Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway

Fimbrial filaments assembled by distinct chaperone pathways share a common mechanism of intersubunit interaction, as elucidated for colonization factor antigen I (CFA/I), archetype of enterotoxigenic Escherichia coli (ETEC) Class 5 fimbriae. We postulated that a highly conserved beta-strand at the major subunit N-terminus represents the donor strand, analogous to interactions within Class I pili. We show here that CFA/I fimbriae utilize donor strand complementation to promote proper folding of and interactions between CFA/I subunits. We constructed a series of genetic variants of CfaE, the CFA/I adhesin, incorporating a C-terminal extension comprising a flexible linker and 10-19 of the N-terminal residues of CfaB, the major subunit. Variants with a donor strand complement (dsc) of >or= 12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor beta-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc19CfaE formed soluble monomers, folded into beta-sheet conformation, displayed adhesion characteristic of CFA/I, and elicited antibodies that inhibited mannose-resistant haemagglutination by ETEC expressing CFA/I, CS4 and CS14 fimbriae. Immunoelectron microscopy indicated that CfaE was confined to the distal fimbrial tip. Our findings provide the basis to elucidate structure and function of this class of fimbrial adhesins and assess the feasibility of an adhesin-based vaccine.     

Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166

Entertoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I. Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae. Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae. CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe. Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes. The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production. Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae.(ABSTRACT TRUNCATED AT 250 WORDS)     

The codon-optimization of cfaE gene and evaluating its high expression capacity and conserved immunogenicity in Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of children diarrhea in the world. Adhesion of ETEC to small intestine is an important virulence trait. One of the most prevalent colonization factors (CFs) in human is CFA/I fimbriae and CfaE which is the required binding factor for adhesion of ETEC to intestinal mucosa.We optimized cfaE gene codons according to codon bias of E. coli to achieve a high level of recombinant protein expression. The optimized gene was expressed in E. coli and rCFaE protein was used for mice immunization. Blocking activity of the obtained antibody was examined by microplate agglutination inhibition test. SDS-PAGE analysis indicated that the optimized sequence of cfaE produces a suitable amount of rCFaE in comparison with native gene sequence. This optimized rCFaE protein could induces strong humoral response in mice and the antibody obtained against rCFaE inhibited the adhesion of ETEC to human group A erythrocytes. It is concluded that codon optimization is a useful approach for obtaining large quantities of recombinant rCFaE protein. With regard to the results of hemagglutination inhibition test, codon optimization and increased production of recombinant protein expressed in E. coli did not affect the immunogenicity potential of CFaE.     

Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine

In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.     

Adaptive evolution of class 5 fimbrial genes in enterotoxigenic Escherichia coli and its functional consequences

Class 5 fimbriae of enterotoxigenic Escherichia coli (ETEC) comprise eight serologically discrete colonization factors that mediate small intestinal adhesion. Their differentiation has been attributed to the pressure imposed by host adaptive immunity. We sequenced the major pilin and minor adhesin subunit genes of a geographically diverse population of ETEC elaborating CFA/I (n = 31), CS17 (n = 20), and CS2 (n = 18) and elucidated the functional effect of microevolutionary processes. Between the fimbrial types, the pairwise nucleotide diversity for the pilin or adhesin genes ranged from 35-43%. Within each fimbrial type, there were 17 non-synonymous and 1 synonymous point mutations among all pilin or adhesin gene copies, implying that each fimbrial type was acquired by ETEC strains very recently, consistent with a recent origin of this E. coli pathotype. The 17 non-synonymous allelic differences occurred in the CFA/I pilin gene cfaB (two changes) and adhesin gene cfaE (three changes), and CS17 adhesin gene csbD (12 changes). All but one amino acid change in the adhesins clustered around the predicted ligand-binding pocket. Functionally, these changes conferred an increase in cell adhesion in a flow chamber assay. In contrast, the two mutations in the non-adhesive CfaB subunit localized to the intersubunit interface and significantly reduced fimbrial adhesion in this assay. In conclusion, naturally occurring mutations in the ETEC adhesive and non-adhesive subunits altered function, were acquired under positive selection, and are predicted to impact bacteria-host interactions.     

A structural basis for sustained bacterial adhesion: biomechanical properties of CFA/I pili

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized the intrinsic biomechanical properties and kinetics of individual CFA/I pili at the single-organelle level, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding, can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.     

