Zhiqiang Lu

Comparative genomic analysis of the Tribolium immune system.

BackgroundTribolium castaneum is a species of Coleoptera, the largest and most diverse order of all eukaryotes. Components of the innate immune system are hardly known in this insect, which is in a key phylogenetic position to inform us about genetic innovations accompanying the evolution of holometabolous insects. We have annotated immunity-related genes and compared them with homologous molecules from other species.ResultsAround 300 candidate defense proteins are identified based on sequence similarity to homologs known to participate in immune responses. In most cases, paralog counts are lower than those of Drosophila melanogaster or Anopheles gambiae but are substantially higher than those of Apis mellifera. The genome contains probable orthologs for nearly all members of the Toll, IMD, and JAK/STAT pathways. While total numbers of the clip-domain serine proteinases are approximately equal in the fly (29), mosquito (32) and beetle (30), lineage-specific expansion of the family is discovered in all three species. Sixteen of the thirty-one serpin genes form a large cluster in a 50 kb region that resulted from extensive gene duplications. Among the nine Toll-like proteins, four are orthologous to Drosophila Toll. The presence of scavenger receptors and other related proteins indicates a role of cellular responses in the entire system. The structures of some antimicrobial peptides drastically differ from those in other orders of insects.ConclusionA framework of information on Tribolium immunity is established, which may serve as a stepping stone for future genetic analyses of defense responses in a nondrosophiline genetic model insect.

Antiviral, anti-parasitic, and cytotoxic effects of 5,6-dihydroxyindole (DHI), a reactive compound generated by phenoloxidase during insect immune response.

Phenoloxidase (PO) and its activation system are implicated in several defense responses of insects. Upon wounding or infection, inactive prophenoloxidase (proPO) is converted to active PO through a cascade of serine proteases and their homologs. PO generates reactive compounds such as 5,6-dihydroxyindole (DHI), which have a broad-spectrum antibacterial and antifungal activity. Here we report that DHI and its spontaneous oxidation products are also active against viruses and parasitic wasps. Preincubation of a baculovirus stock with 1.25 mM DHI for 3 h near fully disabled recombinant protein production. The LC₅₀ for lambda bacteriophage and eggs of the wasp Microplitis demolitor were 5.6 ± 2.2 and 111.0 ± 1.6 μM, respectively. The toxicity of DHI and related compounds also extended to cells derived from insects that serve as hosts for several of the aforementioned pathogens. Pretreatment of Sf9 cells with 1.0 mM DHI for 4 h resulted in 97% mortality, and LC₅₀ values of 20.3 ± 1.2 μM in buffer and 131.8 ± 1.1 μM in a culture medium. Symptoms of DHI toxicity in Sf9 cells included DNA polymerization, protein crosslinking, and lysis. Taken together, these data showed that proPO activation and DHI production is strongly toxic against various pathogens but can also damage host tissues and cells if not properly controlled.

The viral protein EGF1.0 is a dual activity inhibitor of prophenoloxidase activating proteinases 1 and 3 from Manduca sexta

Some pathogens are capable of suppressing the melanization response of host insects, but the virulence factors responsible are largely unknown. The insect pathogen Microplitis demolitor bracovirus encodes the Egf family of small serine proteinase inhibitors. One family member, Egf1.0, was recently shown to suppress melanization of hemolymph in Manduca sexta in part by inhibiting the enzymatic activity of prophenoloxidase activating proteinase 3 (PAP3). However, other experiments suggested this viral protein suppresses melanization by more than one mechanism. Here we report that Egf1.0 inhibited the amidolytic activity of PAP1 and dose-dependently blocked processing of pro-PAP1 and pro-PAP3. Consistent with its PAP inhibitory activity, Egf1.0 also prevented processing of pro-phenoloxidase, serine proteinase homolog (SPH) 1, and SPH2. Isolation of Egf1.0-protein complexes from plasma indicated that Egf1.0 binds PAPs through its C-terminal repeat domain. Egf1.0 also potentially interacts with SPH2 and two other proteins, ferritin and gloverin, not previously associated with the phenoloxidase cascade. Overall, our results indicate that Egf1.0 is a dual activity PAP inhibitor that strongly suppresses the insect melanization response.

