The gasotransmitter hydrogen sulfide (H₂S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H₂S production and metabolism. Here, we investigated the role of H₂S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation–reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

Background: Marinobufagenin, NKA (Na/K‐ATPase) inhibitor, causes vasoconstriction and induces fibrosis via inhibition of Fli1 (Friend leukemia integration‐1), a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP (atrial natriuretic peptide), via a cGMP/PKG1 (protein kinase G1)‐dependent mechanism, reduces NKA sensitivity to marinobufagenin. We hypothesized that VSMC from old rats, due to downregulation of ANP/cGMP/PKG‐dependent signaling, would exhibit heightened sensitivity to the profibrotic effect of marinobufagenin. Methods and Results: Cultured VSMC from the young (3‐month‐old) and old (24‐month‐old) male Sprague–Dawley rats and young VSMC with silenced PKG1 gene were treated with 1 nmol/L ANP, or with 1 nmol/L marinobufagenin, or with a combination of ANP and marinobufagenin. Collagen‐1, Fli1, and PKG1 levels were assessed by Western blotting analyses. Vascular PKG1 and Fli1 levels in the old rats were reduced compared with their young counterparts. ANP prevented inhibition of vascular NKA by marinobufagenin in young VSMC but not in old VSMC. In VSMC from the young rats, marinobufagenin induced downregulation of Fli1 and an increase in collagen‐1 level, whereas ANP blocked this effect. Silencing of the PKG1 gene in young VSMC resulted in a reduction in levels of PKG1 and Fli1; marinobufagenin additionally reduced Fli1 and increased collagen‐1 level, and ANP failed to oppose these marinobufagenin effects, similar to VSMC from the old rats with the age‐associated reduction in PKG1. Conclusions: Age‐associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose marinobufagenin‐induced inhibition of NKA and fibrosis development. Silencing of the PKG1 gene mimicked these effects of aging.

Here, we describe the effects of double knockout (KO) of the cbs and cse genes, which are responsible for H₂S synthesis through the transsulfuration pathway, and KO of the sulfurtransferase gene (dtst1) in Drosophila melanogaster females. The analysis of H₂S production in flies showed minimal levels in the double- and triple-knockout strains. The double- (cbs-/-; cse-/-) and triple- (cbs-/-; cse-/-; dtst-/-) KO flies exhibited a shortened lifespan and reduced fecundity, and showed dramatic changes in Malpighian tubule morphology. The transcriptomic analysis revealed a profound increase in the expression levels of several genes involved in excretory system function in the double-KO and especially the triple-KO flies. Importantly, major groups of differentially expressed genes (DEGs) in the whole bodies of females and ovaries of KO strains included genes responsible for detoxification, reproduction, mitochondrial activity, excretion, cell migration, and muscle system function. The reduced fecundity observed in the double- and triple-KO flies correlated with pronounced changes in the ovarian transcriptome. At the same time, the single knockout of dtst1 increased the flies’ fecundity and lifespan. Our experiments exploring unique Drosophila strains with KO of major H₂S-related genes revealed several new pathways controlled by this ancient adaptogenic system that is involved in various human diseases and aging.

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 lacking key transsulfuration (cbs, cse) and sulfide metabolism (tst1) genes, generated in our laboratory, 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 the immune pathways, highlighting sulfur metabolism as a crucial regulator of homeostasis and innate immunity in Drosophila.

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.

Here, we describe the effect of knockout (KO) of two key genes ( cbs , cse ) responsible for H ₂ S synthesis in the transsulfuration pathway as well as the inactivation of sulfurtransferase gene ( dtst1 ) encoding single domain rhodanese on Drosophila melanogaster genome expression and several life-history traits. The analysis of H ₂ S production in the KO flies showed its minimal level in the strain with double KO ( CBS-/-; CSE-/- ) while flies with triple KO ( CBS -/-; CSE -/-; dTST1 -/-) exhibited higher H ₂ S level and improvements in lifespan. The transcriptomic analysis of fly bodies from the double KO strain with maximum disturbances of fitness components revealed a profound increase in the expression of genes involved in the functioning of the excretory system. Besides, double and triple KO flies exhibited drastic changes in Malpighian tubules’ appearance. Inactivation of genes related to H ₂ S metabolism resulted in altered expression of numerous loci involved in mitochondrial function. While KO of the dtst1 gene did not affect the genome expression, the triple KO flies exhibited more changes in the whole-body transcriptome than double KO flies. Reduced fecundity in double knockouts correlates with pronounced changes in ovarian transcriptome data. Surprisingly, the knockout of the dtst1 gene affected the flies’ memory.