Detection of attachment of enterotoxigenic Escherichia coli (ETEC) to human small intestinal cells by enzyme immunoassay

Abstract Simple immunoassays were developed to study the binding between enterocytes of the small intestine and other cell types, and enterotoxigenic Escherichia coli (ETEC). CFA/I or CFA/II pilus protein or CFA-positive E. coli bacteria were wells of microtitre plates and incubated with vesicles or crude mucus prepared from human brush border enterocytes. Binding of the cell preparations was detected by adding specific rabbit anti-brush border IgG followed by urease-labelled goat anti-rabbit IgG and urea substrate. The binding of purified CFA/I to human or rabbit small intestine, human oral epithelial cells or Caco-2 cells was detected with specific anti-CFA/I IgG. Both human brush border and mucus-derived preparations were able to attach to ETEC. The binding was CFA-specific and strong enough to withstand several washings. In contrast, CFA/I did not bind to small intestinal cells of non-human small intestinal origin, indicating that there may be important differences in affinity between receptors present on human small intestinal cells and cells of non-human small intestinal origin. Antibodies directed against human small intestinal and non-small intestinal cells did not cross-react with either preparation, indicating that receptors between these different cell sources are different. The EIA proved useful during the identification of a newly-recognised 15 kDa bacterial surface component of ETEC strain H10407P, which may function as a putative attachment factor. The EIAs developed in this study were easy to perform and multiple tests could be performed on small samples, including biopsy samples obtained during endoscopy.     

Biomechanical and Structural Features of CS2 Fimbriae of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in under-developed countries often leads to high mortality rates. Isolated ETEC expresses a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II, which are assembled via the alternate chaperone pathway (ACP), are among the most common. Fimbriae are filamentous structures whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical ability to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understanding about the role of fimbriae as virulence factors points to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modeling of its major structural subunit, CotA, reveals structural clues related to the niche in which they are expressed. Using optical-tweezers force spectroscopy, we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity (i.e., the velocity at which the force required for unwinding rises exponentially with increased speed) of 1300 nm/s. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC.     

Epitope Specificity of a Monoclonal Antibody Generated against the Dissociated CFA/I Fimbriae of Enterotoxigenic Escherichia coli

A monoclonal antibody (MAb 84) raised against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli was characterized with regard to antigen binding and epitope specificity. Enzyme-linked immunosorbent assay (ELISA) showed that MAb 84 had higher affinity to CFA/I subunits than to intact CFA/I fimbriae and recognized a Salmonella flagellin carrying an insert corresponding to amino acids 32 to 45 of the CFA/I subunit. Fine epitope mapping based on the Pepscan technique showed that the peptide ³⁹TFESY⁴³, derived from the sequence of the mature CFA/I subunit, was specifically recognized by MAb 84. The ³⁹TFESY⁴³ sequence is probably not accessible on the surface of the native CFA/I fimbriae since MAb 84 did not bind to intact fimbriae as evaluated in inhibition ELISA tests. Moreover, MAb 84 did not agglutinate fimbriated ETEC cells nor inhibit CFA/I-mediated hemagglutination or the adhesion to Caco-2 cells.     

Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways

Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram‐negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone‐usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P‐fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P‐fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.     

Characterization of colonization factor antigen CFA/I from an enterotoxigenic Escherichia coli strain (0128:H12)

CFA/I antigen was isolated and purified from E. coli, mutant 279 B-1-14, serotype 0128:H12, and had the following biochemical and biological features: a) amino-acid content was similar to that of purified antigen prepared from strain H10407; b) latex particles sensitization with purified CFA/I antigen produced bovine and human erythrocytes group A/II hemagglutination in carbohydrates presence; c) purified anti-CFA/I specific antibodies agglutinated CFA/I-positive enterotoxigenic E. coli strains; d) 3H-leucine-labelled CFA/I antigen adhered to rabbits intestinal mucosa at significant values; e) intestinal mucosa pretreating with purified CFA/I antigen, followed by 3H-leucine labelled enterotoxigenic bacteria infection, had a least 3 local effects: 1) intestinal mucosa protection against parental enterotoxigenic bacteria; 2) inhibition of CFA/I-positive bacteria adherence to intestinal mucosa; 3) release of approximately 96% intraluminally inoculated bacteria.     

