TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi

Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.     

Altered tyrosine metabolism and melanization complex formation underlie the developmental regulation of melanization in Manduca sexta

The study of hemolymph melanization in Lepidoptera has contributed greatly to our understanding of its role in insect immunity. Manduca sexta in particular has been an excellent model for identifying the myriad components of the phenoloxidase (PO) cascade and their activation through exposure to pathogen-associated molecular patterns (PAMPs). However, in a process that is not well characterized or understood, some insect species rapidly melanize upon wounding in the absence of added PAMPs. We sought to better understand this process by measuring wound-induced melanization in four insect species. Of these, only plasma from late 5th instar M. sexta was unable to melanize, even though each contained millimolar levels of the putative melanization substrate tyrosine (Tyr). Analysis of Tyr metabolism using substrate-free plasmas (SFPs) from late 5th instar larvae of each species showed that only M. sexta SFP failed to melanize with added Tyr. In contrast, early instar M. sexta larvae exhibited wound-induced melanization and Tyr metabolism, and SFPs prepared from these larvae melanized in the presence of Tyr. Early instar melanization in M. sexta was associated with the formation of a high mass protein complex that could be observed enzymatically in native gels or by PO-specific immunoblotting. Topical treatment of M. sexta larvae with the juvenile hormone (JH) analog methoprene delayed pupation and increased melanizing ability late in the instar, thus linking development with immunity. Our results demonstrate that melanization rates are highly variable in Lepidoptera, and that developmental stage can be an important factor for melanization within a species. More specifically, we show that the physiological substrate for melanization in M. sexta is Tyr, and that melanization is associated with the formation of a PO-containing protein complex.     

In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

Extracellular serine protease cascades mediate immune signaling and responses in insects. In the tobacco hornworm Manduca sexta, nearly 30 serine proteases (SPs) and their homologs (SPHs) are cloned from hemocytes and fat body. Some of them participate in prophenoloxidase (proPO) activation and proSpätzle processing. Here we report the cDNA cloning of hemolymph protease-1b (HP1b), which is 90% identical and 95% similar to HP1a (formerly HP1). The HP1a and HP1b mRNA levels in hemocytes was down- and up-regulated after an immune challenge, respectively. Quantitative real-time polymerase chain reactions revealed their tissue-specific and development-dependent expression, mostly in hemocytes of the feeding larvae. We isolated HP1 precursor (proHP1) from larval hemolymph and observed micro-heterogeneity caused by N-linked glycosylation. Supplementation of the purified proHP1 to plasma samples from naïve larvae or induced ones injected with bacteria caused a small PO activity increase, much lower than those elicited by recombinant proHP1a/b, but no proteolytic cleavage was detected in the zymogens. Incubation of proHP1a/b or their catalytic domains with a cationic detergent, cetylpyridinium chloride, induced an amidase activity that hydrolyzed LDLH-p-nitroanilide. Since LDLH corresponds to the P4–P1 region before the proteolytic activation site of proHP6, we propose that the active but uncleaved proHP1 may cut proHP6 to generate HP6 that in turn activates proPAP1 and proHP8. The catalytic domain of HP1a/b, which by itself does not activate purified proHP6 or hydrolyze LDLH-p-nitroanilide, somehow generated active HP6, HP8, PAP1 and PO in plasma. Together, these results indicate that proHP1 participates in the proPO activation system, although detailed mechanism needs further exploration.     

Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism,mediates immune responses

Tissue damage or pathogen invasion triggers the auto-proteolysis of an initiating serine protease (SP), rapidly leading to sequential cleavage activation of other cascade members to set off innate immune responses in insects. Recently, we presented evidence that Manduca sexta hemolymph protease-1 zymogen (proHP1) is a member of the SP system in this species, and may activate proHP6. HP6 stimulates melanization and induces antimicrobial peptide synthesis. Here we report that proHP1 adopts an active conformation (*) to carry out its function, without a requirement for proteolytic activation. Affinity chromatography using HP1 antibodies isolated from induced hemolymph the 48 kDa proHP1 and also a 90 kDa band (detected by SDS-PAGE under reducing conditions) containing proHP1 and several serpins, as revealed by mass spectrometric analysis. Identification of tryptic peptides from these 90 kDa complexes included peptides from the amino-terminal regulatory part of proHP1, indicating that proHP1* was not cleaved, and that it had formed a complex with the serpins. As suicide inhibitors, serpins form SDS-stable, acyl-complexes when they are attacked by active proteases, indicating that proHP1* was catalytically active. Detection of M. sexta serpin-1, 4, 9, 13 and smaller amounts of serpin-3, 5, 6 in the complexes suggests that it is regulated by multiple serpins in hemolymph. We produced site-directed mutants of proHP1b for cleavage by bovine blood coagulation factor Xa at the designed proteolytic activation site, to generate a form of proHP1b that could be activated by Factor Xa. However, proHP1b cut by Factor Xa failed to activate proHP6 and, via HP6, proHP8 or proPAP1. This negative result is consistent with the suggestion that proHP1* is a physiological mediator of immune responses. Further research is needed to investigate the conformational change that results in conversion of proHP1 to active proHP1*.     

