Identification of a bacterial inhibitor against g-type lysozyme

Abstract

Lysozymes are antibacterial effectors of the innate immune system in animals that hydrolyze peptidoglycan. Bacteria have evolved protective mechanisms that contribute to lysozyme tolerance such as the production of lysozyme inhibitors, but only inhibitors of chicken (c-) and invertebrate (i-) type lysozyme have been identified. We here report the discovery of a novel Escherichia coli inhibitor specific for goose (g-) type lysozymes, which we designate PliG (periplasmic lysozyme inhibitor of g-type lysozyme). Although it does not inhibit c- or i-type lysozymes, PliG shares a structural sequence motif with the previously described PliI and MliC/PliC lysozyme inhibitor families, suggesting a common ancestry and mode of action. Deletion of pliG increased the sensitivity of E. coli to g-type lysozyme. The existence of inhibitors against all major types of animal lysozyme and their contribution to lysozyme tolerance suggest that lysozyme inhibitors may play a role in bacterial interactions with animal hosts.

Edwardsiella tarda MliC, a Lysozyme Inhibitor That Participates in Pathogenesis in a Manner That Parallels Ivy

“…In documented studies of recombinant lysozyme inhibitors, the recombinant proteins were used at the dose of 10 M to 200 M for different analyses (13,17); in our study, the concentration of rMliC used (80 M) relatively high based on the consideration that the protein had undergone reconstitution. Previous studies showed that in the presence of outer membrane permeabilizers or antimicrobials that destabilize the outer membrane of Gram-negative bacteria, lysozymes can effectively kill Gram-negative bacteria (14,16,17,(33)(34)(35). In our study, we found that in the presence of diluted fish serum, which facilitates the action of lysozymes on Gram-negative bacteria, rMliC reduced the killing effect of HEWL The fish were monitored daily for mortality and survival.…”

“…Lysozyme-inhibitory activity of rMliC. Since lysozyme inhibitors in their natural forms are usually difficult to obtain, especially with relatively high purity, these proteins have been studied in the form of recombinant proteins (13,17,20,21,32). In our study, to examine the biological activity of MliC Et , rMliC was purified from E. coli as a Trx-tagged protein.…”

“…To date, inhibitors against all three major types of lysozymes have been identified in Gram-negative bacteria. These inhibitors are known as inhibitor of vertebrate lysozyme (Ivy) proteins, which target C-type lysozyme, periplasmic lysozyme inhibitor of C-type lysozyme (PliC), membrane-associated lysozyme inhibitor of Ctype lysozyme (MliC), periplasmic inhibitor of I-type lysozyme (PliI), and periplasmic inhibitor of G-type lysozyme (PliG) (13)(14)(15)(16)(17). Of these inhibitors, MliC/PliC have been found in various bacterial species, and high-resolution structures of the MliCs/ PliCs of Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, and Brucella abortus have been reported (18)(19)(20)(21).…”

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

“…In addition, knockout of ivy, the gene encoding one of the inhibitors of c-type lysozyme, slightly reduced the survival of Escherichia coli in chicken egg albumen, thus demonstrating a possible ecological significance for this inhibitor (7). Interestingly, we found recently that E. coli and some other Gram-negative bacteria like Salmonella also produce a g-typespecific lysozyme inhibitor (PliG) (22). Since these bacteria closely interact with vertebrate hosts, which can produce both c-and g-type lysozymes, the cooccurrence of c-and g-type inhibitors supports the hypothesis that they are involved in the host interaction of these bacteria.…”

“…Over the last 10 years, specific inhibitors of c-, g-, and i-type lysozymes have been discovered in Gram-negative bacteria (1,15,23,22) and in vitro experiments have shown that they protect the bacteria against the corresponding lysozymes when the outer membrane is permeated by lactoferrin or EDTA (1,6,23,22). In addition, knockout of ivy, the gene encoding one of the inhibitors of c-type lysozyme, slightly reduced the survival of Escherichia coli in chicken egg albumen, thus demonstrating a possible ecological significance for this inhibitor (7).…”