Identifying components of the NF-kappaB pathway in the beneficial Euprymna scolopes-Vibrio fischeri light organ symbiosis

Abstract

The Toll/NF-kappaB pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of Euprymna scolopes was used to identify members of the Toll/NF-kappaB pathway. Full-length transcripts were identified by using 5' and 3' RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-kappaB phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-kappaB pathway in the initiation of the beneficial symbiosis between E. scolopes and Vibrio fischeri.

FIG. 1.

The signaling pathways activating the innate immune responses of plants and animals are similar. NF-κB p105 and Relish have a REL homology domain located at their N terminus (N) and an IκB-like domain at their C terminus. The C terminus domain can be proteolytically cleaved and degraded. Brackets enclose pathway members identified in the present study. The molecules listed in the figure represent full-length sequences. Ai, Argopecten irradians; At, Arabidopsis thaliana; C, C terminus; Cg, Crassostrea gigas; CpG DNA, unmethylated CG dinucleotides prevalent in bacterial DNA; d, drosophila; ECSIT, evolutionarily conserved signaling intermediate in Toll pathways; Es, Euprymna scolopes; IκB, inhibitor of NF-κB; IKK, IκB kinase; Imd, immune deficiency; IRAK, interleukin-1-receptor-associated kinase; ird, immune response deficient; LPS, lipopolysaccharide; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; MyD88, myeloid differentiation primary response gene 88; N, N terminus; ND*, not determined that MAMPs activate this pathway in this superphylum; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; NIK, NF-κB inducing kinase; NOD, nucleotide-binding oligomerization domain protein; o, oyster; PGN, peptidoglycan; PGRP, PGN recognition proteins; R gene, plant resistance genes; REL, proteins containing regions of homology to the rel oncogene, the definition of the NF-κB family of genes; ssRNA, single-stranded RNA; TAB, TAK-binding protein; TAK, transforming growth factor β-activated kinase 1; TLR, Toll-like receptor; Tollip, Toll interacting protein; TRAF, tumor necrosis factor receptor-associated factor.

FIG. 2.

(A) Comparison of Toll-like receptor proteins from E. scolopes, fruit fly, and human. Gray regions indicate the signal peptide, black diagonal shading indicates the LRR region, black regions indicate a transmembrane domain, and black horizontal shading indicates the TIR domain. The number indicates the size of the derived amino acid sequence. Accession numbers are AY956809, NP_524757, and NP_003256, respectively. (B) Unrooted phylogenetic tree produced by the neighbor-joining method, using Kimura two-parameter distances between Toll-like protein sequences of the organisms named on the tree. Gapped positions were included in the analysis. Symbols on the branches represent percentage bootstrap values: +, 90 to 100%; ⋄, 80 to 89%; ⧫, 70 to 79%; ▵, 60 to 69%. Accession numbers clockwise from E. scolopes are as follows: AY956809, XP_393712, AAF86226, AAL37902, NP_524757, AAL37904, XP_393713, BAB85498, AAF86225, NP_649719, NP_523519, NP_733166, AAF86227, NP_003255, NP_112218, NP_003254, NP_006059, NP_003259, NP_942086, NP_001008664, NP_003256, NP_059138, AAF78035, NP_057694, NP_612564, and AAK37544. Abbreviations: Ag, Anopheles gambiae (mosquito); Am, Apis mellifera (honeybee); Bm, Bombyx mori (silk moth); Bt, Bos taurus (cow); Ce, Caenorhabditis elegans (nematode); Dm, Drosophila melanogaster (fruit fly); Es, Euprymna scolopes (Hawaiian bobtail squid); Hs, Homo sapiens (human); Rn, Rattus norvegicus (rat).

FIG. 3.

