PMC

Crystal structures of Y. pestis FyuA and pesticin. (A) The crystal structure of FyuA consists of two domains: the β-barrel in green and the plug domain in red. The TonB box of FyuA was mostly disordered and not observed in the crystal structure. In the periplasmic view of FyuA, the location of the TonB box is not shown for clarity. (B) The crystal structure of pesticin consists of two distinct domains: The predominantly β-sheet N-terminal domain (FyuA binding domain) in purple and encompasses residues 1–164. The mainly α-helical C-terminal domain (residues 168–357) in gold (killing domain). A short linker connects the two domains and is shown in cyan that spans residues 165–167. The TonB box of pesticin was disordered and not observed in the crystal structure. The residues mutated in this study are labelled and shown in stick representation and include residues in the presumed active site of pesticin. (C) To illustrate that the C-terminal domain of pesticin is structurally similar to T4 lysozyme, the crystal structure of T4 lysozyme (green) (PDB code = 2LZM) is represented in the same orientation as the C-terminal domain of pesticin. Similarity between the two proteins is primarily structural; there is little sequence identity except for a partial conservation of active site residues that is apparent after a structural alignment. (D) Superimposition of the pesticin (gold) and T4 lysozyme (green) active sites shows that the catalytic threonine and glutamate residues are conserved in pesticin, whereas the catalytic aspartate in lysozyme is replaced by a proline in pesticin. Therefore, pesticin shares a functional resemblance to T4 lysozyme, but the active sites are not identical. For clarity, representation of secondary structure elements was omitted in D.

Effects of pesticin and yersiniabactin on expression of the HMWPs. SDS-PAGE (7.5% gel) of total-cell proteins of iron-starved strains WA-CS and WA-CS irp1::Kan^r with addition of pesticin and culture supernatant containing yersiniabactin. Lane 1, WA-CS in NBD medium; lane 2, WA-CS in NBD medium with pesticin (1:1,024); lane 3, WA-CS in NBD medium with yersiniabactin supernatant (1:50); lane 4, WA-CS irp1::Kan^r in NBD medium; lane 5, WA-CS irp1::Kan^r in NBD medium with pesticin; lane 6, WA-CS irp1::Kan^r in NBD medium with yersiniabactin supernatant.

Bactericidal assays demonstrate that pesticin and the hybrid lysin kill E. coli cells expressing FyuA. Pesticin or hybrid lysin at a concentration of 100 μg/mL was added to E. coli T7 express cells in PBS at 37 °C while shaking. Control cultures had no proteins added. Samples were removed at several time points, plated, and surviving bacterial colonies were counted. These experiments were repeated at least three times. The standard error of the mean is shown. (A) Cells expressing fyuA are killed by pesticin and the hybrid lysin. (B) Cells expressing fyuA and pim are killed by the hybrid lysin but not by pesticin due to protection conferred by the pesticin immunity protein. Note that the immunity protein cannot protect against killing by the hybrid lysin. (C) E. coli T7 express cells transformed with pET20b (Null) are resistant to killing by pesticin or the hybrid lysin because they do not express fyuA on the cell surface.

Figure 1. Mode of action of pesticin and an engineered hybrid protein: (1) The Pesticin molecule consists of a N-terminal “FyuA binding” and a C-terminal lysozyme-like domain. After traversing the outer membrane pesticin degrades the peptidoglycan structural layer present in the periplasm resulting in cell killing. This translocation depends on an outer membrane protein and virulence factor—FyuA—but the mechanism by which this happens is not yet understood. (2) The lysozyme-like domain of pesticin was substituted for T4 lysozyme to create a hybrid protein. The translocation of a hybrid also results in killing but unlike with pesticin this process is not inhibited by a periplasmic Pim protein. (3) Without additional means of disrupting the OM the externally added T4 lysozyme cannot effect killing. Abbreviations used in the figure: OM, outer membrane; Peri., periplasm; IM, inner membrane; PG, peptidoglycan.