Taxonomic distribution and molecular phylogeny of IRG1, the enzyme producing the innate immune metabolite itaconate.

(A) Itaconate biosynthesis and degradation pathways in nature. Two itaconate biosynthesis pathways produce itaconate from the TCA cycle intermediate cis-aconitate, through either the cis-aconitate decarboxylase (CAD) enzyme IRG1/ACOD1 or the trans-aconitate decarboxylase (TAD) enzyme. Itaconate degradation involves a dedicated citramalyl-CoA lyase CLYBL to generate pyruvate and acetyl-CoA. (B) Taxonomic distribution of IRG1 in eukaryotes. IRG1 is present in Fungi, Lophotrochozoans, and Deuterostomes but is absent in other metazoans. (C) A phylogenetic tree of IRG1 homologs in nature. Bacterial sequences are colored in black, archaeal in green and fungal in orange. High confidence bootstrapping values are shown for the main branches separating the metazoan and A.tereus CAD branches. Branches containing experimentally validated 2-methylcitrate dehydratases are labeled. In the Metazoan branch, sequences were colored based on three lineages, IRG1/ACOD1 (red), IRG1L (IRG1-like, cyan) and Paleo-IRG1 (pIRG1, black). Metazoan proteins that are subsequently studied here are labeled, C. virginica (eastern oyster) pIRG1, B. floridae (lancelet) pIRG1 and D.rerio (zebrafish) IRG1L, in comparison to D.rerio (zebrafish) IRG1/ACOD1 and H.sapiens (human) IRG1/ACOD1.

Phylogeny and synteny for representative metazoan IRG1 sequences.

The tree branches are color-coded for the three lineages including paleo-IRG1 (p IRG1, black), IRG1-like (IRG1L, cyan) and IRG1 (red). The presence of the predicted Mitochondrial Targeting Sequence (MTS) is highlighted by the red dots. Homologous genes in the genomic vicinity spanning the IRG1, IRG1L and IRG1 are color-coded, for which the homologs present in human were annotated by human gene names, or otherwise by Pfam protein domain name. The two copies of pIRG1 in C. virginica are separated by approximately 200 kb, indicated by a dashed line in the syntenic block. Images for experimentally examined species in this report are shown in black, including easter oyster (C. virginica), amphioxus (B. floridae), and zebrafish (D. rerio) IRG1L.

Mitochondrial matrix localization of zebrafish IRG1L.

Limited proteolysis of mitochondria from HEK293T cells expressing zebrafish IRG1L (A) and human IRG1 with engineered mitochondrial targeting sequence (MTS) (B). C, diagram summarizing the representative submitochondrial localization of paleo-IRG1, IRG1L, IRG1/ACOD1 and engineered MTS-ACOD1.

Zebrafish IRG1L is a cis-aconitate decarboxylase producing itaconate.

(A) Recombinant His-tagged zebrafish IRG1L was expressed from E. coli and purified by affinity purification. (B-C) LC-MS analysis confirms itaconate production from cis-aconitate (1mM) by zebrafish IRG1L in a time-, enzyme-dependent manner.

The expression of paleo-IRG1 in eastern oysters is stimulated by Poly(I:C).

(A) oyster anatomy. (B) Quantitative PCR showing unstimulated oyster (n = 5) pIRG1 tissue expression (PHemocytes/other tissue < 0.0001). (C) The pIRG1 expression in the oyster hemocytes is induced Poly(I:C) at 8 hrs shown by qPCR. (D) The time course of pIRG1 expression induction in the oyster hemocytes by Poly(I:C) (10 ug/ml) stimulation. Data are shown as mean ± s.d.

The paleo-IRG1 in amphioxus is implicated in innate immunity.

(A) amphioxus anatomy. (B) Quantitative PCR from amphioxus tissues showing that pIRG1 is barely expressed under unstimulated condition (n =3), but can be strongly induced by both LPS (50 ug/mL; n =3) and Poly (I:C) (1 mg/mL; n = 3) upon oral injection for 24 hrs. Each data point represents tissue from one animal shown as mean ± s.d. The large deviance is likely due to the low and variable mRNA level under basal condition.

Phylogenetic tree and taxonomic distribution of pIRG1/IRG1L/IRG1 sequences in metazoa.

(A) A phylogenetic tree of all metazoan IRG1 sequences. Tree branches were color-coded by three lineages: pIRG1 (black), IRG1L (cyan) and IRG1 (red). Branches with bootstrapping values higher than 70 are highlighted by solid line. Taxa are color-coded on the outer rim of the tree. The sequences with predicted Mitochondrial Targeting Sequence (MTS) were highlighted by red dots. (B) The presence of IRG1 and IRG1L in major mammalian taxa.

The pIRG1 is induced in the eastern oyster hemocytes by innate immune stimuli.

Quantitative PCR showing the IRG1 mRNA level in hemocytes upon 8 hr stimulation of heat-killed E. coli 0111:B4 (HKEB) (1x107 PFU), heat-killed S. aureus (HKSA) (1x107 PFU), zymosan (100 µg/mL) and Poly (I:C) (10 µg/mL). IRG1 is significantly upregulated upon Poly(I:C) stimulation relative to other conditions.