Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family.

Injection of heat-killed bacteria into larvae of the large tenebrionid beetle Zophobas atratus (Insecta, Endopterygota, Coleoptera) results in the appearance in the hemolymph of a potent antibacterial activity as evidenced by a plate growth inhibition assay. We have isolated three peptides (A-C) from this immune hemolymph which probably account for most of this activity. Their primary structures were established by a combination of peptide sequencing and molecular mass determination by mass spectrometry. Peptide A, which is bactericidal against Gram-negative cells, is a 74-residue glycine-rich molecule with no sequence homology to known peptides. We propose the name coleoptericin for this novel inducible antibacterial peptide. Peptides B and C are isoforms of a 43-residue peptide which contains 6 cysteines and shows significant sequence homology to insect defensins, initially reported from dipteran insects. This peptide is active against Gram-positive bacteria. The results are discussed in connection with recent studies on inducible antibacterial peptides present in the three other major orders of the endopterygote clade of insects: the Lepidoptera, Diptera, and Hymenoptera.     

Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval-pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

Isolation and identification of paralytic peptides from hemolymph of the lepidopteran insects Manduca sexta, Spodoptera exigua, and Heliothis virescens

Seven paralytic peptides were isolated and identified from lepidopteran hemolymph. All of these peptides cause rapid, rigid paralysis when injected into Manduca sexta and some other lepidopteran larvae. Each peptide contains 23 amino acid residues including 2 cysteines and the carboxyl termini are acidic. Synthetic peptides in the disulfide or reduced forms, and as carboxyl-terminal acids or amides were equally paralytic. The most potent paralytic peptide, Mas PP I, has the following sequence: H-Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu- Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The two peptides from M. sexta hemolymph are remarkable in that they are autoparalytic (i.e. factors in collected hemolymph that are paralytic when injected into the same larvae).     

Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.     

Viresin. A novel antibacterial protein from immune hemolymph of Heliothis virescens pupae.

Immune hemolymph was collected from fifth instar larvae and 1-day-old pupae of Heliothis virescens after injection of prepupae with live Enterobacter cloacae. Induction of antibacterial activity against Escherichia coli K12 D31 was 7.5 times greater in pupal than in larval immune hemolymph. Lysozyme activity of immune pupal hemolymph against Micrococcus lysodeikticus was 11 times greater when compared with lysozyme activity of immune larval hemolymph. Early pupal immune response with regard to antibacterial activity was much greater than larval immune response in H. virescens. Normal pupal hemolymph showed an increase in antibacterial activity and lysozyme that was induced during metamorphosis. Antibacterial protein was isolated together with lysozyme by gel filtration chromatography and then separated from lysozyme by sequential electrophoresis with a native acid gel and SDS gel. Molecular mass of antibacterial protein was estimated to be 12 kDa. The N-terminal amino acid sequence of 12-kDa protein was different from those of antibacterial molecules found in other insects and has not been identified before. A sample containing 12-kDa protein was negative for immunoblotting with anti-synthetic cecropin B antibody. We have named the novel 12-kDa antibacterial protein viresin. Viresin showed antibacterial activity against several Gram-negative bacteria including E. cloacae but not against Gram-positive bacteria.     

Isolation of a novel basic FGF-binding peptide with potent antiangiogenetic activity

Basic fibroblast growth factor (bFGF), which plays an important role in tumour angiogenesis and progression, provides a potential target for cancer therapy. Here we screened a phage display heptapeptide library with bFGF and identified 11 specific bFGF-binding phage clones. Two of these clones had identical sequence and the corresponding peptide (referred to as P7) showed high homology to the immunoglobulin-like (Ig-like) domain III (D3) of high-affinity bFGF receptors, FGFR1 (IIIc) and FGFR2 (IIIc). The P7 peptide and its corresponding motif in D3 of FGFRs both carried negative charges and shared similar hydrophobic profiles. Functional analysis demonstrated that synthetic P7 peptides mediate strong inhibition of bFGF-induced cell proliferation and neovascularization. Our results demonstrate that the P7 peptide is a potent bFGF antagonist with strong antiangiogenetic activity, and might have therapeutic potential in cancer therapy.     

