Chartchai Krittanai

Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon

A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.

Heterologous expression and characterization of alternatively spliced glutathione S-transferases from a single Anopheles gene.

Three cDNA sequences of glutathione S-transferase (GST), adgst1-2, adgst1-3 and adgst1-4, which are alternatively spliced products of the adgst1AS1 gene, were obtained from fourth instar larvae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide sequences of these three cDNAs share >67% identity and the translated amino acid sequences share 61-64% identity. A comparison of the An. dirus to the An. gambiae enzymes shows that adGST1-2 versus agGST1-4, adGST1-3 versus agGST1-5 and adGST1-4 versus agGST1-3 have 85, 92 and 85% amino acid sequence identity, respectively, which confirms that orthologous isoenzymes occur across anopheline species. These three proteins were expressed at high levels, approximately 15-20 mg from 200 ml of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The subunit sizes of adGST1-2, adGST1-3 and adGST1-4 are 24.3, 23.9 and 25.1 kDa. The recombinant enzymes have high activities with 1-chloro-2,4-dinitrobenzene (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedly low activity with ethacrynic acid and p-nitrophenethyl bromide. adGST1-3 was shown to be the most active enzyme from the kinetic studies. Permethrin inhibition of CDNB activity, at varying concentrations of CDNB, was significantly different, being uncompetitive for adGST1-2, noncompetitive for adGST1-3 and competitive for adGST1-4. In contrast, permethrin inhibition with varying glutathione concentrations was noncompetitive for all three GSTs. Despite the enzymes being splicing products of the same gene and sharing identical sequence in the N-terminal 45 amino acids, these GSTs show distinct substrate specificities, kinetic properties and inhibition properties modulated by the differences in the C-terminus.

Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin

In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to approximately 110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent 3-14, together with a subsequent heating to 95 degrees C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant beta-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and M(r) of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.

Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity.

This study focused on an isolation and characterization of the circulating hemocytes in mud crab, Scylla olivacea. Isolation of specific cell types of hemocytes from crab hemolymph was accomplished by using 60% Percoll density gradient centrifugation. Four separated bands of the hemocytes were successfully obtained. Characterization of these isolated hemocytes by light microscope using trypan blue-rose bengal staining, rose bengal-hematoxilin staining, and phase contrast revealed four distinct types of hemocyte cells. Using their specific morphology and granularity, they were identified as hyaline cell (HC), small granular cell (SGC), large granular cell (LGC) and mixed granular cell (MGC). Transmission electron microscopy (TEM) revealed more details on specific cell size, size of cytoplasmic granule, and nuclear to cytoplasmic ratio, and confirmed the classification. Relative abundance of these cells types in the hemolymph of an adult crab were 15.50±8.22% for HC, 55.50±7.15% for SGC, 13.50±5.28% for LGC, and 15.50±3.50% for MGC. Proteomic analysis of protein expression for each specific cell types by two-dimensional electrophoresis identified two highly abundant proteins, prophenoloxidase (ProPO) and peroxinectin in LGC. Determination of phenoloxidase (PO) activity in each isolated cell types using in vitro and in situ chemical assays confirmed the presence of PO activity only in LGC. Based on an increased PO activity of crab hemolymph during the course of White Spot Syndrome Virus (WSSV) infection, these results suggest that prophenoloxidase pathway was employed for host defense mechanism against WSSV and it may link to the role of large granular hemocyte.

C-terminal hemocyanin from hemocytes of Penaeus vannamei interacts with ERK1/2 and undergoes serine phosphorylation.

