Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.     

ISPsa2, the first mobile genetic element to be described and characterized in the bacterial facultative intracellular pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is a novel, aggressive, facultative Gram-negative bacterium that drastically affects salmon production at different latitudes, with particular impact in southern Chile. Initially, P. salmonis was described as a Rickettsia-like, obligate, intracellular Alphaproteobacteria, but it was reclassified recently as a facultative intracellular Gammaproteobacteria. This designation has prompted the independent growth of the bacterium to a pure state for detailed study of its biology, genetics and epidemiology, properties that are still relatively poorly characterized. The preliminary sequence analysis of a 992-bp fragment of pure P. salmonis DNA allowed us to characterize a novel and complete 863-bp insertion sequence in the bacterial genome (named ISPsa2), which has a novel 16/16bp perfectly inverted terminal repeat flanking a 726-bp ORF that encodes a putative transposase (Tnp-Psa). The coding sequence of the enzyme shares similarities to that described in some Bacillus species and particularly to those of the IS6 family. ISPsa2 carries its own promoter with standard -10 and -35 sequences, suggesting an interesting potential for plasticity in this pathogenic bacterium. Additionally, the presence of ISPsa2 was confirmed from three isolates of P. salmonis collected from different epizootics in Chile in 2010.     

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.     

Characterization of multiple members of the HSP70 family in platyfish culture cells: molecular evolution of stress protein HSP70 in vertebrates

A shift from 28 to 37 degrees C in the incubation temperature of a culture of the platyfish fibroblast cell line, EHS cells (platyfish fibroblast cell line), induced a set of stress proteins. A two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed that the cells expressed three genetically distinct forms of heat-shock protein 70 (HSP70) family proteins: heat-inducible forms of HSP70, the constitutively expressed heat-shock cognate protein 70 (HSC70) and its phosphorylated isoform, and the glucose-regulated protein 78 (GRP78). Three different clones encoding two major isoforms of heat-inducible HSP70, platyfish HSP70-1 and HSP70-2, and of the HSC70 were isolated from a platyfish cDNA library. We compared the deduced amino acid sequences of the platyfish HSP70 and HSC70 proteins with those of other vertebrates. Phylogenetic analysis showed that vertebrate HSP70 could be classified into four cluster groups: (a) fish HSP70, with two isoforms of heat-inducible HSP70 in fish, fish HSP70-1 and HSP70-2; (b) the mammalian testis-specific HSP70-related protein HST70; (c) the mammalian heat-inducible HSP70B'; and (d) the mammalian major histocompatibility complex (MHC)-linked HSP70, including the MHC-linked heat-inducible HSP70 and the testis-specific HSP70-related protein. These findings suggest that vertebrate HSP70 was derived from a single ancestral HSP70 gene during vertebrate evolution and that multiple copies of heat-inducible HSP70 were probably evolved during genetic divergence in fish and higher vertebrates.     

Genetic characterization of the mitochondrial DNA from Lepeophtheirus salmonis (Crustacea; Copepoda). A new gene organization revealed

The mitochondrial DNA (mtDNA) from the salmon louse, Lepeophtheirus salmonis, is 15445 bp. It includes the genes coding for cytochrome B (Cyt B), ATPase subunit 6 and 8 (A6 and A8), NADH dehydrogenase subunits 1-6 and 4L (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), cytochrome c oxidase subunits I-III (COI, COII and COIII), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNAs. Two copies of tRNA-Lys are present in the mtDNA of L. salmonis, while tRNA-Cys was not identified. Both DNA strands contain coding regions in the salmon louse, in contrast to the other copepod characterized Tigriopus japonicus, but only a few genes overlap. In vertebrates, ND4 and ND4L are transcribed as one bicistronic mRNA, and are therefore localized together. The same organization is also found in crustaceans, with the exceptions of T. japonicus, Neocalanus cristatus and L. salmonis that deviate from this pattern. Another exception of the L. salmonis mtDNA is that A6 and A8 do not overlap, but are separated by several genes. The protein-coding genes have a bias towards AT-rich codons. The mitochondrial gene order in L. salmonis differs significantly from the copepods T. japonicus, Eucalanus bungii, N. cristatus and the other 13 crustaceans previously characterized. Furthermore, the mitochondrial rRNA genes are encoded on opposite strands in L. salmonis. This has not been found in any other arthropods, but has been reported in two starfish species. In a phylogenetic analysis, using an alignment of mitochondrial protein sequences, L. salmonis groups together with T. japonicus, being distant relatives to the other crustaceans.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis

