Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTa_A14Q-dmLT or 3xSTa_N12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STa_A14Q-dmLT and CFA/I/II/IV-STa_N12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa_-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTa_N12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa_-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTa_N12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.     

A Chimeric protein of CFA/I,CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti‐CF and anti‐enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti‐CF and antitoxin immunogenicity was then assessed. To achieve high‐level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti‐CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion.

     

Genetic fusion protein 3×STa‐ovalbumin is an effective coating antigen in ELISA to titrate anti‐STa antibodies

Heat‐stable toxin type I (STa)‐ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti‐STa antibodies for ETEC vaccine candidates. STa‐ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti‐STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa‐ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti‐STa antibody titration ELISA. Data showed fusion protein 3×STa‐ovalbumin was effectively expressed and extracted, and anti‐STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa‐ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti‐STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa‐ovalbumin is an effective ELISA coating antigen for anti‐STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.

     

Protection of mice against enterotoxigenic E. coli by immunization with a polyvalent enterotoxin comprising a combination of LTB, STa, and STb

Currently available enterotoxigenic Escherichia coli (ETEC) vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). However, the induction of antitoxic responses against heat-stable enterotoxin a (STa) and b (STb) has merit as these two poorly immunogenic toxins are frequently associated with ETEC strains. In this study, we genetically constructed a trivalent enterotoxin fusion protein (STa–LTB–STb, abbreviated to SLS) in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. Mutagenesis at one disulfide-bridge-forming cysteine in STa led to a dramatic reduction in the STa toxicity of SLS; however, the fusion peptide retained the STb-associated toxicity. Immunization of mice with SLS protein elicited significant antibody responses to LTB, STa, and STb. Significantly, the mice antisera were able to neutralize the biological activity of both STa and STb. In the experiment to assess the protective effect of SLS immunization, the mortality of mice receiving SLS was significantly lower than their control cohorts (P < 0.01) after intraperitoneal challenge with ETEC. These results show that the trivalent fusion enterotoxin SLS has the potential to serve as a useful toxin-based vaccine against ETEC-induced diarrheal disease via a single immunogen.     

产肠毒素大肠杆菌诱导肠上皮细胞凋亡的研究进展

产肠毒素大肠杆菌(enterotoxigenic Escherichia coli, ETEC)是引起人和动物腹泻的重要病原菌之一,其中黏附素和肠毒素是其感染引起腹泻的主要毒力因子。首先,黏附素介导ETEC与宿主小肠上皮细胞的黏附和定殖。随后,定殖的细菌产生肠毒素,导致水、电解质代谢紊乱,最终引起水样腹泻。传统的观点认为ETEC属于非侵袭性大肠杆菌,并不会引起肠上皮细胞凋亡和破坏肠道的屏障结构。但是越来越多的研究证据表明,在体外和体内ETEC感染均可诱导肠上皮细胞凋亡,破坏宿主肠黏膜屏障的完整性,促进疾病发展。本文将就ETEC不同毒力因子诱导细胞凋亡的具体机制、细胞凋亡与疾病发展的相关性以及在临床如何利用抗凋亡治疗预防ETEC感染等方面进行综述,旨为进一步深入阐明ETEC的分子致病机制提供参考,为防治ETEC引起的腹泻提供新策略。     

人源产肠毒素大肠杆菌疫苗的研发进展

腹泻是全球范围内引起5岁以下幼童死亡的第二大病因,而产肠毒素大肠杆菌(ETEC)是引起腹泻的最常见病原菌,其产生的细菌定植因子(CFs)和肠毒素是关键的毒力因子。CFs介导细菌黏附宿主小肠上皮细胞并完成定植,产生热敏肠毒素(LT)和热稳定肠毒素(ST)破坏宿主上皮细胞内的体液平衡,使体液和电介质过量分泌从而导致腹泻。预防ETEC腹泻的首选方法是使用能激发宿主产生抗黏附素免疫力和抗肠毒素免疫力的疫苗,阻断ETEC黏附和定植并中和肠毒素。目前一种名为Dukoral~的霍乱疫苗因能刺激机体产生抗热敏毒素免疫,已经被一些国家批准用于短期保护和预防旅行者腹泻。新型试验性ETEC候选疫苗正在研发中,旨在提供保护期长、反应谱广的抗ETEC感染免疫保护力。本文针对疫苗研发的关键问题和研究现状作一综述,并对未来的研究作出展望。     

