Understanding the mechanism of SAAP-148 and exploring the purification of LptD/E
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Chapter 1: In the race against antimicrobial resistance, De Breij et al. (2018) designed an antibiotic peptide called SAAP-148 derived from the amino acid sequence of the human antimicrobial peptide LL-37. SAAP-148 showed to have high efficacy not only against multidrug-resistant ESKAPE pathogens but also against biofilms and persister cells. Yet, the exact binding mechanism of SAAP-148 is unknown. In this study, we use ssNMR and binding studies to show that the affinity of SAAP-148 is aspecific and it binds to all anionic lipids. Additionally, we use carboxyfluoreiscein leakage assays to study the effect of SAAP-148 on the permeabilization of the lipid vesicles. This data unexpectedly shows that the permeabilization of SAAP-148 is not coherent with the affinity of the negative charge of the lipids. SAAP-148 permeabilizes not only DOPG but DOPC membranes as well. On the other hand, SAAP-148 has only small a perturbation effect on the anionic lipid, cardiolipin. This information can be used to improve the antimicrobial properties of SAAP-148 and in the design of better antibiotics. Chapter 2: Gram-negative bacteria are more resistant to antibiotics as compared to Gram-positive bacteria. One of the reasons for this is the difference is in their cell envelop structure. The outer membrane (OM) of Gram-negative bacteria contains a phospholipid inner leaflet and an almost entirely lipopolysaccharide (LPS) outer leaflet. LPS shields the bacteria from harsh environments and incorporation of LPS is essential for bacterial survival. LPS is incorporated into the membrane by the LPS-transport chain, facilitated by seven proteins (LptABCDEFG). The most accessible part of these proteins is the LptD/E complex. This complex is embedded in the outer membrane of the envelope and performs the final step of the transportation by incorporating the LPS in the outer membrane. An antibiotic, called Murepavadin, binds to this LptD/E complex in Pseudomonas aeruginosa (Pa) and inhibits the incorporation of LPS in the OM. In this study, we give insight into the transformation, expression, and purification of the Pa LptD/E complex that could be used in further studies to investigate the binding of Murepavadin to this complex using solid-state NMR.