Typ IV Secretion Systems (T4SS) in Gram-positive Bacteria

Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G-) origin. Although many medically relevant pathogens are Gram-positive (G+), their conjugation systems have received little attention. We propose that T4SSs of G+ origin have a strikingly different architecture than G- systems due to the distinct structure of the cell envelope lacking the outer membrane and the periplasmic space but featuring a thick, multilayered peptidoglycan. This hypothesis will be tested with two G+ T4S model systems: The conjugative T4SS of the multiple antibiotic resistance plasmid pIP501 has the broadest known host range for plasmid transfer in G+ bacteria including enterococci, staphylococci and streptococci. It is organized in an operon encoding fifteen putative transfer (Tra) proteins. The enterococcal sex-pheromone plasmid pCF10 encodes a partially characterized but distinct T4SS, which will be compared with the pIP501 model system. For four of the pIP501 Tra proteins the functions in the T4SS have been assigned, the relaxase TraA, two ATPases, the VirB4-like protein TraE and the VirD4-like coupling protein TraJ, and the VirB1-like muramidase TraG . Moreover, three of the pIP501 T4SS proteins have been crystallized and the structures of the TraM and TraK C-terminal domains have been solved (Goessweiner-Mohr et al., 2013; Goessweiner-Mohr et al., 2014). In this project, we aim to determine the structure of TraG, an indispensable muramidase of the pIP501 T4SS, the TraM N-terminal domain and/or the full-length TraM protein and the VirB4-like ATPase TraE. In addition we will examine the corresponding proteins of the T4SS of the sex-pheromone responsive Enterococcus plasmid pCF10. The generation of gene-specific knock-outs of the pIP501 and the pCF10 systems will be conducted in our collaborators laboratories and will elucidate further indispensable components of the two T4SSs. The pIP501 and/or pCF10 core complex components will be identified via on-going co-expression experiments. Emerging complexes will be investigated by electron microscopy and crystallography to elucidate the structure of the first core complex of a T4SS derived from a G+ pathogen. This project is highly inter-disciplinary, combining genetic, immunological and functional experiments (knock- outs, complementation, transfer DNA immuno-precipitation (TrIP), opsonophagocytosis assay (OPA), mating assays), which are partly performed in our collaborators laboratories with cloning, biophysical and structural work (solubility screening, CD, SAXS, crystallography, EM). i

Conjugative DNA transfer is the most prevalent mechanism for antibiotic resistance gene dissemination among bacteria. The transfer is mediated by a multi-protein complex, the type IV secretion system (T4SS), which spans the bacterial envelope and acts as a channel for macromolecular secretion. The major goal of this project was the elucidation of the structure-function relationship of essential transfer proteins, which are contained in the tra- operon of the conjugative type IV-like secretion system (T4SS) of plasmid pIP501 of Enterococcus faecalis. This was achieved by a combination of structural methods (crystallography, NMR, electron microscopy), molecular biology methods (cloning and expression of selected proteins, measuring the effect of deletion mutants on the mRNA and the protein expression level) and biochemical methods (in vivo and in vitro cross-linking, expression and isolation of the assembled T4SS, identification of its components by W- blotting). Several components were investigated and their structure and/or function determined (TraH, TraN-DNA complex, TraF). For some additional factors the structure determination is ongoing (e.g. TraM N-terminal domain and TraA). Interactions between the components, which had been investigated in a yeast-two-hybrid study, were reevaluated with a bacterial-two-hybrid (BACTH) assay, yielding the likely composition of a core complex of the conjugative pore. Gene deletion studies (Knock-out mutants) were used to identify essential Tra factors and to characterize a novel repressor of the tra operon, TraN. The previously characterized, essential transfer factor TraM was evaluated as a potential vaccine target showing cross-reactivity against clinically relevant enterococcal and staphylococcal strains. In summary these results contribute to the understanding of the mechanism involved in DNA transfer and exchange of genetic information in this important group of bacteria, which contains several important human pathogens. In the long run, the emerging structural and functional information on this T4SS might enable us to identify further targets for inhibition of the spread of antimicrobial resistance determinants in Gram-positive pathogens.