肠毒素大肠杆菌定居因子CS3和肠毒素融合抗原基因在减毒鼠伤寒沙门氏菌中的共表达

不耐热肠毒素（LT）和耐热肠毒素（ST）是产肠毒素大肠杆菌的主要致病因素,CS3为该菌的优势定居因子,是定居因子CFA/Ⅱ菌毛抗原的共有抗原组分。采用基因操作技术将编码CS3和融合肠毒素蛋白基因转化到减毒鼠伤寒沙门氏菌疫苗侯选株X4072中进行表达。用重组菌株口服免疫小鼠后,免疫动物能产生抗CS3、LT和ST的血清抗体。特别有意义的是,所产生的抗ST抗体能中和天然ST的生物活性。这一结果为研究载体疫苗防治肠毒素大肠杆菌腹泻疾病奠定了基础。     

A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies

A recombinant fusion protein consisting of native Escherichia coli heat-stable enterotoxin (STa) and a dimer of a synthetic IgG-binding fragment (ZZ), derived from Staphylococcus aureus protein A was produced in E. coli. The fusion protein (ZZSTa) was secreted in large quantities into the growth medium and recovered by affinity chromatography on IgG-Sepharose. Rabbits immunized with the fusion protein responded by producing high serum levels of anti-STa antibodies that also effectively neutralized STa toxicity in infant mice. The fusion peptide ZZSTa had a substantially decreased toxicity as compared with native STa. A polymeric form of ZZSTa separated by size fractionation was about 100 times less toxic than the monomeric fusion protein, yet both forms had the same capacity to induce neutralizing antibodies. This suggests that modified non-toxic forms of ZZSTa with retained immunogenicity may be produced and tested for their usefulness as functional components in a vaccine against diarrhoea caused by enterotoxigenic E. coli.     

Little Heterogeneity among Genes Encoding Heat-Labile and Heat-Stable Toxins of Enterotoxigenic Escherichia coli Strains Isolated from Diarrheal Pigs

