Expression of defensins in non-infected araneomorph spiders

Abstract

Defensins are a major family of antimicrobial peptides found throughout the phylogenetic tree. From the spider species: Cupiennius salei, Phoneutria reidyi, Polybetes pythagoricus, Tegenaria atrica, and Meta menardi, defensins belonging to the 'ancestral' class of invertebrate defensins were cloned and sequenced. The deduced amino acid sequences contain the characteristic six cysteines of this class of defensins and reveal precursors of 60 or 61 amino acid residues. The mature peptides consist of 37 amino acid residues, showing up to 70% identities with tick and scorpion defensins. In C. salei, defensin mRNA was found to be constitutively expressed in hemocytes, ovaries, subesophageal nerve mass, hepatopancreas, and muscle tissue. This is the first report presenting and comparing antimicrobial peptides belonging to the family of defensins from spiders.

Fig. 1

C. salei defensin cDNA sequence. The deduced amino acid sequence is presented under the nucleotide sequence. The mature peptide sequence is underlined, the nucleotide sequence of the mature peptide is in bold. Asterisks mark the stop codon. The polyadenylation signal is in bold italics

Fig. 2

RT-PCR analysis of defensin-gene expression in different tissues. Expression was assayed in ovaries (A), subesophageal nerve mass (B), hepatopancreas (C), venom glands (D), heart (E), silk glands (F), hemocytes (G), and muscle (H)

Fig. 3

Spider defensin cDNA sequences. The deduced amino acid sequences are presented under the nucleotide sequences. The mature peptide sequences are underlined, the nucleotide sequence of the mature peptides are in bold. Asterisks mark the stop codons. The polyadenylation signals are in bold italics. a P. pythagoricus defensin, b T. atrica defensin, c P. reidyi defensin, d partial M. menardi defensin

Fig. 4

Alignment of spider defensins with defensins belonging to the ‘ancestral’ group of invertebrate defensins. The conserved cysteine residues are highlighted in black. The characteristic cysteine bonding pattern of arthropod defensins is C1-C4, C2-C5, C3-C6 (C1-C5, C2-C6, C3-C7, C4-C8 for mgd2b, which possesses two additional cysteines). The mgd2b sequence is shown incomplete, the M. menardi sequence was not completely solved (indicated by the dots at the end and the beginning of the sequences). % identities are given compared to C. salei defensin

Fig. 5

Phylogenetic (NJ) tree of invertebrate defensins. The NJ tree was constructed based on amino acid sequences. Bootstrap values based on 1,000 pseudoreplicates are indicated at the left of the respective nodes. Only values >400 are shown. Spider defensins are highlighted in grey. Only complete sequences were used in the alignment, M. menardi partial sequence was excluded. The accession numbers of the ‘ancestral’ class are the same as in Fig. 4. The accession numbers of the ‘insect’ class are: S. calcitrans: O16137; A. mellifera: C55392; P. terraenovae: S12558; A. gambiae: Q17027; A. aegypti: P81603; D. melanogaster: P36192; G. morsitans: AAL34112