Pongsak Khunrae

Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.

The identification and expression of the full-length HtrA2 gene from Penaeus monodon (black tiger shrimp).

HtrA2 is an apoptosis-activating protein to enhance the apoptotic process by preventing the formation of the IAP-caspase complex, thus freeing caspase to trigger the apoptosis pathway. Here, we presented the full-length sequence of HtrA2 from the black tiger shrimp (PmHtrA2). The full-length PmHtrA2 transcript was 1403 bp with a 1338 bp open reading frame encoding 445 amino acids and contains 5 conserved domains, namely, a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a serine protease domain, and a PDZ domain normally found in HtrA2 proteins of other organisms. The mature form of PmHtrA2 was cloned into the pET28b(+) and pET15bThio vectors, and the expression of the protein was compared in Escherichia coli BL21 DE3 and BL21 RIL (CodonPlus) strains. Greater quantities of stable and soluble PmHtrA2 were expressed as a thioredoxin fusion protein in E. coli BL21 RIL (CodonPlus) cells with the recombinant pET15bThio-PmHtrA2 vector. To investigate the expression of PmHtrA2 in shrimp, the crude proteins from several shrimp tissues were imaged by Western blot using the polyclonal antibody specific to the recombinant PmHtrA2. The expression of the 47-kDa immature PmHtrA2 protein could be detected in shrimp lysates from the gills and the muscles. This study is the first to report the full-length PmHtrA2 gene, which is functional in black tiger shrimp and will lead to more focused studies on the function of PmHtrA2 in apoptosis regulation during immune responses to viral infection in shrimp.

Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection

White spot syndrome virus (WSSV) is a major causative agent in shrimp farming. Consequently, RNAi technology is an effective strategy to prevent WSSV infection in shrimp especially dsRNA targeting to rr2 of WSSV. In an effort to develop dsRNA expression in shrimp for control of WSSV infection, we developed a recombinant baculovirus expressing recombinant VP28 as the gene delivery system to carry a gene encoding dsRNA specific to rr2 for triggering the RNAi process in shrimp. The results showed that the recombinant baculovirus harboring VP28 was able to express VP28 indicated by Western blot with polyclonal antibody specific to VP28. VP28 transcript was detected in shrimp hemocytes after co-culture hemocytes with the recombinant baculovirus displaying VP28. In addition, we found that shrimp injected with the recombinant baculovirus displaying VP28 and encoding dsRNA synthetic gene specific to rr2 (Bac-VP28-dsrr2) showed the lowest cumulative mortality (33%) at 14days post infection (dpi) when compared to shrimp injected with baculovirus displaying VP28 (Bac-VP28) (64% cumulative mortality) (p<0.05). According to the results, shrimp injected with Bac-VP28-dsrr2 also showed significantly lower WSSV copies than shrimp injected with Bac-VP28 (p<0.05) along with the down-regulation of rr2 expression at 1, 3 and 7dpi. In conclusion, the Bac-VP28-dsrr2 was effective in prevention of WSSV infection. Therefore, the results obtained here can be applied to the prevention of WSSV infection by mixing the recombinant baculovirus with shrimp feed in the future.

Knockdown of Litopenaeus vannamei HtrA2, an up-regulated gene in response to WSSV infection, leading to delayed shrimp mortality.

HtrA2 is an apoptosis-activating gene that enhances the apoptotic process by preventing the formation of the IAP-caspase complex, thereby freeing caspase to trigger the apoptosis pathway. In this study, we presented the full-length cDNA sequence of HtrA2 from Litopenaeus vannamei (LvHtrA2). The full-length LvHtrA2 was 1335 bp, encoding 444 amino acids. This deduced amino acid sequence contained five conserved domains: a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a trimerization motif, a serine protease domain, and a PDZ domain normally found in the HtrA2 proteins of other organisms. A phylogenetic analysis revealed that LvHtrA2 clustered with the HtrA2 from other invertebrates and was closely related to Penaeus monodon HtrA2 (PmHtrA2). RT-PCR with RNA extracts from L. vannamei revealed that LvHtrA2 expression was found in several tissues, including the lymphoid organs, the haemocytes, the hepatopancreas, the gill, and the stomach, with different expression levels. When determining the role of LvHtrA2 in WSSV infection, it was found that LvHtrA2 transcription was early up-regulated in the WSSV-infected shrimp at 8h post-infection (p.i.) and expression still remained high at 48 h p.i.. It also demonstrated that dsRNA specific to LvHtrA2 reduced the cumulative mortality in the WSSV-infected shrimp compared with the control group. Additionally, depletion of the LvHtrA2 transcripts reduced expression levels for caspase-3 (Cap-3) gene in shrimp. This result could suggest that LvHtrA2 may involved in apoptosis mediated mortality rather than providing immune protection during WSSV infection.

Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.)

Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD(+). Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N(2) immediately prior to X-ray diffraction experiments. The crystals belonged to space group C222(1), with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress.

ICP35 Is a TREX-Like Protein Identified in White Spot Syndrome Virus

ICP35 is a non-structural protein from White spot syndrome virus believed to be important in viral replication. Since ICP35 was found to localize in the host nucleus, it has been speculated that the function of ICP35 might be involved in the interaction of DNA. In this study, we overexpressed, purified and characterized ICP35. The thioredoxin-fused ICP35 (thio-ICP35) was strongly expressed in E. coli and be able to form itself into dimers. Investigation of the interaction between ICP35 and DNA revealed that ICP35 can perform DNase activity. Structural model of ICP35 was successfully built on TREX1, suggesting that ICP35 might adopt the folding similar to that of TREX1 protein. Several residues important for dimerization in TREX1 are also conserved in ICP35. Residue Asn126 and Asp132, which are seen to be in close proximity to metal ions in the ICP35 model, were shown through site-directed mutagenesis to be critical for DNase activity.

Characterization of Bacteriophages Infecting Clinical Isolates of Clostridium difficile

Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0–6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.

C-terminal domain of WSSV VP37 is responsible for shrimp haemocytes binding which can be inhibited by sulfated galactan

ABSTRACT Viral envelope proteins play an important role in facilitating the attachment of viruses to the surface of host cells. Here, we investigated the binding of White Spot Syndrome Virus (WSSV) VP37 to haemocytes of whiteleg shrimp, Litopenaeus vannamei. Three versions of recombinant VP37 proteins, including full length VP37 (VP37(1‐281)), C‐terminal domain VP37 (VP37(111‐281)) and C‐terminal domain disrupted VP37 (VP37(1‐250)) were individually expressed and tested for their haemocytes binding ability. Through an ELISA‐based binding assay, we found that VP37(111‐281) bound to shrimp haemocytes in a similar way to VP37(1‐281), while VP37(1‐250) exhibited a significantly weaker binding. This suggests that the C‐terminal domain of VP37 is required for the binding of VP37 to shrimp haemocytes. Furthermore, we found that the binding of VP37 to shrimp haemocytes was impaired by pre‐incubation of VP37 with sulfated galactan (SG), a sulfated polysaccharide derived from red seaweed (Gracilaria fisheri). Previously, it has been shown that a type of sulfated polysaccharide, heparin, is also present in L. vannamei. To investigate the role of heparin as a receptor for VP37, the binding of VP37 to porcine heparin, whose structure is similar to that found in L.vannamei, was investigated in a Surface Plasmon Resonance (SPR) system. The results showed that VP37 bound strongly to heparin with binding affinity (KD) of 1.0 &mgr;M and the binding was significantly blocked by SG. These findings have lead us to propose that the attachment of WSSV might be mediated by the interaction between VP37 and a heparin‐like molecule presented on the shrimp cells. HighlightsThe bindings of WSSV VP37 proteins to shrimp haemocytes were investigated.C‐terminal domain of VP37 is required for the binding to shrimp haemocytes.SG can block the binding of the C‐terminal domain of VP37 to shrimp haemocytes.The C‐terminal domain of VP37 can interact with heparin.The binding of C‐terminal domain to heparin can be blocked by SG.