Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3',5'-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.     

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.     

LT（K63／R72）, a New Mutant of Escherichia coli Heat-labile Enterotoxin, Exhibits Characteristics More Similar to LT（K63） than LT（R72）

LT(K63), a non-toxic mutant and LT(R72), a low toxic mutant of E. coli heat-labile enterotoxin are frequently used mucosal adjuvants. In many cases, the adjuvanticity of LT(K63) is lower than that of LT(R72), but LT(K63), which induces a mixed Th1/Th2 response, exhibits a higher level of protection than LT(R72) which induces a polarized Th2-type response. To utilize the advantages of both adjuvants, a doublemutation LT(K63/R72) was generated and purified. The characterization results showed that there was no significant difference in production rate and immunogenicity between wild type LT and LT mutants. The results also showed that the toxicity and the trypsin sensitivity of LT(K63/R72) are between that of LT(K63)and LT(R72). Using HPLC, when samples in an OHpak SB-800 column were eluted by denatural buffer(TEAN containing 10 mg/ml SDS), we found the stability of LT(K63/R72) was higher than that of LT(R72)and lower than that of LT(K63). Through further analyzes, we found that LT(K63/R72) exhibits characteristics more closely related to LT(K63) than LT(R72).     

A heat-labile enterotoxin (LT) purified from chicken enterotoxigenic Excherichia coli is identical to porcine LT

We have previously reported that the heat-labile enterotoxin (LTc) isolated from a chicken enterotoxigenic Escherichia coli (ETEC) was identical to LTh produced by human ETEC (Tsuji et al. (1988) FEMS Microbiol Lett. 52, 79-84). In this study, we purified an LTc-like toxin (LTc') from another strain isolated from a chicken that developed diarrhea at a different place and time to the previously reported chicken. Its molecular weight and antigenicity were compared with those of purified LTs from porcine and human ETEC (LTp and LTh). The A subunit of LTc' was identical to those of the purified LTs in mobility on SDS-polyacrylamide gel electrophoresis. The Ouchterlony test demonstrated that LTc' was antigenically identical to LTp. The isoelectric point and amino acid composition of LTc' were also identical to those of LTp. These data suggest that chicken ETEC can be grouped with both the porcine and human types on the basis of the LTs produced.     

In vivo expression of the heat stable (estA) and heat labile (eltB) toxin genes of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro.     

Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia

Background

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller''s and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells.

Methodology/Principal Findings

In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP_bol in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP_bol) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns.

Conclusion/Significance

The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.     

Rectal immunization with rotavirus virus-like particles induces systemic and mucosal humoral immune responses and protects mice against rotavirus infection

To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.     

Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells

Given recent evidence suggesting that the heat-labile enterotoxin (LT) provides a colonization advantage for enterotoxigenic Escherichia coli (ETEC) in vivo, we hypothesized that LT preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterium-host interactions. We present data demonstrating that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-3′,5′-cyclic AMP (cAMP), an inhibitor of protein kinase A, was sufficient to abrogate LT's ability to promote subsequent bacterial adherence. Increased adherence was not due to changes in the surface expression of the host receptor for the K88ac adhesin. Evidence is also presented for a role for bacterial sensing of host-derived cAMP in promoting adherence to host cells.     

Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.     

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.     

Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1–enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.     

Effect of Substitution for Arginine Residues near Position 146 of the A Subunit of Escherichia coli Heat-Labile Enterotoxin on the Holotoxin Assembly

Escherichia coli heat-labile enterotoxin (LT) is a holotoxin which consists of one A and five B subunits. Although B subunit monomers released into periplasm can associate into pentameric structures in the absence of the A subunit, the A subunit accelerates the assembly. To express the function, A subunit constructs the proper spatial structure. However, the regions involved in the construction are unknown. To identify the regions, we substituted arginine residues near position 146 of the A subunit with glycine by oligonucleotide-directed site-specific mutagenesis and obtained the mutants expressing LT(R141G), LT(R143G), LT(R146G), LT(R143G, R146G), LT(R141G, R143G, R146G) and LT(R143G, R146G, R148G). We purified these mutant LTs by using an immobilized d -galactose column and analyzed the purified mutant LTs by SDS-PAGE to examine the amount of A subunit associated with B-subunit oligomer. The substitution of an arginine residue at any position did not induce a significant alteration in the amount of A subunit associated with B-subunit oligomer. However, the substitution of more than two arginine residues induced a significant decrease in the amount of A subunits associated with the B-subunit oligomer. Subsequently, we measured the level of the intracellular B-subunit oligomer of these mutant strains. The measurement revealed that the amount of B-subunit oligomer in cells decreased as the number of substituted arginine residues increased. These results show that all arginine residues near position 146 are important for the construction of the functional A subunit, and thus for holotoxin formation, although each individual arginine residue is not an absolute requirement.     

