecular mechanisms which contribute to the high pathogenicity of C. albicans. This objective was investigated within the context of five work packages:
(1) Identification of general or specific virulence genes associated with infection
(2) Identification of unknown function genes associated with infection
(3) Preparation of a set of mutants lacking infection associated genes with unknown function
(4) Elucidation of the role of these genes during different types of infection
(5) Establishment of an alternative infection model.
Global gene expression data of C. albicans during models of oral, liver and blood infection were analysed. This analysis identified both general and specific aspects of gene expression during infection: for example alternative carbon assimilation appears to occur during all forms of infection whilst genes encoding cell surface proteins are differentially expressed depending on the infection stage (WP1, Wilson et al., 2009). Therefore, a set of 40 previously uncharacterised genes with differential expression profiles during infection were selected (WP2) and the genes of interest removed by targeted gene deletion creating a set of infection-associated gene deletion mutants (WP3). The mutant set was then screened for pathogenic potential in two different (endothelial and epithelial) infection models.
Based on WP1-3, a number of novel candidate pathogenicity factors were identified and then extensively characterised for their role during different types of host pathogen interactions (WP4). For example, SHI1 which encodes a predicted cell surface protein which is unique to C. albicans was characterised. Deletion of SHI1 did not result in any apparent growth defects in vitro. However, the shi1 (deletion) mutant displayed attenuated damage of both endothelial and epithelial cells. Furthermore, deletion of this gene significantly attenuated virulence in an in vivo mouse model of disseminated candidosis. Therefore, SHI1 represents a novel C. albicans virulence factor. Subsequent detailed morphological analysis demonstrated that SHI1 was dispensable for initial hyphal formation by C. albicans but was required for subsequent secondary hyphal growth, hyphal branching and mycelial ramification (secondary hyphal inducer 1). This study therefore provides a novel link between coordinated hyphal branching patterns and pathogenicity in C. albicans (Wilson et al., in preparation).
Finally, the aim of WP5 was to establish a novel infection model for analysing C. albicans-endothelial interactions to determine how the fungus may escape from the circulation. A pump-based system for circulating fungal cells through an endothelial channel, mimicking the human circulatory system, was therefore developed (Wilson & Hube, 2010). Using this model, it was determined that a specific length of hyphae (3-7 µm) optimised binding of fungal cells to endothelium under these conditions. This finding was confirmed by demonstrating that an hgc1 deletion mutant, which cannot form these hyphae could not adhere to endothelium under flow conditions.
In summary, CAINFECTOME has successfully identified two new mechanisms which contribute to the pathogenic potential of C. albicans: the role of very short hyphae in escaping the blood stream and the importance of filament ramification for tissue destruction and virulence. Such detailed elucidation of the infection process will drive forward the development of future diagnostic or therapeutic strategies for use in the clinical setting.
CAINFECTOME aimed to identify novel molecular mechanisms which contribute to the high pathogenicity of C. albicans. This objective was investigated within the context of five work packages:
(1) Identification of general or specific virulence genes associated with infection
(2) Identification of unknown function genes associated with infection
(3) Preparation of a set of mutants lacking infection associated genes with unknown function
(4) Elucidation of the role of these genes during different types of infection
(5) Establishment of an alternative infection model.
Global gene expression data of C. albicans during models of oral, liver and blood infection were analysed. This analysis identified both general and specific aspects of gene expression during infection: for example alternative carbon assimilation appears to occur during all forms of infection whilst genes encoding cell surface proteins are differentially expressed depending on the infection stage (WP1, Wilson et al., 2009). Therefore, a set of 40 previously uncharacterised genes with differential expression profiles during infection were selected (WP2) and the genes of interest removed by targeted gene deletion creating a set of infection-associated gene deletion mutants (WP3). The mutant set was then screened for pathogenic potential in two different (endothelial and epithelial) infection models.
Based on WP1-3, a number of novel candidate pathogenicity factors were identified and then extensively characterised for their role during different types of host pathogen interactions (WP4). For example, SHI1 which encodes a predicted cell surface protein which is unique to C. albicans was characterised. Deletion of SHI1 did not result in any apparent growth defects in vitro. However, the shi1 (deletion) mutant displayed attenuated damage of both endothelial and epithelial cells. Furthermore, deletion of this gene significantly attenuated virulence in an in vivo mouse model of disseminated candidosis. Therefore, SHI1 represents a novel C. albicans virulence factor. Subsequent detailed morphological analysis demonstrated that SHI1 was dispensable for initial hyphal formation by C. albicans but was required for subsequent secondary hyphal growth, hyphal branching and mycelial ramification (secondary hyphal inducer 1). This study therefore provides a novel link between coordinated hyphal branching patterns and pathogenicity in C. albicans (Wilson et al., in preparation).
Finally, the aim of WP5 was to establish a novel infection model for analysing C. albicans-endothelial interactions to determine how the fungus may escape from the circulation. A pump-based system for circulating fungal cells through an endothelial channel, mimicking the human circulatory system, was therefore developed (Wilson & Hube, 2010). Using this model, it was determined that a specific length of hyphae (3-7 µm) optimised binding of fungal cells to endothelium under these conditions. This finding was confirmed by demonstrating that an hgc1 deletion mutant, which cannot form these hyphae could not adhere to endothelium under flow conditions.
In summary, CAINFECTOME has successfully identified two new mechanisms which contribute to the pathogenic potential of C. albicans: the role of very short hyphae in escaping the blood stream and the importance of filament ramification for tissue destruction and virulence. Such detailed elucidation of the infection process will drive forward the development of future diagnostic or therapeutic strategies for use in the clinical setting.