To examine whether the heat-labile enterotoxin gene in porcine enterotoxigenic Escherichia coli (ETEC) strains is as divergent as in human ETEC strains, we sequenced the heat-labile and heat-stable toxin genes from 52 and 33 porcine ETEC strains, respectively. We found that the STa gene is identical, that the LT gene has only two mutations in 4 (of 52) strains, and that both mutations cause a reduction in GM1 binding and toxicity.Enterotoxigenic Escherichia coli (ETEC) strains that colonize small intestines and produce enterotoxins are the major cause of diarrheal disease in humans and animals (8, 16, 18, 21). The key virulence factors of ETEC in diarrhea include enterotoxins and colonization factors or adhesins. Colonization factors or adhesins mediate the attachment of bacteria to host epithelium cells and facilitate bacterial colonization. Enterotoxins disrupt fluid homeostasis and stimulate fluid hyper-secretion in the intestinal epithelial cells that results in diarrhea. Heat-labile toxin (LT) and heat-stable toxin (ST) are the main enterotoxins associated with diarrhea in humans and farm animals, but different LT and ST are produced by human and animal ETEC strains (9, 16).The LT produced by porcine ETEC strains (pLT) or human ETEC strains (hLT) is a holotoxin-structured protein that has one LT_A subunit and five LT_B subunits. Although pLT and hLT are highly homologous in structure and function, these two proteins differ antigenetically (9). Sequence comparative studies showed that the following seven amino acids are different between pLT and hLT: the 4th, 213th, and 237th amino acids of the A subunits and the 4th, 13th, 46th, and 102nd amino acids of the B subunits (6, 7). Similarly, STa (ST type 1) carried by human and porcine ETEC strains is also different. The STa associated with porcine diarrhea (pSTa) is a peptide of 18 amino acids, whereas the STa produced by human ETEC strains (hSTa) is 19 amino acids in length (5, 19). Despite the fact that ETEC constructs expressing pLT or hLT, and pSTa or hSTa, are equivalently virulent in causing diarrhea in gnotobiotic pigs (25), pLT and pSTa are typically expressed by porcine ETEC strains that only cause diarrhea in pigs, whereas hLT and hSTa are exclusively produced by human ETEC strains associated with diarrhea in humans. Although pLT and STb, another porcine-specific ST, were occasionally detected in ETEC strains isolated from human diarrheal patients (3), only infections with hSTa⁺, hLT⁺, or hSTa⁺/hLT⁺ ETEC strains cause diarrhea in humans (17).Interspecies LT have been intensively compared for molecular and immunological characteristics (4, 10, 20, 23). In contrast, intraspecies LT has not been studied much. For a long time, both pLT and hLT were assumed to be highly conserved. However, a very recent study showed that the hLT gene carried by human ETEC strains is considerably divergent (12). After restriction fragment length polymorphism analysis and DNA sequencing of 51 human ETEC strains, Lasaro et al. reported that the human LT gene had seven polymorphic restriction fragment length polymorphism types and 30 nucleotide polymorphic sites and recognized 16 different hLT types (12). To examine whether the LT gene carried by porcine ETEC strains has a similar heterogeneity, we PCR amplified and DNA sequenced the LT genes and also the STa genes of various ETEC strains isolated from diarrheal pigs and analyzed gene sequence conformity.Fifty-two porcine ETEC strains that express LT alone or LT together with other toxins (LT⁺/STb⁺, LT⁺/STb⁺/STa⁺, LT⁺/STb⁺/EAST1⁺, and LT⁺/STa⁺/STb⁺/EAST1⁺) and K88ac or F18 fimbria were selected for the sequencing of the LT gene. Those porcine ETEC strains were isolated from pigs with postweaning diarrhea at different farms in South Dakota, Iowa, Minnesota, Nebraska, and North Dakota. The eltAB gene encoding LT from these 52 strains was PCR amplified with primers pLT-F (5′-ATCCTCGCTAGCATGTTTTAT-3′) and pLT-R (5′-CCCCTCCGGCCGAGCTTAGTT-3′) (25). PCRs were performed in an MJ PT-100 thermocycler (Bio-Rad, Hercules, CA) in a reaction of 50 μl containing 1× Taq DNA polymerase buffer (with Mg²⁺), 0.2 mM deoxynucleoside triphosphate, 0.5 μM each forward and reverse primers, 100 ng of total genomic DNA, and 1 unit Taq DNA polymerase (Applied Biosystems, Foster City, CA). The PCR program contained one cycle of 2 min at 94°C; 30 cycles of 35 s at 94°C, 35 s at 52°C, and 2 min at 72°C; and an extension of 6 min at 72°C. The amplified PCR products were separated on 1% agarose gels (FMC Bioproducts, Rockland, MA) by