Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection

Abstract

The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated M_r of 44.8 kDa and pI of 6.66. Multiple sequence alignment and phylogenetic analysis indicated that OfPAP was orthologous to the PAPs in the other lepidopterans. A large increase of the transcript levels was observed in hemocytes at 4 h post injection (hpi) of killed Bacillus subtilis, whereas its level in integument increased continuously from 4 to 12 hpi in the challenged larvae and began to decline at 24 hpi. After OfPAP expression had been silenced, the median lethal time (LT₅₀) of Escherichia coli-infected larvae (1.0 day) became significantly lower than that of E. coli-infected wild-type (3.0 days, p < 0.01). A 3.5-fold increase in E. coli colony forming units occurred in larval hemolymph of the OfPAP knockdown larvae, as compared with that of the control larvae not injected with dsRNA. There were notable decreases in PO and IEARase activities in hemolymph of the OfPAP knockdown larvae. In summary, we have demonstrated that OfPAP is a component of the PPO activation system, likely by functioning as a PPO activating protease in O. furnacalis larvae.

Keywords: Bacterial challenge; Hemolymph protein; Insect immunity; Melanization; RNA interference.

Fig. 1.. Nucleotide and deduced amino acid sequences and predicted domain structure of O. furnacalis PAP.

(A) The sequence is deposited in GenBank (accession number: GU229936). Nucleotides are numbered from the first one at the 5’-end. The stop codon is marked with an asterisk (*). The polyadenylation signal (AATAAA) and poly (A) tail are marked with short dash lines. The signal peptide sequence is shaded gray. The two clip domains are included in parentheses and highlighted with a wavy line. The SP catalytic triad (H, D, S) at the active site are indicated with “@”. Conserved chymotrypsin-like SP catalytic domain is underlined. (B) Predicted domain structure of OfPAP by BLASTP.

Fig. 2.. Sequence alignment of PAPs from lepidopteran insects.

OfPAP is compared with Danaus plexippus plexippus PPAE3 (OWR45231), M. sexta PAP3 (AAX18637), S. litura PPAE3 (AAW24481) and P. xylostella PAP3a (ANH58162). The ClustalO program was used for alignments. The predicted disulfide linkages between conserved cysteines of the clip domains are shown with yellow lines, respectively. Amino acid residues constituting the catalytic triad (His, Asp and Ser) are shaded pink.

Fig. 3.. Phylogenetic tree of amino acid sequences of PAP from O. furnacalis and other species.

PAPs include various PAP, PPAE or PPAF in O. furnacalis (Of, GU229936), H. armigera (Ha, EF614332), S. litura (Sl, AY677081), Biston betularia (Bb, GU9553224), M. sexta (Ms, AAX18636, AAX18637 and AAZ91696), P. xylostella (Px, AFX82593, ANH58162 and ANH58163), B. mori (Bm, AY061936 and NM_001043367), H. diomphalia (Hd, AB079666), Aedes aegypti (Aa, AF466614), Samia ricini (Sr, BAF43530), Culex quinquefasciatus (Cq, XM_001868385), Tenebrio molitor (Tm, AJ400904). F. chinensis (Fc, JX644447) PPO activating factor (PPAF) was used as an out-group to root the phylogeny. The numbers are neighbor-joining distances. Asterisks indicate the OfPAP and vertical lines indicate two groups of PAPs in the phylogenetic tree.

Fig. 4.. Tissue-specific expression of the OfPAP mRNA in larvae measured by SYBR green qRT-PCR.

The tissues include hemocytes, fat body, midgut and integument collected from day 1, 5^th instar larvae. Relative RNA levels are shown as mean ± SD (n=3).

Fig. 5.. Expression profiles of OfPAP in hemocytes (A), fat body (B) and integument (C) of the 5^th instar naïve larvae or larvae after a B. subtilis or E. coli challenge at different times.

The O. furnacalis rpL8 gene was used as an internal control. Vertical bar represents the mean ± SD (n=3). Significant differences were indicated with “*” at the p<0.05 level, “**” at the p<0.01 level and “***” at the p<0.001 level, respectively. The same below.

Fig. 5.. Expression profiles of OfPAP in hemocytes (A), fat body (B) and integument (C) of the 5^th instar naïve larvae or larvae after a B. subtilis or E. coli challenge at different times.

The O. furnacalis rpL8 gene was used as an internal control. Vertical bar represents the mean ± SD (n=3). Significant differences were indicated with “*” at the p<0.05 level, “**” at the p<0.01 level and “***” at the p<0.001 level, respectively. The same below.

Fig. 6.. Effectiveness of dsRNA-mediated knockdown of OfPAP expression and its impact on survival of O. furnacalis larvae upon bacterial infection.

Effect of dsRNA injection on OfPAP transcript levels in hemocytes (A) and integument (B) at different times. Levels of OfPAP mRNA in relative to rpL8 were determined by qRT-PCR. (C) Survival test of naïve and OfPAP dsRNA-treated larvae. After injection of OfPAP dsRNA, twenty larvae were inoculated with E. coli intra-abdominally (RNAi-infected). Twenty larvae were treated with OfPAP RNAi without bacterial infection (RNAi) and 20 larvae were inoculated with E. coli only (infected). Twenty naïve larvae injected with dsRNA specific to green fluorescent protein (GFP dsRNA) were used as controls. The number of dead larvae was counted daily. Results are expressed as mean ± SD of three independent experiments.

Fig. 6.. Effectiveness of dsRNA-mediated knockdown of OfPAP expression and its impact on survival of O. furnacalis larvae upon bacterial infection.

Effect of dsRNA injection on OfPAP transcript levels in hemocytes (A) and integument (B) at different times. Levels of OfPAP mRNA in relative to rpL8 were determined by qRT-PCR. (C) Survival test of naïve and OfPAP dsRNA-treated larvae. After injection of OfPAP dsRNA, twenty larvae were inoculated with E. coli intra-abdominally (RNAi-infected). Twenty larvae were treated with OfPAP RNAi without bacterial infection (RNAi) and 20 larvae were inoculated with E. coli only (infected). Twenty naïve larvae injected with dsRNA specific to green fluorescent protein (GFP dsRNA) were used as controls. The number of dead larvae was counted daily. Results are expressed as mean ± SD of three independent experiments.

Fig. 7.. Number of viable bacteria (colony forming units or CFU) in hemolymph of OfPAP-silenced larvae after a systemic E. coli infection.

Larvae were challenged with E. coli (2×10⁵ CFUs) following the injection of OfPAP dsRNA. Control larvae were injected with GFP dsRNA or saline solution only. The bacterial CFUs in hemolymph from the knockdown larvae were determined at 6 h after E. coli infection by a modified total plate count method and are shown as mean ± SD from three independent experiments. Asterisks indicate significant difference (p<0.05).

Fig. 8.. Time-dependent changes in PO (A) and IEARase (B) activities of larval hemolymph after RNAi-mediated silencing of OfPAP expression.

Activities are shown as mean ± SD (n=3) from three independent experiments.