Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and several bacterial challenges in mussels

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Abstract

Defensin, mytilin and myticin are antimicrobial peptides (AMP) involved in mussel innate immunity. Their in vitro antibacterial activity is different according to the targeted bacterial species. To determine if this specificity is correlated to different regulations of gene expressions, adult mussels were challenged in vivo with either Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus or by heat shock. RNAs were isolated from circulating hemocytes and AMP mRNAs were quantified by Q-PCR using 28S rRNA as housekeeping gene. In addition, HSP70 gene expression was also quantified as representing non-specific response to stress. In naïve mussels, the three AMP mRNAs were present in dramatically different quantities. Compared to defensin, myticin was expressed 300-fold more and mytilin 30-fold more. HSP70 was found expressed 80-fold more than defensin. AMP genes were differentially regulated according to the challenging bacteria, M. lysodeikticus being the only one inducing down-regulation. Such variations in mRNA quantities were observed immediately after challenging, lasting less than 24 h. Only V. anguillarum effect was observed later, between 12 h and 3 days post-challenge. Compared to their background expression in naïve mussels, the major effect of V. splendidus was the decrease of mytilin and myticin mRNAs, V. anguillarum mainly increased both mytilin and HSP70 mRNAs, whereas M. lysodeikticus almost suppressed defensin mRNA. As expected, heat shock increased HSP70 mRNA, but also myticin mRNA. Consequently, AMP genes responded specifically to the challenges, confirming that at least some of the innate immune mechanisms are specifically orientated.

Introduction

Cationic antimicrobial peptides (AMP) constitute an important component of the innate immune defence. More than 800 sequences of different AMP are stored in database covering all phyla, from plants to animals, including bacteria. AMP are exceptionally diverse in sequence, structure and function, resulting from moderate positive selection on the ability to combat new or altered pathogens [1], [2]. AMP act mainly on Gram-negative bacteria by initially binding to the surface LPS, followed by self-promoted uptake across the outer membrane [3], [4] disrupting the surface membrane integrity, including on protozoa [5]. Meanwhile, it seems that at least some AMP also penetrate into eukaryotic cells without disrupting the cell membrane [6]. Although constitutively expressed, most of the AMP are also induced by contact with pathogens through various cell receptors belonging to the Toll family [7], [8]. Such general up-regulation which aims to protect the host can be reversed by some pathogens, such the entomoparasite, Steinernemae feltiae, in the insect Lepidoptera, Galleria mellonella [9]. In Drosophila, the seven AMP genes are induced not only in surface epithelia, but also in a tissue-specific manner [10], [11].

In bivalve mollusks, the existence and diversity of AMP have been revealed both in Mytilus edulis [12] and Mytilus galloprovincialis [13]. The antibacterial peptides defensin, mytilin and myticin have been completely sequenced. All the mature peptides included eight cysteines engaged in four intramolecular disulfide bonds. Their cDNAs present similar propeptide organisation including a signal peptide followed by the mature sequence and a C-terminal extension [14]. Several isoforms were identified. Only defensin and mytilin genes were completely sequenced, revealing the presence of four exons and one gene copy per genome [15]. Myticin gene was partially sequenced revealing one intron suggesting similar organization [16]. A fourth peptide, mytimycin was only partially sequenced and characterized by the presence of 12 cysteines and a strictly antifungal activity [12].

Several reports concern in vitro bactericidal activity of mollusk hemocytes [17], [18], [19], [20], [21]. In addition, variations of AMP gene expression have been analyzed under conditions of physical stress, bacterial challenge and heat shock. Results inferred from Northern blots suggested that defensin gene can be over-expressed after physical and temperature stress, but reduced following bacterial challenge [15]. Similarly, xenobiotics such as poly aromatic hydrocarbons depressed defensin expression. In contrast, mytilin and myticin expressions were not affected by the various treatments [22].

The present study was undertaken to elucidate if the expression of defensin, mytilin and myticin genes might correspond to the presence of particular bacteria. Adult mussels were in vivo challenged by one injection of Gram-negative bacteria, pathogenic (Vibrio splendidus LGP32) or non-pathogenic (Vibrio anguillarum) for mollusks, or of Gram-positive bacteria (Micrococcus lysodeikticus) irrelevant to marine mollusks. As representative of a non-specific stress, moderate heat shock was applied to naïve mussels. Quantification of specific cDNAs was done in Q-PCR using the SYBR Green chemistry with expression of 28S ribosomic RNA gene considered as housekeeping gene.

Section snippets

Mussels and bacteria

Adult mussels, M. galloprovincialis, were purchased from the marine farm Les Compagnons de Maguelone (Palavas, France) in May–June 2004 and May–June 2005. They were maintained in the laboratory in oxygenated sea water at 20 °C for 1–3 days prior to experimentation. V. splendidus LGP32 is a Gram-negative marine bacteria isolated from juvenile oysters, Crassostrea gigas, during summer 2001 mortalities [23]. V. anguillarum was from Institut Pasteur, France (ATCC 19264). Both Vibrios were grown at 20

Specificity of amplicons

Each pair of primers corresponding to the five genes was tested on the same cDNA template. Resulting amplicons were analyzed for DNA melting temperatures revealing only one symmetrical peak per amplicon that ranged from 80 to 90 °C (Fig. 1A). DNA melting temperatures were measured in numerous Q-PCRs allowing calculation of arithmetical mean values and their respective SE. Such values constituted references to control further amplification assays. Purity of amplicons was also checked on 2%

Discussion

28S rRNA is highly expressed in both untreated and treated mussels, and we previously reported that this expression was not significantly modified by any of the treatments [24]. Generally, 28S rRNA is used as an internal standard gene for mRNA quantifications because its variations are weak and cannot induce a bias in the total amount of RNA. According to some authors, it is more advantageous to consider two types of house keeping genes [26]. Meanwhile, using only one internal standard is

Acknowledgements

V. splendidus strain LGP32 was a gift from Frédérique Le Roux, IFREMER-La Tremblade (France). Authors are grateful to Professor Edwin L. Cooper from University of California-Los Angeles for critical reading of the manuscript and improving the language. CC is part of the International PhD program co-funded by the European Union, the MIUR-Italy (Internazionalizzazione, Art. 7-D.M. 21.06.1999), the Università degli Studi di Palermo-Italy (Biologia Animale Ciclo XVII – Internazionale e Co-tutela di

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