Stable marker on flagellin gene sequences related to arabinose non-assimilating pathogenic Burkholderia pseudomallei

Using PCR-based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15-bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+). This finding led to the development of a PCR-based method in order to differentiate and identify pathogenic B. pseudomallei for epidemiological study. A pair of specific primers was designed covering the 15-bp deletion region at the variable domain. PCR-amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara - isolates of B. pseudomallei, respectively. Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers. The results suggest that the flagellin gene sequences of both B. pseudomallei biotypes in this region are stable and distinct. This method can be applied and useful for the epidemiological study of B. pseudomallei.     

A decrease in cytotoxic and haemolytic activities by inactivation of a single enterotoxin gene in Bacillus cereus Cx5

With the ability to colonize in the guts of a broad range of mosquito larvae, Bacillus cereus Cx5 has a potential to be utilized as a new host cell for the production of mosquito-larvicidal toxins aimed for mosquito control. However, the presence of one single (entFM) and two triple (hblCDA and nheABC) enterotoxin genes were previously confirmed in strain Cx5, raising concerns in its immediate use in the environment. Cx5 cells indeed showed recognizable levels of haemolytic and Vero cell cytotoxic activities. In this study, the single enterotoxin gene in B. cereus Cx5 (entCx5) has been inactivated in order to study the relationship between the presence of this gene and the cytotoxic and haemolytic activities found in the strain. We constructed a gene disruption plasmid, pTentCx5TV2, harbouring a truncated entCx5 gene in the temperature-sensitive shuttle vector, pUCTV2. After introducing the plasmid into B. cereus Cx5, we found that the plasmid was integrated via single crossover into the chromosome as expected at the entCx5 locus, disrupting the gene. Analysis of one mutant strain revealed that Vero cell cytotoxicity and haemolytic activity of the mutant were dramatically decreased compared to that of the wild type strain. This indicates an involvement of the entCx5 gene in haemolytic and Vero cell cytotoxic activities. The results also imply that there is a high possibility to generate an effective, and safe, host cell based on B. cereus Cx5 for the production of mosquito-larvicidal toxin.     

Ex vivo cytotoxicity of the Bacillus thuringiensis Cry4B delta-endotoxin to isolated midguts of Aedes aegypti larvae

The pathological effect of the Bacillus thuringiensis Cry delta- endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.     

Glutamic acid and alanine spacer is not necessary for removal of MFα-1 signal sequence fused to the human growth hormone produced from Pichia pastoris

Human growth hormone (hGH) cDNA was synthesised using codons preferred by Escherichia coli, except for the first 20 amino acids, which were changed to that preferred by Saccharomyces cerevisiae and Pichia pastoris. Polymerase chain reaction (PCR) overlapping approach was employed to create synthetic hGH without glutamic acid-alanine (glu-ala), or with one and two glu-ala spacers (hGH, hGH1 and hGH2, respectively). The necessity of a glu-ala spacer in the cleavage of S. cerevisiae alpha mating factor-1 (MF-1) secretion signal from the synthetic hGH was also investigated. Three types of hGH constructs were integrated into P. pastoris genome, the zeocin-resistant transformants were selected and expression of hGH was determined. A 22-kDa band of secreted hGH was further determined by N-terminal peptide sequencing. The result suggested that the removal of glu-ala from the hGH1 and hGH2 was not efficient and only the hGH construct showed the complete cleavage of the signal sequence, giving a similar N-terminus as the mature hGH. hGH expression was optimized to increase the yield of the protein from the hGH construct (no glu-ala) to 190 mg/l from a 10-ml induction medium.     

Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Achieving high-level expression of the Bacillus thuringiensis Cry4Aa mosquito-larvicidal protein was demonstrated. The 130-kDa Cry4Aa protoxin was overexpressed as an inclusion body in Escherichia coli under the control of the tac promoter together with the cry4Ba promoter. The solubility of the toxin inclusions in carbonate buffer, pH 10.0, was markedly enhanced at a cultivation temperature of 30 degrees C. Elimination of the tryptic cleavage site at Arg-235 in the loop between helices 5 and 6 still retained the high-level toxicity of E. coli cells expressing the Cry4Aa mutant against Aedes aegypti larvae. Trypsin digestion of the R235Q mutant protoxin produced a protease-resistant fragment of ca. 65kDa. A homogeneous product of the 65-kDa trypsin-treated R203Q protein was obtained after size-exclusion chromatography that would pave the way for the further crystallisation and X-ray crystallographic studies.     

Bacillus thuringiensis Cry4A and Cry4B mosquito-larvicidal proteins: homology-based 3D model and implications for toxin activity

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B delta-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel beta-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting alpha4 and alpha5 of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.     

Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.     

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.     

Characterization of Argonaute cDNA from Penaeus monodon and implication of its role in RNA interference

RNA interference (RNAi) has recently become a promising strategy for therapeutic of several viral diseases including those in the black tiger shrimp Penaeus monodon. However, the protein components that play role in RNAi in P. monodon have not yet been identified. Here, we report the cloning and functional characterization of a cDNA encoding Argonaute, a principal constituent of RNAi pathway in P. monodon. P. monodon’s Argonaute (Pem-AGO) exhibited the two signature domains, PAZ and PIWI. Substantial level of Pem-ago expression could be suppressed by double-stranded RNA (dsRNA) that targeted PAZ coding sequence in shrimp primary culture of Oka cells. The Pem-ago depleted cells showed impaired RNAi as the expression of an endogenous gene was rescued from the dsRNA-mediated silencing in these cells. Our results imply that Pem-ago is required for effective RNAi in P. monodon and thus identify the first protein constituent of RNAi machinery in penaeid shrimp.