Identification, cloning and expression of a second gene (vpr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca that displays immunosuppressive activity

Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star™ (DE3) cells was induced by the addition of 0.5 mM IPTG. Cultures were grown at 24 and 37 °C, and VPr1 more readily partitioned into the soluble fraction at 24 °C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed.     

Differential gene expression profiles in the venom gland/sac of Orancistrocerus drewseni (Hymenoptera: Eumenidae)

To determine differential gene expression profiles in the venom gland and sac (gland/sac) of a solitary hunting wasp species, Orancistrocerus drewseni Saussure (1857), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 498 expressed sequence tags (EST) were clustered and assembled into 205 contigs (94 multiple sequences and 111 singletons). About 65% (134) of the contigs had matched BLASTx hits (E≤10^?4). Among these, 115 contigs had similarity to proteins with assigned molecular function in the Gene Ontology database, and most of them (112 contigs, 83%) were homologous to genes from Hymenoptera, particularly to Apis mellifera (98 contigs). The contigs encoding hyaluronidase and phospholipase A2, known to be main components of wasp venoms, were found in high frequencies (27 and 4%, respectively, as judged by the number of ESTs) in the gene ontology category of catalytic activity. Full‐length open reading frames of hyaluronidase and phospholipase A2 were characterized and their abundance in the venom gland/sac was confirmed by quantitative real‐time PCR. Several contigs encoding enzymes, including zinc‐metallopeptidases that are likely involved in the processing and activation of venomous proteins or peptides, were also identified from the library. Discovery of venom gland/sac‐specific genes should promote further studies on biologically active components in the venom of O. drewseni. © 2009 Wiley Periodicals, Inc.     

The role of Pimpla hypochondriaca venom in the suppression of pupal Noctuid host immunity

When pupae of the Tomato moth (Lacanobia oleracea L.) (Lepidoptera: Noctuidae) are injected with venom from the endoparasitoid wasp Pimpla hypochondriaca Retzius (Hymenoptera: Ichneumonidae), they show an increased susceptibility to the fungal entomopathogen Metarhizium anisopliae Sorokin (Fungi Imperfecti: Deuteromycotina). This observation, coupled with the fact that wasp eggs are comparatively unlikely to be encapsulated when implanted into envenomated pupae, suggests that venom of P. hypochondriaca suppresses the cellular immune defense mechanisms of L. oleracea. Injection of host pupae with the venom of P. hypochondriaca has a rapid, adverse effect on the normal respiration pattern of L. oleracea. It is possible that severe disruption to host-metabolism may contribute to a general failure in the hosts' normal immune response repertoire.     

The mode of action of venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae) involves Ca+2‐dependent cell death pathways

The endoparasitoid Pimpla hypochondriaca injects venom during oviposition to condition its lepidopteran hosts. Venom is a complex mixture of proteins and polypeptides, many of which have been identified as enzymes, including phenoloxidase, endopeptidase, aminopeptidase, hydrolase, and angiotensin‐converting enzyme. Constituents of the venom have been shown to possess cytolytic and paralytic activity, but the modes of action of factor(s) responsible for exerting such effects have not been deciphered. In this study, we examined the mode of action of isolated venom using cultured cells (BTI‐TN‐5B1‐4). A series of blockage and inhibition assays were performed using a potent inhibitor (phenylthiourea, PTU) of venom phenoloxidase, and anti‐calreticulin antibodies. Monolayers exposed to venom alone were highly susceptible with more than 84.6±2.3% dead within 15 min. Susceptible cells displayed a retraction of cytoplasmic extensions, rounding, and swelling prior to lysis in more than half (55.7±1.7%) of the dying cells. Within 15 min of exposure to venom, cells displayed qualitative increases in [Ca⁺²]_i as evidenced by staining with the calcium‐sensitive probe fluo‐4 AM, and mitochondrial membrane potential (ΔΨ_m) was undetectable by 5 min post‐treatment with venom. These venom‐mediated changes occurred regardless of whether an external source of calcium was present, or whether venom was pre‐treated with PTU. In contrast, venom toxicity was attenuated by treatment with anti‐calreticulin antibodies. Not only did fewer cells die when exposed to antibody‐treated venom but also cell swelling diminished and no increases in intracellular calcium were detected. A possible mode of action for the venom is discussed. © 2009 Wiley Periodicals, Inc.     

Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae)

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two‐dimensional gel electrophoresis and separation by ion‐exchange and reverse‐phase high‐performance liquid chromatography followed by mass spectrometry using tanden matrix‐assisted laser desorption/ionization with time‐of‐flight (MALDI‐TOF/TOF) mass spectrometry and electrospray ionization‐quadrupole with time‐of‐flight (ESI‐Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10‐ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.     

cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.     