electrophoresis and purified using a QIAquick gel extraction kit according to the manufacturer''s instructions (Qiagen, Valencia, CA). A mixture of purified PCR product (100 to 150 ng) and 10 pmol primer was sent to the Nevada Genomic Center at the University of Nevada for sequencing. Three primers, pLT-F, LT₁₉₂-F (5′-GATTCATCAAGAACAATCCACAGGTG-3′), and LT₁₉₂-R (5′-CCTGTGATTGTTCTTGATGAATC-3′), were used for sequencing the entire eltAB gene.The sequences of the eltAB gene from all 52 porcine ETEC strains were aligned and visually examined. We found that the eltAB gene was nearly identical among the sequenced porcine ETEC strains. Forty-eight (of 52) ETEC strains had identical gene sequences, and only four strains showed heterogeneity. The pathotypes of these four strains were K88/LT/STb, K88/LT/STb/STa, K88/LT/STb/EAST1, and F18/LT/STa/STb/Stx2e. Furthermore, only nucleotides coding two amino acids, the 44th (S44N) and the 60th (S60T) of the eltB gene encoding the B subunit, differed among these four strains. To our surprise, neither of these two substitutions were homologous to the hLT gene nor to any of the hLT types recognized by Lasaro et al. (12). Lasaro et al. showed that 11 of the 15 different hLT types shared some homology with pLT, and some hLT types had as many as four amino acids (K4R and K213E of LT_A and S4T, R13H, or A46E of LT_B; out of seven heterogeneous amino acids) homologous to pLT. Indeed, the hLT6 type differed from the LT of human ETEC prototype {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 in four amino acids (K4R and K213E of LT_A and S4T and A46E of LT_B) (12), but all four of these heterogeneous amino acids were homologous to pLT. Similarly, four of the five amino acids that differed from the prototype hLT in the hLT4 type were identical to pLT. That means that the hLT4 and hLT6 types had only three amino acids heterogeneous to pLT but four different residues compared to the hLT prototype. It seems that hLT4 and hLT6 are more likely pLT rather than hLT. Given that the divergence of the pLT and hLT genes is assumed to be a very recent evolutionary event that occurred 0.9 million years ago (23), it is likely that the hLT gene retains some pLT gene characters (amino acids) that could be of their common ancestor. However, a high homology in the pLT gene certainly seems unparallel to the evolution of the hLT gene. Our further sequence comparison indicated that S44N-substituted pLT_B [pLT_B(S44N)] is homologous to cholera toxin (CT). It has been suggested that the CT and LT genes were derived from the same ancestor but diverged to two lineages about 130 million years ago (23). Then, it is more likely that this pLT_B(S44N) represents a plesiomorphic character, meaning a primitive character that belongs to the common ancestor of CT and LT. The retention of this primitive pLT_B(S44N) by some porcine ETEC strains suggests that the pLT gene could have evolved at a relatively lower rate. Whether such a lower substitution rate of the LT gene in porcine ETEC strains is associated with a lower host exchange rate or a limited travel range in pigs is unclear to us. However, future studies to determine whether an increase in sampling sizes, by including porcine ETEC strains from a greater geographic coverage, could reveal a higher heterogeneity or a greater evolution rate in the pLT gene will be worthwhile.To examine whether the heterogeneity of pS44N and pS60T at the B subunit could affect the biological function of pLT, we cloned the native pLT gene into vector pBR322 (p8458), performed site-directed mutation of the eltAB gene for a substitution of S44N or S60T, and tested these two mutated LT proteins for their binding capability to GM1 receptors and their enterotoxic activity in stimulating intracellular cyclic AMP (cAMP) in cells. Primers pBRNheI-F2 (5′-CAGCATCGCCATTCACTATG-3′) and pBREagI-R (5′-AGATGACGACCATCAGGGAC-3′) were designed to amplify the porcine eltAB gene. The amplified eltAB gene products and vector pBR322 were digested with NheI and EagI (New England Biolabs, Beverly, MA), separated by gel electrophoresis, purified with the QIAquick gel extraction kit, and then ligated with T4 DNA ligase (Promega, Madison, WI). Two microliters of the T4-ligated products were introduced into 25 μl of TOPO cells (Invitrogen, Valencia, CA) in a standard electroporation. Antibiotic-selected colonies were initially screened by PCR, and positive colonies were sequenced to ensure that the cloned gene was in the reading frame. The verified clone was