A novel dispersin protein in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is a diarrheal pathogen defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture. We have previously shown that EAEC strains secrete a 10-kDa protein that is immunogenic in a human EAEC challenge model. We report here that this protein is encoded by a gene (called aap) lying immediately upstream of that encoding the AggR transcriptional activator, and that aap is under AggR control. The product of aap has a typical signal sequence and is secreted to the extracellular milieu, where it remains noncovalently attached to the surface of the bacterium. EAEC aap mutants aggregate more intensely than the wild-type parent in a number of assays, forming larger aggregates and fewer individual bacteria. Infection of colonic biopsies with wild-type EAEC strain 042 and its aap mutant revealed more dramatic autoagglutination of the mutant compared with the wild-type parent. Our data suggest that the aap gene product participates in formation of a surface coat that acts to disperse the bacteria, thus partially counteracting aggregation mediated by aggregative adherence fimbriae. We have therefore named the aap gene product “dispersin,” and we propose that it may be representative of a functional class of colonization factors. Since dispersin is expressed in vivo, is highly immunogenic, and is present in most EAEC strains, it holds considerable promise as an EAEC immunogen.

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen that has been associated with endemic and epidemic diarrheal illness both in developing and industrialized countries (1, 2). The pathogenesis of EAEC infection is thought to comprise adherence to the intestinal mucosa, most likely of both the small and the large intestines, followed by elaboration of one or more enterotoxins (3). These toxins include the heat stable toxin-like EAST1 and plasmid-encoded (Pet) toxin, the latter of which is responsible for mucosal cytotoxicity in explanted colonic tissue (1, 2, 4–6). Adherence of EAEC to the intestinal mucosa is characterized by the presence of a thick biofilm (2, 7), which may mediate persistence of this organism in the human intestine.

The defining feature of EAEC remains its distinctive aggregative adherence (AA) pattern observed in the HEp-2 adherence assay, in which the bacteria are coincubated with HEp-2 cells in cell culture medium for 3 hours. In this assay, EAEC adhere to the surface of HEp-2 cells, to the glass substratum, and to each other in a characteristic stacked-brick formation (8). We have shown that AA to HEp-2 cells in well-characterized EAEC strains requires a member of the aggregative adherence fimbriae (AAF) family (9, 10). AAF/I and AAF/II are each encoded on an approximately 100-kb virulence plasmid (designated pAA) (11, 12). The proven human pathogenic strain 042 requires AAF/II fimbriae for adherence to colonic explants (10).

Despite the importance of AAF fimbriae in adherence, we have observed that the majority of EAEC strains lack AAF/I and AAF/II (6). However, our data also suggest that most EAEC strains in our collection carry the pAA plasmid, as evidenced by the presence of several other conserved plasmid loci. Most prominent among these is a transcriptional activator of the AraC class, designated AggR, which is required for expression of both AAF/I and AAF/II, but which is also present in a large percentage of EAEC strains that do not express any identified AAF. A second, cryptic locus (originally designated aspU) was found in 80% of EAEC strains in our collection (6). Here, we characterize the aspU locus, which lies immediately upstream of aggR in EAEC 042. This gene encodes a secreted low–molecular weight protein that appears to coat the bacterial surface and thereby promote dispersal of EAEC on the intestinal mucosa. Importantly, the protein, designated dispersin herein, corresponds to a previously cryptic protein against which EAEC-challenged volunteers demonstrated secretory IgA responses (9).

Characterization of the aap locus. We have previously reported that aggR, encoding a transcriptional activator of AAF expression, is encoded downstream of the fimbrial subunit (aafA) on pAA2 in prototype EAEC strain 042 (Figure 1). Sequencing of plasmid DNA upstream of aggR revealed an open reading frame (ORF) terminating 815 nucleotides from the aggR start codon. The ORF was 348 nucleotides in length, encoding a predicted protein product of 116 amino acids. SignalP analysis strongly predicted a signal sequence with cleavage after position 21. The predicted N-terminal amino acid sequence of the mature species was identical to the N-terminus of a cryptic protein we had previously reported to be secreted from EAEC strain 17-2, and initially surmised to be a fimbrial subunit (ref. 9; GenBank accession no. A49221). The predicted mature protein product of aap is 10.2 kDa in size. Neither the nucleotide sequence of aap nor its deduced amino acid sequence displays significant identity to any other protein or gene in the databases.

