Maureen J. Gorman

Serine proteases as mediators of mosquito immune responses

Serine proteases regulate several invertebrate defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization of pathogen surfaces. These processes require the presence of serine proteases in the hemolymph where they can rapidly activate immune pathways in response to pathogen detection. Hemolymph coagulation in the horseshoe crab is controlled by several serine proteases, including two that are pathogen recognition molecules and two in the clip domain family of serine proteases. The antimicrobial peptide synthesis and melanization pathways include clip domain proteases as well as other, uncharacterized serine proteases. We have identified five serine proteases from the hemolymph of the mosquito, Anopheles gambiae. One, Sp22D, is a large protease with potential pathogen binding domains. Sp22D is expressed in three tissues that have immune functions (midgut epithelium, fat body, and hemocytes), and its transcript abundance increases after immune challenge. Sp14A, Sp14D1, and Sp14D2 are clip domain serine proteases that are similar to enzymes with presumed roles in melanization or antimicrobial peptide synthesis. They undergo changes in transcript abundance in response to infection with bacteria or malaria parasites, and they reside in a chromosomal region that has been associated with melanization of parasites. Sp18D, also a clip domain protease, is similar to a Manduca protease with a likely role in immunity, but immune challenge does not affect its mRNA abundance.

4 – PHENOLOXIDASES IN INSECT IMMUNITY

Phenoloxidases are present as zymogens in insect hemolymph, and they become activated upon wounding or infection as part of the innate immune response. These enzymes are similar to mammalian tyrosinases in their ability to use reactive sites containing copper atoms to catalyze two types of reactions that require molecular oxygen as a substrate. They can hydroxylate tyrosine to form dihydroxyphenylalanine, and they oxidize o -diphenols to form quinones. The quinones undergo additional reactions leading to synthesis of melanin, which is deposited on the surface of encapsulated parasites, hemocyte nodules, and wound sites. The melanin itself and reactive chemical species produced during melanin synthesis appear to help kill invading pathogens and parasites. Rather than having sequence similarity to mammalian tyrosinases, insect prophenoloxidases (proPOs) are homologous with arthropod hemocyanins and insect hexamerin storage proteins. ProPOs are synthesized primarily by hemocytes and released into plasma by cell lysis. They are activated by proteolytic cleavage at a specific site near their amino-terminus through the action of hemolymph serine proteases containing amino-terminal clip domains. These proPO activating proteases are themselves activated as part of a protease cascade stimulated by recognition of microbial infection. At least in some cases, activation of proPO also requires the participation of serine protease homolog (SPH) cofactors, which lack proteolytic activity. The proPO activation cascade is regulated by plasma serine protease inhibitors, including members of the serpin superfamily, and active phenoloxidase (PO) may be directly inhibited by proteinaceous factors. Such regulation is essential because the products of PO activity are potentially toxic to the host. Further research is required to gain a more detailed understanding at a molecular level of the assembly of plasma protein complexes leading to proPO activation and melanin deposition on foreign surfaces and the regulation of these processes.

Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade

Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1–3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of ∼32 and ∼37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.

Bacterial challenge stimulates innate immune responses in extra‐embryonic tissues of tobacco hornworm eggs

Innate immunity protects juvenile and adult vertebrates and invertebrates against potential pathogens; however, it is unknown when developing embryos become immune competent and just how they are guarded from infection. To address these questions, we studied the effect of immune challenge on early stage eggs of the tobacco hornworm, Manduca sexta. We detected many immune‐related proteins and mRNAs in naive eggs. Upon immune challenge, antimicrobial protein genes were up‐regulated, and antibacterial activity increased. Antimicrobial protein mRNAs and lysozyme were present in the extra‐embryonic tissues of immune‐challenged eggs; in addition, melanization in response to bacteria occurred in the yolk but not embryonic tissues. We conclude that the extra‐embryonic tissues of early stage M. sexta eggs are immune competent and likely protect the developing embryo from infection. We suggest that innate immune responses of extra‐embryonic tissues may be a common mechanism for protecting early embryos.