selected as a pLT recombinant strain and designated strain 8458. To construct mutant strains, two pairs of primers, LT_B44-F (5′-ATCATTACATTTAAGAACGGCGAA-3′) and LT_B44-R (5′-TTCGCCGTTCTTAAATGTAATGAT-3′) and LT_B60-F (5′-CAACATATAGACACCCAGAAAAAAGCC-3′) and LT_B60-R (5′-GGCTTTTTTCTGGGTGTCTATATGTTG-3′), were used for site-directed mutation at nucleotides coding the 44th and 60th amino acids of the LT_B subunit, respectively. Briefly, the amplified products from two separate PCRs, one using pBRNheI-F2 with LT_B44-R or LT_B60-R and the other using pBREagI-R with LT_B44-F or LT_B60-F, with recombinant pLT plasmid p8458 as the DNA template, were overlapped in a third splicing overlap extension PCR to produce mutated pLT genes. The splicing overlap extension PCR products were digested with NheI and EagI restriction enzymes and ligated into vector pBR322 for the p8647 (S44N) and p8649 (S60T) plasmids. Plasmids p8647 and p8649 were separately introduced into TOPO 10 E. coli cells (Invitrogen) for mutant strains 8647 (S44N) and 8649 (S60T).Equivalent amounts of cells from overnight-grown cultures of the recombinant (8458) and two mutant (8647 and 8649) strains were used for total protein preparation by using bacterial protein extraction reagent (B-PER in phosphate buffer; Pierce, Rockford, IL). Both pelleted protein samples (periplasmic proteins) and culture supernatant samples (outer-membrane secreted proteins) were used in a GM1 enzyme-linked immunosorbent assay (ELISA) to examine whether a substitution at the 44th or 60th amino acid would affect the binding of LT to GM1 receptors. Anti-CT rabbit antiserum (1:5,000; Sigma) and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5,000; Sigma, St. Louis, MO) were used as the primary and secondary antibodies as described previously (2, 14, 24). GM1 ELISA data indicated optical density (OD) values from the pellet samples of strains 8548, 8647, and 8649 and phosphate-buffered saline of 0.677 ± 0.004, 0.616 ± 0.001, 0.647 ± 0.004, and 0.006 ± 0, whereas the OD values of the supernatant samples which were vacuum concentrated were 0.949 ± 0.008, 0.726 ± 0.004, 0.660 ± 0.005, and 0.05 ± 0.002, respectively (Fig. ​(Fig.1).1). Statistical analysis using the Student t test with two-tailed distribution indicated that the binding of the pellet samples from the native and the mutated LT to GM1 was not significantly different (P = 0.10 and P = 0.45, respectively). However, the GM1 binding from the supernatant samples of the LT mutant strains was significantly lower than that of the LT recombinant strain (P < 0.01 and P < 0.01, respectively).Open in a separate windowFIG. 1.GM1 ELISA to detect LT proteins expressed by the pLT recombinant (8458) and mutant [8647(S44N) and 8649(S60T)] strains. Protein samples from the pellet and vacuum-concentrated supernatants of overnight-grown cultures were used in the GM1 ELISA. Each sample was assayed in triplicate to calculate OD means and standard deviations. Anti-CT serum (1:5,000) was used as the primary antibody and goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G (1:5,000) was used as the secondary antibody. OD values were measured after a 20-min reaction with peroxidase substrates (KPL, Gaithersburg, MD) at a wavelength of 405 nm.Our GM1 ELISA data indicated that the supernatant sample of the recombinant strain expressing a native LT had a greater GM1 binding activity. This could suggest that the recombinant strain had more LT protein crossing the outer membrane and being secreted in the supernatant than either mutant strain or that mutations at the B subunit negatively affected the binding of LT proteins to GM1 receptors. It has been reported that a single amino acid mutation of the LT_B or CT_B subunit resulted in lower GM1 binding activity, especially mutations of residues from the binding pocket (13, 15, 22). When amino acid 33 or 88 of the CT_B subunit was replaced, both mutants failed to bind or bound poorly to GM1 (22), and when a substitution at residue 57 of its B subunit occurred, this CT mutant showed 1.5-log-lower GM1 binding than the native CT (1, 13). Similarly, when amino acid 46 or 47 of the B subunit was replaced, both LT mutants exhibited lower GM1 binding activity than the wild-type LT strain (13). However, in contrast to our observation that our 8647 and 8649 mutant strains showed lower GM1 binding activity in the supernatant, Mudrak et al. indicated that the T47A mutant strain had more LT protein detected in the supernatant than the wild-type strain (13). Whether and how a