Characterization of a sensory complex involved in antimicrobial peptide resistance

In their habitats, microorganisms are often in competition for limited nutrients. In order to succeed, many Gram-positive bacteria resort to production of peptide antibiotics. Therefore, resistance mechanisms against these compounds are essential. The first step of ensuring survival is the perception of the harmful drugs and mediation of resistance against it. In recent years, a group of ABC-transporters have been recognized as important resistance determinate against antimicrobial peptides. The expression of these transporters is generally regulated by a two-component system, which in most cases is encoded next to the transporter. Together they are described as detoxification modules. The permeases of the transporters are characterized by a large extracellular domain, while the histidine kinases lack an obvious input domain. One of the best understood examples is the BceRS-BceAB system of Bacillus subtilis, which mediates resistance against bacitracin, mersacidin and actagardine. For this system it was shown that the histidine kinase is not able to detect the substrate directly and instead has an absolute requirement for the transporter in stimulus perception. This describes a novel mode of signal transduction in which the transporter is the actual sensor and therefore regulates its own expression. To date, mechanistic details for this unique mode of signal transduction remain unknown. Several other examples have been described for transport proteins that have acquired additional sensing or regulatory functions beyond solute transport, and these have been designated trigger transporters. For these bifunctional transporters a direct protein-protein interaction with membrane-integrated or soluble components of signal transduction relays has been postulated. However, for most sensor/co-sensor pairs, conclusive proof of such an interaction is lacking, and so far little is known about the sites that might mediate contacts between the putative protein interfaces and how communication is achieved. Based on sequence and architectural similarities, we identified over 250 BceAB-like transporters in the protein database, which occurred almost exclusively in Firmicutes bacteria. To whether the regulatory interplay between the ABC transporter and the two-component system was a common theme in these antimicrobial peptide resistance modules, we carried out a phylogenetic study of these identified systems. We identified a clear coevolutionary relationship between transport permeases and histidine kinases. Furthermore, we identified conserved putative response regulator binding sites in the promoter regions of the transporter operons. Additionally, we were able to provide a tool to identify TCSs for transporters lacking a regulatory system in their genomic neighbourhood, which was based on the coclustering of histidine kinases and transporter permeases. These findings also suggested the existence of a sensory complex between BceAB-like transporters and BceS-like histidine kinases. To further investigate the signaling mechanism, we performed a random mutagenesis of the transport permease BceB with the aim to identify regions or residues within the transporter that are involved in signaling and/or resistance. With this approach we were able to identify mutations that affected either the ability for signaling or mediation of resistance. This showed a partial genetic separation of the two qualities, which could be achieved by single amino acid replacements. These results provide first insights into the signaling mechanism of the Bce system. In order to analyse the proposed communication between two-component system and ABC transporter, we further characterized their interactions by in vivo and in vitro approaches. We could demonstrate that the transporter BceAB is indeed able to interact directly with the histidine kinase. Because it was unknown how the signal perception by BceAB-type transporters occurs, we next analyzed substrate binding by the transporter permease BceB and could show direct binding of bacitracin by BceB. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the activity of the histidine kinase. In summary this thesis clearly shows the existence of a sensory complex comprised of BceRS-like two-component systems and BceAB-like ABC transporters and provides first functional insights into the mechanism of stimulus perception, signal transduction and antimicrobial resistance mechanism employed by these wide spread detoxification systems against antimicrobial peptides.