Interactions between Escherichia coli and the plant‐parasitic nematode Meloidogyne javanica

Aims: To determine the potential of the plant‐parasitic nematode Meloidogyne javanica to serve as a temporary reservoir for Escherichia coli. Methods and Results: The adhesion to and persistence of E. coli on the surface of M. javanica were evaluated at different times and temperatures. A pure culture of green fluorescent protein (GFP) tagged E. coli was mixed with ca. 1000 J2 M. javanica for 2 h at 25°C. The nematodes were then washed and the rate of the adhesion of the bacteria to the nematodes was determined by counting the viable nematode‐associated E. coli, and by fluorescence microscopy. A dose‐dependent adhesion rate was observed only at a bacterium to nematode ratio of 10⁴–10⁶ : 1. The adhesion of E. coli to the nematodes was also tested over a 24 h‐period at 4°C, 25°C and 37°C. At 4°C and 37°C, maximal adhesion was observed at 5 h; whereas at 25°C, maximal adherence was observed at 8 h. Survival experiments showed that the bacteria could be detected on the nematodes for up to 2 weeks when incubated at 4°C and 25°C, but not at 37°C. Conclusions: Under laboratory conditions, at 4°C and 25°C, M. javanica could serve as a temporary vector for E. coli for up to 2 weeks. Significance and Impact of the Study: These findings support the hypothesis that, in the presence of high concentrations of E. coli, M. javanica might serve as a potential vehicle for the transmission of food‐borne pathogens.     

Expression of biologically active recombinant antifreeze protein His-MpAFP149 from the desert beetle (<Emphasis Type="Italic">Microdera punctipennis dzungarica</Emphasis>) in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An insect antifreeze protein gene Mpafp149 was cloned by the RT-PCR approach from the desert beetle Microdera punctipennis dzungarica. Sequence analysis revealed that this gene encoding a protein of 120 amid acids and this protein showed 65–76% homology with other insect antifreeze proteins, the deduced amino acid sequence displays very high similarities in those regions that contain tandem the 12-residue repeats (TCTxSxxCxxAx) domain and the TCT motif. Mpafp149 gene was cloned into pET-28a vector and expressed in Escherichia coli. A single-step purification based on specific binding of histidine residues was achieved. The purified His-MpAFP149 was SDS–PAGE analyzed, showing an atypical migration with molecular weight of about 24 kDa. The expression of His-MpAFP149 was confirmed by Western blot with specific binding to anti-GST-MpAFP149 antibody. The thermal hysteresis activity of the purified recombinant protein was 0.915°C at 0.09 mg/ml, and the supercooling point was −9.6°C at 0.03 mg/ml. In vitro antifreeze activity assay by measuring the survival rate of bacteria at −7 and −20°C respectively, with the protection of His-MpAFP149 showed that the His-MpAFP149 fusion protein was able to enhance the freeze resistance of bacteria.     

Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host,Pieris rapae (Lepidoptera: Pieridae)

Pteromalus puparum is a predominant endoparasitoid wasp of Pieris rapae. Its venom is the only active factor injected into host associated with oviposition. In this report, we explored whether the venom alone from this wasp affects the endocrine system of its host or not. We monitored the changes of hemolymph juvenile hormone (JH; only JH III detected), ecdysteroid, and juvenile hormone esterase activity (JHE) over 72 h in parasitized and venom‐microinjected P. rapae pupae. Non‐parasitized and PBS‐microinjected P. rapae served as controls. Results showed that JH titers were significantly higher in parasitized and venom‐microinjected pupae than that in control pupae during 24 to 72 h. After 12 h, JH titers were significantly promoted by parasitization and venom microinjection. JHE activities of non‐parasitized and PBS‐microinjected pupae were significantly higher than that of parasitized and venom‐microinjected pupae, which was with a peak at 12 h (parasitized pupae) or 24 h (venom‐microinjected pupae) during 6 to 48 and 12 to 36 h, respectively. The hemolymph titers of ecdysteroid in non‐parasitized and PBS‐microinjected pupae increased rapidly during 12 to 36 h with a peak at 36 h, and were higher than treatments before 48 h, while presenting a significant difference at 24 to 48 h between the treatments and controls. The results demonstrate that venom alone of this parasitoid wasp can disrupt its host's endocrine system. © 2009 Wiley Periodicals, Inc.     

Effects of temperature on the growth and reproduction characteristics of Bemisia tabaci B‐biotype and Trialeurodes vaporariorum

The development period, survival rate, longevity and fecundity of two whiteflies, Bemisia tabaci B‐biotype and Trialeurodes vaporariorum (Homoptera: Aleyrodidae) were compared under different temperature laboratory conditions (15°C, 18°C, 21°C and 24°C). Egg development of B. tabaci B‐biotype was significantly longer compared with that of T. vaporariorum at 15°C, 18°C and 24°C. Significantly longer pseudo‐pupae development and lower survival rate were found in B. tabaci B‐biotype at 15°C compared with those at 18°C, 21°C and 24°C. Significantly higher fecundity was found in B. tabaci B‐biotype at 24°C compared with that at 15°C, 18°C and 21°C. However, the fecundity of T. vaporariorum was significantly lower at 24°C relative to that at 15°C, 18°C and 21°C. Significantly shorter 1st instar larval development was found in T. vaporariorum compared with that of B. tabaci at 15°C and 18°C. Significantly longer 2nd instar larval development was found in B. tabaci and T. vaporariorum at 15°C compared with that at 18°C, 21°C and 24°C. However, significantly shorter 3rd instar larval development was found in T. vaporariorum compared with that of B. tabaci at 15°C, 18°C and 24°C. The adaptive divergence of tolerance to relatively low temperature may be an important factor that results in the interspecific differentiation between the seasonal dynamics of these two whiteflies in China.     

