(613f) Could Class IIb Bacteriocins Induce Pore Formation? Investigation through Microsecond Long Atomistic Molecular Dynamic Simulation

Antibiotic resistance poses an alarming threat to public health and modern society at large. In our times, it is an unfortunate reality that resistance has been found to all commercially available antibiotics, even to the so-called “last resort” ones [1,2]. Different government-issued reports warn that we are approaching a “post-antibiotic era”, where even minor bacterial infections could be deadly [1,2]. As the number of cases of antibiotic resistance is increasing, the effective utilization of conventional antibiotics is rapidly decreasing. To overcome this challenge, alternative drugs should be examined. One promising alternative source of new antibiotic drugs that has been investigated extensively of late is the use of antimicrobial peptides (AMPs) [3]. Bacteriocins are antimicrobial peptides (AMPs) produced by bacteria that show great potential as novel antibiotic agents [4]. They are active at lower concentrations and they exhibit higher specificity against their targets, compared to other AMPs [5]. In order to most powerfully utilize the full potential of bacteriocins and use them as a platform to develop new antibacterial agents, we must develop an understanding of their mechanism of action.

We study Plantaricin EF (PlnEF), a two-peptide class IIb bacteriocin, that is comprised of Plantaricin E (PlnE) and Plantaricin F (PlnF). It has been well-documented that class IIb bacteriocins induce cell death through membrane leakage [6]. Although, the complete mechanism of action of class IIb bacteriocins is still unclear, it has been suggested that these peptides act through a receptor-mediated mechanism of action. To elucidate the initial interaction of the peptides with the membrane, we have previously performed molecular dynamics simulations of the two-peptide bacteriocin PlnEF on the surface of a model lipid bilayer [7]. Next, we employed an extensive mutational analysis and created fusion peptides that blocked the activity of each of the terminai of each peptide in order to identify the orientation of the peptides in a transmembrane conformation [8]. These experiments suggested that PlnE and PlnF create an antiparallel dimer when located inside a membrane. We were then able to design a transmembrane model of the dimer embedded in the bilayer.

The dimer showed a remarkable stability over a 1 μs long atomistic molecular dynamics simulation. We analyzed the dimer structure and identified important amino acids that are responsible for the interaction between the peptides and the anchoring of the dimer in the membrane. We further investigated the impact of the of the dimer’s presence to the rest of the system (the membrane, water, and ions). Most importantly, we showed that the transmembrane PlnEF dimer can form a small torroidal pore that allows water permeation and suggests possible ion conduction. This is the first time (to our knowledge) that it has been demonstrated in atomistic detail that a LAB bacteriocin with narrow antimicrobial activity range, can form pores on its own. We believe this finding could be of immense importance to the designing of new antibiotic agents, as it would steer the search for better bacteriocins toward peptides that form stable pores, interact more strongly with the membrane in specific regions, and increase water or ion permeability.

References:

[1] S. Reardon, WHO warns against “post-antibiotic” era, Nature. (2014).

[2] Antimicrobial Resistance: Tackling a Crisis for the Future Health and Wealth of Nations, in: Rev. Antimicrob. Resist. Chaired by Jim O’Neill, 2014.

[3] D.H. Lloyd, Alternatives to conventional antimicrobial drugs: a review of future prospects., Vet. Dermatol. 23 (2012) 299–304, e59-60.

[4] P.D. Cotter, R.P. Ross, C. Hill, Bacteriocins - a viable alternative to antibiotics?, Nat. Rev. Microbiol. 11 (2013) 95–105.

[5] J. Nissen-Meyer, I.F. Nes, Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action, Arch. Microbiol. 167 (1997).

[6] J. Nissen-Meyer, C. Oppegård, P. Rogne, H.S. Haugen, P.E. Kristiansen, The Two-Peptide (Class-IIb) Bacteriocins: Genetics, Biosynthesis, Structure, and Mode of Action, in: Prokaryotic Antimicrob. Pept., Springer New York, New York, NY, 2011: pp. 197–212.

[7] P.K. Kyriakou, B. Ekblad, P.E. Kristiansen, Y.N. Kaznessis, Interactions of a class IIb bacteriocin with a model lipid bilayer, investigated through molecular dynamics simulations, Biochim. Biophys. Acta - Biomembr. 1858 (2016) 824–835.

[8] B. Ekblad, P.K. Kyriakou, C. Oppegård, J. Nissen-Meyer, Y.N. Kaznessis, P.E. Kristiansen, et al., Structure–Function Analysis of the Two-Peptide Bacteriocin Plantaricin EF, Biochemistry. 55 (2016) 5106–5116.