Mechanisms of intrinsic resistance to antimicrobial peptides of Edwardsiella ictaluri and its influence on fish gut inflammation and virulence

Abstract

The genus Edwardsiella comprises a genetically distinct taxon related to other members of the family Enterobacteriaceae. It consists of bacteria differing strongly in their biochemical and physiological features, natural habitats, and pathogenic properties. Intrinsic resistance to cationic antimicrobial peptides (CAMPs) is a specific property of the genus Edwardsiella. In particular, Edwardsiella ictaluri, an important pathogen of the catfish (Ictalurus punctatus) aquaculture and the causative agent of a fatal systemic infection, is highly resistant to CAMPs. E. ictaluri mechanisms of resistance to CAMPs are unknown. We hypothesized that E. ictaluri lipopolysaccharide (LPS) plays a role in both virulence and resistance to CAMPs. The putative genes related to LPS oligo-polysaccharide (O-PS) synthesis were in-frame deleted. Individual deletions of wibT, gne and ugd eliminated synthesis of the O-PS, causing auto-agglutination, rough colonies, biofilm-like formation and motility defects. Deletion of ugd, the gene that encodes the UDP-glucose dehydrogenase enzyme responsible for synthesis of UDP-glucuronic acid, causes sensitivity to CAMPs, indicating that UDP-glucuronic acid and its derivatives are related to CAMP intrinsic resistance. E. ictaluri OP-S mutants showed different levels of attenuation, colonization of lymphoid tissues and immune protection in zebrafish (Danio rerio) and catfish. Orally inoculated catfish with O-PS mutant strains presented different degrees of gut inflammation and colonization of lymphoid tissues. Here we conclude that intrinsic resistance to CAMPs is mediated by Ugd enzyme, which has a pleiotropic effect in E. ictaluri influencing LPS synthesis, motility, agglutination, fish gut inflammation and virulence.

Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut

“…E. ictaluri LPS O-PS mutants exhibited different levels of virulence, tissue colonization, and intestinal gut inflammation in orally inoculated catfish (18). Indeed, the E. ictaluri wild type causes diarrhea-like symptoms in orally infected fish, in contrast to E. ictaluri LPS mutant strains (18). This observation correlates with the current idea that fish recognize the O-PS of the LPS instead of the lipid A (19) and that fish recognize LPS at the intestinal level.…”

“…The E. ictaluri typical O-chain was found to be an unbranched linear polymer of a repeating tetrasaccharide unit composed of D-glucose, 2-acetamido-2-deoxy-D-galactose, and D-galactose in a 1:2:1 ratio having the (41). The E. ictaluri O-PS biosynthesis enzymes are encoded by four genes, wibT, galF, gne, and ugd, located in the O-PS gene cluster (17,18). As mentioned previously, we determined that catfish orally inoculated with the E. ictaluri wild type developed diarrhea-like symptoms, in contrast to fish inoculated with LPS-defective (⌬wibT, ⌬gne, and ⌬ugd) mutants (18).…”

“…LPS is an important virulence factor for E. ictaluri (16)(17)(18). The E. ictaluri LPS gene cluster has been identified by transposon mutagenesis (16,17) and was recently fully described (18).…”

“…The E. ictaluri LPS gene cluster has been identified by transposon mutagenesis (16,17) and was recently fully described (18). E. ictaluri LPS O-PS mutants exhibited different levels of virulence, tissue colonization, and intestinal gut inflammation in orally inoculated catfish (18).…”

“…The E. ictaluri O-PS biosynthesis enzymes are encoded by four genes, wibT, galF, gne, and ugd, located in the O-PS gene cluster (17,18). As mentioned previously, we determined that catfish orally inoculated with the E. ictaluri wild type developed diarrhea-like symptoms, in contrast to fish inoculated with LPS-defective (⌬wibT, ⌬gne, and ⌬ugd) mutants (18). Intestinal diseases often lead to disruption of the intestinal epithelial barrier either through attachment and internalization-mediated effector molecule release or through stimulation of host inflammatory responses that ultimately compromise junctional integrity (42).…”