Impfstoffe für alle? Dodecin als Impfstoffplattform.

The Corona pandemic has painfully taught us the threat of new pathogens in a globalized world and how vital modern vaccines are. Platform technologies play an important role in the discovery of new vaccines as reducing the time for the development dramatically - time that saves lives. Here, we present the protein Dodecin and how it may be utilized as a versatile platform technology to produce cheap and robust new vaccines for everyone in all parts of the world.

Dodecin as carrier protein for immunizations and bioengineering applications.

In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod's ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).

Comparative biochemical and structural analysis of the flavin-binding dodecins from Streptomyces davaonensis and Streptomyces coelicolor reveals striking differences with regard to multimerization.

Dodecins are small flavin-binding proteins that are widespread amongst haloarchaeal and bacterial species. Haloarchaeal dodecins predominantly bind riboflavin, while bacterial dodecins have been reported to bind riboflavin-5'-phosphate, also called flavin mononucleotide (FMN), and the FMN derivative, flavin adenine dinucleotide (FAD). Dodecins form dodecameric complexes and represent buffer systems for cytoplasmic flavins. In this study, dodecins of the bacteria Streptomyces davaonensis (SdDod) and Streptomyces coelicolor (ScDod) were investigated. Both dodecins showed an unprecedented low affinity for riboflavin, FMN and FAD when compared to other bacterial dodecins. Significant binding of FMN and FAD occurred at relatively low temperatures and under acidic conditions. X-ray diffraction analyses of SdDod and ScDod revealed that the structures of both Streptomyces dodecins are highly similar, which explains their similar binding properties for FMN and FAD. In contrast, SdDod and ScDod showed very different properties with regard to the stability of their dodecameric complexes. Site-directed mutagenesis experiments revealed that a specific salt bridge (D10-K62) is responsible for this difference in stability.

Characterization of the small flavin-binding dodecin in the roseoflavin producer Streptomyces davawensis.

Genes encoding dodecin proteins are present in almost 20 % of archaeal and in more than 50 % of bacterial genomes. Archaeal dodecins bind riboflavin (vitamin B2), are thought to play a role in flavin homeostasis and possibly also help to protect cells from radical or oxygenic stress. Bacterial dodecins were found to bind riboflavin-5'-phosphate (also called flavin mononucleotide or FMN) and coenzyme A, but their physiological function remained unknown. In this study, we set out to investigate the relevance of dodecins for flavin metabolism and oxidative stress management in the phylogenetically related bacteria Streptomyces coelicolor and Streptomyces davawensis. Additionally, we explored the role of dodecins with regard to resistance against the antibiotic roseoflavin, a riboflavin analogue produced by S. davawensis. Our results show that the dodecin of S. davawensis predominantly binds FMN and is neither involved in roseoflavin biosynthesis nor in roseoflavin resistance. In contrast to S. davawensis, growth of S. coelicolor was not reduced in the presence of plumbagin, a compound, which induces oxidative stress. Plumbagin treatment stimulated expression of the dodecin gene in S. davawensis but not in S. coelicolor. Deletion of the dodecin gene in S. davawensis generated a recombinant strain which, in contrast to the wild-type, was fully resistant to plumbagin. Subsequent metabolome analyses revealed that the S. davawensis dodecin deletion strain exhibited a very different stress response when compared to the wild-type indicating that dodecins broadly affect cellular physiology.

Flavin Storage and Sequestration by Mycobacterium tuberculosis Dodecin.

Dodecins are small flavin binding proteins occurring in archaea and bacteria. They are remarkable for binding dimers of flavins with their functional relevant aromatic isoalloxazine rings deeply covered. Bacterial dodecins are widely spread and found in a large variety of pathogens, among them Pseudomonas aeruginosa, Streptococcus pneumonia, Ralstonia solanacearum, and Mycobacterium tuberculosis ( M. tuberculosis). In this work, we seek to understand the function of dodecins from M. tuberculosis dodecin. We describe flavin binding in thermodynamic and kinetic properties and achieve mechanistic insight in dodecin function by applying spectroscopic and electrochemical methods. Intriguingly, we reveal a significant pH dependence in the affinity and specificity of flavin binding. Our data give insight in M. tuberculosis dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in M. tuberculosis may specifically be important for flavin homeostasis during the elaborate lifestyle of this organism, which calls for the evaluation of this protein as drug target.