mutation at amino acid 44 or 60 of the B subunit affects the formation, stability, or secretion of the mutant LT proteins will be studied in the future.To examine whether the lower GM1 binding activity of the supernatant samples from the mutant strains was caused by a lower LT production, we conducted an ELISA by directly coating an ELISA plate with total proteins from the pellet and supernatant samples of each strain (without GM1) and by using anti-CT antiserum to quantify the LT protein. ELISA data showed that the OD values of strains 8458, 8647, and 8649 were 0.209 ± 0.005, 0.225 ± 0.009, and 0.21 ± 0 in the supernatant samples and 0.571 ± 0.025, 0.614 ± 0.060, and 0.616 ± 0.026 in the pellet samples, respectively. A Student t test indicated that there were no significant differences between the recombinant strain and the mutant strains in the OD values for the pellet and supernatant protein samples (P = 0.26 and P = 0.84, respectively, for the supernatant samples; P = 0.34 and P = 0.10, respectively, for the pellet samples). These data suggested that a similar amount of LT proteins was produced among these three strains.A single amino acid substitution of the B subunit can result in a reduction in not only GM1 binding but also toxicity for the mutated LT proteins (11, 13, 22). To study whether the mutation of S44N or S60T at the B subunit affected pLT toxicity, we measured the recombinant and mutant strains for their stimulation of intracellular cAMP levels in T-84 cells by using a cAMP competitive enzyme immunoassay (EIA) kit (Invitrogen) by following the manufacturer''s instructions. Briefly, 1 × 10⁵ T-84 cells were seeded in each well of a 24-well plate. After removing the Dulbecco''s modified Eagle medium (DMEM/F12; Gibco/Invitrogen, Grand Island, NY), 75 μl of overnight-grown (in 4AA medium) supernatant of the recombinant or each mutant strain (in triplicate) was added to each well. The cells were lysed with 100 μl of 0.1 M HCl after 2 h of incubation and then neutralized. A total of 100 μl of lysis supernatant was mixed with kit-supplied conjugates and antibody reagents, and the mixture was added to each well of the supplied EIA plate. After incubation on a shaker at 500 rpm at room temperature for 2 h, the plate was washed and dried by blotting, and p-nitrophenyl phosphate substrate solution was added. The OD was measured at 405 nm after 20 min of development. Data from the cAMP ELISA indicated that cAMP levels in T-84 cells incubated with supernatant samples from strains 8458, 8647, and 8649 (from equivalent amounts of cells) were 2.3 ± 0.1, 0.46 ± 0.05, and 0.35 ± 0.01 pmol/ml, respectively (Fig. ​(Fig.2).2). Data clearly indicated that the mutations of S44N and S60T reduced the LT toxic activity. Knowing that it is the A subunit that determines the toxicity of LT and CT, whereas the LT_B and CT_B subunits mediate the binding of the toxin to the host GM1 receptors, we thought that substitution at the B subunits would not affect toxicity. However, we believe that mutations at the B subunits could alter LT protein structure and reduce the binding of the holotoxin to the host GM1 receptors, thus resulting in the reduction of toxic activity.Open in a separate windowFIG. 2.Intracellular cAMP ELISA to detect the toxicity of native LT and mutated LT proteins. Supernatants (in 4AA medium) of overnight-grown cultures from the 8458 (recombinant), 8647 (S44N), and 8649 (S60T) strains were used to stimulate an increase in intracellular cAMP levels in T-84 cells by using a cyclic GMP EIA kit (Invitrogen).The estA gene encoding STa from 33 STa-positive porcine ETEC strains was also sequenced for conformity. This porcine estA gene was PCR amplified using primers pSTaSfcI-F2 and STaEagI-R under conditions described previously (25). The PCR products were purified and sequenced with pSTaSfcI-F2 primer. The sequencing data showed that all sampled STa genes were identical and of porcine origin.Sequence data from our study clearly indicated that both LT and STa expressed by porcine ETEC strains are porcine specific. The LT gene of porcine ETEC strains showed little heterogeneity, and the STa gene is identical. Information from this study will be helpful for a prevalence study of toxin genes among porcine ETEC strains and toxin gene evolution and possibly instructive in antitoxin vaccine development. However, future studies with increasing sampling sizes and a greater geographic coverage will be helpful to understand divergence in the LT and STa genes among porcine ETEC strains.     

