A thioredoxin response to the WSSV challenge on the Chinese white shrimp, Fenneropenaeus chinensis

Thioredoxin (TRX) is involved in cell redox homeostasis. In addition, it is responsible for maintaining proteins in their reduced state. In our study, a Fenneropenaeus chinensis thioredoxin (FcTRX) gene was identified from the Chinese white shrimp. The full length of FcTRX was 777 bp, including a 60 bp 5′ untranslated region (UTR), a 318 bp open reading frame (ORF) encoding a 105 amino acids protein, and a 399 bp 3′ UTR. FcTRX contained a TRX domain with a conserved motif of Cys-Gly-Pro-Cys (CGPC). No signal peptide was predicted by SMART analysis. The molecular mass and pI of FcTRX were 12 kDa and 4.62, respectively. FcTRX is a widely distributed gene, and its mRNA is detected in hemocytes, hearts, hepatopancreas, gills, stomach, and intestine from an unchallenged shrimp. The expression level of FcTRX was the highest in hepatopancreas, where it was down-regulated to the lowest level at 12 h white spot syndrome virus (WSSV) challenge. In the gills, it went up to the highest level at 6 h. Western blot showed that FcTRX protein in hepatopancreas challenged with WSSV was down-regulated from 2 h to 12 h and then restored to the level similar to that of unchallenged shrimp at 24 h. In the gills challenged with WSSV, the FcTRX protein was up-regulated from 6 h to 24 h. Our research indicated its possible role in the anti-WSSV innate immunity of shrimps.     

Structure and activity of the Pseudomonas aeruginosa hotdog-fold thioesterases PA5202 and PA2801

The hotdog fold is one of the basic protein folds widely present in bacteria, archaea and eukaryotes. Many of these proteins exhibit thioesterase activity against fatty acyl-CoAs and play important roles in lipid metabolism, cellular signalling and degradation of xenobiotics. The genome of the opportunistic pathogen Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (at 1.70 and 1.75 ? for PA5202 and PA2801 respectively) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long α-helix (Asp(57) in PA5202 and Glu(35) in PA2801). Alanine residue replacement mutagenesis of PA5202 identified four residues (Asn(42), Arg(43), Asp(57) and Thr(76)) that are critical for its activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis, suggesting a functional link between PA5202 activity and pyocyanin production. Thus the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.     

Identification of three different types of serine proteases (one SP and two SPHs) in Chinese white shrimp

Serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, embryonic development, blood coagulation, and immune defense responses. In this paper, we identify one SP and two SPHs, including a masquerade SPH (FcMas), a CUB domain containing SP (FcCUBSP), and a single domain containing SPH (FcSPH2) in Chinese white shrimp, Fenneropenaeus chinensis. FcMas has a Gly-rich region formed by three repeats of LGGQGGG, a clip domain and a C-terminal SP-like domain. Absence of Ser catalytic residue results in the loss of serine protease activity of FcMas, which then functions as an SPH. FcCUBSP has a signal peptide, followed by a CUB domain and an SP domain. FcSPH2 has a signal peptide and an SP-like domain. Loss of one catalytic residue (H) makes FcSPH2 catalytically inactive, which is considered an SPH. Phylogenetic analysis shows that FcMas and other SPHs from shrimp or insect are classified into one group. FcSPH2 is grouped in the chymotrypsin family. RT-PCR results show that FcMas mRNA is mainly distributed in hemocytes and gills. FcCUBSP is only detected in gills, whereas FcSPH2 is found in hepatopancreas only. QRT-PCR is used to analyze changes of FcMas, FcCUBSP and FcSPH2 in some tissues challenged with white spot syndrome virus (WSSV) or Vibrio. FcMas in hemocytes is down-regulated by WSSV or Vibrio challenge, and down-regulated by WSSV in gills. However, it is up-regulated upon Vibrio challenge in gills. FcCUBSP in gills and FcSPH2 in hepatopancreas are up-regulated upon WSSV or Vibrio challenge. Results suggest the roles of FcMas, FcCUBSP and FcSPH2 in shrimp's innate immunity.     

