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

Scheme of three-step PCR to prepare DNA fragments for chromosomal gene replacement.

<p>In a first step, an antibiotic resistance cassette is amplified using primers carrying 5′ end 50 bp extensions homologous to the upstream (primer 2) and downstream (primer 1) region of the target inhibitor gene in APEC. The resulting PCR product is then used in a second step in combination with two other primers (primer 3 and primer 4) to separately amplify a larger part of the downstream and the upstream regions of the target gene. This results in two products which consist of the resistance marker cassette flanked by an upstream or a downstream 200 bp (or more) region homologous to the target gene. In a third step these two products are combined and amplified, resulting in a fusion product that has a large upstream and downstream homology region at either side of the resistance marker, and that is used for gene replacement.</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

Strains and plasmids.

<p>Strains and plasmids.</p

Goose-Type Lysozyme Inhibitor (PliG) Enhances Survival of Escherichia coli in Goose Egg Albumen ▿

The goose-type lysozyme inhibitor PliG enhances the survival of Escherichia coli in goose but not in chicken egg white, which contains goose- and chicken-type lysozymes, respectively. These results indicate that both the type of host lysozyme and the type of bacterial lysozyme inhibitor may affect bacterium-host interactions