Expression fusion immunogen by live attenuated Escherichia coli against enterotoxins infection in mice

Previous epidemiological studies have shown that enterotoxins from enterotoxigenic Escherichia coli (ETEC) appear to be the most important causes of neonatal piglet and porcine post-weaning diarrhoea (PWD). Thus, it is necessary to develop an effective vaccine against ETEC infection. In the present study, the Kil cassette was inserted into the pseudogene yaiT by homologous recombination to create an attenuated E. coli double selection platform O142(yaiT-Kil). After that, PRPL-Kil was replaced with a fusion gene (LTA1-STa₁₃-STb-LTA2-LTB-STa₁₃-STb) to establish oral vaccines O142(yaiT::LTA1-STa₁₃-STb-LTA2-LTB-STa₁₃-STb) (ER-T). Subsequently, BALB/c mice were orally immunized with ER-T. Results showed that serum IgG and faecal sIgA responded against all ETEC enterotoxins and induced F41 antibody in BALB/c mice by orogastrically inoculation with recombinant E. coli ER-T. Moreover, the determination of cellular immune response demonstrated that the stimulation index (SI) was significantly higher in immunized mice than in control mice, and a clear trend in the helper T-cell (Th) response was Th2-cell (IL-4) exceed Th1-cell (IFN-γ).Our results indicated that recombinant E. coli ER-T provides effective protection against ETEC infection.     