(A) Comparison of PGRP proteins from E. scolopes, bay scallop, fruit fly, and human sources. Gray regions indicate the signal peptide, black regions indicate a transmembrane domain, and black diagonal shading indicate the PGRP domain. The number indicates the size of the derived amino acid sequence. Reading from left to right and down from the top, accession numbers are AY956811, AY956812, AY956813, AY956814, AAR92030, NP_572727, NP_731576, NP_005082, and AAL05629, respectively. (B) Unrooted phylogenetic tree produced by the neighbor-joining method, using Kimura two-parameter distances between PGRP protein sequences of the organisms named on the tree. Gapped positions were included in the analysis. Symbols on the branches represent percentage bootstrap values: +, 90 to 100%; ⋄, 80 to 89%; ⧫, 70 to 79%. Values below 70% are not shown. Accession numbers clockwise from E. scolopes PGRP1 are as follows: AY956811, AY956812, AAR92030, AAL05629, XP_395941, NP_731576, AAG32064, NP_005082, NP_729468, AAG23736, NP_788467, AAK00295, NP_572727, BAA77210, CAD89136, CAD89193, NP_443123, EAA10910, AY956814, and AY956813. (C) Alignment of the PGRP domains of the EsPGRPs and those of other organisms. Residues critical to PGRP function are highlighted. Accession numbers from top to bottom are as follows: AY956811, AY956812, AY956813, AY956814, NP_729468, NP_005082, NP_731576, NP_572727, and AAL05629. Abbreviations: Ag, Anopheles gambiae (mosquito); Ai, Argopecten irradians (bay scallop); Am, Apis mellifera (honeybee); Bm, Bombyx mori (silk moth); Dm, Drosophila melanogaster (fruit fly); Es, Euprymna scolopes (Hawaiian bobtail squid); Hs, Homo sapiens (human).

FIG. 4.

(A) Comparison of IRAK4-like proteins from E. scolopes, rat, fruit fly, and tomato. Black regions indicate the serine/threonine protein kinase domain, and gray regions indicate the alpha helices of a possible death domain. Number indicates size of the derived amino acid sequence. The accession numbers are AY956810, XP_217026, NP_476971, and T07416, respectively. (B) Unrooted phylogenetic tree produced by the neighbor-joining method, using Kimura two-parameter distances between IRAK4 protein sequences of the organisms named on the tree. Gapped positions were included in the analysis. The numbers on the branches are percent bootstrap values. Accession numbers clockwise from E. scolopes are as follows: AY956810, NP_057207, NP_084202, XP_217026, NP_502587, NP_476971, XP_311931, AAF76311, T07416, and AAK11568. Abbreviations: Ag, Anopheles gambiae (mosquito); Ce, Caenorhabditis elegans (nematode); Dm, Drosophila melanogaster (fuit fly); Es, Euprymna scolopes (Hawaiian bobtail squid); Hs, Homo sapiens (human); Le, Lycopersicon esculentum (tomato); Lh, Lycopersicon hirsutum (tomato); Mm, Mus musculus (mouse); Rn, Rattus norvegicus (rat).

FIG. 5.

(A) Comparison of TRAF6 proteins from E. scolopes, zebrafish, human, and fruit fly with characteristic domains indicated. Black hatched shading indicates the RING finger domain, gray regions indicate the TRAF-type zinc finger domains, and black regions indicate the TRAF homology domain. The number indicates size of the derived amino acid sequence. The accession numbers are AY956816, NP_956115, NP_004611, and AAD47895, respectively. (B) Unrooted phylogenetic tree produced by the neighbor-joining method, using Kimura two-parameter distances between TRAF protein sequences of the organisms named on the tree. Gapped positions were included in the analysis. Numbers on the branches are percentage bootstrap values. Accession numbers clockwise from E. scolopes are as follows: AY956816, NP_956115, NP_004611, P70196, XP_230377, XP_311879, AAD34345, AAD47895, AAD34346, NP_991325, NP_004286, NP_033449, XP_220640, CAE88928, CAC82653, CAD69021, NP_066961, XP_231032, NP_989550, NP_004610, NP_035763, CAD57164, NP_035762, and Q13114. Abbreviations: Ag, Anopheles gambiae (mosquito); Ca, Carassius auratus (goldfish); Dm, Drosophila melanogaster (fruit fly); Dr, Danio rerio (zebrafish); Es, Euprymna scolopes (Hawaiian bobtail squid); Gg, Gallus gallus (chicken); He, Hydractinia echinata (hydrozoa); Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Om, Oncorhynchus mykiss (rainbow trout); Rn, Rattus norvegicus (rat).

FIG. 6.

