Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin

Chimeric proteins exhibiting antigenic determinants of the heat-labile enterotoxin (LT) and heat-stable (STa) enterotoxins on the same molecule may provide a means to obtain immunoprophylactic and diagnostic reagents for Escherichia coli-caused diarrhea. We recently showed that fusion of two different lengths of the STa gene to the C end of the A-subunit of LT (LTA) results in LTA::STa fusion proteins as monitored by GM1-ELISA [Sanchez et al.: FEBS Lett. 208 (1986) 194-198]. Here we determine the approximate molecular size of the LTA::STa fusion proteins and provide further evidence of their hybrid nature by immunoblot analysis. Using this technique we also demonstrate that to obtain detectable amounts of these recombinant proteins it is essential to coexpress them with the respective B-subunit of LT (LTB). We propose that this dependence on coexpression reflects the association between the LTA::STa hybrids and LTB subunits. The resulting LTA::STa/LTB complexes were found in the E. coli periplasm. This indicated that the exported hybrids, once associated with LTB, were stabilized and formed molecules that behaved essentially as native LT. The protective effect exerted by the B-subunit might conceivably be extended to other LTA-derived hybrid proteins, thus allowing the fusion of other foreign peptides to LTA and their subsequent recovery in the same fashion.     

大肠杆菌 LT B亚基基因表达载体对犬细小病毒VP2 DNA疫苗免疫应答的增强作用

【目的】通过融合基因表达载体和共免疫基因表达载体研究大肠杆菌不耐热肠毒素(LT)B亚基基因对犬细小病毒VP2DNA疫苗免疫应答的影响。【方法】提取大肠杆菌44815菌株基因组DNA,通过PCR方法从基因组DNA中扩增LTB基因,同时采用PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2的主要抗原表位基因(VP2-70,编码70个氨基酸)。将上述基因分别连接到含有人CD5信号肽序列的载体pcDNA-CD5sp上,分别构建成它们的分泌型真核表达载体,pcDNA-CD5sp-LTB和pcDNA-CD5sp-VP2-70。再利用酶切连接的方法构建LTB与VP2-70融合的真核表达载体pcDNACD5sp-LTB-VP2-70。然后用pcDNACD5sp-VP2-70(VP2-70组)、pcDNACD5sp-LTB-VP2-70(VP2-LTB融合组)、pcDNA-CD5sp-LTB/pcDNACD5sp-VP2-70(VP2-LTB共免疫组)和pcDNA3.1A(空载体对照组)分别免疫小鼠。免疫后用间接ELISA检测不同时间小鼠血清的抗体水平,用MTT方法检测小鼠免疫5周后脾脏淋巴细胞的增殖活性。【结果】经过测序表明本研究扩增的LTB和VP2基因序列和构建的相关表达载体结构正确。通过Western-blot检测证明构建的表达载体均能介导相应基因在真核细胞进行分泌表达。ELISA检测结果表明,3组实验组小鼠接受VP2DNA疫苗免疫后均能产生特异的体液免疫应答反应,特别是VP2-LTB基因融合组小鼠的抗体水平在第5周时高达1:5120,明显高于其它两组(P<0.01)。3组免疫小鼠抗体的亚型均表现IgG1抗体水平明显高于IgG2a抗体水平(P<0.01)。淋巴细胞增殖实验结果表明,在ConA的刺激下,3组免疫小鼠的淋巴细胞刺激指数均明显高于对照组(P<0.01),说明VP2DNA疫苗能够引起淋巴细胞的增殖。但3组免疫小鼠之间的刺激指数没有明显差异(P>0.05)。【结论】在小鼠体内,LTB基因表达载体可明显提高CPVVP2DNA疫苗的体液免疫应答水平。     

Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae

Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.     

Release of heat-labile enterotoxin subunits by Escherichia coli.

Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant. The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release. In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes. The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane.     

ARF binds the C-terminal region of the Escherichia coli heat-labile toxin (LTA1) and competes for the binding of LTA2

Cholera toxin (CT) and the heat-labile enterotoxin (LT) from Escherichia coli are highly related in terms of structure and biochemical activities and are the causative agents of cholera and traveler's diarrhea, respectively. The pathophysiological action of these toxins requires their activity as ADP-ribosyltransferases, transferring the ADP-ribose moiety from NAD onto the stimulatory, regulatory component of adenylyl cyclase, Gs. This reaction is highly dependent on the protein cofactor, termed ADP-ribosylation factor (ARF), that is itself a 20 kDa regulatory GTPase. In this study, we define sites of interaction between LTA and human ARF3. The residues identified as important to ARF binding include several of those previously shown to bind to the A2 subunit of the toxin and those important to the organization of two flexible loops, previously implicated as regulators of substrate entry. A model for how ARF acts to enhance the catalytic activity is proposed. A critical portion of the overlap between ARF and LTA(2) in binding LTA(1) includes a short region of sequence homology between LTA(2) and the switch II region of ARF. LTA(2) also interacted with ARF effectors in two-hybrid assays, and thus, we discuss the possibility that the LTA(2) subunit may function in cells as a partial ARF mimetic to compete for the binding of ARF to LTA(1) or regulate aspects of the toxin's transport from the cell surface to the ER.     

