Detection and genetic profiling of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infections in wild berried freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> collected for hatchery production

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) has been detected widely in penaeid culture facilities in Asia and the Americas. IHHNV infection on sub-adult and postlarvae of the giant freshwater prawn, Macrobrachium rosenbergii which had caused up to 80% mortalities was first reported in Southeast Taiwan in 2006. In Malaysia, although, there has been no report on IHHNV infections in M. rosenbergii, preliminary work suggests that there is an urgent need to setup a screening protocol for IHHNV for both wild and cultured populations. In this study, polymerase chain reaction based screening was carried out on 30 randomly sampled berried wild M. rosenbergii before and after spawning. All samples did not showed any sign of IHHNV infection. However, the results showed that 20% of the samples were IHHNV positive. Sequence analysis of the amplified band using NCBI-BLAST showed that the putative IHHNV sequence had 98% nucleotide sequence (388 bp) identity with the IHHNV isolate AC-05-005 non-structural protein 1 gene and seven other IHHNV strains in the data bank further affirming the suggestion on the presence of IHHNV in wild freshwater prawn populations in Malaysia.     

Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Molecular identification and expression of heat shock cognate 70 (<Emphasis Type="Italic">HSC</Emphasis>70) in the pacific white shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80–99.6% homology with 12 diverse species’ HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multitissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of β-actin. Furthermore, quantitative real-time experiments showed that HSC70 was upregulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of the white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps’ cytoprotection. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 265–274. The text was submitted by the authors in English.     

Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> de Man

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.     

Proteasome inhibition induces <Emphasis Type="Italic">hsp30</Emphasis> and <Emphasis Type="Italic">hsp70</Emphasis> gene expression as well as the acquisition of thermotolerance in <Emphasis Type="Italic">Xenopus laevis</Emphasis> A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.     

Association of the Rv0679c protein with lipids and carbohydrates in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>/<Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

The Rv0679c gene in Mycobacterium tuberculosis H37Rv encodes a protein with a predicted molecular mass of 16,586 Da consisting of 165 amino acids which contains a putative N-terminal signal sequence and a consensus lipoprotein-processing motif. Globomycin treatment, Triton X-114 separation and mass spectrometry analyses clarified a property of the Rv0679c protein as a lipoprotein. In addition, trifluoromethanesulphonic acid treatment of the lysate revealed an association of the recombinant Rv0679c protein with carbohydrates. The Rv0679c protein homolog of Mycobacterium bovis BCG was also expressed as the protein associated with lipids and carbohydrates. In Western blot analysis, each of the protein homolog and Lipoarabinomannan (LAM) was detected as a similar pattern by anti-Rv0679c and anti-LAM antibodies, respectively. Interestingly, the Rv0679c protein was detected in commercially available LAM purified from M. tuberculosis. Inhibition assay of LAM synthesis in M. bovis BCG by ethambutol showed an altered migration pattern of the Rv0679c protein to low molecular mass similar to that of LAM. The results suggest that the Rv0679c protein exists as a tight complex with LAM in M. tuberculosis/M. bovis BCG.     

Efficient expression in <Emphasis Type="Italic">E. coli</Emphasis> of an enantioselective nitrile hydratase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The genes encoding an enantioselective nitrile hydratase (NHase) from Rhodococcus erythropolis AJ270 have been cloned and an active NHase has been produced in Escherichia coli. Maximal activity was found when the genes encoding the α- and β-subunits were transcribed as one unit and the gene encoding the P44k activator protein as a separate ORF on a single replicon. Addition of n-butyric acid and FeSO₄ could improve NHase activity. Coexpression of the GroEL-GroES chaperone proteins increased activity in the absence of P44k protein but had no effect in the presence of P44k. The recombinant enzyme was highly enantioselective in the synthesis of S-(+)-3-benzoyloxy- 4-cyanobutyramide from the prochiral substrate 3-benzoyloxyglutaronitrile.     

Cloning and expression of <Emphasis Type="Italic">Bacillus thuringiensis cry</Emphasis>11 crystal protein gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The six most toxic Pakistani isolates of Bacillus thuringiensis (SBS Bt-23, 29, 34, 37, 45 and 47), which were previously characterized for their toxicity against larvae of mosquito, Anopheles stephensi, and the presence of cry4 gene, were used for cry11 (cry4D) gene amplification. A 1.9-kb DNA fragment of cry11 gene was PCR-amplified, cloned in expression vector pT7-7, and then used for transformation of E. coli BL21C. The optimum expression was obtained with 1 mM IPTG at 37°C for 3 h. This gene showed different percentage homologies at protein level with scattered mutations in the toxic region. Biotoxicity assay of recombinant protein showed that Cry11 of SBS Bt 45 (DAB Bt 5) was the most toxic protein against third instar larvae of mosquito, A. stephensi, and has potentiality of a bioinsecticide against mosquitoes.     

Expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile enterotoxin B subunit in transgenic watercress (<Emphasis Type="Italic">Nasturtium officinale</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB) gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein. G_M1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to G_M1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized LTB subunits formed biologically active pentamers.     

Regulation of the <Emphasis Type="Italic">htpX</Emphasis> Gene of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> and Its Expression in <Emphasis Type="Italic">E. coli</Emphasis>

Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies. In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions. This gene was induced after a temperature upshift to 37°C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts. Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E. coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37°C or 45°C in all culture media tested, but not at 28°C in minimal culture medium. The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting.     

The stress responsive and morphologically regulated <Emphasis Type="Italic">hsp90</Emphasis> gene from <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> is essential to cell viability

Background  

Paracoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target.     

Larval excretory-secretory products from the parasite <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> modulate HSP70 protein expression in defence cells of its snail host, <Emphasis Type="Italic">Biomphalaria glabrata</Emphasis>

Synthesis of heat shock proteins (HSPs) following cellular stress is a response shared by many organisms. Amongst the HSP family, the ∼70 kDa HSPs are the most evolutionarily conserved with intracellular chaperone and extracellular immunoregulatory functions. This study focused on the effects of larval excretory-secretory products (ESPs) from the parasite Schistosoma mansoni on HSP70 protein expression levels in haemocytes (defence cells) from its snail intermediate host Biomphalaria glabrata. S. mansoni larval stage ESPs are known to interfere with haemocyte physiology and behaviour. Haemocytes from two different B. glabrata strains, one which is susceptible to S. mansoni infection and one which is resistant, both showed reduced HSP70 protein levels following 1 h challenge with S. mansoni ESPs when compared to unchallenged controls; however, the reduction observed in the resistant strain was less marked. The decline in intracellular HSP70 protein persisted for at least 5 h in resistant snail haemocytes only. Furthermore, in schistosome-susceptible snails infected by S. mansoni for 35 days, haemocytes possessed approximately 70% less HSP70. The proteasome inhibitor, MG132, partially restored HSP70 protein levels in ESP-challenged haemocytes, demonstrating that the decrease in HSP70 was in part due to intracellular degradation. The extracellular signal-regulated kinase (ERK) signalling pathway appears to regulate HSP70 protein expression in these cells, as the mitogen-activated protein-ERK kinase 1/2 (MEK1/2) inhibitor, U0126, significantly reduced HSP70 protein levels. Disruption of intracellular HSP70 protein expression in B. glabrata haemocytes by S. mansoni ESPs may be a strategy employed by the parasite to manipulate the immune response of the intermediate snail host.     

Enhanced Thermotolerance of <Emphasis Type="Italic">E. coli</Emphasis> by Expressed OsHsp90 from Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A gene encoding the rice (Oryza sativa L.) 90-kDa heat shock protein (OsHsp90) was introduced into Escherichia coli using the pGEX-6p-3 expression vector with a glutathione-S-transferase (GST) tag to analyze the possible function of this protein under heat stress for the first time. We compared the survivability of E. coli (BL21) cells transformed with a recombinant plasmid containing GST-OsHsp90 fusion protein with control E. coli cells transformed with the plasmid containing GST and the wild type BL21 under heat shock after isopropyl β-d-thiogalactopyranoside induction. Cells expressing GST-OsHsp90 demonstrated thermotolerance at 42, 50, and 70°C, treatments that were more harmful to cells expressing GST and the wild type. Further studies were carried out to analyze the heat-induced characteristics of OsHsp90 at 42, 50, and 70°C in vitro. When cell lysates from E. coli transformants were heated at these heat stresses, expressed GST-OsHsp90 prevented the denaturation of bacterial proteins treated with 42°C heat shocks, and partially prevented that of proteins treated at 50 and 70°C; meanwhile, cells expressing GST-OsHsp90 withstood the duration at 50°C. These results indicate that OsHsp90 functioned as a chaperone, binding to a subset of substrates, and maintained E. coli growth well at high temperatures.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> heat shock protein 70 is able to suppress the thermosensitivity of an <Emphasis Type="Italic">Escherichia coli</Emphasis> DnaK mutant strain

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp70 was able to suppress the thermosensitivity of an Escherichia coli dnaK756 strain, but not that of the corresponding deletion strain (dnaK52) or dnaK103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK756 strain. We also identified two key amino acid residues (V401 and Q402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp70 and PfHsp70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp40 chaperone machinery.     

