Role of lysozyme inhibitors in the virulence of avian pathogenic Escherichia coli

Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors

A New Family of Lysozyme Inhibitors Contributing to Lysozyme Tolerance in Gram-Negative Bacteria

Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host

Structural and functional analysis of bacterial lysozyme inhibitors

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

Are bacterial lysozyme inhibitors important in bacteria-host interactions?

status: publishe

Structural characterization of the PliG lysozyme inhibitor family

Several Gram-negative bacteria protect themselves against the lytic action of host lysozymes by producing specific proteinaceous inhibitors. So far, four different families of lysozyme inhibitors have been identified including Ivy (Inhibitor of vertebrate lysozyme), MliC/PliC (Membrane associated/periplasmic inhibitor of C-type lysozyme), PliI and PliG (periplasmic inhibitors of I- and G-type lysozymes, respectively). Here we provide the first crystallographic description of the PliG family. Crystal structures were obtained for the PliG homologues from Escherichia coli, Salmonella enterica serotype Typhimurium and Aeromonas hydrophila. These structures show that the fold of the PliG family is very distinct from that of all other families of lysozyme inhibitors. Small-angle X-ray scattering studies reveal that PliG is monomeric in solution as opposed to the dimeric PliC and PliI. The PliG family shares a highly conserved SG(x)xY sequence motif with the MliC/PliC and PliI families where it was shown to reside on a loop that blocks the active site of lysozyme leading to inhibition. Surprisingly, we found that in PliG this motif is not well exposed and not involved in the inhibitory action. Instead, we could identify a distinct cluster of surface residues that are conserved across the PliG family and are essential for efficient G-type lysozyme inhibition, as evidenced by mutagenesis studies.status: publishe

Structure based discovery of small molecule suppressors targeting bacterial lysozyme inhibitors

The production of lysozyme inhibitors, competitively binding to the lysozyme active site, is a bacterial strategy to prevent the lytic activity of host lysozymes. Therefore, suppression of the lysozyme-inhibitor interaction is an interesting new approach for drug development since restoration of the bacterial lysozyme sensitivity will support bacterial clearance from the infected sites. Using molecular modelling techniques the interaction of the Salmonella PliC inhibitor with c-type lysozyme was studied and a protein-protein interaction based pharmacophore model was created. This model was used as a query to identify molecules, with potential affinity for the target, and subsequently, these molecules were filtered using molecular docking. The retained molecules were validated as suppressors of lysozyme inhibitory proteins using in vitro experiments revealing four active molecules.status: publishe

Strains and plasmids.

<p>Strains and plasmids.</p

Mortality curves of 1-day old chickens upon subcutaneous infection with APEC strains.

<p>Number of surviving animals up to 7 days post infection with APEC CH2 (•), APEC CH2 pACYC177 (empty plasmid control) (○), APEC inhibitor knock-out (▪) and the corresponding complemented APEC inhibitor knock-out strain (□). Time points where the number of survivors with the inhibitor knock-out was significantly different from that with the wild-type are marked with ‘*’ and the corresponding p-value.</p

Serum resistance of different APEC strains.

a<p> <i>Relative growth is the increase in plate count (N_{3 h}/N_{0 h}) in serum expressed relative to the increase in plate count in heat-inactivated serum ( = 100%). N_{3 h}/N_{0 h} ranged between 240 and 347 in heat-inactivated serum. Mean values+standard deviations for three independent cultures are shown. Significant differences (p<0.05) with the wildtype APEC CH2 strain are indicated with an asterisk.</i></p