CTB/CS3融合蛋白与GM1结合能力和免疫原性的分析

免疫霍乱毒素B亚单位（CTB）或肠毒素大肠杆菌（ETEC）定居因子CS3可使人体对ETEC的侵染有保护作用.为探索研制ETEC双组分亚单位疫苗的可行性,利用大肠杆菌诱导表达系统表达了CTB与CS3的融合蛋白（CTB/CS3）.蛋白质印迹结果表明,诱导表达的29 ku蛋白具有CTB和CS3蛋白双重抗原性.经Ni-NTA亲和层析纯化获得重组蛋白CTB/CS3,复性的重组蛋白可以部分形成五聚体并保留了与神经节苷脂GM1的结合能力.动物实验表明,融合蛋白CTB/CS3具有CTB和CS3蛋白的双重免疫原性,同时,CTB的免疫载体作用提高了CS3的免疫强度.     

动物源产肠毒素大肠杆菌(ETEC)黏附素研究进展

动物源产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是引起动物(尤其是幼龄动物)腹泻的主要病原菌。已知黏附素和肠毒素是ETEC中两种重要的毒力因子,在致病性中两者缺一不可。其中黏附素结合到宿主易感肠上皮细胞是ETEC感染的第一步,也是最重要的关键步骤。动物源ETEC的菌毛黏附素主要包括K88、K99、987P、F18、F17和F41等。人们从20世纪60年代就开始了ETEC菌毛黏附素的相关研究,包括菌毛的基因、结构组成、生物合成、菌毛表达的调控机制以及黏附素和宿主受体相互作用等,这些研究基础有助于我们深入了解ETEC病原菌的感染机理;并且在疾病诊断和新疫苗的开发中具有重大意义。     

Ingestion of transgenic carrots expressing the <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile enterotoxin B subunit protects mice against cholera toxin challenge

Diarrheal diseases caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are worldwide health problems that might be prevented with vaccines based on edible plants expressing the B subunit from either the cholera toxin (CTB) or the E. coli heat labile toxin (LTB). In this work we analyzed the immunity induced in Balb/c mice by ingestion of three weekly doses of 10 μg of LTB derived from transgenic carrot material. Although the anti-LTB serum immunoglobulin G (IgG) and intestinal IgA antibody responses were higher with 10 μg-doses of pure bacterial recombinant LTB (rLTB), the transgenic carrot material also elicited significant serum and intestinal antibody responses. Serum anti-LTB IgG1 antibodies predominated over IgG2a antibodies, suggesting that mainly Th2 responses were induced. A decrease of intestinal fluid accumulation after cholera toxin challenge was observed in mice immunized with either rLTB or LTB-containing carrot material. These results demonstrate that ingestion of carrot-derived LTB induces antitoxin systemic and intestinal immunity in mice and suggest that transgenic carrots expressing LTB may be used as an effective edible vaccine against cholera and ETEC diarrhea in humans.     

Molecular mechanisms of enterotoxigenic Escherichia coli infection

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in developing countries, and perennially the most common cause of traveller's diarrhea. ETEC constitute a diverse pathotype that elaborate heat-labile and/or heat-stable enterotoxins. Recent molecular pathogenesis studies reveal sophisticated pathogen–host interactions that might be exploited in efforts to prevent these important infections. While vaccine development for these important pathogens remains a formidable challenge, extensive efforts that attempt to exploit new genomic and proteomic technology platforms in discovery of novel targets are presently ongoing.     

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.     

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

Oral immunization of mice with plant-derived fimbrial adhesin FaeG induces systemic and mucosal K88ad enterotoxigenic Escherichia coli-specific immune responses

The importance of adhesins in pathogenicity has resulted in them being useful targets in the defense against bacterial infections. To produce edible vaccines against piglet diarrhea caused by enterotoxigenic Escherichia coli (ETEC), plants were genetically engineered to produce recombinant fimbrial adhesin FaeG. To evaluate the efficacy of the edible vaccine FaeG in mice, the soluble protein extracts were examined by about 15 microg recombinant FaeG for each oral immunization dose per mouse. After four doses of vaccination, both IgG and IgA antibodies specific to K88ad fimbriae were elicited in serum, and specific IgA antibodies were also evoked in feces of the immunized mice. Moreover, visible K88ad ETEC agglutination by the specific serum from the immunized mice was observed, implying the antibody was highly specific and effective. Results from an in vitro villous-adhesion assay further confirmed that serum antibodies of the immunized mice could inhibit K88ad ETEC from adhering to pig intestinal receptors, further demonstrating the oral immune efficacy of the plant-derived FaeG. This study provides a promising, noninvasive method for vaccinating swine by feeding supplements of transgenic plant. Moreover, the low cost and ease of delivery of this edible ETEC vaccine will facilitate its application in economically disadvantaged regions.     

Escherichia coli Expressing EAST1 Toxin Did Not Cause an Increase of cAMP or cGMP Levels in Cells, and No Diarrhea in 5-Day Old Gnotobiotic Pigs

Background

Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.

Methodology/Principal Findings

In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.

Conclusion/Significance

Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.     

Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.