A putative cell surface receptor for white spot syndrome virus is a member of a transporter superfamily

White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral disease in shrimp and has a wide host range among crustaceans. In this study, we identified a surface protein, named glucose transporter 1 (Glut1), which could also interact with WSSV envelope protein, VP53A. Sequence analysis revealed that Glut1 is a member of a large superfamily of transporters and that it is most closely related to evolutionary branches of this superfamily, branches that function to transport this sugar. Tissue tropism analysis showed that Glut1 was constitutive and highly expressed in almost all organs. Glut1's localization in shrimp cells was further verified and so was its interaction with Penaeus monodon chitin-binding protein (PmCBP), which was itself identified to interact with an envelope protein complex formed by 11 WSSV envelope proteins. In vitro and in vivo neutralization experiments using synthetic peptide contained WSSV binding domain (WBD) showed that the WBD peptide could inhibit WSSV infection in primary cultured hemocytes and delay the mortality in shrimps challenged with WSSV. These findings have important implications for our understanding of WSSV entry.     

Functional characterization of thioesterase superfamily member 1/Acyl-CoA thioesterase 11: implications for metabolic regulation

Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is highly expressed in brown adipose tissue and limits energy expenditure in mice. Them1 is a putative fatty acyl-CoA thioesterase that comprises tandem hot dog-fold thioesterase domains and a lipid-binding C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. To better define its role in metabolic regulation, this study examined the biochemical and enzymatic properties of Them1. Purified recombinant Them1 dimerized in solution to form an active fatty acyl-CoA thioesterase. Dimerization was induced by fatty acyl-CoAs, coenzyme A (CoASH), ATP, and ADP. Them1 hydrolyzed a range of fatty acyl-CoAs but exhibited a relative preference for long-chain molecular species. Thioesterase activity varied inversely with temperature, was stimulated by ATP, and was inhibited by ADP and CoASH. Whereas the thioesterase domains of Them1 alone were sufficient to yield active recombinant protein, the START domain was required for optimal enzyme activity. An analysis of subcellular fractions from mouse brown adipose tissue and liver revealed that Them1 contributes principally to the fatty acyl-CoA thioesterase activity of microsomes and nuclei. These findings suggest that under biological conditions, Them1 functions as a lipid-regulated fatty acyl-CoA thioesterase that could be targeted for the management of metabolic disorders.     

Shrimps survive white spot syndrome virus challenge following treatment with Vibrio bacterin

Taking an innovative approach, a vaccination study using five bacterial strains viz. Vibrio campbelli (B60), V. alginolyticus (B73), V. parahaemolyticus-like (B79), V. parahaemolyticus (R8) and V. harveyi (RG203) was conducted in Penaeus monodon against white spot syndrome virus (WSSV) infection, considered as one of the serious pathogens of shrimps. Oral challenge with shrimps infected with WSSV showed a relative percentage survival of 5 and 47% in the P. monodon juveniles vaccinated with V. parahaemolyticus and V. harveyi, respectively. Results showed that there is a possibility of specifically immunising the shrimps against WSSV using bacterin prepared out of Vibrio harveyi isolates taken from shrimps infected with WSSV. Also, there was a level of protection attained by the shrimps due to immunisation with Vibrio strains.     

Crystal structure of human thioesterase superfamily member 2

Hotdog-fold has been identified in more than 1000 proteins, yet many of which in eukaryotes are less studied. No structural or functional studies of human thioesterase superfamily member 2 (hTHEM2) have been reported before. Since hTHEM2 exhibits about 20% sequence identity to Escherichia coli PaaI protein, it was proposed to be a thioesterase with a hotdog-fold. Here, we report the crystallographic structure of recombinant hTHEM2, determined by the single-wavelength anomalous dispersion method at 2.3A resolution. This structure demonstrates that hTHEM2 indeed contains a hotdog-fold and forms a back-to-back tetramer as other hotdog proteins. Based on structural and sequence conservation, the thioesterase active site in hTHEM2 is predicted. The structure and substrate specificity are most similar to those of the bacterial phenylacetyl-CoA hydrolase. Asp65, located on the central alpha-helix of subunit B, was shown by site-directed mutagenesis to be essential to catalysis.     

Identification of a Hotdog Fold Thioesterase Involved in the Biosynthesis of Menaquinone in Escherichia coli

Escherichia coli is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in Escherichia coli, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine Escherichia coli hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the ydiI gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in ΔybgC and ΔentH mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.     