Figure 1

Map of aap-aggR loci from strains 042 (producing AAF/II) and 17-2 (producing AAF/I). The aap-aggR locus is highly conserved between the two strains, despite divergence of the fimbrial loci. The aap-aggR intergenic region is 99% identical in the two strains, except for a 50-nucleotide insertion present in the 17-2 sequence. IS, insertion sequence. Values percent indicate nucleotide homology between 042 and 17-2 sequences.

The aap-aggR region was remarkably conserved between AAF/I–encoding strain 17-2 (GenBank accession no. Z32523) and AAF/II–encoding strain 042 (Figure 1), despite the fact that the organizations of AAF/I and AAF/II biogenesis genes are dramatically different (12, 22). To assess the conservation of the aap-aggR locus among EAEC strains, PCR was performed on total genomic DNA from a selection of EAEC strains previously hybridized with an aggR probe (6). Primers were chosen from aggR and aap in strain 042 such that the PCR product would span the intergenic region. All strains known to hybridize with the aggR probe yielded identical 2-kb fragments; all probe-negative strains did not yield a PCR product (not shown).

Since aap and aggR are separated by less than 1 kb, we asked whether or not the two genes were transcriptionally linked. RT-PCR was performed using the aap upstream primer and the aggR downstream primer from the experiments above. This RT-PCR reaction did not yield a product, suggesting that the two are not transcribed polycistronically (not shown).

aap transcription is AggR-dependent. AggR is required for transcriptional activation of AAF/I and AAF/II genes, and its proximity to aap suggested that AggR might also be required for aap expression. To test this hypothesis, we performed RT-PCR for the aap transcript in strains 042 and 042aggR (Figure 2). A product of the predicted size was seen in 042 but was absent in 042aggR. To confirm a requirement for AggR, we complemented 042aggR with aggR cloned under control of the arabinose-dependent promoter in pBAD30. Restoration of aap expression was observed in 042(pBADaggR) when cells were grown in the presence of arabinose (ara-inducing conditions) but not when they were grown in the presence of glucose (repressing conditions).

Figure 2

RT-PCR for aap transcript in 042, 042aggR, and 042aggR(pBADaggR). Cultures were grown to late log phase in either L-broth with 0.5% glucose or L-broth with 0.5% arabinose. RT-PCR was performed as described in Methods, and products were separated on 1% agarose gels. The aap transcript migrates as a 0.3-kb species as predicted.

Localization of the aap gene product. The aap gene product has a strongly predicted signal sequence, and previous data have shown that the protein can be found in bacterial supernatants (9). However, quantitative immunoblot experiments suggested that most of the Aap protein pelleted with the bacterium. Experiments were performed to resolve this discrepancy (Figure 3). Broth cultures of 042 were heated to 60°C for 20 minutes or subjected to vigorous vortexing. Both heat and shear forces remove bacterial fimbriae (23). Heat treatment of the culture slightly increased the amount of protein found in the supernatant; vortexing did not release Aap. In contrast, addition of Triton X-100 to a final concentration of 0.1% released the majority of Aap into the supernatant fraction. These data suggest that the aap gene product is a peripheral membrane protein, which is noncovalently attached to the bacterial outer membrane.

Figure 3

Localization of the aap gene product. Western immunoblot analysis was performed on 042 grown in L-broth and subjected to addition of 0.1% Triton X-100 detergent, incubation at 60°C for 20 minutes, or vigorous vortexing for 10 minutes. Cultures were grown to late log phase, and bacteria were pelleted by centrifugation. Pellets (P) were prepared by boiling whole pellet in SDS-PAGE buffer and separating 10% of the sample derived from a 1.0-ml culture. Supernatant (S) was prepared by TCA precipitation. Western immunoblots were performed by standard methods using highly specific polyclonal antiserum to Aap protein. Aap migrates at the predicted 10-kDa size.

