Synthetic proteins in human pathogenic yeasts

The numbers of infectious diseases caused by human pathogenic yeasts have been continuously increasing over recent years. Their high morbidity and mortality have turned them into a serious public health problem. An efficient treatment is particularly complicated by systemic mycoses, which spread throughout the whole body, or emerging resistance to antibiotics. The most prevalent causative of systemic mycoses in humans is the pathogenic yeast Candida albicans, which can elicit severe infections if the immune system of its host is suppressed by, for example, operations, chemotherapy or diseases. Candida albicans features a multitude of mechanisms which lead to pathogenicity. These mechanisms are mediated by virulence factors, proteins with various functions in the cell. Virulence factors are essential for pathogenicity and therefore appear to be a promising target for the development of therapeutics. However, this requires profound knowledge of the molecular characteristics and physiological interaction networks of cell proteins. As techniques to study protein interaction networks in vivo are scarce, especially for C. albicans, scientists at the Fraunhofer IGB developed a new method to analyze protein-protein interactions with artificial amino acids.

The protein of interest is modified in only one building block, which is one amino acid that is replaced by an artificial amino acid. This artificial amino acid confers new physicochemical properties to the protein of interest. Artificial amino acids belong to the scientific area of synthetic biology and there are currently over 300 artificial amino acids available. These synthetic amino acids offer scientists a variety of applications for proteins, for example, to facilitate analyses or add entirely new properties. The artificial amino acid p-azidophenylalanine (Fig. 2), a derivative of the natural amino acid phenylalanine, is particularly suitable for the study of molecular interactions. The azido-group does not occur naturally in proteins and can be activated by UV light to form a stable covalent bond with molecules in close vicinity. If the modified, synthetic protein interacts with another protein in the cell, the interaction can be captured by UV photo crosslinking under physiological conditions and is stable for further purification or identification of the interacting partner.

In order to conduct in vivo interaction studies, the synthetic amino acid must be incorporated into proteins. A synthetic biology method, the so-called expanded genetic code, can be used to achieve this. The artificial amino acid is incorporated during cellular protein synthesis specifically and with unrivalled efficiency into the protein of interest, at the desired position, mediated by special biomolecules, tRNAs and tRNA synthetases.

After extensive basic research at the Fraunhofer IGB, we were able to successfully apply the expanded genetic code methodology to the human pathogenic yeast Candida albicans. The necessary tRNA and tRNA synthetases were specifically modeled for the incorporation of artificial amino acids in C. albicans. Moreover, we were not only able to demonstrate the general applicability of the method with a model protein, but also with the central virulence factor Tup1 (Fig. 3). Therefore, the position-specific incorporation of an artificial amino acid into a eukaryotic pathogen has been achieved for the first time. Furthermore, we could, for the first time, characterize a physiological interaction of the virulence factor Tup1 by means of the synthetic label. The thus-modified C. albicans strains can now be applied to extensive interaction studies, such as in virulence factors. The investigation of host-pathogen interactions is also possible.

The system developed here is additionally suitable for protein-DNA or protein-metabolite interactions. After having demonstrated its general applicability, it is likewise conceivable to expand the genetic code of Candida albicans with other artificial amino acids and thereby further extend the range of molecular tools for the investigation of virulence mechanisms.

We are grateful to the Ministry of Science, Research and the Arts Baden-Württemberg for a state graduate scholarship (Landesgraduiertenförderung) to fund the project "Erweiterung des genetischen Codes zur Analyse von Protein-Protein-Interaktionen im humanpathogenen Pilz Candida albicans”.