Molecular and functional characterization of ILYS-5, a major invertebrate lysozyme of Caenorhabditis elegans.

To overcome bacterial invasion and infection, animals have evolved various antimicrobial effectors such as antimicrobial peptides and lysozymes. Although C. elegans is exposed to a variety of microbes due to its bacterivorous lifestyle, previous work on the components of its immune system mainly based on the description of transcriptional changes during bacterial challenges. Very few effector components of its immune system have been characterized so far. To investigate the role of lysozymes in terms of antibacterial defense and digestion, we studied a member of the widely neglected family of C. elegans invertebrate lysozymes (ILYS). We focused on the so far virtually undescribed ILYS-5, which we purified from protein extracts of C. elegans tracing its peptidoglycan-degrading activity and localized the tissue expression of the gene in vivo using a translational reporter construct. We recombinantly synthesized ILYS-5 and determined the physicochemical activity optimum and the antibacterial spectrum of a lysozyme from C. elegans for the first time. With an activity optimum at low ionic strength (≤100 mM) and at acidic pH (≤ pH 4.0), ILYS-5 is likely to be involved in killing and digestion of bacteria within acidified phagolysosomes and acidic regions of the gut, presumably secreted by lysosome-like vesicles. This notion is supported by potent activity against various live Gram-positive and Gram-negative bacteria. Notably, members of the natural associated microbiome of C. elegans are substantially less susceptible to ILYS-5. Ablation of the ilys-5 gene resulted in reduction of lifespan and fertility when cultured on the standard food bacterium Escherichia coli OP50, whereas exposure of the ilys-5 knock-out mutant to the host-associated bacterium Pseudomonas lurida MYb11 did not have a clear effect. These findings indicate a role of ILYS-5 in immunity and nutrition and a co-evolved adaptation of host and bacteria to the mutualistic nature of their interaction.

PPARγ-dependent peptidoglycan recognition protein 3 (PGlyRP3) expression regulates proinflammatory cytokines by microbial and dietary fatty acids.

PGlyRPs recognize bacterial peptidoglycan and function in antibacterial innate immunity. Focusing on the interference between nutrition and recognition pattern proteins, free fatty acids (FFA) of dietary and bacterial sources may exert their immunological response through modulating the expression level of the PGlyRPs in enterocytes. PGlyRP3 was the only PGlyRPs member expressed in Caco2 cells. In silico analysis showed that the promoter of PGlyRP3 has some PPRE regions that, as tested by EMSA, bind physically to the PPARγ-RXRα complex. PGlyRP3 gene expression was induced by PPARγ ligands including GW1929 and some FFA. Overexpression of PGlyRP3 in Caco2 cells down regulated the expression of the inflammatory cytokines IL-8, IL-12 and TNF-α, while its silencing increased the expression of these cytokines. FFA that induced the PGlyRP3 inhibited the tested cytokines. Silencing of PGlyRP3 gene caused the same FFA to increase the cytokine gene expression. A negative regulation of NF-κB pathway, including up-regulation of Iκß-α and down regulation of NF-κB and COX-2, is involved in the anti-inflammatory effects of PGlyRP3. In conclusion, PPARγ mediates a modulation of PGlyRP3 gene expression, which is involved in inhibiting inflammation through negative regulation of NF-κB pathway.