Electron microscopy of aap mutants. The aap gene was inactivated in strain 042 by single-crossover homologous recombination of suicide plasmid pJP5603. This construct was complemented by cloning aap into plasmid pQE70, under control of the lac promoter, and was designated pAap. By Immunogold electron microscopy, the surface layer formed a reticulated structure (Figure 4a), which occasionally was visualized between adjacent bacteria (arrows in Figure 4a). 042aap did not stain with the antiserum.

Figure 4

Electron microscopy of 042 and mutants. (a) Immunogold transmission electron microscopy for Aap protein. Black arrows indicate surface protein detected by anti-Aap antibody. Strains examined by scanning electron microscopy are 042 (b and c), 042aafA (d), 042aap (e–g), and 042aap(pAap) (h). In i, 042aap was incubated with 3 mg/ml purified Aap protein prior to fixing and staining. White arrows indicate AAF fimbriae in all specimens. All bars indicate 1 μm.

Scanning electron microscopy was also performed on 042 and several mutants (Figure 4, b–i). Under the growth conditions of the assay, 042 expresses only the AAF/II fimbriae, as null mutants in the fimbrial subunit are afimbriate (Figure 4d). AAF/II extends from the bacterial cell as thin fibers (Figure 4, b and c), joining into thicker bundles and often separating again before attaching the bacterium to the substratum. AAF/II–mediated interbacterial adherence commonly occurred at distances of 2 μm or more (Figure 4c). This appearance was consistent with previous reports (10).

In contrast, 042aap agglutinated more intimately than its wild-type parent, with attachment at a distance seen very rarely (Figure 4, e–g). Interbacterial adherence typically featured bacteria in parallel, with membrane fusions between two bacteria commonly observed (Figure 4f). Moreover, the AAF fimbriae were not observed to extend from the surface of the bacteria but rather lay along the bacterial cell surface, often wrapping around the bacterium (Figure 4, f and g). The complemented aap mutant exhibited an intermediate phenotype (Figure 4h), with looser aggregation than that of 042aap. The addition of purified Aap protein to 042aap resulted in partial rescue of the wild-type phenotype (Figure 4i).

Altered adherence and autoagglutination in an aap mutant. Because of its location near adherence-related genes in EAEC strains and the appearance of 042aap under scanning electron microscopy, we suspected that the aap gene product may play a role in EAEC autoagglutination and adherence. Indeed, stationary-phase broth culture of 042aap exhibited differences in spontaneous settling of bacterial aggregates to the bottom of the culture tube (Figure 5). This difference was reversed in 042aap(pAap) (Figure 5a). Increased aggregation of 042aap was confirmed by transmission electron microscopy (Figure 5b). Aggregation of bacteria in broth cultures was quantitated using the settling assay described by Hasman et al. (18). A typical result of this assay is depicted in Figure 6.

Figure 5

Autoagglutination of 042 and mutants in L-broth. L-broth cultures were grown overnight at 37°C with shaking, then permitted to settle undisturbed at room temperature for 1 hour. (a) Broth cultures revealing a pellet at the bottom of the culture tube correlating with autoagglutination of bacterial cells. (b) Transmission electron microscopy of 042 and 042aap cultures. Ten microliters of L-broth cultures from a were withdrawn prior to incubation on the bench-top and processed for negative staining. Bars, 1 μm.

Figure 6

Spontaneous settling of 042 and mutants. Overnight L-broth cultures containing 0.2 mM IPTG were permitted to settle undisturbed at room temperature. At 15-minute intervals, a 100-μl sample was withdrawn from the top of each broth culture and the OD₆₀₀ of the withdrawn sample was determined. Rate of clearing of the uppermost portion of the culture correlates with rate of spontaneous settling, a measure of agglutination. Samples were tested in triplicate. Bars represent SEM.

