Michael R. Kanost

Immune pathways and defence mechanisms in honey bees Apis mellifera.

Social insects are able to mount both group‐level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome‐wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one‐third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens.

Innate immune responses of a lepidopteran insect, Manduca sexta

Summary:  Many innate immune mechanisms are conserved throughout the animal kingdom. Manduca sexta, a widely used model for insect biochemical research, employs these mechanisms to defend against invading pathogens and parasites. We have isolated from M. sexta hemolymph a group of proteins (hemolin, peptidoglycan recognition proteins, β‐1,3‐glucan recognition proteins, and C‐type lectins), which serve as a surveillance mechanism by binding to microbial surface molecules (e.g. peptidoglycan, lipopolysaccharide, lipoteichoic acid, and β‐1,3‐glucan). The binding triggers diverse responses such as phagocytosis, nodule formation, encapsulation, melanization, and synthesis of anti‐microbial peptides/proteins. Some of these responses are mediated and coordinated by serine proteinase cascades, analogous to the complement system in mammals. Our current research is focused on the proteolytic activation of prophenoloxidase (proPO) – a reaction implicated in melanotic encapsulation, wound healing, and protein cross‐linking. We have isolated three proPO‐activating proteinases, each of which requires serine proteinase homologs as a cofactor for generating active phenoloxidase. The proteinases and proteinase‐like molecules, containing one to two clip domains at their amino‐terminus, are acute‐phase proteins induced upon an immune challenge. Inhibitory regulation of the proteinases by serpins and association of the proteinase homologs with a bacteria‐binding lectin are important for ensuring a localized defense response. Additional serine proteinases expressed in M. sexta hemocytes and fat body have been discovered. Future research efforts will be aimed at elucidating the proteinase cascade for proPO activation and investigating the roles of proteinases in other immune responses such as processing of plasmatocyte‐spreading peptide.

Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti (Aa), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa, the malaria vector Anopheles gambiae (Ag), and the fruit fly Drosophila melanogaster (Dm). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

Serine proteinase inhibitors in arthropod immunity

Arthropod hemolymph contains proteins with serine proteinase inhibitory activity. These inhibitors may exist in plasma or in hemocyte granules. Serine proteinase inhibitors from the Kazal, Kunitz, alpha-macroglobulin, and serpin families have been identified in arthropod hemolymph and have been characterized biochemically. Two new families of low molecular weight serine proteinase inhibitors have recently been discovered: one in silkworms (the Bombyx family) and another in locusts and a crayfish. The serine proteinase inhibitors in arthropod hemolymph are likely to function in protecting their hosts from infection by pathogens or parasites. Some may inhibit fungal or bacterial proteinases. Others probably have roles in regulating endogenous proteinases involved in coagulation, prophenol oxidase activation, or cytokine activation.

Insect Haemolymph Proteins

Publisher Summary This chapter discusses the structure and function of major groups of hemolymph proteins that are common to all insects, storage proteins, lipoproteins, vitellogenins, and inducible antibacterial proteins. It also discusses some proteins and peptides that are present in smaller amounts, sometimes occurring only in a few insect species. With the development of microsequencing techniques that can provide information about the sequence of amino acids at the N-terminal end of an intact protein on a sample of less than 20 picomoles, it has become feasible to use one- or two-dimensional polyacrylamide gel electrophoresis (PAGE) a the protein purification method of choice. In many cases, proteins can be transferred directly from gels to derivatized paper or other media and spots cut from the medium can be inserted directly into the sequencer. If necessary, proteins on the medium can be cleaved to peptides, which can be separated by high-pressure liquid chromatography (HPLC) or by PAGE.

Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationships

Serine proteases (SPs) and serine protease homologs (SPHs) constitute the second largest family of genes in the Drosophila melanogaster genome. Eighty-four SPs comprise less than 300 amino acid residues, and a significant portion of them are probably digestive enzymes. Some larger SPs may contain one or more regions important for protein-protein interactions, including clip domains, low-density lipoprotein receptor class A repeats, and scavenger receptor cysteine-rich domains. We identified 37 clusters of SP or SPH genes, which probably evolved from relatively recent gene duplication and sequence divergence. A majority of the SPs may be trypsin-like and activated by cleavage after a specific arginine or lysine residue. Among the 147 SPs and 57 SPHs studied, 24 SPs and 13 SPHs contain at least one regulatory clip domain. A multiple sequence alignment of the clip domains provided further information on structural conservation of these regulatory modules. Detailed sequence comparison led to an improved classification system for SPs containing clip domains. These analyses have established a framework of information about evolutionary relationships among the Drosophila SPs and SPHs, which may facilitate research on these proteins as well as homologous molecules from other invertebrate species.

Immulectin, an inducible C-type lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase

Immulectin, a C-type lectin from the tobacco hornworm, Manduca sexta, was cloned from a larval fat body cDNA library. The immulectin cDNA encodes a 309 residue polypeptide. Immulectin synthesis was induced by injection of killed gram-positive or gram-negative bacteria or yeast. After injection of bacteria, immulectin mRNA appeared in fat body and immulectin protein was detected in hemolymph. Immulectin contains two carbohydrate recognition domains. The carboxyl-terminal carbohydrate recognition domain is most similar (36% identity) to a lipopolysaccharide-binding protein from the American cockroach, Periplaneta americana. It also shares 26-35% identity to carbohydrate recognition domains of various mammalian C-type lectins. Two immulectin isoforms were identified in the hemolymph of bacteria-injected larvae. Recombinant immulectin agglutinated gram-positive and gram-negative bacteria and yeast. Addition of recombinant immulectin to M. sexta plasma stimulated activation of phenol oxidase. A combination of immulectin with lipopolysaccharide from E. coli activated phenol oxidase more rapidly and to a higher level than immulectin alone, whereas lipopolysaccharide by itself had little effect on phenol oxidase activation. Immulectin synthesized in response to bacterial or fungal infection may help to trigger protective responses in M. sexta in a manner similar to mannose-binding protein, a C-type lectin that functions in the mammalian innate immune system.

Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm, Manduca sexta

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection.

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1‐specific RNA interference (RNAi) disrupted all three types of moult (larval–larval, larval–pupal and pupal–adult) and greatly reduced whole‐body chitin content. Exon‐specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval‐pupal and pupal‐adult moults, whereas splice variant 8b was required only for the latter. TcCHS2‐specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi‐mediated down‐regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.

Pattern recognition proteins in Manduca sexta plasma.

Recognition of nonself is the first step in mounting immune responses. In the innate immune systems of both vertebrates and arthropods, such recognition, termed pattern recognition, is mediated by a group of proteins, known as pattern recognition proteins or receptors. Different pattern recognition proteins recognize and bind to molecules (molecular patterns) present on the surface of microorganisms but absent from animals. These molecular patterns include microbial cell wall components such as bacterial lipopolysaccharide, lipoteichoic acid and peptidoglycan, and fungal beta-1,3-glucans. Binding of pattern recognition proteins to these molecular patterns triggers responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, and synthesis of antimicrobial peptides. In this article, we describe four classes of pattern recognition proteins, hemolin, peptidoglycan recognition protein, beta-1,3-glucan recognition proteins, and immulectins (C-type lectins) involved in immune responses of the tobacco hornworm, Manduca sexta.