Development of an in vivo rat subcutaneous Candida albicans biofilm model: characterization and application in the search of new anti-biofilm molecules.

Candida albicans exists as a commensal within the human body. It possesses different virulence factors that give this microorganism an opportunity to cause mucosal or deep tissue infections, especially in critically ill patients, with an impaired immune system. One of the most important virulence factors is the capacity to adhere to different types of biomaterials and subsequently, the development of mature biofilms. Biofilms are characterized by multilayer structures composed of Candida cells in different morphological stages covered with extracellular material. Fungal biofilms have been predominantly studied in vitro but such studies do not take into consideration the interaction of the pathogen with the immune system of the host. Therefore, the development of in vivo biofilm model systems remains crucial. The knowledge about in vivo C. albicans biofilms should bring a better insight into the effect of the host on biofilm formation. In this study, we successfully developed and characterized a novel in vivo C. albicans subcutaneous biofilm model. This system showed to be an easy tool that allows the analysis of multiple biofilms retrieved from one animal. Additionally, the biofilm architecture obtained from the devicesincubated subcutaneously, showed to be in an agreement with the biofilms acquired with the more sophisticated central venous catheter model. The subcutaneous in vivo model proved to be a reliable system that can be further used in the analysis of C. albicans foreign body infections. The treatment of biofilm-associated infections remains very difficult, especially because of theextracellular polymeric material that covers the biofilm and that at the same time, serves as a protective barrier against antifungals. The therapy of such infections often fails and the device must be removed, or replaced. Therefore, it is important to generate novel antifungal drugs and develop novel technologies that lead to reduction or complete eradication of these infections. In this work we showed that anidulafungin, one of the newest echinocandins, was effective against mature C. albicans biofilms formed in vivo in our subcutaneous biofilm model. On the other hand, fluconazole failed to reduce mature biofilm in vitro and in vivo. With these experiments weshowed that our in vivo model system can be used to test the antibiofilm capacity of novel antifungals. Besides the antifungal therapy with compounds, we introduced a novel method based on the induced aggregation of a protein of choice by expressing or supplying a small interfering peptide. In our studies we have used the Als3 protein as a proof of principle. The use of specific Als3 aggregator constructs resulted in the loss of function of Als3 as shown by reduced adhesion to plastic as well as to epithelial cells and reduced biofilm formation. Apart from expression cassetteswe also used several peptides with high-amyloid potential to cause ß-aggregation of Als3. One peptide (F9) resulted in excellent activity against C. albicans adhesion and mature biofilm development on biomaterials. Strikingly, it also caused reduction in adhesion and invasion of C. albicans to epithelial cells. Addition of F9 to C. albicans cells resulted in a significantly reduced amount of Als3 on hyphal cells. The results obtained in this study clearly show the potential use of peptide based therapeutics. In conclusion, this thesis described different aspects of biofilms caused by the leading human fungal pathogen C. albicans. We defined a new in vivo biofilm model that is an easy tool for antifungal testing and will form the basis for further analysis of fungal biofilms. In addition, we developed a so-called PROTEINi technology in C. albicans and showed that a short specific interfering peptide can inactivate its target protein in a specific way. Our results with Als3 clearly show the potential use of such platform for future antifungal research.status: publishe