Structure and evolution of the Ivy protein family, unexpected lysozyme inhibitors in Gram-negative bacteria

Abstract

Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect. This feature guided the identification of Ivy orthologues in other Gram-negative bacteria. The structure of the evolutionary distant Pseudomonas aeruginosa Ivy orthologue was also determined in complex with hen egg white lysozyme, and its antilysozyme activity was confirmed. Ivy expression protects porous cell-wall E. coli mutants from the lytic effect of lysozyme, suggesting that it is a response against the permeabilizing effects of the innate vertebrate immune system. As such, Ivy acts as a virulence factor for a number of Gram-negative bacteria-infecting vertebrates.

Fig. 1.

Stereoviews of the Ivyc structure. (a) Cα trace of the Ivyc monomer (residues 2–128). (b) Ribbon structure of the Ivyc dimer.

Fig. 2.

The Ivyc structure. (a) Cartoon representation of the Ivyc–HEWL complex. Monomers of the Ivyc dimer are colored blue and red, and the two lysozyme molecules are colored orange. The Ivyc H60 residues responsible of the HEWL inhibition are represented as light blue balls and sticks. The HEWL D52 residue is colored in yellow, and the E35 is in purple with balls and sticks representation. These two residues are part of the HEWL active site and make hydrogen bonds with the Ivyc H60 residues. The black arrows line up the surface contact area between the Ivyc dimer and each lyzosyme molecule. (b) Cartoon representation of the superimposition of the Ivy molecules in the complex structure (red and pink) and the three isolated Ivyc molecules (yellow, blue, purple) superimposed with one Ivyc molecule from the complex (red). Secondary structure elements are marked on the structure. (c) Stereoview of the electron density map of the Ivyc protruding loop penetrating the HEWL active site. The two F_o − F_c electronic density map is contoured at 1.0 σ. Residues are colored by types: basic residues in cyan, acidic residues in red, polar residues in light green, hydrophobic residues in yellow, cysteine residues in green, and aromatic residues in purple. Ivyc H60 and HEWL E35 and D52 are labeled. (d) Interaction of the Ivyc loop (CKPHDC) with the lysozyme active site. Ivyc is represented as a blue ribbon; H60, C57, and C62 are yellow balls and sticks; and the lysozyme active site as molecular surface is colored according to residue types (acidic, red; polar, green; hydrophobic, white; basic, blue).

Fig. 3.

Phylogenetic analysis of the Ivy protein family. The phylogenetic tree suggests that an ancestor of Ivyc existed in both the gamma and beta divisions (in yellow) but was subsequently lost in some of the gamma (e.g., Salmonella) and beta division clades (e.g., most Neisseriaceae and Bordetella). The anomalous position of the branch leading to the few alpha species (in red) in which Ivy is identified strongly suggests an acquisition by horizontal transfer from Enterobacteria, as was probably the case for B. cepacia (loss of the Burkholderia-derived gene, acquisition from Enterobacteria). Ivyc paralogous sequences exhibiting the noncanonical loop motif (in green) appear to have emerged from a duplication within and limited to the Pseudomonas clade (followed by the loss of the original Ivyc orthologue, except for P. aeruginosa).

Fig. 4.

Effect of increasing concentrations of HEWL on E. coli mutants' growth kinetics. (a) E. coli MG1655 ΔtolBΔivy. (b) E. coli MG1655 ΔtolBΔivy transformed with the pET26 plasmid expressing the Ivyc protein in the periplasm. For E. coli MG1655 ΔtolB (JC864) as a control see SI Fig. 6.