红螯光壳螯虾白斑综合症血液病理学研究

采用Wright-Geimsa染色法和电镜技术对人工感染的红螯光壳螯虾(Cherax quadricarinatus)白斑综合症(White spot syndrome,WSS)血液病理学进行了研究。结果显示:患病螯虾血细胞总数、透明细胞(AH)数量极显著减少(P<0.01),大颗粒细胞(LGH)极显著增加(P<0.01);病毒感染后3种血细胞大小均有增加趋势,透明细胞和大颗粒细胞的核质比(NP)较感染病毒前极显著下降(P<0.01)。显微病理学变化主要表现为血涂片中血细胞明显减少,病变、破损或解体的细胞增多,至濒死期螯虾血液呈典型的溶血状态。超微病理学变化表现为血细胞受到了损伤。高尔基体变形、线粒体结构模糊破损;核膜变形核固缩、细胞核高度异染色质化;濒临死亡的螯虾血细胞细胞器和染色质溶解,胞浆水肿,细胞溶解坏死。在患病螯虾的血细胞核中清晰可见WSSV粒子。     

Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus

This study focused on apoptosis in various tissues of the black tiger shrimp Penaeus monodon following white spot syndrome virus (WSSV) injection. The study included: (1) light microscopy (LM) and transmission electron microscopy (TEM) of various tissues; (2) fluorescent LM of nuclear DNA by staining with 4, 6-diamidine-2-phenyl indole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labelling (TUNEL) techniques; and (3) determination of caspase-3 activity. Juvenile P. monodon were injected with WSSV, and several tissues of ectodermal and mesodermal origin were studied at different intervals after injection. The total haemocyte count had decreased to one-tenth of its original level 60 h after WSSV injection. By LM, extensive destruction by WSSV was observed in the stomach epithelium, gills, hematopoietic tissue, hemocytes and the heart, but the most severely affected tissue was the subcuticular epithelium. TEM revealed that at 6 h post-injection (p.i.) the chromatin of infected nuclei was marginated, and by 24 h p.i. the nuclei were filled with enveloped and non-enveloped WSSV virions. At later stages of the infection, the nucleus extruded WSSV particles. Chromatin margination and nuclear condensation and fragmentation (i.e. signs of apoptosis) were observed as early as 6 h p.i. in all affected tissues, but occurred in cells without WSSV virions rather than in cells with virions. The occurrence of apoptosis was supported by data obtained using TUNEL and by DAPI-staining and progressed from 6 to 60 h p.i. In addition, caspase-3 activity in WSSV-infected shrimp was about 6-fold higher than that in uninfected shrimp. The data strongly suggests that apoptosis occurs following WSSV infection in P. monodon, but the extent to which it contributes to shrimp mortality requires further investigation.     

The BH1999 protein of Bacillus halodurans C-125 is gentisyl-coenzyme A thioesterase

In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a k(cat)/K(m) of 1.6 x 10(6) M(-1) s(-1) and the hydrolysis of 3-hydroxybenzoyl-CoA with a k(cat)/K(m) of 3.0 x 10(5) M(-1) s(-1). All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.     

Vaccination of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV)

Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.     

A Kazal type serine proteinase SPIPm2 from the black tiger shrimp Penaeus monodon is capable of neutralization and protection of hemocytes from the white spot syndrome virus

A Kazal type serine proteinase SPIPm2 is abundantly expressed in the hemocytes and shown to be involved in innate immune response against white spot syndrome virus (WSSV) in Penaeus monodon. The SPIPm2 is expressed and stored in the granules in the cytoplasm of semigranular and granular but not the hyaline hemocytes. Upon WSSV challenge and progression of infection, the SPIPm2 was secreted readily from the semigranular and granular hemocytes. The more they secreted the SPIPm2, the less they were distinguishable from the hyaline cells. The WSSV-infected cells were either semigranular or granular hemocytes or both and depleted of SPIPm2. The rSPIPm2 was able to temporarily and dose-dependently neutralize the WSSV and protect the hemocytes from viral infection judging from the substantially less expression of WSSV late gene VP28. The antiviral activity was very likely due to the binding of SPIPm2 to the components of viral particle and hemocyte cell membrane.