We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small... more We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small DNA virus of shrimp, from wild Penaeus stylirostris. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The left, mid, and right ORFs on the complementary (plus) strand have potential coding capacities of 666 amino acids (aa) (75.77 kDa), 363 aa (42.11 kDa), and 329 aa (37.48 kDa), respectively. The overall genomic organization is similar to that of the mosquito brevidensoviruses. The left ORF most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest aa sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. A search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae, and we therefore propose to rename this virus Penaeus stylirostris densovirus (PstDNV).
The 184-kb Bacilus anthracis plasmid pXO0, which is required for virulence, contains three genes ... more The 184-kb Bacilus anthracis plasmid pXO0, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), kef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pagklacZ transcriptional construct to measure pag promoter activity, we cloned in BaciUus subtilis a gene (alxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located aIxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted Mr of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcriptiontranslation analysis. The cloned airA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. 3-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.
Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE)... more Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC 50 ] ؍ 12 ng/ml) Ն RAAR (EC 50 ؍ 18 ng/ml) > FTKR (EC 50 ؍ 24 ng/ml) > STRR (EC 50 ؍ 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC 50 ؍ 2.9 ng/ml) and PA (EC 50 ؍ 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.
To isolate novel cellular factors that are activated or repressed upon WSV infection, the RNA fin... more To isolate novel cellular factors that are activated or repressed upon WSV infection, the RNA fingerprints of healthy and WSV infected blue shrimp ( Penaeus stylirostris) were compared using the mRNA differential display technique. Thirty-two unique differentially expressed, and one constitutively expressed, cDNA sequences were retrieved. Six of 32 cDNAs showed similarities with the database entries: cDNA 10G32-142 to a shrimp arginine kinase, 22C48-201 to shrimp mitochondrial ATPase gene; 22C47-197, 21G49-203 and 20A55-268 to shrimp ESTs and 20G50-206 to a WSV gene, ORF 116. The constitutively expressed gene showed significant similarity to a yeast elongation factor 1-alpha gene. The expression of a subset of differentially expressed genes (13 of 32) was further evaluated by real-time RT-PCR. Ten of 13 genes showed statistically significant changes in expression between healthy and WSV infected animals suggesting that these genes may play an important role in WSV pathogenesis.
White spot syndrome virus (WSSV) is currently the most important viral pathogen infecting penaeid... more White spot syndrome virus (WSSV) is currently the most important viral pathogen infecting penaeid shrimp worldwide. Although considerable progress has been made in characterizing the WSSV genome and developing detection methods, information pertaining to host genes involved in WSSV pathogenesis is limited. We examined the potential of cDNA microarray analysis to study gene expression in WSSV-infected shrimp. Shrimp cDNAs were printed as low-density arrays on glass slides and were hybridized with Cy3/Cy5 labeled probes derived from RNA isolated from healthy and WSSV-infected shrimp. Genes that code for proteins that are relevant to crustacean immunity, structural proteins, as well as proteins of unknown function were among those whose mRNA expression was altered upon WSSV infection. To validate the microarray data, the temporal expression of three differentially expressed genes, an immune gene (C-type lectin-1), a structural gene (40S ribosomal protein), and a gene involved in lipid metabolism (fatty acid binding protein) was measured in healthy and WSSV-infected shrimp by real-time RT-PCR. The data suggest that WSSV infection alters the expression of a wide array of cellular genes, and provides a framework for further studies aimed at identifying genes whose function may provide insight into the mechanism of WSSV infection in shrimp.
A rapid and highly sensitive real-time PCR detection and quantification method for infectious hyp... more A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the Gene-Amp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (C T) values over a range of 1 to 10 5 copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp -actin gene was amplified in parallel with the viral DNA. The C T values of IHHNV-and WSV-infected samples were used to determine absolute viral copy numbers from the standard C T curves of these viruses. For each virus and its -actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100-to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.
SummaryComparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in t... more SummaryComparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc‐binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site‐directed mutagenesis that substituted Ala for either of the residues (H‐686 and H‐690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E‐687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E‐720 and E‐721 with Ala had no effect on LF activity. LF bound 65Zn both in solution and on protein blots. The 65Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.