Molecular characterization of five serine protease genes cloned from Anopheles gambiae hemolymph.

We identified five new serine protease cDNAs from the hemolymph of the malaria vector, Anopheles gambiae. All five show sequence similarity to genes thought to be involved in vertebrate or invertebrate defense responses. Sp14A, Sp14D2 and Sp22D demonstrate changes in transcript abundance in response to bacteria injections. Sp14A and Sp14D2, as well as the previously characterized Sp14D1, are induced by infection with the malaria parasite, Plasmodium berghei. These three proteases, along with Sp18D, are related to a group of secreted proteases that have amino-terminal clip domains and trypsin-like substrate specificity. BLAST results and phylogenetic analyses group Sp14A, Sp14D1 and Sp14D2 with the Drosophila protease EASTER, and three prophenoloxidase activating enzymes from other insects. EASTERs substrate is SPAETZLE, a ligand involved in embryogenesis but also in activating anti-microbial peptide synthesis. Their similarity to EASTER and immune inducibility suggest that one of these proteases may activate a SPAETZLE-like ligand during anti-parasite responses in mosquitoes. Alternatively, as potential prophenoloxidase activators, Sp14A, Sp14D1 or Sp14D2 may play a role in melanotic encapsulation of Plasmodium.

Sp22D: a multidomain serine protease with a putative role in insect immunity.

Serine proteases play critical roles in a variety of insect immune responses; however, few of the genes that code for these enzymes have been cloned. Here, we describe the molecular characterization of a serine protease gene from the mosquito Anopheles gambiae. Sp22D codes for a 1322 amino acid polypeptide with a complex domain organization. In addition to the carboxy terminal serine protease catalytic domain, Sp22D contains two putative chitin binding domains, a mucin-like domain, two low density lipoprotein receptor class A domains, and two scavenger receptor cysteine rich domains. A typical signal peptide sequence and a lack of potential transmembrane helices suggest that Sp22D is secreted. Sp22D is expressed constitutively in three immune-related cell types: adult hemocytes, fat body cells, and midgut epithelial cells. Wounding induces no changes in transcript abundance, but within 1h after injection of bacteria, Sp22D mRNA increases 1.5-fold. Based on domain organization, tissue distribution, and transcriptional up-regulation in response to immune challenge, we suggest that Sp22D has an immune function. In addition, we predict that Sp22D is secreted into the hemolymph where it may interact with pathogen surfaces and initiate an immune response.

Tyrosine hydroxylase is required for cuticle sclerotization and pigmentation in Tribolium castaneum.

Newly synthesized insect cuticle is soft and pale but becomes stronger (sclerotized) and often darker (pigmented) over several hours or days. The first step in the sclerotization and pigmentation pathways is the hydroxylation of tyrosine to produce 3,4-dihydroxyphenylalanine (DOPA). Tyrosine hydroxylase (TH) is known to catalyze this reaction during pigmentation, but a role for TH in sclerotization has not been documented. The goal of this study was to determine whether TH is required for cuticle sclerotization in the red flour beetle, Tribolium castaneum. We used quantitative RT-PCR to verify that TH expression occurs at the time of cuticle tanning and immunohistochemistry to confirm that TH is expressed in the epithelial cells underlying sclerotized cuticle. In addition, we found that a reduction in TH function (mediated by RNA interference) resulted in a decrease in cuticle pigmentation and a decrease in the hardness of both pigmented and colorless cuticle. These results demonstrate a requirement for TH in sclerotization as well as brown pigmentation of insect cuticle.

