Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 protective roles have been well documented by in vivo targeted disruption of defensin gene which resulted in the death of A. gambiae after Gram-positive bacterial infection [31]. Insect defensins consist of 30-50 amino acids linked by three or four disulfide bridges. They share a common CSab structural motif with some func- tionally related scorpion neurotoxins targeting various ion channels, protease inhibitors, and even a plant sweet taste peptide [8,32]. In A. mellifera, there are only two defensins named defensin1 and defensin2 [33]. Amdefensin2 is a CITD with similar sequence, gene organization and molecular size to those from other insects. However, amdefensin1, also known as royalisin, has a lineage-specific carboxyl- terminal extension. Defensins with clear orthologous relationship with these two defensins have been identified in the N. vitripennis genome based on sequence similarity and identical precursor organization (signal peptide, pro- peptide and mature peptide) by our group [25]. These two defensins have undergone gene duplication to form a mul- tigene family containing five members (navidefensin1-1, 1-2, 2-1, 2-2 and 2-3). A detailed analysis of their exon- intron organization allowed identifying N. vitripennis- specific intron loss and gain (Figure 1). For example, navi- defensin1-1 has a phase 2 intron at the residue lysine following the CXC motif, which is conserved with amde- fensin1, while navidefensin1-2 lacks this intron. 3’-intron loss or gain has been frequently observed in a large num- ber of genes by a possible reverse transcription-mediated mechanism [34]. Whether dynamic evolution of introns is associated with the carboxyl-terminal extension in defen- sin1 remains an open question [35]. Three-dimensional (3D) models of navidefensin1-1 and navidefensin2-1 were constructed by comparative modeling with sapecin’s structure (PDB: 1L4V) [36] as a template, which confirmed their typical CSab folding, as identified by an a-helix and a two-stranded b-sheet. Residues spanning S15-S23 in navidefensin1-1 and S18- A26 in navidefensin2-1 form an a-helix with separate hydrophobic (A16, A17 and L22 in navidefensin1-1 or A19, A21, V22, L25, and A26 in navidefensin2-1) and hydrophilic (S15, N20, and S23 in navidefensin1-1 or S18 and R23 in navidefensin2-1) surfaces. Two b-stands (residues G28 to C31 and C36 to R39 in navidefensin1-1 or G31 to C34 and C39 to R42 in navidefensin2-1) consti- tute an antiparallel sheet with a g-core region which has been considered as a functional determinant responsible for antimicrobial activity of some defensins [37]. Because of no suitable templates to build a model of the extended carboxyl-terminus of navidefensin1-1, alterna- tively an ab initio prediction method was employed. In both methods, a conserved CSab core was successfully predicted (Verify3D values > 0.2). For navidefensin1-1, the ab initio method predicted an a-helix in its Page 3 of 19 carboxyl-terminus (residues K44 to R50) (Figure 2B). The existence of an extra glycine in the carboxyl-terminus of navidefensin1-1 suggests that Phe51 is likely amidated to provide a force for the helical stability, as predicted in bee defensin1 [33,38]. (2) Nasonins In addition to the defensins mentioned above, we identified a new DLP subfamily without a propeptide, which contains fourteen members (named nasonin-1 to -14) (Figure 1). Sequence analysis revealed that nasonins are more closely related to two known non-classical defensins than CITDs in precursor organi- zation, n-loop size and sequence similarity, one being antifungal termicin isolated from termites [39,40]; another being defensin1 from the Mexican scorpion Centruroides limpidus limpidus [41]. Intriguingly, such similarity was also observed between nasonins and a scorpion K+ channel toxin cobatoxin [42,43] (Figure 1). These observations suggest that nasonins could have diverse functional features. For nasonin-2 and nasonin-6, their defensin unit has undergone 4 to 5 internal repeats, as described in fungal DLPs [32]. What’s the potential function of such repeats and how these repeats occur will be further discussed below. We also predicted the structure of nasonin-1 by com- parative modeling using scorpion neurotoxin cobatoxin (PDB: 1PJV) [43] as a template. Although having a shorter n-loop than CITDs, nasonins can also adopt a typical CSab architecture (Figure 2B). The reliability of this model was evaluated by Verify3D with a score of 0.248. Overall, nasonin-1 more resembles structurally termicin (PDB: 1MM0) [44] and cobatoxin (Figure 2B), but they differ in molecular surface charge distribution (data not shown), which could explain functional diver- sity of these peptides, as observed between human beta- defensin2 and the snake toxin crotamine [45]. Navidefensin3 is a unique non-classical defensin because it has identical precursor organization and gene structure to navidefensin1 and 2 but similar n-loop size and amino acid sequence to nasonins. In fact, several non-classical insect defensins with a short n-loop were also found in Lepidoptera insects (e.g. gallerimycin, spo- doptericin and the B. mori defensins) [46-48]. In the tree presented in Figure 2A, navidefensin3 is clustered together with nasonins rather than navidefensins, sug- gesting that this DLP is an evolutionary link between these two multigene subfamilies of defensins. Its lack in A. mellifera is consistent with the absence of the naso- nin subfamily in this species. (3) Navitricins We also found two unique DLPs (named navitricin-1 and -2) in the N. vitripennis genome, which possess 8 cysteines with an identical alignment pattern to the family III members of fungal DLPs [32]. Analysis of genomic sequences identified a phase 1 intron in the mature peptide-coding region of navitricins (Figure 3A).