Egf1.5 is a second phenoloxidase cascade inhibitor encoded by Microplitis demolitor bracovirus

The three-member Egf gene family from the polydnavirus Microplitis demolitor bracovirus (MdBV) encodes novel proteins distinguished by a shared cysteine-rich motif. Prior studies determined that one family member, Egf1.0, inhibits melanization of hemolymph from the moth Manduca sexta by disabling phenoloxidase activating proteinases (PAPs). Here we characterized a second family member, Egf1.5, which shares an identical cysteine-rich motif with Egf1.0, but possesses an extended C-terminal repeat domain. Similar to Egf1.0, Egf1.5 inhibited processing and the amidolytic activity of PAP1 and PAP3 from M. sexta. Egf1.5 also bound PAP1, PAP3 and serine proteinase homolog 2 (SPH2). Comparative studies indicated that Egf1.5 and Egf1.0 similarly inhibited melanization of plasma from two lepidopterans (Pseudoplusia includens and Helicoverpa zea) that are permissive hosts for M. demolitor and MdBV, and two lepidopterans (M. sexta and Bombyx mori) that are nonpermissive hosts. Expression studies showed that transcript abundance of egf1.5 and egf1.0 was also similar in MdBV-infected P. includens and H. zea. Taken together, our results indicate that Egf1.5 and Egf1.0 are functionally similar paralogs.

Regulation of melanization by glutathione in the moth Pseudoplusia includens

The phenoloxidase (PO) cascade regulates the melanization of hemolymph, which serves as a conserved humoral immune response in insects and other arthropods. The reductant glutathione (GSH) has long been used to inhibit melanization of hemolymph from insects but whether GSH levels in hemolymph are sufficient to play a physiological role in regulating melanization is unknown. Here, we characterized the abundance and effects of GSH on the melanization of plasma from larval stage Pseudoplusia includens (Lepidoptera: Noctuidae). GSH concentration in newly collected plasma from day two fifth instars ranged from 50 to 115 microM, while the titer of tyrosine, a substrate for the PO cascade, was 141 microM. GSH titers rapidly declined in plasma after collection from larvae, but no melanin formation occurred until GSH levels fell below 20 microM. Added GSH dose-dependently blocked melanization while PO substrates overrode GSH inhibition. Experiments conducted in the absence of oxygen and presence of PO cascade inhibitors further suggested that depletion of GSH from plasma was primarily due to formation of reactive intermediates produced by activated PO. Additional studies identified hemocytes as a potential source of plasma GSH. Hemocyte lysates recycled oxidized glutathione (GSSG) into GSH using NADPH, while intact hemocytes released GSH into the medium. These results suggest that in addition to protease cascade-related mechanisms that regulate phenoloxidase, GSH exerts another level of control on melanization of insect hemolymph.

Manipulation of the silkworm immune system by a metalloprotease from the pathogenic bacterium Pseudomonas aeruginosa

ABSTRACT Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 &Dgr;lasB mutant and found that PO activity in hemolymph of the PAO1 &Dgr;lasB‐infected larvae was significantly higher than that in the wild type‐infected. The mutant‐inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 &Dgr;lasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the &Dgr;lasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection. HighlightsPseudomonas aeruginosa expresses and releases elastase B in the hemolymph after infection of larval silkworm.Elastase B compromises prophenoloxidase activation and melanization of the silkworm hemolymph.Elastase B upregulates expression of antimicrobial peptide gloverin in the silkworm.Elastase B contributes to the growth of P. aeruginosa in the silkworm and pathogenicity of P. aeruginosa to the host.