(A) The characteristic domains of IKKγ exhibited by the derived amino acid sequence from E. scolopes. The underlined region represents a helical structure containing coiled coil regions (gray shading) and a leucine zipper motif with specific leucines (and hydrophobic isoleucine) marked with an asterisk. The C-terminal zinc finger motif is shaded in black with characteristic amino acids labeled with a “#” symbol. (B) Unrooted phylogenetic tree produced by the neighbor-joining method, using Kimura two-parameter distances between IKK protein sequences of the organisms named on the tree. Gapped positions were included in the analysis. Numbers on the branches are percentage bootstrap values. *, The original description does not indicate assignment to α, β, or γ classes of IKK. Accession numbers clockwise from E. scolopes are as follows: AY956817, NP_499831, AAG02485, AAC05683, NP_001269, AAH18243, NP_001547, NP_034676, AAG02486, NP_989567, AAH63986, NP_776779, NP_003630, and AAM54725. Abbreviations: Bt, Bos taurus (cow); Ce, Caernorhabditis elegans (nematode); Cg, Crassostrea gigas (Pacific oyster); Dm, Drosophila melanogaster (fruit fly); Dr, Danio rerio (zebrafish); Es, Euprymna scolopes (Hawaiian bobtail squid); Gg, Gallus gallus (chicken); Hs, Homo sapiens (human); Mm, Mus musculus (mouse).

FIG. 7.

(A) Comparison of IκB family molecules from E. scolopes, rat, and fruit fly. Black diagonal shading represents the N-terminal regulatory region, with the serines of the phosphorylation site and the lysines of the possible ubiquitin conjugation sites underlined and highlighted in boldface. The ankyrin repeat domains are shaded in gray, and the PEST-rich region is shaded in black. The number indicates the size of the derived amino-acid sequence. The accession numbers are AY956818, XP_343066, and NP_476942, respectively. (B) Unrooted phylogenetic tree produced by the neighbor-joining method, using Kimura two-parameter distances between IκB protein sequences of the organisms named on the tree. Gapped positions were included in the analysis. The numbers on the branches are percent bootstrap values. The accession numbers clockwise from E. scolopes are as follows: AY956818, CAC85086, Q91974, NP_035037, XP_343066, Q08353, CAD12786, O00221, NP_032716, AAB58262, NP_002494, NP_035038, and NP_476942. Abbreviations: At, Arabidopsis thaliana (thale cress); Bt, Bos taurus (cow); Dm, Drosophila melanogaster (fruit fly); Es, Euprymna scolopes (Hawaiian bobtail squid); Gg, Gallus gallus (chicken); Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Om, Oncorhynchus mykiss (rainbow trout); Rn, Rattus norvegicus (rat); Ss, Sus scrofa (pig).

FIG. 8.

(A) Comparison of REL family proteins from E. scolopes, oyster, human, and fruit fly. The RHD is indicated by gray shading and contains the following: the conserved residues of the DNA-binding motif (black horizontal shading) underlined and highlighted in boldface; the immunoglobulin-like plexin domain, found in all REL family molecules, indicated by black diagonal shading; and the nuclear localization signal indicated by solid black shading, with conserved residues underlined and highlighted in boldface. The unshaded region that is C-terminal to the RHD represents the transactivation domain. The number indicates size of the derived amino acid sequence. Accession numbers are AY956819, AAK72691, NP_068810, and NP_724052, respectively. (B) Unrooted phylogenetic tree produced by the neighbor-joining method, using Kimura two-parameter distances between REL protein sequences of the organisms named on the tree. Gapped positions were included in the analysis. Numbers on the branches are percentage bootstrap values. Accession numbers clockwise from E. scolopes are as follows: AY956819, AAK72690, AAK72691, Q04865, NP_068810, NP_033071, XP_238994, XP_223688, P16236, NP_033072, Q01201, BAB47172, BAB47173, XP_310177, AAP23055, AAG22858, NP_724052, and NP_724054. Abbreviations: Ag, Anopheles gambiae (mosquito); Am, Apis mellifera (honeybee); Cg, Crassostrea gigas (Pacific oyster); Dm, Drosophila melanogaster (fruit fly); Es, Euprymna scolopes (Hawaiian bobtail squid); Hr, Halocynthia roretzi (ascidian); Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Rn, Rattus norvegicus (rat); Tc, Tribolium castaneum (beetle); XI, Xenopus laevis (African clawed frog).

FIG. 9.

Putative signaling pathway of the evolutionarily conserved Toll/NF-κB pathway utilizing the E. scolopes transcripts identified in the present study. Black shading indicates identification of full-length transcripts, and gray shading indicates identification of partial length transcripts (see Tables 1 and 2). Dashed lines indicate a pathway in which not all of the signaling intermediates have been found in E. scolopes.