Tissue expression profile and transcriptional responses of abalone defensin after bacteria challenge by qRT-PCR

To examine whether defensin mRNA was constitutively expressed as in other mollusks, qRT-PCR was used to analyze 108 bp fragment of abalone defensin. The transcripts of abalone defensin were detected in all the tissues examined including hemocytes, gills, mantle, muscle, digestive tract and hepatopancrease (fig. 34A). The highest constitutive expression (8.5-fold) of abalone defensin was detected in mantle compared to that of hemocytes. Defensin mRNA expression was significantly (p<0.05) higher in mantle and hepatopancrease than other analyzed tissues. After healthy abalones were experimentally challenged with bacteria, (V.

alginolyticus, V. parahemolyticus and L. monocytogenes) transcriptional response of defensin was determined in hemocytes, gills and digestive tract by quantitative real time RT-PCR (fig.

34 B,C,D). Transcriptional level of defensin was up-regulated significantly (p<0.05) in hemocytes during 48 h p.i. (except at 12 h) of bacteria mixture. The highest defensin induction-fold (3.8) was detected in hemocyte at 24 h p. i. compared to PBS control at 3 h. In hemocytes, defensin transcriptional up-regulation was higher at 24 and 48 h p.i than early stage. Neither the gill nor the digestive tract was induced defensin transcripts significantly at early stage (before 24 h) of p.i. In gills defensin transcripts were significantly induced (1.4-fold) only at 24 h while digestive tract showed the late phase response with highest expression fold (2.3-fold) at 48 h p.i. of bacteria. Even though, the transcriptional response was different in hemocytes, gills and digestive tract, present results have shown that abalone defensin expression could be induced by bacteria infection.

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Fig. 34. Tissue expression profile and transcriptional responses of abalone defensin after bacteria challenge by qRT-PCR. The relative defensin expression fold was calculated by the 2^-∆∆CT method using abalone ribosomal protein as a reference gene. Relative expression fold of each tissue was compared to that of hemocyte to determine the tissue specific expression.

A) tissue expression analysis; B) gills; C) digestive tract; D) hemocytes; Data are presented as mean relative expression ± standard deviation (SD) for three replicate real-time reactions from pooled tissue of three individual abalones at each time point. The bars represent the standard deviation (n=3). Gl-gill; Mn-mantle; Ms-muscle; Dg-digestive tract; Hp-hepatopancreas; Hm-hemocyte; cont-control (PBS).

4.3.1.4. Discussion

In the present study, we report the identification and molecular characterization of a defensin cDNA from disk abalone. The defensin family peptides share several characteristic features of small cationic AMPs, including molecular mass below 10 kDa, net positive (+) charge, high hydrophobic ratio, amphipathic structure (α helix, β sheet or α helix/ β sheet structure) and three to four disulfide bonds that all collectively to be important for their antibacterial action (Froy et al., 2003; Bulet et al., 2004). In agreement with the general features of AMPs, the abalone defensin amino acid sequence displays a typical distribution of positive charge (+5), hydrophobic residue ratio (46%), lower molecular mass (4.9 kDa) and α helical structure. It was described (Froy et al., 2003) that, invertebrate defensins consist of six cysteine residues in a pattern of C-X5-16-C-X3-C-X9-10-C-X4-7-C-X1 (Xio et a;., 2004) and which are formed disulfide linkages of C1-C4, C2-C5 and C3-C6.We found that six cysteine residues presented in abalone defensin are arranged in the pattern of C-X16-C-X3-C-X9-C-X4 -C-X1 which is in agreement with the consensus invertebrate defensin. Additionally, abalone defensins shares the classical disulfide linkage structure (C1-C4, C2-C5, C3-C6) similar to invertebrate defensins. However, the number of cysteine residue presented in abalone defensin is in contrast to that of mussel defensins (MGD1 and MGD2), oyster defensin (CgDef) due to two additional cysteine residues present in those peptide sequences. Froy and Gurevits (2003) showed that high degree of identity among mature defensins from mollusk and arthropods. Multiple and pairwise sequence analysis results from present study also reveals that abalone defensin shares higher homology to arthropod defensin family than that of mollusks. The presence of invertebrate defensin family domain, sequence similarity, phylogenetic relationship and other common features suggest that abalone defensin could be a new member of invertebrate defensin family and closely related to arthropod defensins.

Several reports described that AMPs are expressed either constitutively or induced upon pathogenic challenge (Iwanaga et al., 1998; Imler et al., 2000). In lower order vertebrate zebrafish, several β-defensin homologues were constitutively expressed in gills, gonad, gut, kidney, muscle, skin and spleen (Zou et al., 2007). In Pacific oyster, the expression of Cg-defh1 is considerably low while Cg-defh2 is constitutively expressed in gills and mantle than the hemocytes (Gonzales ey al., 2004). The highest expression of Pacific oyster Cg-Def has been shown in mantle at a higher mRNA level (20-60-fold). In our results also mantle appears as the prominent tissue for expressing defensin in non-stimulated abalones. Additionally,

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digestive tract and hepatopancreas and it is more similar to Cg-Def expression in gills, hemocytes, muscle. The expression results of abalone defensin and Cg-Def are in contrast with those reported in mussel (M. galloprovencialis) defensin, which is only produced in hemocytes (Mitta et al., 1999). Selsted and Ouellet (Selsted et al., 2005) have been discussed that in early life forms, defensins and other AMP provided simple but efficient mechanisms for host resistance to microbial infection and during the gene diversification organism retained specific genes, which their products have additional advantages to the host. In general, expression of the genes encoding AMPs is induced by bacterial challenge (Hoffmann et al., 1996). Up-regulation of defensin transcripts were detected in scallop Argopecten irradians hemolymph (Zhao et al., 2007), C. gigas (Cg-defh2) in gills and mantle (Gonzales et al., 2007), M. galloprovincialis gills and mantle (Hubert et al., 1996), C. verginica gills (Seo et al., 2004) by bacterial challenge. For an example, expression of the scallop defensin transcripts in hemolymph has been gradually increased during 4-32 h post challenge of Vibrio anguilarum. Eventhough the induced level (fold) and the time points are not perfectly matched; these results are consistent with the transcriptional induction of abalone defenisn in hemocytes, gill and digestive tract. This could be due to variation of infected bacterial dose, physiological status of animal, culturing conditions during the bacterial challenge and level of constitutive expression before the bacterial challenge. There are few observations on either decrease or no change of transcriptional responses of mollusk defensins against bacterial infection. For an example, C. gigas Cg-defh2 (hemocyte) was significantly decreased after bacterial challenge (Gonzales et al., 2007), while C. gigas mantle Cg-Def transcript level was unaffected by bacteria challenge (Gueguen et al., 2006). Similar to those at some extent abalone defensin transcription level has been decreased in hemocytes at 12 h p.i. This may be due to mobilization effect of hemocytes towards the bacterial colonization or infected site.

Present study results indicate that transcription of abalone defensin is un-changed at early stages in gills and digestive tract after bacterial infection. This preliminary result would suggest that transcriptional change of abalone defensin in gills and digestive tract is not highly responded to bacterial mixture used in this study. Hence, more detailed study of abalone defensin gene expression with response to various microbial challenges is required to understand the tissue specific differences of transcriptional regulation. The constitutive and ubiquitous expression of abalone defensin in hemocytes as well as other tissues with transcriptional up-regulation against bacterial infection would suggest that it has an important immune function in host defense against pathogens.