In Vitro Cellular & Developmental Biology – Animal, Jun 1, 2001
Creation of a shrimp cell line has been an elusive goal. This failure may be due to the compositi... more Creation of a shrimp cell line has been an elusive goal. This failure may be due to the composition of the cell culture medium, which may be inadequate to support primary cultured cells. Shrimp hemolymph should contain the nutritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shrimp, Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Shrimps and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino acids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Levels of hemolymph components were compared to 2xL-15 with 20% fetal bovine serum, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2 x L-15 medium. In contrast, other FAAs were up to 50 times higher in the 2 x L-15 medium than in the hemolymph. To mimic more closely the hemolymph composition, we created two new media based on either the 0.2 x L-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by 3H-thymidine uptake and 35S-methionine uptake assays. The ovary cells of P. stylirostris cultured in either of the new media formed monolayers, while the cells cultured in 2 x L-15 medium did not. Despite these differences, there was no evidence of sustained DNA or protein synthesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division.
Proceedings of the National Academy of Sciences of the United States of America, Oct 28, 1997
A challenge for subunit vaccines whose goal is to elicit CD8 ؉ cytotoxic T lymphocytes (CTLs) is ... more A challenge for subunit vaccines whose goal is to elicit CD8 ؉ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA ؉ LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varie... more Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varied markedly with respect to a-(1,3)-glucan content. When yeast cell walls were fractionated by standard techniques, the avirulent strains contained up to 1,000-fold less a-(1,3)-glucan than did their virulent parents. No at-(1,3)glucan could be detected on the surface of the avirulent strain yeast cells if we used a mouse monoclonal antibody that recognized this polymer. A similar relationship between virulence and oa-(1,3)-glucan has been described for Paracoccidioides brasiliensis. a-(1,3)-Glucan is also found in several other pathogenic fungi and may thus be an important common virulence determinant.
Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death i... more Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.
The anthrax toxin complex contains two catalytic proteins, edema factor (EF) and lethal factor (L... more The anthrax toxin complex contains two catalytic proteins, edema factor (EF) and lethal factor (LF), which must be translocated to the cytosol of eukaryotic cells to cause toxicity. The third protein component of the toxin, protective antigen (PA), achieves this internalization. PA binds to cell surface receptors, is cleaved after the sequence Argl64-Lysl65-Lysl66-Argl67 by a cell surface protease, and thereby exposes a binding site to which EF or LF bind with high affinity. The complexes enter endosomes, which then become acidified, causing transfer of LF and EF to the cytosol.
Real-time RT-PCR using SYBR Green chemistry uses a green fluorescence dye, SYBR Green I, that bin... more Real-time RT-PCR using SYBR Green chemistry uses a green fluorescence dye, SYBR Green I, that binds to double stranded DNA (dsDNA) and exhibits enhancement of fluorescence upon binding to the DNA. The indiscriminate binding ability of SYBR Green I dye to dsDNA often results in non-specific products. We have shown that increasing the amplicon size from Â/50 to Â/75 Á/ 100 bp increases the specificity due to higher melting temperature of the amplicon and also enhances the sensitivity of detection of real-time RT-PCR using SYBR Green chemistry while detecting two RNA viruses in laboratory-challenged shrimp, the Taura syndrome virus (TSV), and yellow head virus (YHV). The increased sensitivity of the larger amplicon over the smaller amplicon varied from 1.6 to 6.82-fold (with a median value of 4-fold) for the TSV-infected samples, and 1.80 Á/10.27-fold (with a median value of 4-fold) for the YHV-infected samples. The longer amplicon also has a higher Tm value compared with the shorter amplicon (75.6 vs. 72.0 8C for TSV, and 81.3 vs. 72.5 8C for YHV). The increased melting temperature of the longer amplicon compared with the shorter amplicon will enable easier discrimination of a specific product from a primer dimer or other non-specific products. The improved method for the detection of TSV and YHV will be applicable not only to the detection of other viral pathogens but also to the quantitative measurement of cellular gene expression by real-time SYBR Green RT-PCR.
Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins pre... more Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway. This review focuses on the usefulness of bacterial toxins for delivering antigens to the MHC class I pathway. Several toxins naturally translocate into the cytosol, where they mediate their cytopathic effects, and the mechanisms by which this occurs has been elucidated. Molecular characterization of these toxins identified the functional domains and enabled the generation of modified proteins that were no longer toxic but retained the ability to translocate into the cytosol. Thus, these modified toxins could be examined for their ability to carry peptides or whole proteins into the cytosolic processing pathway. Of the toxins studied-diphtheria, pertussis, Pseudomonas, and anthrax-the anthrax toxin appears the most promising in its ability to deliver large protein antigens and its efficiency of translocation. Human Immunology 54, 129-136 (1997). Published by Elsevier Science Inc. ABBREVIATIONS 2m 2 microglobulin CTL cytotoxic T lymphocyte CYA pertussis adenylate cyclase DT diphtheria toxin EF anthrax toxin edema factor ER endoplasmic reticulum LF anthrax toxin lethal factor MHC major histocompatibility complex PA anthrax toxin protective antigen PE Pseudomonas exotoxin PT pertussis toxin TAP transporter associated with antigen processing TCR T cell receptor
Pattern recognition proteins such as lipopolysaccharide and -1,3-glucan binding protein (LGBP) p... more Pattern recognition proteins such as lipopolysaccharide and -1,3-glucan binding protein (LGBP) play an important role in the innate immune response of crustaceans and insects. Random sequencing of cDNA clones from a hepatopancreas cDNA library of white spot virus (WSV)-infected shrimp provided a partial cDNA (PsEST-289) that showed similarity to the LGBP gene of crayfish and insects. Subsequently full-length cDNA was cloned by the 5-RACE (rapid amplification of cDNA ends) technique and sequenced. The shrimp LGBP gene is 1,352 bases in length and is capable of encoding a polypeptide of 376 amino acids that showed significant similarity to homologous genes from crayfish, insects, earthworms, and sea urchins. Analysis of the shrimp LGBP deduced amino acid sequence identified conserved features of this gene family including a potential recognition motif for -(133) linkage of polysaccharides and putative RGD cell adhesion sites. It is known that LGBP gene expression is upregulated in bacterial and fungal infection and that the binding of lipopolysaccharide and -1,3-glucan to LGBP activates the prophenoloxidase (proPO) cascade. The temporal expression of LGBP and proPO genes in healthy and WSV-challenged Penaeus stylirostris shrimp was measured by real-time quantitative reverse transcription-PCR, and we showed that LGBP gene expression in shrimp was upregulated as the WSV infection progressed. Interestingly, the proPO expression was upregulated initially after infection followed by a downregulation as the viral infection progressed. The downward trend in the expression of proPO coincided with the detection of WSV in the infected shrimp. Our data suggest that shrimp LGBP is an inducible acute-phase protein that may play a critical role in shrimp-WSV interaction and that the WSV infection regulates the activation and/or activity of the proPO cascade in a novel way. Invertebrates lack a true adaptive immune system and rely instead on various innate immune responses against invading pathogens (12). Both cellular and humoral immunity play important roles in defense against microorganisms in invertebrates. The cellular immune responses include encapsulation, phagocytosis, and nodule formation (24), whereas the humoral responses include the clotting cascade, the synthesis of a wide array of antimicrobial peptides, and the phenoloxidase (PO)activating system (prophenoloxidase [proPO] system) (11, 15, 34). Bacteria and fungi have molecules such as lipopolysaccharide (LPS) and -1,3-glucans on their surfaces that are recognized by nonself recognition molecules and elicit an immune reaction in both vertebrates and invertebrates. Proteins that recognize the LPS and -1,3-glucans are known as pattern recognition proteins (PRPs) (16). In mammals, the complex of bacterial LPS and the plasma LPS binding protein binds to CD14, which transduces the signal through toll-like receptors and activates the NF-B signaling cascade (13, 21, 22, 49). In insects and crustaceans, LPS and -1,3-glucans, upon binding to LPS and glucan binding protein, activate proPO, the coagulation cascade, and the genes for antibacterial effector proteins (11, 15, 33).
