Ramírez-Gómez F et al. (2009), LPS-induced genes in intestinal tissue of the s...

ECB-ART-41207

PLoS One 2009 Jul 08;47:e6178. doi: 10.1371/journal.pone.0006178.

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LPS-induced genes in intestinal tissue of the sea cucumber Holothuria glaberrima.

Ramírez-Gómez F , Ortiz-Pineda PA , Rivera-Cardona G , García-Arrarás JE .

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Metazoan immunity is mainly associated with specialized cells that are directly involved with the immune response. Nevertheless, both in vertebrates and invertebrates other organs might respond to immune activation and participate either directly or indirectly in the ongoing immune process. However, most of what is known about invertebrate immunity has been restricted to immune effector cells and little information is available on the immune responses of other tissues or organs. We now focus on the immune reactions of the intestinal tissue of an echinoderm. Our study employs a non-conventional model, the echinoderm Holothuria glaberrima, to identify intestinal molecules expressed after an immune challenge presented by an intra-coelomic injection of lipopolysaccharides (LPS). The expression profiles of intestinal genes expressed differentially between LPS-injected animals and control sea water-injected animals were determined using a custom-made Agilent microarray with 7209 sea cucumber intestinal ESTs. Fifty (50) unique sequences were found to be differentially expressed in the intestine of LPS-treated sea cucumbers. Seven (7) of these sequences represented homologues of known proteins, while the remaining (43) had no significant similarity with any protein, EST or RNA database. The known sequences corresponded to cytoskeletal proteins (Actin and alpha-actinin), metabolic enzymes (GAPDH, Ahcy and Gnmt), metal ion transport/metabolism (major yolk protein) and defense/recognition (fibrinogen-like protein). The expression pattern of 11 genes was validated using semi-quantitative RT-PCR. Nine of these corroborated the microarray results and the remaining two showed a similar trend but without statistical significance. Our results show some of the molecular events by which the holothurian intestine responds to an immune challenge and provide important information to the study of the evolution of the immune response.

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Species referenced: Echinodermata
Genes referenced: a2m ahcy cd109 LOC100887844 LOC100893217 LOC100893907 LOC100894118 LOC579000 LOC590297 LOC590371 LOC592971 mtf1

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	Figure 2. Sequence alignment of the metabolic genes found in the array.Weblogo is also shown to denote residue conservation levels. A. Alignment of Ahcys from H. glaberrima, Bos Taurus (77735583), Aedes aegypti (157118541) Sus scrofa (AJ427478.2) and Xenopus tropicalis (113205576). Numerals represent the active site of the enzyme; plus signs represent NAD+ binding sites. The AdoHcyase domain (cl09931) is enclosed between black arrowheads. B. Alignment of Gnmts from H. glaberrima, Homo sapiens (NM_018960.4), Mus musculus (15679953), Sus scrofa (NM_001110419.1) and Danio rerio (NM_212816.1). Numerals marks AdoMet binding sites. Black arrowheads enclose the AdoMet_MTases superfamily domain (cd02440). C. Alignment of GAPDHs from H. glaberrima, Homo sapiens (NM_002046.3), Gallus gallus (NM_204305.1), Mus musculus (NM_008084.2), and Danio rerio (NM_001115114.1). The black arrowhead represents the ending of the C-terminal domain of GAPDH (pfam02800).
	Figure 3. Multiple sequence alignment of Aplha Actinins from H. glaberrima, Homo sapiens (CH471098.1), Mus musculus (AY036877.1), Gallus gallus (NM_205323.1) and Danio rerio (NM_001037573.1).Enclosed in black arrowheads is the first calponin homology domain (cd00014) of the protein. Numerals denote putative actin binding sites.
	Figure 4. Semiquantitative relative RT-PCR validation of 11 selected sequences.For each sequence a gel image shows the PCR amplification of the gene and the control NADH (second lower band), except for Hg_Act1, where the actin band is lower than that of NADH. Each lane represents RT-PCR products from RNA pooled from three different animals. Graph bars indicate the averaged OD ratios between each gene and NADH for three different experiments (each with a different pool of animals). Lines represent standard deviation. Asterisks represent t-test significance (P<0.05, *P<0.01).
	Figure 5. Semiquantitative relative RT-PCR of 6 immune associated genes.For each gene a representative gel image is shown, each lane represents RNA from a single individual. Graph bars indicate the averaged OD ratios between each gene and NADH for three different experiments (with three different animals). Lines represent standard deviation. Asterisks represent t-test significance (**P<0.01). Mtf1: Melanotransferrin 1 (accession number GQ243222); A2M: alpha-2-macroglobulin-like; Kaz1: Kazal-type serine proteinase inhibitor 1; Cath: Cathepsin; Ft: Ferritin (acc. No. EU010238).

References [+] :

Adams, Immunology of the gut and liver: a love/hate relationship. 2008, Pubmed