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.