To understand molecular immune response of Penaeus vannamei during Taura syndrome virus (TSV) infection, expression and functional proteomics studies were performed on hemocyanin, which is a major abundant protein in shrimp hemocytes. Two-dimensional electrophoresis (2-DE) revealed up-regulation of several C-terminal fragments of hemocyanin, whereas the N-terminal fragments were down-regulated during TSV infection. 2-D Western blot analysis showed that the C-terminal hemocyanin fragments had more acidic isoelectric points (pI), whereas the N-terminal fragments had less acidic pI. Further analysis by NetPhos showed a greater number of serine phosphorylation sites in the C-terminal hemocyanin. Additionally, motif scan using Scansite revealed ERK D-domain, which is required for activation of ERK1/2 effector kinase, as a kinase-binding site at the 527th valine in the C-terminal hemocyanin, whereas neither motif nor functional domain was found in the N-terminus. Co-immunoprecipitation confirmed the interaction between the C-terminal hemocyanin and ERK1/2. 1-D Western blot analysis showed that ERK1/2 was also up-regulated during TSV infection. Our findings demonstrate for the first time that ERK1/2 signaling pathway may play an important role in molecular immune response of P. vannamei upon TSV infection through its interaction with the C-terminal hemocyanin.

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery

Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non‐leptospirosis patients with a negative MAT were pooled and used as the negative control. 2‐D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI‐TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti‐human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications.

Mud crab susceptibility to disease from white spot syndrome virus is species-dependent

BackgroundBased on a report for one species (Scylla serrata), it is widely believed that mud crabs are relatively resistant to disease caused by white spot syndrome virus (WSSV). We tested this hypothesis by determining the degree of susceptibility in two species of mud crabs, Scylla olivacea and Scylla paramamosain, both of which were identified by mitochondrial 16 S ribosomal gene analysis. We compared single-dose and serial-dose WSSV challenges on S. olivacea and S. paramamosain.FindingsIn a preliminary test using S. olivacea alone, a dose of 1 × 106 WSSV copies/g gave 100% mortality within 7 days. In a subsequent test, 17 S. olivacea and 13 S. paramamosain were divided into test and control groups for challenge with WSSV at 5 incremental, biweekly doses starting from 1 × 104 and ending at 5 × 106 copies/g. For 11 S. olivacea challenged, 3 specimens died at doses between 1 × 105 and 5 × 105 copies/g and none died for 2 weeks after the subsequent dose (1 × 106 copies/g) that was lethal within 7 days in the preliminary test. However, after the final challenge on day 56 (5 × 106 copies/g), the remaining 7 of 11 S. olivacea (63.64%) died within 2 weeks. There was no mortality in the buffer-injected control crabs. For 9 S. paramamosain challenged in the same way, 5 (55.56%) died after challenge doses between 1 × 104 and 5 × 105 copies/g, and none died for 2 weeks after the challenge dose of 1 × 106 copies/g. After the final challenge (5 × 106 copies/g) on day 56, no S. paramamosain died during 2 weeks after the challenge, and 2 of 9 WSSV-infected S. paramamosain (22.22%) remained alive together with the control crabs until the end of the test on day 106. Viral loads in these survivors were low when compared to those in the moribund crabs.ConclusionsS. olivacea and S. paramamosain show wide variation in response to challenge with WSSV. S. olivacea and S. paramamosain are susceptible to white spot disease, and S. olivacea is more susceptible than S. paramamosain. Based on our single-challenge and serial challenge results, and on previous published work showing that S. serrata is relatively unaffected by WSSV infection, we propose that susceptibility to white spot disease in the genus Scylla is species-dependent and may also be dose-history dependent. In practical terms for shrimp farmers, it means that S. olivacea and S. paramamosain may pose less threat as WSSV carriers than S. serrata. For crab farmers, our results suggest that rearing of S. serrata would be a better choice than S. paramamosain or S. olivacea in terms of avoiding losses from seasonal outbreaks of white spot disease.

Quantitative analysis of time-series fluorescence microscopy using a spot tracking method: application to Min protein dynamics

The dynamics of MinD protein has been recognized as playing an important role in the accurate positioning of the septum during cell division. In this work, spot tracking technique (STT) was applied to track the motion and quantitatively characterize the dynamic behavior of green fluorescent protein-labeled MinD (GFP-MinD) in an Escherichia coli system. We investigated MinD dynamics on the level of particle ensemble or cluster focusing on the position and motion of the maximum in the spatial distribution of MinD proteins. The main results are twofold: (i) a demonstration of how STT could be an acceptable tool for MinD dynamics studies; and (ii) quantitative findings with parametric and non-parametric analyses. Specifically, experimental data monitored from the dividing E. coli cells (typically 4.98 ± 0.75 µm in length) has demonstrated a fast oscillation of the MinD protein between the two poles, with an average period of 54.6 ± 8.6 s. Observations of the oscillating trajectory and velocity show a trapping or localized behavior of MinD around the polar zone, with average localization velocity of 0.29 ± 0.06 µm/s; and flight switching was observed at the pole-to-pole leading edge, with an average switching velocity of 2.95 ± 0.31 µm/s. Subdiffusive motion of MinD proteins at the polar zone was found and investigated with the dynamic exponent, α of 0.34 ± 0.16. To compare with the Gaussian-based analysis, non-parametric statistical analysis and noise consideration were also performed.

