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.     

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.     

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.     

Off‐pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone‐usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor‐strand complementation (DSC) and cleft‐mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone‐subunit complex and the cleft‐mediated anchorage of the subunit C‐terminus additionally assist in subunit refolding. Surprisingly, over‐stabilization of the chaperone‐subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off‐pathway self‐polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly.     

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.     

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)     

Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of non-pathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cell-surface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination.     

Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo

Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.     

Crystal structure of enterotoxigenic Escherichia coli colonization factor CS6 reveals a novel type of functional assembly

Coli surface antigen 6 (CS6) is a widely expressed enterotoxigenic Escherichia coli (ETEC) colonization factor that mediates bacterial attachment to the small intestinal epithelium. CS6 is a polymer of two protein subunits CssA and CssB, which are secreted and assembled on the cell surface via the CssC/CssD chaperone usher (CU) pathway. Here, we present an atomic resolution model for the structure of CS6 based on the results of X‐ray crystallographic, spectroscopic and biochemical studies, and suggest a mechanism for CS6‐mediated adhesion. We show that the CssA and CssB subunits are assembled alternately in linear fibres by the principle of donor strand complementation. This type of fibre assembly is novel for CU assembled adhesins. We also show that both subunits in the fibre bind to receptors on epithelial cells, and that CssB, but not CssA, specifically recognizes the extracellular matrix protein fibronectin. Taken together, structural and functional results suggest that CS6 is an adhesive organelle of a novel type, a hetero‐polyadhesin that is capable of polyvalent attachment to different receptors.     

The three-dimensional structure of CFA/I adhesion pili: traveler's diarrhea bacteria hang on by a spring

To survive the harsh environment of a churning intestinal tract, bacteria attach to the host epithelium via thin fibers called pili (or fimbriae). Enterotoxigenic Escherichia coli bacteria expressing colonization factor antigen I (CFA/I) pili and related pili are the most common known bacterial cause of diarrheal disease, including traveler's diarrhea. CFA/I pili, assembled via the alternate chaperone pathway, are essential for binding and colonization of the small bowel by these pathogenic bacteria. Herein, we elucidate unique structural features of CFA/I pili that appear to optimize their function as bacterial tethers in the intestinal tract. Using transmission electron microscopy of negatively stained samples in combination with iterative three-dimensional helical reconstruction methods for image processing, we determined the structure of the CFA/I pilus filament. Our results indicate that strong end-to-end protein interactions and weak interactions between the coils of a sturdy spring-like helix provide the combination of strength, stability, and flexibility required to sustain bacterial adhesion and incite intestinal disease. We propose that CFA/I pili behave like a spring to maintain attachment to the gut lining during vortex mixing and downward flow of the intestinal contents, thereby persisting long enough for these bacteria to colonize the host epithelium and cause enteric disease.     

Structure and secretion of CofJ,a putative colonization factor of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera‐like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co‐regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large β‐sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side‐chains at one end and structural homology to the pore‐forming proteins perfringolysin O and α‐haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.     

The possession of coli surface antigen CS6 by enterotoxigenicEscherichia coli of serogroups O25, O27, O148, and O159: a possible colonization factor?

A total of 134 enterotoxigenicEscherichia coli (ETEC) of serogroups O25, O27, O148, and O159 were tested in the enzyme-linked immunosorbent assays for the colonization factor antigens I (CFA/I), CFA/II (coli surface antigens CS1, 2 and 3) and putative colonization factor (PCF) 8775 (CS4, 5 and 6). CS6 was detected without CS4 or CS5 in 94% of the strains of serogroup O25, 86% of strains of serogroup O27, 87% of strains of serogroup O148, and 29% of strains of serogroup, O159. The frequency with which CS6 occurs in ETEC of common serotypes without the antigens CS4 or CS5 suggests that it might be a colonization factor.     

Antigen-Specific Memory B-cell Responses to Enterotoxigenic Escherichia coli Infection in Bangladeshi Adults

Background

Multiple infections with diverse enterotoxigenic E. coli (ETEC) strains lead to broad spectrum protection against ETEC diarrhea. However, the precise mechanism of protection against ETEC infection is still unknown. Therefore, memory B cell responses and affinity maturation of antibodies to the specific ETEC antigens might be important to understand the mechanism of protection.