Characterization of the Ryanodine Receptor-Ca2+ Release Channel from the Thoracic Tissues of the Lepidopteran Insect Heliothis virescens

The existence of invertebrate forms of the RyR has recently been confirmed (Takeshima et al., 1994, Puente et al., 2000). However, information on the functional properties of this insect RyR is still limited. We report the functional characterization of a RyR from the thoracic muscle of H. virescens (Scott-Ward et al., 1997). A simple purification protocol produced membranes from homogenized prefrozen H. virescens thoracic muscle with a [³H]-ryanodine binding activity of 1.19 ± 0.21 pmol/mg protein (mean ±se; n= 4). [³H]-Ryanodine binding to the H. virescens receptor was dependent on the ryanodine concentration in a hyperbolic fashion with a K _D of 3.82 nm (n= 4). [³H]-ryanodine binding was dependent on [Ca²⁺] in a biphasic manner and was stimulated by 1 mm ATP. Millimolar caffeine did not stimulate [³H]-ryanodine binding to H. virescens membranes in the presence of either nanomolar or micromolar Ca²⁺. A protein of at least 400 KDa was recognized in H. virescens membrane proteins by a specific anti-H. virescens RyR antibody. Discontinuous density sucrose gradient fractionation of microsomal membranes produced vesicles suitable for single-channel studies. Ca²⁺-sensitive, Ca²⁺-permeable channels were successfully inserted into artificial lipid bilayers from H. virescens membrane vesicles. The H. virescens RyR-channel displayed a Ca²⁺ conductance of ∼110 pS and underwent a persistent and characteristic modification of ion handling and gating following addition of 100 nm ryanodine. The gating of H. virescens channels was sensitive to ATP and ruthenium red in a manner similar to mammalian RyR. This is the first report to describe the single channel and [³H]-ryanodine binding properties of a native insect RyR. Received: 3 July 2000/Revised: 17 October 2000     

Isolation and molecular characterization of a novel strain of Bacillus with antifungal activity from the sorghum rhizosphere

We looked for bacterial strains with antifungal activity in the sorghum rhizosphere. A prescreening procedure to search for hemolytic activity among the isolated strains allowed us to detect good fungitoxic activity in a bacterial isolate that we named UM96. This bacterial isolate showed strong growth inhibition in bioassays against the pathogens Diaporthe phaseolorum, Colletotrichum acutatum, Rhizoctonia solani, and Fusarium oxysporum. The supernatant of isolate UM96 also showed strong hemolytic activity, which was not observed in the protease-treated supernatant. However, the supernatant that was treated with protease had similar antagonistic effects to those exhibited by the supernatant that was not treated with this enzyme. These results suggest that a bacteriocin-like compound is responsible for the hemolytic activity; whereas, as far as antifungal effect is concerned, an antibiotic of nonribosomal origin, such as a lipopeptide, might be acting. Further molecular characterization by partial 16S rDNA sequencing placed isolate UM96 in a cluster with Bacillus amyloliquefaciens; however, the highest identity match found in databases of Bacillus species was 91% identity. This suggests that Bacillus sp UM96 might be a novel species.     

Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens

Summary Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin β₁, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of culturedHeliothis virescens midgut cells; clusters of immunostained integrin β₁-like material, indicative of activated integrin, were detected on aggregating midgut columnar cells. Growth factor-like peptides (midgut differentiation factors 1 and 2 [MDF1 and MDF2]), isolated from conditioned medium containingManduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells toBacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150–200% increase in the numbers of midgut stem and differentiating cells. However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics. MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells. However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered. Products mentioned in this article are not endorsed by the U.S. Department of Agriculture.     

Cloning and characterization of a microsomal epoxide hydrolase from Heliothis virescens

Epoxide hydrolases (EHs) are α/β-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs). In this study we obtained a full-length cDNA, hvmeh1, from the generalist feeder Heliothis virescens that encoded a highly active EH, Hv-mEH1. Of the 10 different EH substrates that were tested, Hv-mEH1 showed the highest specific activity (1180 nmol min^?1 mg^?1) for a 1,2-disubstituted epoxide-containing fluorescent substrate. This specific activity was more than 25- and 3900-fold higher than that for the general EH substrates cis-stilbene oxide and trans-stilbene oxide, respectively. Although phylogenetic analysis placed Hv-mEH1 in a clade with some lepidopteran JH metabolizing EHs (JHEHs), JH III was a relatively poor substrate for Hv-mEH1. Hv-mEH1 showed a unique substrate selectivity profile for the substrates tested in comparison to those of MsJHEH, a well-characterized JHEH from Manduca sexta, and hmEH, a human microsomal EH. Hv-mEH1 also showed unique enzyme inhibition profiles to JH-like urea, JH-like secondary amide, JH-like primary amide, and non-JH-like primary amide compounds in comparison to MsJHEH and hmEH. Although Hv-mEH1 is capable of metabolizing JH III, our findings suggest that this enzymatic activity does not play a significant role in the metabolism of JH in the caterpillar. The ability of Hv-mEH1 to rapidly hydrolyze 1,2-disubstituted epoxides suggests that it may play roles in the metabolism of fatty acid epoxides such as those that are commonly found in the diet of Heliothis.     

