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.     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

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.     

Virulence factor gene profiles of Escherichia coli isolates from clinically healthy pigs

Nonpathogenic, intestinal Escherichia coli (commensal E. coli) supports the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports, E. coli isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive E. coli isolates from swine with no clinical signs of intestinal disease. The isolation of E. coli from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this E. coli population. Additionally, we traced the occurrence of hemolytic E. coli over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic E. coli isolates without virulence genes or with only the fedA gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic E. coli serogroups. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations and that high numbers of E. coli cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.     

A recombinant Escherichia coli heat-stable enterotoxin b (STb) fusion protein eliciting neutralizing antibodies

Abstract STb is a heat-stable enterotoxin elaborated by enterotoxigenic Escherichia coli strains associated with weaning piglets and is responsible for diarrhoea in those animals. The maltose binding protein (MBP) of E. coli was used as a carrier for STb, a poorly immunogenic molecule. Constructions were produced where the gene coding for mature STb toxin (MBP-STb) and a fragment of the gene spanning the major epitopic region of STb (AA8–AA30)(MBP-STb₂) were fused to malE gene coding for MBP. The fusion proteins accumulated in the periplasm and were detected with a polyclonal antibody raised against the purified toxin. MBP-STb induced secretion in the biological model whereas MBP-STb₂ was non-toxic. Immunization of rabbits evoked an antibody response to STb for these two fusion proteins. However, only MBP-STb elicited antibodies that effectively neutralized the toxicity of pure STb toxin as determined in the rat loop assay.     

Production of enterotoxins by strains of Escherichia coli isolated from pigs

The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO. Vero and Hela cells and ILT. More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains). No correlation between intensity of ILT and particular serotype was observed. Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin. The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs. Strains of E. coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes. The cytotoxic activity of supernatants of E. coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed. This suggests the production by the strains of enterotoxin LT and cytotoxin VT. Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs. This suggests the production by these isolates of a toxin (toxins) differing from the E. coli enterotoxins.     

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.     

Escherichia coli cytotoxins and enterotoxins.

Vero cell cytotoxins and cytotonic enterotoxins produced by E. coli are toxic proteins, which have been implicated in a number of specific diseases in humans and animals. Nomenclature for these toxins is complicated by the existence of different names for the same toxin. The Vero cell cytotoxins are called verotoxins because they are lethal for Vero cells in culture; they are also known as Shiga-like toxins (SLTs) because they are clearly related to Shiga toxin in structure, amino acid sequence, mechanism of action, and biological activity. SLTs belong to two classes. SLT-I is identical with Shiga toxin and is in a class by itself (class I). The other SLTs are closely related to each other and form a second class (class II). Class II SLTs include SLT-II, SLT-IIv, SLT-IIvha, SLT-IIvhb, and SLT-IIva. All SLTs that have been investigated are A-B subunit protein toxins, whose A subunits possess N-glycosidase activity against 28S rRNA and cause inhibition of protein synthesis in eukaryotic cells. These toxins are enterotoxic as well as cytotoxic. SLTs produced in the intestine are absorbed into the blood stream and affect vascular endothelial cells in target organs. They may also have a direct toxic effect on enterocytes. Diseases in which E. coli SLTs have been implicated include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans and edema disease in pigs. Variation in receptor specificities among SLTs may be the reason for different disease syndromes in different host species. The E. coli enterotoxins belong to three distinct classes: heat-labile enterotoxin (LT), heat-stable enterotoxin type I or type a (STI, STa), and heat-stable enterotoxin type II or type b (STII, STb). There is clear evidence that these cytotonic enterotoxins play an essential role in diarrheal disease. LT is an A-B subunit protein toxin, closely related to cholera toxin. Following binding of LT to receptors in enterocytes the A subunit is internalized. The enzymatically active A subunit transfers ADP-ribose from NAD to a GTP-dependent adenylate cyclase regulatory protein, thereby elevating intracellular levels of adenylate cyclase. The increased levels of cyclic AMP cause stimulation of A kinase and lead to hypersecretion of electrolytes and fluid. STI is a small peptide of 18 or 19 amino acids. It binds to receptors in enterocytes and stimulates particulate guanyl cyclase. Elevated intracellular cyclic GMP stimulates G kinase, resulting in increased Cl- secretion and impaired absorption of Na+Cl-. STII is a peptide toxin whose mechanism of action is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)     

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。     

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.