Mortality among hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was associated with infections by a Piscirickettsia salmonis-like organism (WSPSLO). Infected fish had no consistent external signs other than pale gills, lethargy and impaired swimming behavior. Internally, the kidney and spleen were enlarged, and some fish had livers with multiple pale foci. Smears from infected kidney, liver, and spleen stained with Wright-Giemsa had intracytoplasmic coccoid organisms, often in pairs, that ranged in size from 0.5 to 1.0 microm. Microscopic lesions included multifocal hepatic, renal, and splenic necrosis, and intralesional macrophages often contained the WSPSLO. The bacterium was isolated from infected fish on cell lines of salmonid (CHSE-214) and white seabass (WSBK) origin. The WSPSLO induced plaque formation and destroyed the cell monolayers within 10 to 14 d incubation at temperatures of 15 and 20 degrees C. The bacterium retained infectivity for cell lines up to 14 d at 4 and 13 degrees C, up to 7 d at 20 degrees C, but it was inactivated at 37 and 56 degrees C within 24 and 1 h, respectively. Freezing at -20 degrees C reduced infectivity by 100-fold. Dehydration and resuspension in distilled water completely inactivated the bacterium. In contrast, the WSPSLO retained nearly all of its infectivity for CHSE-214 cells following a 72 h period in seawater at 20 degrees C. Polyclonal rabbit antibodies made to the WSPSLO reacted specifically in indirect fluorescent antibody tests (IFAT) with the bacterium in cell cultures and smears from infected fish tissues. Tissue smears from infected salmon or CHSE-214 cells with P. salmonis reacted weakly with the anti-WSPSLO serum. Conversely, polyclonal anti-P. salmonis serum produced a weakly positive reaction with the WSPSLO from infected CHSE-214 cells. The WSPSLO as propagated in CHSE-214 cells was highly virulent for juvenile coho salmon Oncorhynchus kisutch, inducing 80% mortality within 10 d of intraperitoneal injection of 10(2.5)-50% tissue culture infectious doses per fish. We conclude that the bacterium from white seabass possesses antigenic differences from P. salmonis yet possesses virulence for salmon equal to known strains of P. salmonis.     

Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding

The human HSP70 gene was modified in vitro using oligonucleotide-directed mutagenesis to add sequences encoding a peptide from the testis-specific form of human lactate dehydrogenase (LDH) to the carboxy terminus of HSP70. The peptide-tagged HSP70 can be distinguished from the endogenous HSP70 protein using an LDH peptide-specific antiserum in indirect immunofluorescence assays of cells transiently transfected with an expression vector containing the tagged HSP70 gene regulated by the human HSP70 promoter. A series of deletion mutants within the HSP70 protein coding region were generated. Using double-label indirect immunofluorescence with the LDH peptide-specific antiserum and HSP70-specific mAbs, we compared the intracellular distribution of the deletion mutants to that of endogenous HSP70. We have determined that sequences in the carboxy terminus of HSP70 are necessary for proper nucleolar localization after heat shock. In contrast, sequences in the amino terminus of HSP70 are responsible for the ATP-binding ability of the protein. Mutants that were unable to bind ATP, however, still displayed nucleolar association, indicating that ATP binding is apparently not required for interaction with substrate. Additional support that HSP70 appears to be composed of at least two domains follows from the results of trypsin digestions of wild type and mutant HSP70. Protease digestion of the mutant HSP70 proteins identified a region of HSP70 that, when deleted, affected HSP70 conformation.     

Isolates of Piscirickettsia salmonis from Scotland and Ireland show evidence of clonal diversity

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.     

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.     

Isolation of a gene encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe

We have isolated cDNA and genomic clones encoding a mitochondrial HSP70 protein from Schizosaccharomyces pombe. Nucleotide sequence analysis indicates that the encoded protein is homologous to the HSP70s of other organisms. The highest degree of amino acid conservation is with the proteins encoded by the Escherichia coli dnaK gene, the SSC1 gene of Saccharomyces cerevisiae and the MTP70 gene of Trypanosoma cruzi, the latter two having recently been shown to be located in the mitochondria. Western-blot analysis with immunoglobulin G raised against a peptide corresponding to the C terminus of the SSP1 protein indicates a 70-kDa protein which is associated with the mitochondria.     

柔嫩艾美耳球虫HSP基因的克隆、表达及鉴定

为研究柔嫩艾美耳球虫热激蛋白(Heat shock proteins,HSPs)的生物学特性,应用RACE和RT-PCR技术,从柔嫩艾美耳球虫子孢子中首次克隆获得了EtHSP的全长cDNA(GenBank Accession No.FJ911605)。EtHSP包含一个1455 bp的开放阅读框,编码484个氨基酸,预测表达蛋白的分子量大小为53.5 kD。应用Real-time PCR对柔嫩艾美耳球虫不同发育阶段(未孢子化卵囊、孢子化卵囊、子孢子和裂殖子)表达量进行分析,发现该基因在子孢子阶段的表达明显高于其他阶段。同时,构建了原核表达重组质粒pET28a(+)-EtHSP,转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE及Western blotting分析。结果显示,重组质粒pET28a(+)-EtHSP在大肠杆菌中以包涵体形式表达,经1 mmol/L IPTG诱导6 h后的表达量最高,该蛋白可被抗柔嫩艾美耳球虫的多克隆抗血清识别,表明该蛋白具有较好的反应原性。本研究结果为进一步研究该基因的生物学功能奠定了基础。     

Cloning of the HSP70 gene from Halobacterium marismortui: relatedness of archaebacterial HSP70 to its eubacterial homologs and a model for the evolution of the HSP70 gene.

