Interaction of Antimicrobial Peptides with Model Microbial Cell Membranes

Abstract

The discovery of antibiotics revolutionised healthcare, greatly reducing the incidence of bacterial infections. Unfortunately, however, bacteria are under strong selective pressure to evolve resistance to antibiotics, and this, along with the over- and mis-use of antibiotics and the dearth of development of new antibiotics, mean the world is now facing an antimicrobial resistance crisis. Antimicrobial peptides (AMPs) are a type of antimicrobial agent that often act by disrupting the bacterial cell membrane, which allows less potential for resistance. Most classes of AMPs exhibit regular secondary structure, and these are also the most well understood. Some, however, in particular non-ribosomally synthesised AMPs, do not form secondary structure, and may include non-natural amino acids, be lipidated, or form cycles. This thesis focuses on laboratory synthesised variants of two types of non-ribosomally synthesised AMPs, battacins and polymyxins. The overarching goal is to understand which chemical features of AMPs are most important for their activity both to rationalise the differences in activity of existing variants as well as enable better prediction of even more active variants. Molecular dynamics (MD) simulations are used to probe the peptide-membrane interactions of battacin (chapter 2 and 3) and polymyxin (chapter 4) analogues at an atomic level of detail, and, in chapter 5, Markov state models (MSMs) are used to characterise the major structural states of two battacin analogues and quantify the transition rates between them in order to further understand their behaviour. Chapter 1 contains an introduction to antimicrobial resistance and AMPs, including their different types, modes of action, and clinical usages. It then briefly introduces microbial cell membranes, before seguing into a detailed description of how MD simulations work. Finally, there is a brief contextual summary of MSMs, which are described in further detail in chapter 5. Chapter 2 comprises a research article published in the journal ACS Omega (Chakraborty et al., 2021) describing the use of MD simulations to investigate the mode of action of two novel linear battacin analogues, an octapeptide and a pentapeptide. These two AMPs are unusual in that they have activity against both Gram negative and Gram positive model species (De Zoysa et al., 2015). The simulations of single copies of these peptides in the presence of model E. coli and S. aureus membranes showed that, in agreement with studies of other AMPs, the positively charged 2,4- diaminobutyric (Dab) residues were important for binding to the membrane surface, with the importance of the positive charge confirmed by simulation of uncharged NH2-Dab analogues. Insertion of the hydrophobic portions of the lipid moieties and, to a lesser extent, hydrophobic amino acid side chains, into the membrane core also made an important contribution to the peptide-membrane interaction energy, with such insertion also having been observed for other AMPs. Chapter 3 investigates four additional linearised battacin analogues, again in the presence of both E. coli and S. aureus membrane models due to experimental evidence of their activity against both Gram negative and Gram positive bacteria (Yim et al., 2020). Here, in addition to simulating single peptide molecules with the membrane models, multiple peptide molecules in solution and in the presence of the membrane models were also simulated. The two analogues with linear acyl chains were found to aggregate in solution, which appeared to slow their interaction with the membrane due to some molecules in each aggregate being occluded from the membrane surface. Overall, however, the results were similar to those observed in chapter 2, with positively charged residues being important for interactions with the membrane surface, and penetration of the hydrophobic portion of the lipid(s) and, to a lesser extent, hydrophobic amino acid side chains, providing an energetically favourable anchor. The tert-butyl benzoate alkyl group of one analogue had substantially more favourable interactions with the membrane compared to the linear acyl chains, although it is possible that this was in part due to the lack of inter-peptide interactions that might provide alternative binding partners for the linear chains. Chapter 4 describes the use of MD simulations to investigate polymyxins, another type of AMP. As with the peptides investigated in Chapter 3, a host of analogues of the naturally occurring polymyxin B3 were synthesised and tested experimentally. Both single and multiple molecules of a select subset of these were simulated in solution and in the presence of the model E. coli IM. A similar pattern was observed for the polymyxins as for the battacin analogues described in chapters 2 and 3: positively charged residues such as Dab are important for binding to the membrane surface, and lipid groups are required for anchoring and membrane disruption. Too many lipid tails is not necessarily helpful though: aggregation in solution was observed for the triply-lipidated analogue, providing an explanation for its lack of activity experimentally. Chapter 5 contains a detailed background to MSMs, followed by their use to characterise the dynamics of two of the battacin analogues from chapter 3, the negative control, Ba-S, and the highly active Ba-t4. A set of features derived from the internal coordinates of the peptides during 1500 ns of MD simulation were extracted and subjected to time-lagged independent component analysis (tICA) to identify the features related to the slowest time scale motions. These were then discretised using the Common nearest neighbour (CNN) clustering algorithm in a hierarchical, iterative manner. Lastly, the core-sets of features identified by clustering were used to build MSMs. Unfortunately, a quality check revealed the core-sets and thus the MSMs to be of poor quality, due to either insufficient sampling in the MD simulations and/or poor discretisation. The MSMs were therefore not able to be interpreted with respect to differences in behaviour resulting from the different chemical structures of the two peptides or their environment. Altogether, the work presented in this thesis has confirmed the importance of two factors known to be key to AMP activity: the presence of positively charged moieties that form attractive Coulombic interactions with the negatively charged surface of bacterial cell membranes, and the requirement for one or more lipid groups. For the non-ribosomally synthesised AMPs studied here, the positively charge is typically provided by non-natural amino acids such as Dab. Unfortunately, these are also the cause of the nephrotoxicity of the non-ribosomally synthesised AMPs that have been used clinically, polymyxins B and E, so there is a need for analogues that reproduce the favourable membrane interactions while reducing toxicity. The lipids can be hydrocarbon chains, acyl groups, or geometrically more complex alkyl groups. More than two such groups can be detrimental to AMP activity, however, as they can stimulate aggregation of the AMPs in solution that can then reduce their propensity to interact with the membrane. Long hydrocarbon chains can have the same effect. Hydrophobic amino acid side chains, such as the benzyl group of Phe, insert to a lesser degree compared with the attached lipids, and appear to be insufficient for high levels of activity.