     

Genes coding for Shiga-like toxin and heat-stabile enterotoxin in porcine strains of Escherichia coli

Besides diarrheagenic enterotoxigenic Escherichia coli (ETEC) that produce classical heat stable and/or heat labile enterotoxins (STs, LTs) and the class of Shiga-like toxin-producing entero-hemorrhagic E. coli (EHEC), a new category of E. coli is defined sharing similarities with ETEC and EHEC. DNA hybridization studies indicate that some E. coli serovars from porcine origin harbor genes encoding cytotonic ST and cytotoxic Shiga-like toxin. The presence of two potent toxins might contribute to the virulence of such strains and should be taken into consideration when bio-assays are performed.     

Acquisition and maintenance of enterotoxin plasmids in wild-type strains of Escherichia coli

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.     

Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli

Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences of K88-positive and F18-positive porcine ETEC strains and examined the phylogenetic distribution of clinical porcine ETEC strains and their plasmid-associated genetic content. The genomes of porcine ETEC strains UMNK88 and UMNF18 were both found to contain remarkable plasmid complements containing known virulence factors, potential novel virulence factors, and antimicrobial resistance-associated elements. The chromosomes of these strains also possessed several unique genomic islands containing hypothetical genes with similarity to classical virulence factors, although phage-associated genomic islands dominated the accessory genomes of these strains. Phylogenetic analysis of 78 clinical isolates associated with neonatal and porcine diarrhea revealed that a limited subset of porcine ETEC lineages exist that generally contain common toxin and fimbrial profiles, with many of the isolates belonging to the ST10, ST23, and ST169 multilocus sequencing types. These lineages were generally distinct from existing human ETEC database isolates. Overall, most porcine ETEC strains appear to have emerged from a limited subset of E. coli lineages that either have an increased propensity to carry plasmid-encoded virulence factors or have the appropriate ETEC core genome required for virulence.     

Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E. coli toxins

Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII). The results with the EDP and SLTII probes were identical. All VTEC hybridized with these two probes, while non VTEC did not. All 133 E. coli were negative for the SLTI probe. Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP. The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins. Only the STb probe was hybridized by 36 of them. Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.     

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。     

Biological properties of enterotoxigenic Escherichia isolated from patients with acute intestinal diseases of unknown etiology

As the result of the comparative examination of adult patients with acute enteric diseases and normal adults, 173 E. coli enterotoxigenic strains were isolated (161 strains from the patients and 12 strains from normal persons). 83% of the isolated enterotoxigenic E. coli (ETEC) produced two enterotoxins: thermolabile (LT) and thermostable (ST). Enterotoxigenicity was most pronounced in the strains of ETEC belonging to the prevaling variant ST + LT +. The enterotoxigenic properties of ETEC were highly stable: the production of ST and LT in the strains remained unchanged after their storage for up to 4 years. The isolated ETEC comprised 48 serogroups and 61 strains. The strains belonging to the same seroval had a similar degree of toxigenicity. The strains belonging to different serovars considerably differed in the activity of their enterotoxins. The production of two kinds of enterotoxins in the isolated E. coli strains was inter-related: the strains with a high activity of ST were, as a rule, good producers of LT.     

Prosequence switching: An effective strategy to produce biologically active E. coli heat-stable enterotoxin STh

Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.     

Pregnancy interrupting effects of some bacterial toxins.

Embryotoxic properties of Shigella dysenteriae and Clostridium perfringens toxins, of E. coli endotoxin, V. cholerase and E. coli enterotoxins were compared in mice. E. coli endotoxin has embryotoxic effects at all stages of pregnancy. E. coli enterotoxin V. cholerae enterotoxin and Shigella dysenteriae toxin are most effective mainly at earlier stages of pregnancy. Clostridium perfringens toxin has no embryotoxic effect.     

Mode of action of heat-stable Escherichia coli enterotoxin. Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.