We investigate the interplay of proteins in functional complexes that mediate the transport of proteins and peptides across cell membranes. Bacterial toxins that act inside human cells serve as ideal models of highly specialised and extremely efficient protein transport machines.

These modular proteins have different functional subunits for receptor binding (B) and membrane transport (T), via which they transport their enzymatically active subunit (A) into the cytosol of human cells. There, the A subunit modifies its specific substrate, which leads to cell damage and the clinical symptoms of toxin-mediated severe diseases such as diptheria, whooping cough (pertussis), anthrax, tetanus or botulism.

Due to this special structure and mode of action, bacterial protein toxins are the most toxic substances known.

Our fundamental research work on the mode of action of bacterial toxins has concrete pharmacological applications in the areas described below in the search for pharmacological toxin inhibitors and the development of molecular transporters for targeted drug delivery in immune and cancer cells.

For various medically relevant toxins such as diphtheria toxin, pertussis toxin, anthrax toxin or the clostridial enterotoxins, we were able to show that certain subunits in endosomes form transmembrane pores that mediate the transport of unfolded enzyme subunits across endosome membranes. Specific motifs of the translocating proteins bind directly to cellular chaperones (Hsp90, Hsp70) and peptidyl-prolyl cis/trans isomerases, resulting in functional protein transport machines that mediate membrane transport. Of particular interest to us are the mechanisms that lead to the temporally and spatially coordinated aggregation of such functional multiprotein complexes in cells, their structures and their role in the unfolding and refolding of translocating proteins.

Bacterial protein toxins are the most toxic substances known and direct triggers of numerous life-threatening diseases in humans, which is why they are important drug targets and the development of pharmacological toxin inhibitors is of high scientific and medical relevance. If it is possible to inhibit the uptake of the enzymatically active A subunit of the toxins into the cytosol of the target cell, the substrate modification and thus the cell damage, which is the basis of the clinical symptoms, does not occur.

This is particularly important as the existing antibacterial agents ("antibiotics") are only effective against bacteria, but not against the toxins already released by the bacteria. Furthermore, toxin inhibitors may represent attractive pharmacological options against (multi)resistant toxin-producing pathogens such as Clostridioides difficile. We are pursuing the following approaches in this project area:

- Search for toxin inhibitors in the human peptidome; e.g. defensins (in SFB 1279)

- Development of customised synthesis of toxin inhibitors that specifically inactivate structural parts of the toxins; e.g. synthetic toxin pore blockers (dendrimers etc.)

- Novel use of established drugs approved for other applications as toxin inhibitors; e.g. bacitracin, cyclosporin A (CsA), FK605 (tacrolimus), chloroquine, cyclodextrins

Many biological agents such as proteins, enzymes, peptides or nucleic acids ("biologics") show very good and specific efficacy against their respective target molecules, but cannot be used therapeutically because their drug targets ("drug tragets") are located in the cytosol of certain cells or in the central nervous system and therefore cannot be reached by these macromolecules because they cannot cross cell membranes. The development of molecular transport systems for the targeted and effective introduction of such active substances into the cell interior and their controlled release in the cytosol is therefore of great importance and the subject of our research work.

Our findings on cellular protein transport have direct pharmacological application in the development of novel modular "nanomachines" for cell type-selective transport and controlled release of therapeutic (macro)molecules (e.g. proteins, peptides) into the cytosol of human cells. The optimal protein subunits for receptor recognition, membrane transport and enzyme activity are assembled into new hybrid molecules, whereby recombinant protein transporters and semi-synthetic molecular complexes are generated using biotin/avidin technology.

The transporters are used in interdisciplinary and translational projects, including the Ulm CRCs 1149 and 1279, to investigate pathophysiological mechanisms of trauma, infectious and tumour diseases and to develop novel pharmacological approaches against such diseases.

Molecular, biochemical and functional analysis of Shiga toxin and subtilase subunits of enterohaemorrhagic Escherichia coli (H. Barth/ H. Schmidt)

Inhibition of bacterial protein toxins, in particular the toxins of Clostridioides difficile, by factors and cleavage products of the human complement system (H. Barth/M. Huber-Lang)

Multifunctional peptide nanocarriers for regulating cell migration in the microenvironment of metastatic breast cancer cells (T. Weil/H. Barth; C01 in SFB 1279)

Biohybrid transporters for cell-specific transport and controlled release of pharmacologically active peptides (H. Barth/J. Michaelis; C02 in SFB1279)

Cellular and molecular effects of trauma-induced damage to the distal respiratory epithelium (H. Barth/M. Frick; A05 in SFB1149)

Endogenous peptides as novel inhibitors of the Bordetella pertussis toxin (H. Barth; A06 in SFB 1279)