Structural and functional analysis of bacterial lysozyme inhibitors

Abstract

Lysozymes are antibacterial effectors of the innate immune system in animals that hydrolyse peptidoglycan, the major constituent of the bacterial cell wall. In the animal kingdom three major different lysozyme types can be distinguished, namely chicken (c-), goose (g-) and invertebrate (i-) type lysozymes. Whereas c- and g-type lysozymes are found in all vertebrate and some invertebrate phyla, i-type lysozyme occurs only in invertebrate organisms. Bacteria have in turn evolved protective mechanisms that contribute to lysozyme tolerance such as the production of specific lysozyme inhibitors. Only inhibitors of c- and i- type lysozyme were known at the start of this study and, therefore the first objective of this research was to investigate the existence of bacterial g-type lysozyme inhibitor(s).A screening of periplasmic extracts from various Gram-negative bacteria for g-type lysozyme inhibition revealed the presence of a potential g-type lysozyme inhibitor in Escherichia coli. Purification by chromatographic techniques and identification by tandem mass spectrometry led to the discovery of a novel periplasmic protein from E. coli that is responsible for the g-type lysozyme inhibition. This protein is designated as PliG (Periplasmic lysozyme inhibitor against g-type lysozyme). Based on surface plasmon resonance analysis, PliG binds with very high affinity to salmon g-type lysozyme (SalG) (ka = 2.02 ± 0.47 x 106 (1/Ms)), but not to hen egg white lysozyme (c-type) or Venerupis phillipinarum lysozyme (i-type). G-type lysozyme from goose eggs and from more remote taxonomic groups known to produce g-type lysozymes (urochordates and molluscs) are also inhibited by PliG.With the discovery of PliG, at least one inhibitor family against each lysozyme type in the animal kingdom is now known, and PliG was used together with a lysozyme inhibitor of c-type lysozyme (Ivy) and i-type lysozyme (PliI) to develop a novel toolbox for lysozyme profiling in animal body fluids or tissues such as hemolymph, saliva, egg white, . This toolbox was elaborated as a serial array of three affinity chromatography columns containing PliI, PliG and Ivy as an immobilized ligand. After successful validation, this array was used to profile the lysozyme content of blue mussel (Mytilus edulis) hemolymph, revealing the presence of active i-type and also g-type lysozyme, as well as an uncharacterized novel bacteriolytic protein. In a next part of the work, the structure-function relationship of PliG was investigated using site-directed mutagenesis combined with inhibition assays and protein-protein interaction analysis. Unexpectedly, these studies indicated that the conserved SGxY motif, which PliG shares with the c-type lysozyme inhibitor family MliC/PliC and the i-type inhibitor family PliI and which is crucial for the interaction of these inhibitors with c- and i-type lysozyme respectively, is not implicated in the interaction with g-type lysozyme. However, three other amino acids (Y47, R115 and R119), pinpointed as candidate interacting amino acids by the crystal structure of the PliG-SalG complex, were confirmed to be important for the inhibition capacity of PliG towards SalG by site directed mutagenesis.The biological function of PliG was analysed using E. coli strains in which the pliG gene was deleted and subsequently reintroduced at another genomic location for genetic complementation. Using these strains, it was established that PliG enhances survival of E. coli upon challenge with g-type lysozyme when the outer membrane is permeabilized by EDTA or by introducing a tolA mutation. PliG-mediated protection against g-type lysozyme in E. coli was subseqently confirmed in more natural settings such as goose egg white and mussel hemolymph, both of which contain g-type lysozyme. These results together with previous observations in our research group for c- and i-type lysozyme inhibitors support the notion that bacterial lysozyme inhibitors have evolved as virulence factors or colonization factors which provide protection against animal host lysozymes.To provide more direct evidence for such function, Avian Pathogenic E. coli (APEC) were used in two animal model systems, the chicken and the zebrafish. APEC, like all other E. coli, are known to produce two inhibitors against c-type lysozyme (Ivy and MliC) and one against g-type lysozyme (PliG). In the chicken model, the virulence of the mliC knock-out mutant was cleary attenuated and could be fully restored by genetic complementation, whereas knock-out of ivy or pliG had no effect on virulence. In the fish model, on the other hand, PliG was needed for full virulence of APEC, but not MliC or Ivy.In conclusion, this thesis represents an important advance of the existing insights on bacterial lysozyme inhibitors in several aspects. First, it strengthens the concept of lysozyme inhibitors as a unique group of proteins in Gram-negative bacteria by describing for the first time a lysozyme inhibitor targeting specifically g-type lysozymes. Second, using some model systems, it provides experimental support for a role of lysozyme inhibitors in the bacterial colonization of animal hosts producing lysozyme and in pathogenesis. As such, this work establishes bacterial lysozyme inhibitors as a novel type of determinants of bacteria-host interactions, and it opens an interesting perspective on new avenues of research and on potential applications in this field.nrpages: 193status: publishe