High yield enzymatic conversion of intravascular leukotriene A4 in blood-free perfused lungs

Experiments to investigate the fate of intravascularly administered leukotriene (LT) A4, an unstable intermediate of LT generation, were performed in isolated, ventilated, and blood-free perfused rabbit lungs. LT extracted from the lung effluent were separated by different reverse phase and straight phase HPLC procedures as methylated and nonmethylated compounds. Identity of eluting LT was confirmed by UV spectrum analysis and immunoreactivity. Pulmonary artery injection of 75 to 300 nmol of LTA4 resulted in the rapid appearance of cysteinyl-LT as well as LTB4 in the recirculating perfusate. The yield of these enzymatically generated LTA4 metabolites vs non-enzymatic hydrolysis products (6-trans-LTB4, 5-trans-epi-LTB4, 5,6-dihydroxyeicosatetraenoic acids) ranged above 90%. Experiments with application of tritiated LTA4 showed exclusive origin of the detected LT from the exogenously applied precursor. The time course of cysteinyl-LT appearance in the perfusate suggested metabolism of LTC4 via LTD4 to LTE4, whereas there was no evidence for LTB4 omega-oxidation. In the dose range of LTA4 used, the enzymatic conversion of this LT precursor did not approach saturation. Collectively, these data indicate that the intact pulmonary vasculature contains a hitherto not described capacity for enzymatic conversion of intravascularly offered LTA4 to both cysteinyl-LT and LTB4. This may be of biological significance for a putative transcellular biosynthesis of LT in the pulmonary microcirculation upon contact with LTA4 feeder cells, such as activated granulocytes.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Escherichia coli heat-labile enterotoxin genes are flanked by repeated deoxyribonucleic acid sequences.

The enterotoxin regions of the heat-labile and heat-stable enterotoxin (LT+ ST+) plasmid, pJY11, originating in a clinically isolated Escherichia coli strain, have been isolated as various-sized deoxyribonucleic acid (DNA) fragments by using cloning vehicles. The structure of the LT+ region and its neighboring DNA regions was studied by utilizing these recombinant plasmids. The LT+ region consisted of at least two genes, toxA and toxB, which could complement each other in trans. The toxA- and toxB-encoded polypeptides (LT subunits A and B, respectively) were identified by their immunological cross-reactivity with Vibrio cholerae enterotoxin subunit A or B. These tox genes and the promoter(s) were localized with respect to the restriction endonuclease cleavage map. The LT+ region was flanked by repeated DNA sequences (designated as beta). Another tox gen(s), encoding ST (designated as toxS), which was also flanked by inverted, repeated DNA sequences (designated as alpha), was located between one of the beta sequences and the LT+ region. These novel DNA structures (beta-alpha-toxS-alpha-toxA-toxB-beta) suggest the possibility that the LT+ region is on a transposon containing an ST transposon within the structure.     

Determination of the contribution of cysteinyl leukotrienes and leukotriene B4 in acute inflammatory responses using 5-lipoxygenase- and leukotriene A4 hydrolase-deficient mice

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.     

Overlapping genes in the heat-labile enterotoxin operon originating from Escherichia coli human strain

Summary We have determined the nucleotide sequence at the distal end of the heat-labile enterotoxin subunit A (LTA) gene (toxA) originating from human enterotoxigenic Escherichia coli. The sequenced region covers the entire LT-A2 region and a part of the LT-A1 region. In confirming our previous prediction based on product analysis of clones toxA regions, the data suggest the overlapping of the distal end (5-TTA TGA) of toxA with the proximal end (5-ATG AAT) of the LT subunit B gene (toxB), in the sequence 5-TTATGAAT. Some additional characteristics of the LT operon as well as of the products are discussed.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A4, B4, C4, D4 and E4 have potent myotropic activity on guinea-pig lung parenchymal strip in vitro. The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB4, the tissues became desensitized to LTB4 but were still responsive to histamine, LTA4, LTC4, LTD4 and LTE4. When LTD4 was infused continuously, the lung strips contracted to LTB4 and histamine but were no longer responsive to LTA4, LTC4, LTD4 and LTE4. Furthermore, FPL-55712 (10 ng ml-1 - 10 ug ml-1) produced dose-dependent inhibitions of LTA4, LTC4, LTD4 and LTE4 without inhibiting the contraction to LTB4 and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB4 and another for LTD4; LTA4, LTC4 and LTE4 probably act on the LTD4 receptor.     

Cistrons encoding Escherichia coli heat-labile toxin.

The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter.     

Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene

In vitro mutagenesis of the LTA gene, encoding the A subunit of the Escherichia coli heat-labile enterotoxin, has been used to obtain A subunits deficient in enzymic activity. One inactive A-subunit mutant which contained two amino acid substitutions, was shown to associate with native B subunits to form a holotoxoid lacking toxin activity. A serine to phenylalanine mutation appears to be responsible for the loss of toxicity.     

Escherichia coli heat-labile enterotoxin. Nucleotide sequence of the A subunit gene

We report the complete DNA sequence of the Escherichia coli elt A gene, which codes for the A subunit of the heat-labile enterotoxin, LT. The amino acid sequence of the LT A subunit has been deduced from the DNA sequence of elt A. The LT A subunit starts with methionine, ends with leucine, and comprises 254 amino acids. The computed molecular weight of LT A is 29,673. The A subunit of cholera toxin (CT A) has been shown to be structurally and functionally related to the LT A subunit. Comparison of the primary structure of LT A with the known partial amino acid sequence of CT A indicates that the 2 polypeptides share considerable homology throughout their sequences. The NH2-terminal regions exhibit the highest degree of homology (91%), while the COOH-terminal region, containing the sole cystine residue in each toxin is less conserved (approximately 52%). Alignment of homologous residues in the COOH-terminal regions of LT A and CT A indicates that a likely site for proteolytic cleavage of LT A is after Arg residue 188. The resulting A2 polypeptide would be 46 amino acids long, would contain a single cysteine residue, and have Mr = 5261. The elt A nucleotide sequence further predicts that the LT A protein is synthesized in a precursor form, possessing an 18-amino acid signal sequence at its NH2 terminus.     

Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.     

Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.     

Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).     

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.