A receptor-binding site as revealed by the crystal structure of CfaE, the colonization factor antigen I fimbrial adhesin of enterotoxigenic Escherichia coli

CfaE is the minor, tip-localized adhesive subunit of colonization factor antigen I fimbriae (CFA/I) of enterotoxigenic Escherichia coli and is thought to be essential for the attachment of enterotoxigenic E. coli to the human small intestine early in diarrhea pathogenesis. The crystal structure of an in cis donor strand complemented CfaE was determined, providing the first atomic view of a fimbrial subunit assembled by the alternate chaperone pathway. The in cis donor strand complemented variant of CfaE structure consists of an N-terminal adhesin domain and a C-terminal pilin domain of similar size, each featuring a variable immunoglobulin-like fold. Extensive interactions exist between the two domains and appear to rigidify the molecule. The upper surface of the adhesin domain distal to the pilin domain reveals a depression consisting of conserved residues including Arg(181), previously shown to be necessary for erythrocyte adhesion. Mutational analysis revealed a cluster of conserved, positively charged residues that are required for CFA/I-mediated hemagglutination, implicating this as the receptor-binding pocket. Mutations in a few subclass-specific residues that surround the cluster displayed differential effects on the two red cell species used in hemagglutination, suggesting that these residues play a role in host or cell specificity. The C-terminal donor strand derived from the major subunit CfaB is folded as a beta-strand and fits into a hydrophobic groove in the pilin domain to complete the immunoglobulin fold. The location of this well ordered donor strand suggests the positioning and orientation of the subjacent major fimbrial subunit CfaB in the native assembly of CFA/I fimbriae.     

The fimbriae of human enterotoxigenic Escherichia coli strain 334 are related to CS5 fimbriae

Escherichia coli strain 334 is a human enterotoxigenic strain of serotype O15:H11 which had previously been shown to produce 'attachment pili'. These fimbriae were compared with other colonization factors. From strain 334 a mannose-resistant haemagglutination positive colony 334A and a mannose-resistant haemagglutination negative variant 334C were isolated. By electron microscopy the fimbriae of strain 334A were shown to have a helical structure resembling coli-surface-associated antigen (CS5) fimbriae. An antiserum was raised to strain 334A and absorbed with a fimbriae-negative variant of that strain, 334C. By immuno-electron microscopy this antiserum was shown to coat fimbriae of strain 334A but not CS5 fimbriae produced by strain E17018A. Conversely, CS5 antiserum did not coat the fimbriae produced by strain 334A. No antigenic cross-reaction was detected between these intact fimbriae when anti-strain 334A serum and CS5 antiserum were used in immunodiffusion tests. By enzyme-linked immunosorbent assays (ELISAs) the fimbriae of strain 334A were shown to be antigenically unrelated to most other human ETEC adhesins, namely colonization factor antigens (CFA/I, CFA/III and CFA/IV), coli-surface-associated antigens (CS1, CS2, CS3, CS4, CS6 and CS17) and putative colonization factors (PCFO159:H4 and PCFO166). However, a heated suspension of strain 334A reacted weakly with CS5 antiserum in an ELISA. By SDS-PAGE the fimbriae of strain 334A were shown to consist of subunits of similar size to CS5 subunits, that is about 21.5 kDa. Western immunoblotting revealed that the subunits of 334A and CS5 fimbriae shared common epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal–mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.     

Immunoprotective oral whole cell vaccine for enterotoxigenic Escherichia coli diarrhea prepared by in situ destruction of chromosomal and plasmid DNA with colicin E2

Abstract Vaccine regimens which mimic actual infection with bacterial enteropathogens should offer the best opportunity for successful long-term immunoprotection against diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC) or Vibrio cholerae . Based on this principle, we designed and tested an oral whole cell anti-ETEC vaccine consisting of intact cells of ETEC strain H-10407 (ST⁺LT⁺; O78: H11: CFA/I) which were rendered incapable of replication by treatment with a potent DNA endonuclease, colicin E2. Young healthy volunteers were administered two oral doses of either placebo or approx. 3 × 10¹⁰ vaccine cells. In a double-blind study, 9 of 10 vaccinees responded with an increase in CFA/I-specific intestinal IgA antibody, determined as percent of total IgA. Challenge with virulent strain H-10407 (5 × 10⁹ living cells) produced diarrhea in 8 of 9 (89%) of the placebo-treated volunteers and in 2 of 10 (20%) of the vaccinees. Thus, the colicin E2-killed whole cell vaccine afforded both a significant intestinal immune response and significant protection against challenge with the virulent organism. The data presented here suggest that for this vaccine preparation an intestinal anti-CFA/I IgA response is a good indicator of a protective immune response, which most likely involves antibody responses to a number of antigens in addition to CFA/I. We conclude that the colicin E2 method for preparing an oral anti-ETEC vaccine merits further study and that this method may also be applicable to other enteropathogens.     

