Análisis proteómico diferencial de la matriz extracelular de biopelículas formadas por aislados clínicos de Candida albicans

The incidence of human fungal infections has greatly increased in the last twenty years. Candida albicans is the leading cause of human systemic as well as superficial mycoses. In healthy individuals, C. albicans acts as a commensal organism. However, in immunosuppressed patients it acts as an opportunistic pathogen. The commensal to pathogen shift of C. albicans depends on the loss of the balance between the immune system and the fungus. Approximately 80 percent of human infections are due to the formation of biofilms. Biofilms are microorganism communities that develop on biotic or non-biotic surfaces covered by a complex tridimensional structure of extracellular matrix. Biofilm formation is an outstanding clinical problem because their microorganisms are more resistant to both antimicrobial agents and the immune response. In this regard, most Candida infections are associated with biofilm formation. Amongst the different virulence factors of C. albicans (morphogenesis, phenotypical change, quorum sensing, hydrolytic enzyme secretion, adhesion, and hydrophobicity), biofilm formation and hydrophobicity have been, and are still currently, considered as outstanding factors. In this PhD thesis, both properties of C. albicans have been thoroughly analyzed in 448 clinical isolates of C. albicans from patients with systemic candidiasis. The hydrophobicity study of these 448 isolates showed that no significant differences were found among the 88 percent of the isolates or between the clinical isolates and the control strand C. albicans SC5314. Regarding the biofilm formation, 70 percent of the isolates had low or no biofilm formation ability. Moreover, there was no correlation between hydrophobicity and biofilm formation capacity. The biofilm matrix has a major role in its growth, it is involved in the adhesion interactions, is responsible for its mechanical stability, and controls cell dispersion. In addition, it acts as a digestive system providing nutrients for its constituent cells as well as a protective barrier against both antifungal agents and host defenses. Regarding these functional activities, among the cellular components of the biofilm, proteins deserve special attention due to both their potential enzymatic activity and as targets for other molecules. In the proteomic analysis of the ethanolic extracts from of the extracellular matrix of the biofilms of nine clinical isolates and the control strain, 905 proteins were identified. Most of that proteins (mean 708) were present in the isolates with higher biofilm formation capacity; 148 in those with moderate biofilm formation capacity, and 143 in the isolates with poor biofilm formation capacity. Fifty seven proteins were common to all nine studied isolates and the control strain. That proteins are multifunctional species, from different cellular locations, and involved in different pathways, some of which deserve special interest, such as the defense against chemical or biological injuries, as well as their involvement in protein binding, transport and metabolism. Most of these proteins (93%) have negative hydropathic indexes which is concomitant with the fact that biofilms are considered hydrogels. Moreover, only 10 out of the 57 proteins had a signal peptide, which may mean that most of them get the extracellular matrix by a non-classic secretion way. Glx3, one of the 57 common proteins, is also one of the major proteins. The study of mutants for the GLX3 gene showed that its absence produces a series of effects on the integrity of the cell wall and increases its sensitivity to agents that alter the assembly of the cell wall components, as well as to zymolyase and the oxidative stress, effects that are partially recovered by the reintegration of this gene. In addition, the null mutant for the GLX3 gene is avirulent in murine models. Due to these effects, further studies should investigate this GlX3 null mutant as a potential immunological tool.