Neuropeptide Y reduces the expression of <Emphasis Type="Italic">PLCB2</Emphasis>, <Emphasis Type="Italic">PLCD1</Emphasis> and selected <Emphasis Type="Italic">PLC</Emphasis> genes in cultured human endothelial cells

The ability of mycobacteria to grow and invade target tissues is the key component in the process of Mycobacterium bovis infection. Therefore, analysis of the proteins responsible for cell invasion will assist clinicians in combating bovine tuberculosis. The Mb1514 gene of M. bovis encodes a hypothetical invasion protein (designated here as MbINV protein), whose function has not yet been directly identified. In this study, the Mb1514 gene from M. bovis was cloned, and expressed in E. coli. The recombinant MbINV protein (a single band of approximately 28 kDa) was purified for biological analysis. Our data demonstrated that recombinant MbINV protein significantly inhibited the viability of RAW264.7 macrophages in a dose-dependent manner (P < 0.05), and induced cell necrosis, indicating that the protein is toxic. MbINV protein infection significantly enhanced the mRNA expression levels of TNF-α, IL-1β, and NOS2 (P < 0.01), suggesting that MbINV protein may be one of the virulence factors which directly interact with macrophages and modulate the host immune response to M. bovis. An invasion inhibition assay showed that MbINV-inhibited M. bovis invasion of RAW264.7 cells in a concentration-dependant manner, demonstrating it is an invasion protein.     

The HSP70 heat shock response in the Antarctic fish <Emphasis Type="Italic">Harpagifer antarcticus</Emphasis>

Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) genes, plus GRP78 (Glucose-regulated protein 78 kDa) were surveyed for expression levels via Q-PCR after both an acute 2-h heat shock experiment and a time course assay in the Antarctic plunderfish Harpagifer antarcticus. In general, down regulation of all genes was observed during the course of the heat shock experiments. This thermally induced down regulation was particularly acute for the GRP78 gene, which at one time point was more than 100-fold down regulated. These results demonstrate the loss of the heat shock response in H. antarcticus, a basal member of the Notothenioidei. This finding is discussed with reference to the survival of Notothenioids during observed ocean warming and also the reorganisation of cellular protein mechanisms of species living in extreme environments.     

<Emphasis Type="Italic">Pm37</Emphasis>, a new broadly effective powdery mildew resistance gene from <Emphasis Type="Italic">Triticum </Emphasis><Emphasis Type="Italic">timopheevii</Emphasis>

Powdery mildew is an important foliar disease in wheat, especially in areas with a cool or maritime climate. A dominant powdery mildew resistance gene transferred to the hexaploid germplasm line NC99BGTAG11 from T. timopheevii subsp. armeniacum was mapped distally on the long arm of chromosome 7A. Differential reactions were observed between the resistance gene in NC99BGTAG11 and the alleles of the Pm1 locus that is also located on chromosome arm 7AL. Observed segregation in F_2:3 lines from the cross NC99BGTAG11 × Axminster (Pm1a) demonstrate that germplasm line NC99BGTAG11 carries a novel powdery mildew resistance gene, which is now designated as Pm37. This new gene is highly effective against all powdery mildew isolates tested so far. Analyses of the population with molecular markers indicate that Pm37 is located 16 cM proximal to the Pm1 complex. Simple sequence repeat (SSR) markers Xgwm332 and Xwmc790 were located 0.5 cM proximal and distal, respectively, to Pm37. In order to identify new markers in the region, wheat expressed sequence tags (ESTs) located in the distal 10% of 7AL that were orthologous to sequences from chromosome 6 of rice were targeted. The two new EST-derived STS markers were located distal to Pm37 and one marker was closely linked to the Pm1a region. These new markers can be used in marker-assisted selection schemes to develop wheat cultivars with pyramids of powdery mildew resistance genes, including combinations of Pm37 in coupling linkage with alleles of the Pm1 locus.     

Expression of <Emphasis Type="Italic">Ruminococcus albus</Emphasis> Xylanase Gene (<Emphasis Type="Italic">xyn</Emphasis>A) in <Emphasis Type="Italic">Streptococcus bovis</Emphasis> 12-U-1

The objective of this study was to ligate the xylanase gene A (xynA) isolated from Ruminococcus albus 7 into the promoter and signal-peptide region of the lichenase [β-(1,3-1,4)-glucanase] gene of Streptococcus bovis JB1. This fusion gene was inserted into the pSBE11 vector, and the resulting recombinant, plasmid pXA, was used to transform S. bovis 12-U-1 cells. The transformant, S. bovis 12UXA, secreted the xylanase, which was stable against freeze-thaw treatment and long-time incubation at 37°C. The introduction of pXA and production of xylanase did not affect cell growth, and the xylanase produced degraded xylan from oat-spelt and birchwood. Received: 24 June 2002 / Accepted: 7 October 2002