DISCOVERING 3-HYDROXYANTHRANILIC ACID'S ROLE IN C. ELEGANS IMMUNITY

Abstract

Investigating our prior characterization of C. elegans haao-1 knockdown, which yielded a significant phenotypic lifespan extension, we performed follow-up analyses geared towards identifying the mechanism behind lifespan extension and regulation of 3-hydroxyanthranilic acid (3HAA). We observed through microscopy that the accumulation of 3HAA in the gut region may provide an interface of interaction between bacteria and 3HAA, potentially implying an intermediate immune role or an antibacterial property that extends lifespan. The lifespan of RNAi knockdown screens with innate immune genes in C. elegans (daf-16, dbl-1, pmk-1) suggests that 3HAA to mediate its effect independently of previously identified primary innate immune response pathways. 3HAA metabolism occurs in the tryptophan-kynurenine pathway where haao-1 is a gene that codes for an enzyme responsible for catalyzing the conversion of 3HAA into α-Amino-β-carboxymuconate-ε-semialdehyde and kynu-1 is a gene that codes for an enzyme responsible for catalyzing the conversion of 3-hydroxykynurenine (3HK) into 3HAA. Lifespan experiments between wildtype (WT) worms and our haao-1 knockout worms on non-pathogenic (Escherichia coli OP50) vs. pathogenic bacteria (Pseudomonas aeruginosa PA14) retained the extended lifespan in comparison to WT, showing that this lifespan extension may be mediated by nonspecific mechanisms and that beneficial effects are retained in more severe pathogenic challenge. We further explored the interface between microbial interaction with 3HAA using our Systematic Imaging of C. elegans Killing Organisms (SICKO) pipeline, which allows us to quantify colonization/infection area in the worm intestine using fluorescently labeled bacteria diet and fluorescent microscopy. SICKO showed a reduction in overall infection area and onset of infection in haao-1 relative to WT. Coupling this information with localization of KYNU-1 and HAAO-1 fused to fluorescent proteins implies a tissue separated model of metabolism where 3HAA is not just an intermediate product in the production of NAD, but also plays a role some pro-immune role separate from presently identified mechanisms. It is implied that 3HAA itself is antimicrobial and may have potential as an anti-microbial small molecule drug.