Characterization of the multicopper oxidase gene family in Anopheles gambiae

The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including diphenols, and several oxidases with specific substrates such as iron, copper or ascorbic acid. We have identified five putative MCO genes in the genome of Anopheles gambiae and have cloned cDNAs encompassing the full coding region for each gene. MCO1 mRNA was detected in all developmental stages and in all of the larval and adult tissues tested. We observed an increase in MCO1 transcript abundance in the midguts and Malphighian tubules of adult females following a blood meal and in adult abdominal carcasses in response to an immune challenge. Two alternatively spliced isoforms of MCO2 mRNA were identified. The A isoform of MCO2 was previously detected in larval and pupal cuticle where it probably catalyzes sclerotization reactions (He, N., Botelho, J.M.C., McNall, R.J., Belozerov, V., Dunn, W.A., Mize, T., Orlando, R., Willis, J.H., 2007. Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry. Insect Biochem. Mol. Biol. 37, 135-146). The B isoform was transcriptionally upregulated in ovaries in response to a blood meal. MCO3 mRNA was detected in the adult midgut, Malpighian tubules, and male reproductive tissues; like MCO1, it was upregulated in response to an immune challenge or a blood meal. MCO4 and MCO5 were observed primarily in eggs and in the abdominal carcass of larvae. A phylogenetic analysis of insect MCO genes identified putative orthologs of MCO1 and MCO2 in all of the insect genomes tested, whereas MCO3, MCO4 and MCO5 were found only in the two mosquito species analyzed. MCO2 orthologs have especially high sequence similarity, suggesting that they are under strong purifying selection; the A isoforms are more conserved than the B isoforms. The mosquito specific group shares a common ancestor with MCO2. This initial study of mosquito MCOs suggests that MCO2 may be required for egg development or eggshell tanning in addition to cuticle tanning, while MCO1 and MCO3 may be involved in metal metabolism or immunity.

An easter-like serine protease from Anopheles gambiae exhibits changes in transcript abundance following immune challenge

The nucleotide and deduced amino acid sequence of a serine protease (AgSp14D1) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 360 amino acid protein that contains two domains and has the highest sequence similarity to the Drosophila melanogaster serine protease easter and to prophenol oxidase activating enzyme (pPAE) from Manduca sexta. The catalytic domain is at the carboxy terminus and has the conserved serine, histidine and aspartic acid residues found in serine proteases as well as six cysteines common to invertebrate enzymes. The amino terminus contains critical cysteines that define a clip (=disulphide knot) domain which places this gene product in a subfamily of regulatory serine proteases that includes not only easter and pPAE but also the Drosophila proteins masquerade, stubble and snake as well as proclotting enzyme and factor B from the horseshoe crab. In situ hybridization to the polytene chromosomes detects a single band at 14D and Southern analysis with a probe from the 5′ end of the gene confirms the single copy status of this gene. Northern analysis reveals changes in transcript abundance during development and following blood feeding. Interestingly, this analysis also shows an increase in transcript levels following wounding or injection of bacteria.

The role of surface characteristics in eliciting humoral encapsulation of foreign bodies in Plasmodium-refractory and -susceptible strains of Anopheles gambiae

A refractory strain of the mosquito, Anopheles gambiae, melanotically encapsulates and kills many species of malaria parasites, whereas susceptible strains allow the parasites to develop normally. To study the role of surface characteristics in eliciting this immune response, 27 types of chromatography beads that differed in matrix type, charge, functional group, and functional group density were assayed for degree of melanotic encapsulation in refractory and susceptible mosquitoes. Overall, two glucan-based matrices, Sephadex (dextran) and cellulose, stimulated the strongest responses, regardless of functional group. Substituting matrix hydroxyl groups with functional groups on Sephadex and cellulose beads decreased the level of encapsulation. These results demonstrate that glucans induce melanotic encapsulation in An. gambiae. Beads with agarose, polystyrene, and acrylic matrices, and most methacrylate-based beads elicited little or no melanization; however, epoxide-methacrylate beads were encapsulated, demonstrating that glucans are not essential for eliciting a response. Comparisons between the two strains demonstrated that refractory mosquitoes melanized many bead types to a greater degree than did susceptible mosquitoes. On this basis, we propose that an important difference between the two strains is that one of the enzymes involved in the melanization pathway functions at a higher level in the refractory strain. Finally, of all beads tested, only 85% substituted CM-Sephadex beads were virtually unmelanized in susceptible mosquitoes but highly melanized in the refractory strain; thus, a specific surface microenvironment is necessary to demonstrate this effect.