In the conventional 3-hour HEp-2 adherence assay, 042aap displayed dramatically larger aggregates than did its wild-type parent (Figure 7, a and b). This phenotype was also reversed in 042aap(pAap) (Figure 7c). When Aap protein was added to the HEp-2 adherence assay simultaneously with bacterial inoculation, adherence by 042aap returned to levels similar to those seen with wild-type (Figure 7d). Addition of BSA (Figure 7e) with the inoculum or addition of Aap after inoculation (not shown) did not have any effect on adherence. Quantitation of bacterial adherence by plate count revealed a dose-response relationship to IPTG concentration in 042aap(pAap), with two-log reduction in adherence upon addition of 0.05 mM IPTG. This concentration of IPTG does not affect growth rate of the test bacteria (not shown).

Figure 7

Role of aap in epithelial cell adherence. Assays were performed for 3 hours in DMEM–0.5% glucose with 0.2 mM IPTG at 37°C in atmosphere. (a) 042; (b) 042aap; (c) 042aap(pAap). (d and e) The standard HEp-2 cell adherence assay was performed using strain 042aap, but with pure Aap (d) or pure BSA (e) added concurrently with the bacterial inoculum. Each protein was added to a final concentration of 0.1 mg/ml. (f) Quantitation of HEp-2 cell adherence on confluent monolayers growing in DMEM without IPTG (042 and 042aap) or with indicated IPTG concentration [042aap(pAap)]. Bacteria were removed from glass using 1% Triton X-100 and quantitated by viable count. Assays were performed in triplicate, and means are illustrated in histograms with error bars indicating SE. *P < 0.05 vs. 042 by one-tailed t test of log-transformed data; **P < 0.05 vs. 042aap by one-tailed t test of log-transformed data.

When a similar adherence assay was performed in the absence of HEp-2 cells (Figure 8), 042aap produced more abundant adherence to glass than did its parent, whereas adherence of 042aap(pAap) was similar to that of wild-type. Quantitation of this assay (Figure 8d) also revealed a dose-response relationship to IPTG concentration.

Figure 8

Glass adherence assay. (a–c) Assay was performed for 3 hours in DMEM–0.5% glucose with 0.1 mM IPTG at 37°C in atmosphere. (a) 042; (b) 042aap; (c) 042aap(pAap). (d) Quantitation of adherence grown in DMEM without IPTG (042 and 042aap) or with the indicated concentration of IPTG [(042aap(pAap)]. Bacteria were removed from glass using 1% Triton X-100 and quantitated by viable count. Assays were performed in triplicate, and means are illustrated in histograms with error bars indicating SEM. *P < 0.05 vs. 042aap by one-tailed t test of log-transformed data.

Roles for aap in mucosal adherence and mucous layer penetration. We have previously shown that EAEC strain 042 adheres abundantly to the surface of human intestinal biopsy sections in culture (24, 25). To investigate the role of the aap gene product in mucosal adherence, 042, 042aap, and 042aap(pAap) were incubated with fresh colonic biopsy specimens for 6 hours (Figure 9). Wild-type 042 adhered as previously described, producing a biofilm on the absorptive surface of the colonic mucosa, while sparing the colonic glands. Notably, 042 exhibited both aggregates and individual bacteria, very similar to the scanning electron microscopic images from silica chip experiments shown in Figure 4. In contrast, 042aap adhered in larger, tighter aggregates with very few single bacteria observed on the surface of the mucosa.

Figure 9

In vitro organ culture assay of 042 (a) and 042aap (b). Bacterial cultures were incubated with colonic biopsies from normal children for 6 hours; then specimens were fixed, stained, and examined by scanning electron microscopy. White arrows (a) denote loose adherence of 042; gray arrows (b) show tight aggregates typical of 042aap. Bars, 10 μm.

We hypothesized that decreasing the number of AAF-mediated aggregates could serve to facilitate the penetration of the 042 through the intestinal mucous layer, on the assumption that single bacteria would be more adept at swimming through the mucous gel. To test this hypothesis, we adapted a previously published method (19) in which bacteria are placed at the top of a column composed of purified mucin in water. These experiments revealed that 042aap was more than an order of magnitude less efficient at penetrating the mucin column than was the wild-type parent strain (Figure 10).

