Knockouts of Sulfur Metabolism Genes Induce Chronic Inflammation and Immune Dysregulation in Drosophila melanogaster

Hydrogen sulfide (H2S) is a vital gasotransmitter essential for maintaining redox homeostasis and modulating inflammatory responses. Herein, we examined a collection of Drosophila melanogaster knockout (KO) lines generated in our laboratory, lacking key genes involved in transsulfuration (cbs, cse) and sulfide metabolism (tst1), to elucidate the role of this adaptive system in immunity. Genetic ablation of these pathways results in profound H2S deficiency and hyperhomocysteinemia, leading to chronic oxidative stress. This leads to constitutive activation of major immune pathways—including IMD, Toll, and JAK-STAT—even in the absence of infection, a hallmark of chronic inflammation. Indeed, transcriptomic analysis and qRT-PCR studies revealed significant upregulation of these pathways in double (cbs; cse) and triple (cbs; cse; tst1) KO flies under control conditions. Following septic injury with Bacillus subtilis, double and triple KO flies exhibited increased expression of antimicrobial peptides (AMPs) and pattern recognition receptors compared to control and TST1-/- single KO lines. Notably, double and triple KO lines showed more prolonged immune activation. The triple KO flies exhibited the worst survival rate following septic injury. These results demonstrate that H2S deficiency causes chronic inflammation through the sustained activation of immune pathways, highlighting sulfur metabolism as a crucial regulator of homeostasis and innate immunity in Drosophila.