Isolation and identification of a novel endophytic bacterial strain with antifungal activity from wild blueberryVaccinium uliginosum

A strain of endophytic bacterium with a good anti-fungal ability was first isolated fromVaccinium uliginosum. It was identified asSerratia marcescens using the 16s rDNA sequence homology and through its physiological and biochemical characteristics. Through the alignment and cladistic analysis of homologous nucleotide sequences of knownSerratia marcescens, it was found to be a novel subspecies ofS. marcescens. An extensive suppressive spectrum of this endophytic strain ofS. marcescens against plant pathogenic fungi was observed. In particular, it inhibited the growth of the causal agents of blueberry leaf spot and of other ten plant fungal pathogens. The inhibition rate, among the different fungi, ranged from 22.7–70.5%. The study indicated the potential use of thisS. marcescens endophytic strain in the biological control of blueberry’s leaf spot as well as many other plant diseases. To our knowledge, this was the first time that an endophyticS. marcescens was isolated from a wild blueberry in China, and may also represent a novel introduction to the world.     

Isolation and characterization of a novel Bacillus strain from coffee phyllosphere showing antifungal activity

AIMS: The isolation and characterization of a novel coffee-associated Bacillus mojavensis strain, designated as strain AB1, and its survival on the coffee phyllosphere. METHODS AND RESULTS: A pair of 16S rDNA primers was designed to amplify a highly variable region within the 16S rDNA gene of Bacillus spp., with the purpose of identifying the AB1 isolate through PCR and sequence analysis. By this method, AB1 was identified as a strain of B. mojavensis. Bioassays were carried out to characterize the broad spectrum antifungal activity of AB1. Plant colonization studies revealed that AB1 could colonize the coffee phyllosphere better than Bacillus thuringiensis. CONCLUSIONS: These studies suggest that AB1 could be a new strain of B. mojavensis. AB1 is also shown to have antifungal activity against a wide spectrum of pathogenic fungi. The antifungal metabolite of AB1 has been partially characterized as a thermostable, protease- and alkali-resistant substance that is secreted into the surrounding medium. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as is known, this is the first strain of B. mojavensis which has been identified as inhabiting the coffee phyllosphere. The study highlights the potential use of AB1 as an antifungal agent in the coffee crop and as a delivery agent of the insecticidal toxin of B. thuringiensis to the coffee phyllosphere. The 16S rRNA identification strategy discussed could also be used in the identification of other new Bacillus strains.     

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301–9], with some modifications. A DEAE-Sepharose, equilibrated with 20 mM Tris–HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS–Tricine gel electrophoresis with a molecular mass of approximately 6 kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314 bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.     

Structural,immunocytochemical and initial biochemical characterization of NAOH-extracted gap junctions from an insect,Heliothis virescens

Subcellular fractions enriched in gap junctions with an ultrastructure similar to those in intact insect tissue have been obtained by extracting crude membranes from the tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) with 2.5 mM NaOH. n-Octyl--d-glucopyranoside (OG) was used to further purify integral membrane proteins in the NaOH-extracted fractions. A polyclonal antibody (R16) is described that specifically labels nonextracted and NaOH-extracted gap junctions in cell fractions by electron microscope immunocytochemistry. R16 immunostaining of sectioned Heliothis testis at the light-microscope level yields a pattern of immunoreactivity consistent with the distribution of gap junctions in the tissue. R16 identifies a 40-kDa protein as a candidate gap junction protein on immunoblots of crude membrane, NaOH-extracted and NaOH/OG-extracted fractions.     

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.     

Isolation of a novel legumin-like lectin with potent hemagglutinating activity from seeds of the Chinese chestnut Castanea mollisima

A novel mannose- and glucose-specific lectin with high hemagglutinating activity was isolated from seeds of the Chinese chestnut Castanea mollisima. The lectin possessed a molecular mass of 140 kDa and was made up of two subunits, one with a molecular mass of 31 kDa and another with a molecular mass of 32 kDa. They exhibited substantial homology in N-terminal sequence to the storage protein legumin. The lectin was unstable in the presence of acid and alkali and at temperatures above 50 degrees C, but it was unaffected by various salts. The lectin was purified with a procedure involving ion exchange chromatography on CM-Sepharose, Q-Sepharose and Resource Q and gel filtration on Superose 12.