Heat shock induces the synthesis of a set of proteins in Halobacterium marismortui whose molecular sizes correspond to the known major heat shock proteins. By using the polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of the 70-kDa heat shock protein (HSP70) family, we have successfully cloned and sequenced a gene fragment containing the entire coding sequence for HSP70 from H. marismortui. HSP70 from H. marismortui shows between 44 and 47% amino acid identity with various eukaryotic HSP70s and between 51 and 58% identity with its eubacterial and archaebacterial homologs. On the basis of a comparison of all available HSP70 sequences, we have identified a number of unique sequence signatures in this protein family that provide a clear distinction between eukaryotic organisms and prokaryotic organisms (archaebacteria and eubacteria). The archaebacterial (viz., H. marismortui and Methanosarcina mazei) HSP70s have been found to contain all of the signature sequences characteristic of eubacteria (particularly the gram-positive bacteria), which suggests a close evolutionary relationship between these groups. In addition, detailed analyses of HSP70 sequences that we have carried out have revealed a number of additional novel features of the HSP70 protein family. These include (i) the presence of an insertion of about 25 to 27 amino acids in the N-terminal quadrants of all known eukaryotic and prokaryotic HSP70s except those from archaebacteria and the gram-positive group of bacteria, (ii) significant sequence similarity in HSP70 regions comprising its first and second quadrants from organisms lacking the above insertion, (iii) highly significant similarity between a protein, MreB, of Escherichia coli and the N-terminal half of HSP70s, (iv) significant sequence similarity between the N-terminal quadrant of HSP70 (from gram-positive bacteria and archaebacteria) and the m-type thioredoxin of plant chloroplasts. To account for these and other observations, a model for the evolution of HSP70 proteins involving gene duplication is proposed. The model proposes that HSP70 from archaebacteria (H. marismortui and M. mazei) and the gram-positive group of bacteria constitutes the ancestral form of the protein and that all other HSP70s (viz., other eubacteria as well as eukaryotes) containing the insert have evolved from this ancient protein.     

Characterization of proteases in the skin mucus of Atlantic salmon (Salmo salar) infected with the salmon louse (Lepeophtheirus salmonis) and in whole-body louse homogenate

As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17-22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.     

Infectivity of a Scottish isolate of Piscirickettsia salmonis for Atlantic salmon Salmo salar and immune response of salmon to this agent

A Scottish isolate of Piscirickettsia salmonis (SCO-95A), previously shown by intraperitoneal injection to have a lethal dose (LD50) of < 2 x 10(3) infectious rickettsial units, was tested for virulence by bath challenge, surface application to the skin, or dorsal median sinus injection. Atlantic salmon Salmo salar post-smolts were used in all experiments, and exposure to 1 x 10(5) tissue culture infective doses (TCID) of P. salmonis ml(-1) for 1 h in a bath challenge resulted in only 1 mortality, 18 d later, in 10 exposed fish. Application of 2.5 x 10(6) TCID of P. salmonis SCO-95A to paper discs on the skin failed to induce any mortalities within 42 d. Intraperitoneally, fish were administered vaccines containing 10(9) heat-inactivated (100 degrees C, 30 min) or 10(9) formalin-inactivated P. salmonis SCO-95A in adjuvant, with a control group receiving phosphate-buffered saline (PBS) in adjuvant. After an induction period of over 6 mo fish were challenged by injection of P. salmonis into the dorsal median sinus. Mortalities in the control group reached 81.8% and the heat-inactivated and formalin-inactivated vaccines gave significant protection from P. salmonis, with relative percentage survivals of 70.7 and 49.6%, respectively. The nature of the protective antigen is unknown, but could be lipopolysaccharide or a heat-stable outer membrane protein. Fish that survived a dorsal median sinus challenge of P. salmonis or were cohabitants showed a strong immune response to P. salmonis.     

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy

A cell line derived from the tailfin of the marine teleost yellowtail fish Seriola quinqueradiata was established to examine cellular temperature regulation in an ectothermic animal. Three cytosolic members of the HSP70 family, heat-shock cognate proteins HSC70-1, HSC70-2 and heat-shock protein HSP70, were isolated from cultured yellowtail cells as stress-responsive biomarkers. Expression of hsp70 was heat-inducible, in contrast to the hsc70-1 gene product, which was expressed constitutively. In addition, expression of hsc70-2 was only induced under severe heat-shock conditions. Subcellular fractionation and immunocytochemistry showed localization of HSC70/HSP70 in the lysosomes, indicating that chaperone-mediated autophagy is induced by heat shock. Thus, chaperone-mediated autophagy is assisted by HSC70/HSP70, and heat-inducible expression of the genes encoding these proteins may be responsible for survival and adaptation under heat-shock conditions in fish cells.