Non-replicating oral whole cell vaccine protective against enterotoxigenic Escherichia coli (ETEC) diarrhea: stimulation of anti-CFA (CFA/I) and anti-enterotoxin (anti-LT) intestinal IgA and protection against challenge with ETEC belonging to heterologous serotypes

An oral killed (non-replicating) whole-cell anti-ETEC vaccine was prepared by treating enterotoxigenic Escherichia coli strain H-10407 (ST + LT +; 078: H11: CFA/I) with a 100%-lethal amount of colicin E2. Colicin E2 is a potent DNA endonuclease which enters the target bacterial cells without disrupting cellular integrity. Thus the vaccine consists of intact cells lacking chromosomal and plasmid DNA but possessing a normal complement of antigens, including CFA/I and enterotoxin(s), unaltered by chemical- or heat-treatment. Young healthy volunteers were administered two oral doses, one month apart, of approximately 3 x 10(10) vaccine cells. Of 22 vaccinees, 17 (77.3%) showed an intestinal anti-CFA/I IgA response and 19 (86.4%) showed an increase in intestinal anti-LT IgA. Twenty of 22 (90.9%) vaccinees had antibody responses to either CFA/I, LT, or both antigens, demonstrating that colicin E2-treated CFA-positive E. coli cells are an efficient vehicle in terms of delivery of antigens to the gut immune system. We previously demonstrated protection of vaccinees against challenge with the living homologous ETEC (strain H-10407). In this study, two groups of 8 vaccinees were challenged with a diarrheagenic dose of virulent ST + LT + ETEC of heterologous serotype; one group was challenged with a CFA/I-positive 063: H- strain and the other group was challenged with a CFA/II-positive 06: H16 strain. Approximately 75% efficacy was achieved in both challenge groups. None of the 16 vaccinees who had responded to both CFA/I and LT became ill upon challenge while both of the vaccinees who had not responded to either antigen did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tight Conformational Coupling between the Domains of the Enterotoxigenic Escherichia coli Fimbrial Adhesin CfaE Regulates Binding State Transition

CfaE, the tip adhesin of enterotoxigenic Escherichia coli colonization factor antigen I fimbriae, initiates binding of this enteropathogen to the small intestine. It comprises stacked β-sandwich adhesin (AD) and pilin (PD) domains, with the putative receptor-binding pocket at one pole and an equatorial interdomain interface. CfaE binding to erythrocytes is enhanced by application of moderate shear stress. A G168D replacement along the AD facing the CfaE interdomain region was previously shown to decrease the dependence on shear by increasing binding at lower shear forces. To elucidate the structural basis for this functional change, we studied the properties of CfaE G168D (with a self-complemented donor strand) and solved its crystal structure at 2.6 Å resolution. Compared with native CfaE, CfaE G168D showed a downward shift in peak erythrocyte binding under shear stress and greater binding under static conditions. The thermal melting transition of CfaE G168D occurred 10 °C below that of CfaE. Compared with CfaE, the atomic structure of CfaE G168D revealed a 36% reduction in the buried surface area at the interdomain interface. Despite the location of this single modification in the AD, CfaE G168D exhibited structural derangements only in the adjoining PD compared with CfaE. In molecular dynamics simulations, the G168D mutation was associated with weakened interdomain interactions under tensile force. Taken together, these findings indicate that the AD and PD of CfaE are conformationally tightly coupled and support the hypothesis that opening of the interface plays a critical modulatory role in the allosteric activation of CfaE.     

Immunoprotective oral whole cell vaccine for enterotoxigenic Escherichia coli diarrhea prepared by in situ destruction of chromosomal and plasmid DNA with colicin E2

Vaccine regimens which mimic actual infection with bacterial enteropathogens should offer the best opportunity for successful long-term immunoprotection against diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC) or Vibrio cholerae. Based on this principle, we designed and tested an oral whole cell anti-ETEC vaccine consisting of intact cells of ETEC strain H-10407 (ST+LT+; O78:H11:CFA/I) which were rendered incapable of replication by treatment with a potent DNA endonuclease, colicin E2. Young healthy volunteers were administered two oral doses of either placebo or approx. 3 X 10(10) vaccine cells. In a double-blind study, 9 of 10 vaccinees responded with an increase in CFA/I-specific intestinal IgA antibody, determined as percent of total IgA. Challenge with virulent strain H-10407 (5 X 10(9) living cells) produced diarrhea in 8 of 9 (89%) of the placebo-treated volunteers and in 2 of 10 (20%) of the vaccinees. Thus, the colicin E2-killed whole cell vaccine afforded both a significant intestinal immune response and significant protection against challenge with the virulent organism. The data presented here suggest that for this vaccine preparation an intestinal anti-CFA/I IgA response is a good indicator of a protective immune response, which most likely involves antibody responses to a number of antigens in addition to CFA/I. We conclude that the colicin E2 method for preparing an oral anti-ETEC vaccine merits further study and that this method may also be applicable to other enteropathogens.