Spatial and temporal distribution of Patched-related protein in the Drosophila embryo

Patched-related (Ptr) encodes a protein with 12 potential transmembrane domains and a sterol-sensing domain that is closely related in predicted topology and domain organization to Patched, the canonical receptor of the Hedgehog pathway. Here we describe the production of an antibody specific for Drosophila Ptr and analyse its spatial and temporal distribution in the embryo. We find that at early developmental stages Ptr is predominantly localized at cell periphery but later on it becomes strongly and almost exclusively expressed in hemocytes. Interestingly Ptr null mutant embryos died without hatching. Our findings suggest that Ptr plays an essential function in Drosophila development, perhaps as a new receptor of embryonic hemocytes.     

Gene expression and molecular characterization of a novel C-type lectin,encapsulation promoting lectin (EPL), in the rice armyworm,Mythimna separata

Insect cellular immune reactions differ depending on the target species. Phagocytosis is activated to scavenge microorganisms such as bacteria and fungi. On the other hand, larger invaders such as parasitoid wasps are eliminated by activation of encapsulation. In this study, we hypothesized that novel determinants regulate cellular immunities independent of surface molecular pattern recognition involving pattern recognition receptors (PRRs). Immune-related genes differentially expressed depending on the treated material size were screened in larval hemocytes of the rice armyworm, Mythimna separata. Consequently, we identified a novel C-type lectin gene up-regulated by injection of large beads but not small beads of identical material. Examination of in vitro effect of the recombinant protein on the immune reactions clarified that the protein activated encapsulation reaction, while it suppressed phagocytosis. These results suggest that this novel C-type lectin designated “encapsulation promoting lectin (EPL)” regulates cellular immunity by a novel immune target size-recognition mechanism.     

Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease

Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-residue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae.     

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta

Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R³⁶⁶ at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R⁴¹⁰ as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg³⁶⁶ by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg⁴¹⁰ for trypsin-like proteases, Gly⁴⁰⁶ and Ala⁴⁰⁹ for the elastase and Thr⁴⁰⁴ for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0–50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae.     

The ACBP gene family in Rhodnius prolixus: Expression,characterization and function of RpACBP-1

The acyl-CoA-binding proteins (ACBP) constitute a family of conserved proteins that bind acyl-CoA with high affinity and protect it from hydrolysis. Thus, ACBPs may have essential roles in basal cellular lipid metabolism. The genome of the insect Rhodnius prolixus encodes five ACBP genes similar to those described for other insect species. The qPCR analysis revealed that these genes have characteristic expression profiles in insect organs, suggesting that they have specific roles in insect physiology. Recombinant RpACBP-1 was able to bind acyl-CoA in an in vitro gel-shift assay. Moreover, heterologous RpACBP-1 expression in acb1Δ mutant yeast rescued the multi-lobed vacuole phenotype, indicating that RpACBP-1 acts as a bona fide acyl-CoA-binding protein. RpACBP-1 knockdown using RNAi caused triacylglycerol accumulation in the insect posterior midgut and a reduction in the number of deposited eggs. The amount of stored triacylglycerol was reduced in flight muscle, and the incorporation of fatty acids in cholesteryl esters was increased in the fat body. These results showed that RpACBP-1 participates in several lipid metabolism steps in R. prolixus.     

Nodules from Fynbos legume Virgilia divaricata have high functional plasticity under variable P supply levels

Legumes have the unique ability to fix atmospheric nitrogen (N₂) via symbiotic bacteria in their nodules but depend heavily on phosphorus (P), which affects nodulation, and the carbon costs and energy costs of N₂ fixation. Consequently, legumes growing in nutrient-poor ecosystems (e.g., sandstone-derived soils) have to enhance P recycling and/or acquisition in order to maintain N₂ fixation. In this study, we investigated the flexibility of P recycling and distribution within the nodules and their effect on N nutrition in Virgilia divaricata Adamson, Fabaceae, an indigenous legume in the Cape Floristic Region of South Africa. Specifically, we assessed tissue elemental localization using micro-particle-induced X-ray emission (PIXE), measured N fixation using nutrient concentrations derived from inductively coupled mass-spectrometry (ICP-MS), calculated nutrient costs, and determined P recycling from enzyme activity assays. Morphological and physiological features characteristic of adaptation to P deprivation were observed for V. divaricata. Decreased plant growth and nodule production with parallel increased root:shoot ratios are some of the plastic features exhibited in response to P deficiency. Plants resupplied with P resembled those supplied with optimal P levels in terms of growth and nutrient acquisition. Under low P conditions, plants maintained an increase in N₂-fixing efficiency despite lower levels of orthophosphate (Pi) in the nodules. This can be attributed to two factors: (i) an increase in Fe concentration under low P, and (ii) greater APase activity in both the roots and nodules under low P. These findings suggest that V. divaricata is well adapted to acquire N under P deficiency, owing to the plasticity of its nodule physiology     

A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonah_m) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonah_m reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.