Purification,Partial Characterizations,and N‐Terminal Amino Acid Sequence of a Procoagulant Protein FV‐2 from Daboia Russelli Siamensis (Myanmar) Venom

In this study, we purified and characterized the procoagulant protein FV‐2 from Daboia russelli siamensis (Myanmar) venom using ion‐exchange chromatography on CM‐Sephadex C‐50 and gel filtration on Superdex^TM G‐75 column. The activation of factor X and prothrombin was determined, respectively, by specific chromogenic substrates. The fibrinogen‐clotting activity, thermal stability, and pH stability were also determined. The N‐treminal sequence was determined by the National Center of Biomedical Analysis of China. In the end, FV‐2 was achieved with a molecular weight of 13,608.0 Da. It could activate factor X, but did not affect prothrombin or fibrinogen. The suitable pH was 6.5–7.5, and the suitable temperature ranged from 25 to 60°C. The N‐terminal sequence was Asn‐Phe‐Phe‐Gln‐Phe‐Ala‐Glu‐Met‐Ile‐Val‐Lys‐Met‐Thr‐Gly‐Lys. Taken together, our studies suggest that FV‐2 is a factor X–activating enzyme, which can activate factor X to factor Xa, but it has no effect on prothrombin and fibrinogen.     

A defensin‐like antimicrobial peptide from the venoms of spider,Ornithoctonus hainana

The defensin‐like antimicrobial peptides have been characterized from various other arthropods including insects, scorpions, and ticks. But no natural spider defensin‐like antimicrobial peptides have ever been isolated from spiders, except couple of cDNA and DNA sequences of five spider species revealed by previous genomic study. In this work, a defensin‐like antimicrobial peptide named Oh‐defensin was purified and characterized from the venoms of the spider, Ornithoctonus hainana. Oh‐defensin is composed of 52 amino acid (aa) residues including six Cys residues that possibly form three disulfide bridges. Its aa sequence is MLCKLSMFGAVLGV PACAIDCLPMGKTGGSCEGGVCGCRKLTFKILWDKKFG. By BLAST search, Oh‐defensin showed significant sequence similarity to other arthropod antimicrobial peptides of the defensin family. Oh‐defensin exerted potent antimicrobial activities against tested microorganisms including Gram‐positive bacteria, Gram‐negative bacteria, and fungi. The cDNA encoding Oh‐defensin precursor was also cloned from the cDNA library of O. hainana. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Using temperature and time criteria to control the effectiveness of continuous thermal sanitation of piggery effluent in terms of set microbial indicators

Aim: To determine the minimal conditions (temperature–time), necessary to achieve set sanitation targets for selected microbial indicators during the continuous thermal treatment of pig slurry. Methods and Results: The effectiveness of thermal treatment between 55 and 96°C was studied using Escherichia coli, enterococci, sulfite‐reducing Clostridia (SRC), mesophilic culturable bacteria (MCB), F+‐specific and somatic phages. Identification of SRC and MCB was performed using 16S rRNA gene analysis. Ten minutes at 70°C or 1 h at 60°C was sufficient to reduce the vegetative bacteria by 4–5 log₁₀, but it had little effect on somatic phages nor on spore formers, dominated by Clostridium sp. At 96°C, somatic phages were still detected, but there was a reduction of 3·1 log₁₀ for SRC and of 1·4 log₁₀ for MCB. At 96°C, Clostridium botulinum was identified among the thermotolerant MCB. Conclusion: Only those hygienic risks relating to mesophilic vegetative bacteria can be totally eliminated from pig slurry treated at 60°C (60 min) or 70°C (<10 min). Significance and Impact of the Study: Hygiene standards based on the removal of the indicators E. coli and enterococci can easily be met by treatment as low as 60°C (enabling, a low‐cost treatment using heat recovery). However, even at 96°C, certain pathogens may persist.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: Evolution of venom group II phospholipase A2's may imply gene duplications

The sequence coding for a snake venom phospholipase A₂ (PLA₂), BJUPLA₂, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA₂'s. A striking feature of the cDNA is the high sequence conservation of the 5 and 3 untranslated regions in cDNAs coding for PLA₂'s from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA₂ with group II PLA₂'s in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA₂'s.The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X76289.Correspondence to: A.M. Moura-da-Silva     

Isolation and characterization of new poly(3HB)‐accumulating star‐shaped cell‐aggregates‐forming thermophilic bacteria

Aims: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3‐hydroxybutyrate), poly(3HB), at elevated temperatures. Methods and Results: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram‐negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star‐shaped cell‐aggregates (SSCAs) during growth; only strain MW10 consisted of free‐living rod‐shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii , Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8 g l^?l (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. Conclusions: New PHA‐accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. Significance and Impact of the Study: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA‐accumulating bacteria and also by thermophilic bacteria. PHA‐producing thermophiles can significantly reduce the costs of fermentative PHA production.