Methodology

In this study, we investigated the heat labile toxin B subunit (LTB) and colonization factor antigens (CFA/I and CS6) specific IgA and IgG memory B cell responses in Bangladeshi adults (n = 52) who were infected with ETEC. We also investigated the avidity of IgA and IgG antibodies that developed after infection to these antigens.

Principal Findings

Patients infected with ETEC expressing LT or LT+heat stable toxin (ST) and CFA/I group or CS6 colonization factors developed LTB, CFA/I or CS6 specific memory B cell responses at day 30 after infection. Similarly, these patients developed high avidity IgA and IgG antibodies to LTB, CFA/I or CS6 at day 7 that remained significantly elevated at day 30 when compared to the avidity of these specific antibodies at the acute stage of infection (day 2). The memory B cell responses, antibody avidity and other immune responses to CFA/I not only developed in patients infected with ETEC expressing CFA/I but also in those infected with ETEC expressing CFA/I cross-reacting epitopes. We also detected a significant positive correlation of LTB, CFA/I and CS6 specific memory B cell responses with the corresponding increase in antibody avidity.

Conclusion

This study demonstrates that natural infection with ETEC induces memory B cells and high avidity antibodies to LTB and colonization factor CFA/I and CS6 antigens that could mediate anamnestic responses on re-exposure to ETEC and may help in understanding the requirements to design an effective vaccination strategies.     

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.     

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.     

肠毒素大肠杆菌定居因子CFA/Ⅰ和CS6在减毒福氏志贺氏菌中的共表达

定居因子CFA/I和CS6是肠毒素大肠杆菌 (ETEC)中重要的两种优势抗原 ,是ETEC疫苗研制的首选组分。采用基因重组技术将二者构建在以asd基因为选择标记的重组质粒上 ,与asd基因缺失突变型减毒福氏志贺氏菌FWL0 1构成宿主 载体平衡致死系统。实验结果表明 ,重组疫苗候选株能够稳定表达CFA/I和CS6抗原 ,并可在菌体表面形成相应菌毛。重组菌口服免疫BALB/c小鼠后 ,可诱生相应的抗CFA/I和CS6的特异性血清抗体IgG和分泌型抗体sIgA ,说明以志贺氏菌为载体 ,可以构建同时表达多个定居因子抗原的ETEC多价菌苗     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

The role of CFA/I in adherence and toxin delivery by ETEC expressing multiple colonization factors in the human enteroid model

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low-to-middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC that causes moderate-to-severe diarrhea and plays an important role in pathogenesis. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and strain-dependent differences in CFA/I and CS21 expression. The critical role of CFA/I in adherence by ETEC strains expressing CFA/I and CS21 was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting a limitation of this static aerobic infection model. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the lack of decrease in transepithelial electrical resistance and the lack of IL-8 cytokines produced during infection. Taken together, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the human enteroid model has significant utility for the study of ETEC pathogenesis and evaluation of vaccine-induced functional antibody responses.     

Enterotoxigenic Escherichia coli in children with acute diarrhoea and controls in Teresina/PI, Brazil: distribution of enterotoxin and colonization factor genes

Aims: To investigate the distribution of the genes that encode enterotoxins and the colonization factors (CF) types as well as the antibiotic susceptibility profile of enterotoxigenic Escherichia coli (ETEC) isolated from children from the Brazilian Northeast. Methods and Results: We conducted a 3·5‐year prospective study that involved 250 children with and 150 without diarrhoea, aged 1–60 months, from low‐income families in Teresina/Brazilian Northeast. All samples were assayed for E. coli, enterotoxin and CF genes and antimicrobial susceptibility by microbiological methods and PCR. ETEC strains were isolated from 9·2% children with and 4·0% without diarrhoea. Infection was more common in children aged 6–24 months in rainy months. elt⁺/CFA/IV⁺ and elt⁺/CS14⁺ were the most frequent genotypes. Susceptibility to nalidixic acid, ciprofloxacin and gentamicin and resistance to ampicillin, cephalothin and sulfamethoxazole–trimethoprim were common. Conclusions: elt ⁺ isolates and ETEC strains harbouring genes encoding CFA/IV and CS/14 were the most common ETEC found in Brazilian Northeast. Significance and Impact of the Study: Our data, the first generated for north‐eastern Brazilian children, may be important for the development of an effective vaccine and for facilitation of an empirical choice of antibiotic treatment or prophylaxis for traveller’s diarrhoea in the area studied.     

Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103–HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.