BaciNus anthracis produces three proteins which combine to form two toxic activities. Protective ... more BaciNus anthracis produces three proteins which combine to form two toxic activities. Protective antigen (PA,
We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small... more We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small DNA virus of shrimp, from wild Penaeus stylirostris. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The left, mid, and right ORFs on the complementary (plus) strand have potential coding capacities of 666 amino acids (aa) (75.77 kDa), 363 aa (42.11 kDa), and 329 aa (37.48 kDa), respectively. The overall genomic organization is similar to that of the mosquito brevidensoviruses. The left ORF most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest aa sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. A search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae, and we therefore propose to rename this virus Penaeus stylirostris densovirus (PstDNV).
The 184-kb Bacilus anthracis plasmid pXO0, which is required for virulence, contains three genes ... more The 184-kb Bacilus anthracis plasmid pXO0, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), kef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pagklacZ transcriptional construct to measure pag promoter activity, we cloned in BaciUus subtilis a gene (alxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located aIxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted Mr of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcriptiontranslation analysis. The cloned airA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. 3-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.
Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE)... more Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC 50 ] ؍ 12 ng/ml) Ն RAAR (EC 50 ؍ 18 ng/ml) > FTKR (EC 50 ؍ 24 ng/ml) > STRR (EC 50 ؍ 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC 50 ؍ 2.9 ng/ml) and PA (EC 50 ؍ 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.
To isolate novel cellular factors that are activated or repressed upon WSV infection, the RNA fin... more To isolate novel cellular factors that are activated or repressed upon WSV infection, the RNA fingerprints of healthy and WSV infected blue shrimp ( Penaeus stylirostris) were compared using the mRNA differential display technique. Thirty-two unique differentially expressed, and one constitutively expressed, cDNA sequences were retrieved. Six of 32 cDNAs showed similarities with the database entries: cDNA 10G32-142 to a shrimp arginine kinase, 22C48-201 to shrimp mitochondrial ATPase gene; 22C47-197, 21G49-203 and 20A55-268 to shrimp ESTs and 20G50-206 to a WSV gene, ORF 116. The constitutively expressed gene showed significant similarity to a yeast elongation factor 1-alpha gene. The expression of a subset of differentially expressed genes (13 of 32) was further evaluated by real-time RT-PCR. Ten of 13 genes showed statistically significant changes in expression between healthy and WSV infected animals suggesting that these genes may play an important role in WSV pathogenesis.
White spot syndrome virus (WSSV) is currently the most important viral pathogen infecting penaeid... more White spot syndrome virus (WSSV) is currently the most important viral pathogen infecting penaeid shrimp worldwide. Although considerable progress has been made in characterizing the WSSV genome and developing detection methods, information pertaining to host genes involved in WSSV pathogenesis is limited. We examined the potential of cDNA microarray analysis to study gene expression in WSSV-infected shrimp. Shrimp cDNAs were printed as low-density arrays on glass slides and were hybridized with Cy3/Cy5 labeled probes derived from RNA isolated from healthy and WSSV-infected shrimp. Genes that code for proteins that are relevant to crustacean immunity, structural proteins, as well as proteins of unknown function were among those whose mRNA expression was altered upon WSSV infection. To validate the microarray data, the temporal expression of three differentially expressed genes, an immune gene (C-type lectin-1), a structural gene (40S ribosomal protein), and a gene involved in lipid metabolism (fatty acid binding protein) was measured in healthy and WSSV-infected shrimp by real-time RT-PCR. The data suggest that WSSV infection alters the expression of a wide array of cellular genes, and provides a framework for further studies aimed at identifying genes whose function may provide insight into the mechanism of WSSV infection in shrimp.
A rapid and highly sensitive real-time PCR detection and quantification method for infectious hyp... more A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the Gene-Amp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (C T) values over a range of 1 to 10 5 copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp -actin gene was amplified in parallel with the viral DNA. The C T values of IHHNV-and WSV-infected samples were used to determine absolute viral copy numbers from the standard C T curves of these viruses. For each virus and its -actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100-to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.
SummaryComparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in t... more SummaryComparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc‐binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site‐directed mutagenesis that substituted Ala for either of the residues (H‐686 and H‐690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E‐687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E‐720 and E‐721 with Ala had no effect on LF activity. LF bound 65Zn both in solution and on protein blots. The 65Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.
