Structural Basis of Pore Formation in the Mannose Phosphotransferase System by Pediocin PA-1

Abstract

Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides that have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like (or class IIa) bacteriocins (PLBs) exhibit antibacterial activity against several Gram-positive bacterial strains by forming pores in the cytoplasmic membrane of target cells with a specific receptor, the mannose phosphotransferase system (man-PTS). In this study, we report the cryo-electron microscopy structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1, the first and most extensively studied representative PLB, at resolutions of 3.12 and 2.45 Å, respectively. The structures revealed that the binding of pediocin PA-1 opens the Core domain of man-PTS away from its Vmotif domain, creating a pore through the cytoplasmic membranes of target cells. During this process, the N-terminal β-sheet region of pediocin PA-1 can specifically attach to the extracellular surface of the man-PTS Core domain, whereas the C-terminal half penetrates the membrane and cracks the man-PTS like a wedge. Thus, our findings shed light on a design of novel PLBs that can kill the target pathogenic bacteria. IMPORTANCE Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans, who become infected by ingesting contaminated food products (i.e., dairy, meat, fish, and vegetables): the disease has a fatality rate of 33%. Pediocin PA-1 is an important commercial additive used in food production to inhibit Listeria species. The mannose phosphotransferase system (man-PTS) is responsible for the sensitivity of Listeria monocytogenes to pediocin PA-1. In this study, we report the cryo-EM structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1 at resolutions of 3.12 and 2.45 Å, respectively. Our results facilitate the understanding of the mode of action of class IIa bacteriocins as an alternative to antibiotics.

Keywords: antibiotic resistance; antimicrobial peptides; bacteriocins; man-PTS; mannose phosphotransferase; pediocin PA-1; pediocin-like/class IIa bacteriocins.

FIG 1

Cryo-EM structures of man-PTS from Listeria monocytogenes and its complex with antilisterial bacteriocin pediocin PA-1. (A) The structure of the bacteriocin-free man-PTS trimer is represented as viewed from the extracellular side of the membrane, with each protomer differently colored. man-PTS is spatially organized into the Vmotif and Core domains. (B) The Vmotif domain is viewed within the plane of the membrane, with parts of the structural components on one protomer removed for clarity. Three stabilizing interactions between protomers are identifiable. (C) The color-coded domain architecture of ManY and ManZ. ManY and ManZ from E. coli have a similar fold, except for loop L78Z. Regions γ and γ+, which were successively inserted into L78Z, resulted in ManZs from Listeria monocytogenes and Lactococcus garvieae. (D) The Vmotif domain is color-coded based on the domain architecture as in panel C. Region γ+ from Lactococcus garvieae is indicated by a dashed loop. Loop L78Z is stabilized through one end by stacking the effect of extTM9Y onto the neighboring TM9Y and the other end by helical bundles of region γ. (E) Top view of a man-PTS complex of Listeria monocytogenes with pediocin PA-1. Pediocin PA-1s are shown as surface representations colored in yellow. Man-PTS is color-coded in accordance with panel C. (F) Surface representation of the interface between the Vmotif and Core domains in the bacteriocin-free man-PTS. (G) Surface representation of the interface between the Vmotif domain and Core domain in the bacteriocin-bound man-PTS. The transmembrane pore is shown in green mesh as generated with HOLE software (51). (H) The electrostatic potential surface representation of the interface between the Vmotif domain and Core domain. (I) The pore radii along the potential transport path are shown. The minimal radius of the pore is approximately 1.5 Å.

FIG 2

Structure and orientation in membrane of pediocin PA-1. (A) Sequence alignment of pediocin-like bacteriocins from the four representative subgroups (8). The pediocin PA-1 peptide chain is divided into three regions: a cationic, hydrophilic, and highly conserved N-terminal region, a less-conserved more hydrophobic C-terminal α-helical region, and a hairpin-like C-terminal tail. (B) An illustration and electrostatic potential surface depiction of the structure and orientation of pediocin PA-1 in membranes. The N-terminal β-sheet has two distinct hydrophobic/hydrophilic surfaces.

FIG 3

The recognition of the N-terminal region of pediocin PA-1 by man-PTS. (A) The highly conserved residues are hydrophobic, including the disulfide bond from the hydrophobic surface, whereas the other surface is hydrophilic, without any observable interactions with the receptor, man-PTS. (B) The N-terminal part of pediocin PA-1 is attached to the Core domain of man-PTS, primarily through hydrophobic interactions.

FIG 4

Interactions between the C-terminal region of pediocin PA-1 and man-PTS. (A) The interaction of the C-terminal portion of pediocin PA-1 and man-PTS is shown schematically. The central α-helix region is shown in the helical wheel. (B) The specific interactions between man-PTS and the central α-helix, as well as the C-terminal tail of pediocin PA-1. The transmembrane pore is shown in green mesh. (C) Enlarged view of the interactions between the central α-helix of pediocin PA-1 and man-PTS. (D) Interactions between the C-terminal tail of pediocin PA-1 and man-PTS. (E) Bactericidal activity of the wild type (WT) and various lmManZ mutants and the C-terminal tail of pediocin PA-1. Serial dilutions of overnight cultures were spotted (5 μL) on LB plates.

FIG 5

The proposed mechanism of pediocin PA-1 antibacterial activity. (A) An elevator-type mechanism of sugar transportation across membranes. The Core domain moves vertically against a stationary Vmotif domain. (B) Once the pediocin PA-1 binding site on the Core domain of the receptor is exposed, the N-terminal region of pediocin PA-1 specifically recognizes the Core domain for binding. Subsequently, the C-terminal wedge-like region is embedded between the Vmotif and Core domains to form a pore. The immunity protein potentially blocks the channel from the cytoplasmic side to protect the producing strain.