Yellow head virus infection in black tiger shrimp reveals specific interaction with granule-containing hemocytes and crustinPm1 as a responsive protein

Yellow head virus (YHV) causes acute infections and mass mortality in black tiger shrimp culture. Our study aims to investigate molecular interaction between YHV and circulating hemocytes of Penaeus monodon at early infection. Total shrimp hemocytes were isolated by Percoll gradient centrifugation and identified by flow cytometric analysis. At least three types of hemocyte cells were identified as hyaline, semi-granular, and granular hemocytes. Experimental infection of YHV in shrimp culture demonstrated drastic changes in total and each hemocyte cell counts. Immunohistochemistry analysis demonstrated interaction and replication of YHV mainly with the granule-containing hemocytes and little to none in hyaline cell. These granule-containing hemocytes are proposed to be YHV targets providing the first line of defense to viral infection. Protein expression profiling of granule-containing hemocytes revealed several immune-responsive proteins including antimicrobial protein crustins (crustinPm1 and crustinPm4), alpha-2-macroglobulin, and kazal-type serine proteinase inhibitor. During an early phase of YHV infection at 6 hpi crustinPm1 illustrated a significant increase of mRNA and protein expression level in plasma. The results suggest that an antimicrobial crustinPm1 may participate in shrimp defense mechanism against YHV, especially on the granule-containing hemocytes.

Comparative proteome analysis of rubber latex serum from pathogenic fungi tolerant and susceptible rubber tree (Hevea brasiliensis)

UNLABELLED Many cultivated rubber trees (Hevea brasiliensis) are invaded by various Phytophthora species fungi, especially in tropical regions which result in crop yield losses. Comparative proteome analysis coupled with liquid chromatography electrospray/ionization (LC-ESI) mass spectrometry identification was employed to investigate the relative abundance of defense related proteins in Phytophthora sp. susceptible (RRIM600) and tolerant (BPM24) clones of rubber tree. Proteome maps of non-rubber constituent of these two model clones show similar protein counts, although some proteins show significant alterations in their abundance. Most of the differentially abundant proteins found in the serum of BPM24 illustrate the accumulation of defense related proteins that participate in plant defense mechanisms such as beta-1,3-glucanase, chitinase, and lectin. SDS-PAGE and 2-D Western blot analysis showed greater level of accumulation of beta-1,3-glucanase and chitinase in latex serum of BPM24 when compared to RRIM600. A functional study of these two enzymes showed that BPM24 serum had greater beta-1,3-glucanase and chitinase activities than that of RRIM600. These up-regulated proteins are constitutively expressed and would serve to protect the rubber tree BPM24 from any fungal invader. The information obtained from this work is valuable for understanding of defense mechanisms and plantation improvement of H. brasiliensis. BIOLOGICAL SIGNIFICANCE Non-rubber constituents (latex serum) have almost no value and are treated as waste in the rubber agricultural industry. However, the serum of natural rubber latex contains biochemical substances. The comparative proteomics analysis of latex serum between tolerant and susceptible clones reveals that the tolerant BPM24 clone contained a high abundance of several classes of fungal pathogen-responsive proteins, such as glucanase and chitinase. Moreover, other proteins identified highlighted the accumulation of defensive-associated proteins participating in plant fungal immunity. The isolation of beta-1,3-glucanase, chitinase, and lectin from latex serum should be further investigated and may provide a therapeutic application. This investigation will lead to possible use of latex serum as a great biotechnological resource due to the large quantity of serum produced and the biochemicals contained therein.