The prevalence and molecular typing of enterotoxigenic Escherichia coli strains isolated from diarrheic stools in Malatya, Turkey

This study was performed from June 2002 to November 2003 year in Malatya, eastern Turkey. Stools of 172 diarrheic patients and 90 healthy controls were analysed for enterotoxigenic Escherichia coli (ETEC). Heat-labile (LT) and heat-stable (ST) toxins were investigated by passive latex agglutination and enzyme immunoassay, respectively. Nine ETEC strains were isolated from 172 diarrheic stools (5.2%). Seven of the ETEC strains (10.1%) were isolated from 69 children in the 0-5 year age group. Two of these pediatric isolates were ST positive (2.9%) and five were LT positive (7.2%). ETEC was not isolated in the 6-18 year age group. Two ST producing E. coli strains were detected in diarrheic adult patients (> 18 years). In the 90 controls, two ETEC strains were detected (2.2%). One of them was a LT producer (1.1%) and the other was a ST producer (1.1%). E. coli strains producing both toxins simultaneously were not observed. ETEC positivity was higher in the diarrheic group than in the control group but statistically not significant (p > 0.05). The rate of resistance among ETEC strains to cefuroxime axetil, ampicillin, piperacillin, and trimethoprim-sulfamethoxazole was 72.7%, 54.5%, 45.5%, and 36.4%, respectively whereas the resistance rate to the same antibiotics in non-ETEC strains was 14%, 62%, 54%, and 66%, respectively. All ETEC isolates were intermediately resistant to cephalothin and fully susceptible to other antibiotics tested. Typing of the ETEC strains was done by arbitrary primed polymerase chain reaction (AP-PCR). Only two LT strains of the 11 typed strains had a unique profile. The remaining nine were mixed LT and ST strains and divided into two groups. The first group had three strains having a similarity coefficient ranging from 70-90%. The other one had six strains, five of them were similar and one was subtype isolate. It can be concluded that ETEC strains might be considerably important enteropathogens especially in pediatric patients in the 0-5 year age group. High clonal relation indicated that ETEC strains were epidemiologically related.     

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.     

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.     

Common antigenic determinant in the receptor binding sites of Escherichia coli enterotoxin and cholera toxin

Abstract A mutant (TUH No. 9) of a porcine strain of enterotoxigenic Escherichia coli (ETEC) produces as abnormal B subunit (B') of heat-labile enterotoxin (LT), which has aspartate instead of glycine at residue 33 from the N-terminus and does not bind to the receptor, GM1 ganglioside. The antigenicities of the receptor-binding site of LT were analyzed.
The antibody, which could not bind to the B' subunit in the anti-B subunit of porcine LT(LTp)-serum, could bind to cholera toxin (CT), LTp and LT produced by a human ETEC strain (LTh), suggesting that it recognizes a common epitope of LTp, LTh and CT. Thus glycine at residue 33 from the N-terminus in the B subunit of CT, LTh and LTp may be related to the common epitope of these three toxins. The bindings of CT, LTh and LTp to the antibody were inhibited by the GM1 ganglioside.
These data indicate that the antibody recognizes a common epitope in the receptor (GM1 ganglioside)-binding site of CT, LTh and LTp.     

Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC)

Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit’s potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.     

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.     

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines

Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.     

Exopolysaccharides Synthesized by Lactobacillus reuteri Protect against Enterotoxigenic Escherichia coli in Piglets

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter⁻¹ of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that E. coli is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P = 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent in situ hybridization analysis demonstrated that reuteran significantly (P = 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.