In Vitro Cellular & Developmental Biology – Animal, Jun 1, 2001
Creation of a shrimp cell line has been an elusive goal. This failure may be due to the compositi... more Creation of a shrimp cell line has been an elusive goal. This failure may be due to the composition of the cell culture medium, which may be inadequate to support primary cultured cells. Shrimp hemolymph should contain the nutritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shrimp, Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Shrimps and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino acids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Levels of hemolymph components were compared to 2xL-15 with 20% fetal bovine serum, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2 x L-15 medium. In contrast, other FAAs were up to 50 times higher in the 2 x L-15 medium than in the hemolymph. To mimic more closely the hemolymph composition, we created two new media based on either the 0.2 x L-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by 3H-thymidine uptake and 35S-methionine uptake assays. The ovary cells of P. stylirostris cultured in either of the new media formed monolayers, while the cells cultured in 2 x L-15 medium did not. Despite these differences, there was no evidence of sustained DNA or protein synthesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division.
Proceedings of the National Academy of Sciences of the United States of America, Oct 28, 1997
A challenge for subunit vaccines whose goal is to elicit CD8 ؉ cytotoxic T lymphocytes (CTLs) is ... more A challenge for subunit vaccines whose goal is to elicit CD8 ؉ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA ؉ LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varie... more Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varied markedly with respect to a-(1,3)-glucan content. When yeast cell walls were fractionated by standard techniques, the avirulent strains contained up to 1,000-fold less a-(1,3)-glucan than did their virulent parents. No at-(1,3)glucan could be detected on the surface of the avirulent strain yeast cells if we used a mouse monoclonal antibody that recognized this polymer. A similar relationship between virulence and oa-(1,3)-glucan has been described for Paracoccidioides brasiliensis. a-(1,3)-Glucan is also found in several other pathogenic fungi and may thus be an important common virulence determinant.
Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death i... more Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.
The anthrax toxin complex contains two catalytic proteins, edema factor (EF) and lethal factor (L... more The anthrax toxin complex contains two catalytic proteins, edema factor (EF) and lethal factor (LF), which must be translocated to the cytosol of eukaryotic cells to cause toxicity. The third protein component of the toxin, protective antigen (PA), achieves this internalization. PA binds to cell surface receptors, is cleaved after the sequence Argl64-Lysl65-Lysl66-Argl67 by a cell surface protease, and thereby exposes a binding site to which EF or LF bind with high affinity. The complexes enter endosomes, which then become acidified, causing transfer of LF and EF to the cytosol.
Real-time RT-PCR using SYBR Green chemistry uses a green fluorescence dye, SYBR Green I, that bin... more Real-time RT-PCR using SYBR Green chemistry uses a green fluorescence dye, SYBR Green I, that binds to double stranded DNA (dsDNA) and exhibits enhancement of fluorescence upon binding to the DNA. The indiscriminate binding ability of SYBR Green I dye to dsDNA often results in non-specific products. We have shown that increasing the amplicon size from Â/50 to Â/75 Á/ 100 bp increases the specificity due to higher melting temperature of the amplicon and also enhances the sensitivity of detection of real-time RT-PCR using SYBR Green chemistry while detecting two RNA viruses in laboratory-challenged shrimp, the Taura syndrome virus (TSV), and yellow head virus (YHV). The increased sensitivity of the larger amplicon over the smaller amplicon varied from 1.6 to 6.82-fold (with a median value of 4-fold) for the TSV-infected samples, and 1.80 Á/10.27-fold (with a median value of 4-fold) for the YHV-infected samples. The longer amplicon also has a higher Tm value compared with the shorter amplicon (75.6 vs. 72.0 8C for TSV, and 81.3 vs. 72.5 8C for YHV). The increased melting temperature of the longer amplicon compared with the shorter amplicon will enable easier discrimination of a specific product from a primer dimer or other non-specific products. The improved method for the detection of TSV and YHV will be applicable not only to the detection of other viral pathogens but also to the quantitative measurement of cellular gene expression by real-time SYBR Green RT-PCR.
Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins pre... more Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway. This review focuses on the usefulness of bacterial toxins for delivering antigens to the MHC class I pathway. Several toxins naturally translocate into the cytosol, where they mediate their cytopathic effects, and the mechanisms by which this occurs has been elucidated. Molecular characterization of these toxins identified the functional domains and enabled the generation of modified proteins that were no longer toxic but retained the ability to translocate into the cytosol. Thus, these modified toxins could be examined for their ability to carry peptides or whole proteins into the cytosolic processing pathway. Of the toxins studied-diphtheria, pertussis, Pseudomonas, and anthrax-the anthrax toxin appears the most promising in its ability to deliver large protein antigens and its efficiency of translocation. Human Immunology 54, 129-136 (1997). Published by Elsevier Science Inc. ABBREVIATIONS 2m 2 microglobulin CTL cytotoxic T lymphocyte CYA pertussis adenylate cyclase DT diphtheria toxin EF anthrax toxin edema factor ER endoplasmic reticulum LF anthrax toxin lethal factor MHC major histocompatibility complex PA anthrax toxin protective antigen PE Pseudomonas exotoxin PT pertussis toxin TAP transporter associated with antigen processing TCR T cell receptor
Pattern recognition proteins such as lipopolysaccharide and -1,3-glucan binding protein (LGBP) p... more Pattern recognition proteins such as lipopolysaccharide and -1,3-glucan binding protein (LGBP) play an important role in the innate immune response of crustaceans and insects. Random sequencing of cDNA clones from a hepatopancreas cDNA library of white spot virus (WSV)-infected shrimp provided a partial cDNA (PsEST-289) that showed similarity to the LGBP gene of crayfish and insects. Subsequently full-length cDNA was cloned by the 5-RACE (rapid amplification of cDNA ends) technique and sequenced. The shrimp LGBP gene is 1,352 bases in length and is capable of encoding a polypeptide of 376 amino acids that showed significant similarity to homologous genes from crayfish, insects, earthworms, and sea urchins. Analysis of the shrimp LGBP deduced amino acid sequence identified conserved features of this gene family including a potential recognition motif for -(133) linkage of polysaccharides and putative RGD cell adhesion sites. It is known that LGBP gene expression is upregulated in bacterial and fungal infection and that the binding of lipopolysaccharide and -1,3-glucan to LGBP activates the prophenoloxidase (proPO) cascade. The temporal expression of LGBP and proPO genes in healthy and WSV-challenged Penaeus stylirostris shrimp was measured by real-time quantitative reverse transcription-PCR, and we showed that LGBP gene expression in shrimp was upregulated as the WSV infection progressed. Interestingly, the proPO expression was upregulated initially after infection followed by a downregulation as the viral infection progressed. The downward trend in the expression of proPO coincided with the detection of WSV in the infected shrimp. Our data suggest that shrimp LGBP is an inducible acute-phase protein that may play a critical role in shrimp-WSV interaction and that the WSV infection regulates the activation and/or activity of the proPO cascade in a novel way. Invertebrates lack a true adaptive immune system and rely instead on various innate immune responses against invading pathogens (12). Both cellular and humoral immunity play important roles in defense against microorganisms in invertebrates. The cellular immune responses include encapsulation, phagocytosis, and nodule formation (24), whereas the humoral responses include the clotting cascade, the synthesis of a wide array of antimicrobial peptides, and the phenoloxidase (PO)activating system (prophenoloxidase [proPO] system) (11, 15, 34). Bacteria and fungi have molecules such as lipopolysaccharide (LPS) and -1,3-glucans on their surfaces that are recognized by nonself recognition molecules and elicit an immune reaction in both vertebrates and invertebrates. Proteins that recognize the LPS and -1,3-glucans are known as pattern recognition proteins (PRPs) (16). In mammals, the complex of bacterial LPS and the plasma LPS binding protein binds to CD14, which transduces the signal through toll-like receptors and activates the NF-B signaling cascade (13, 21, 22, 49). In insects and crustaceans, LPS and -1,3-glucans, upon binding to LPS and glucan binding protein, activate proPO, the coagulation cascade, and the genes for antibacterial effector proteins (11, 15, 33).
BaciNus anthracis produces three proteins which combine to form two toxic activities. Protective ... more BaciNus anthracis produces three proteins which combine to form two toxic activities. Protective antigen (PA,
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