AbstractCell wall biosynthesis is an important target of antimicrobial peptides (AMPs), which in light of the imminent shortage of antibiotics are considered promising candidates for future treatment of infections. Yet, numerous Gram-positive bacteria have developed specific resistance systems against many AMPs. These systems feature so-called BceAB-like ATP-binding cassette transporters consisting of ten transmembrane helices and a characteristic extracellular domain of around 200 amino acids.
So far, the mechanisms behind substrate recognition and binding as well as the nature of the physiological substrate of the transporters remained elusive. This is a major impediment to our understanding of the resistance mechanism, and multiple theories have been proposed regarding these important questions. The targets of AMPs are crucial membrane-anchored intermediates of the lipid II cycle. One hypothesis suggests that the transporters expel the AMP from the membrane, in which case the physiological substrate should be the AMP itself or the AMP bound to its cellular target. Alternatively, the transporters may flip the drug target to the cytoplasmic face of the membrane to remove it from access by the AMP.
Here, we investigate the substrate specificity of the transporter BceAB of Bacillus subtilis. We focus on characterising the binding capacity of its large extracellular domain in vitro and further aim to identify the physiological substrate of BceAB using in vivo approaches. Combining the findings of biochemical, biophysical and physiological assays conducted for this study, we propose the complex formed between the AMP and its cellular target to be the physiological substrate of BceAB, rather than the unbound AMP or the drug target alone.
Considering this result in the context of previous findings and the literature enabled us to gain valuable insights into the potential resistance mechanism of BceAB-like transporters that play such a crucial role in antimicrobial resistance.
|Date of Award||29 May 2019|
|Supervisor||Christopher Pudney (Supervisor) & Susanne Gebhard (Supervisor)|