Figure 10

Penetration of 042 and 042aap through mucus. Columns of 10 mg/ml mucin in HBSS were prepared in tuberculin syringes, and the bacterial inocula were applied to the top of the column. The columns were incubated at 37°C for 75 minutes. After this time, the mucin suspension was drained from the column in 0.1-ml fractions, and the number of 042 (black bars) or 042aap (white bars) was determined by quantitative plating in triplicate. Bars represent SD. Fraction 10 represents the bottom of the column, fraction 1 the top. *P < 0.05 by Student’s t test of log-transformed data.

Secretion of Aap. Unresolved by the above experiments is the mechanism of Aap translocation past the outer membrane. We observed above that the predicted aap gene product possesses several attributes characteristic of bacterial pilin subunits, including a C-terminal glycine residue that mediates interaction with a PapD-like chaperone protein (26). We have previously shown that AAF/II expression is dependent on the PapD-like chaperone AafD and the PapC usher homolog AafC (12). To assess a possible role for the AafC/AafD secretion system in Aap expression, we assayed Aap in supernatants of 042 with insertional mutations in the aafD or aafC genes. Both mutants secreted Aap (Figure 11). Since aafD and aafC mutants are afimbriate (12), this experiment provides further verification that, though secreted, the Aap protein is not incorporated into the AAF organelle.

Figure 11

Western immunoblots for Aap performed on supernatants (S) or pellets (P) of overnight L-broth cultures with or without 0.1% Triton X-100. Positions of molecular weight markers are shown at right.

We have previously shown that most EAEC strains in our collection harbor a partially conserved virulence plasmid (designated pAA), which typically encodes the transcriptional activator AggR. Here, we show that aggR is part of a highly conserved two-gene cluster, which also encodes a novel low–molecular weight secreted protein that mediates dispersal of EAEC on the colonic mucosa. In recognition of this phenotype, we have herein designated this gene aap (anti-aggregation protein). We now further suggest the more descriptive name “dispersin” for the aap gene product. Our data suggest that dispersin is secreted to the environment, but that it remains noncovalently associated with the bacterial cell surface.

Several roles for dispersin in adherence and colonization are plausible. All enteric pathogens must adhere to the epithelial surface. But at the same time, it may be beneficial to negatively modulate adherence to permit dispersal across the epithelial surface. Knutton et al. have shown that the bundle-forming pilus of enteropathogenic E. Coli (EPEC) mediates both aggregation and dispersal, which occur sequentially (27). In this way, EPEC can adhere and multiply early in the pathogenetic sequence, thereby establishing a foothold on the epithelium, but the bacteria then disperse individual progeny, which are free to establish new foci of infection. This paradigm is intuitively beneficial and therefore may be a property of many other mucosal pathogens. Indeed, Benitez et al. have suggested that the Hap mucinase of Vibrio cholerae may serve as an enzymatic “detachase,” mutation of which results in increased density of bacterial colonization but also attenuation of virulence (28).

We also hypothesized that dispersin may play a role in penetration of the intestinal mucous blanket, expecting that large aggregates would be retarded in translocation through a viscous gel. Indeed, using a purified mucin gel column assay, we showed that the dispersin mutant penetrated more slowly than the wild-type parent. The human colonic mucosa is covered with a continuous mucous gel approaching 1 mm in thickness (29), which provides a formidable barrier against colonic pathogens. Single bacteria may be more adept at swimming through this layer; alternatively, biophysical characteristics conferred by the dispersin protein coat could promote mucous layer penetration. Whatever their precise roles, Aap, Hap, and some component of bundle-forming pili are apparently the first examples of bacterial “dispersins,” which may facilitate efficient colonization via bacterial dispersal. Thorough elucidation of the roles of these dispersins in vivo has not yet been accomplished, because of the limited availability of good whole-animal models, but is clearly worthy of investigation.

We can propose one potential mechanism by which dispersin may promote dispersal. AAF fimbriae are highly hydrophobic (10), and this hydrophobicity would favor strong autoagglutination in an aqueous environment. The predicted amino sequence of dispersin is more hydrophilic than that of AAF/II, and thus the presence of dispersin on the surface of the bacterial cell may ameliorate surface hydrophobicity. Scanning electron microscopy data are consistent with this hypothesis. Absence of dispersin results in striking alteration in the morphology of the AAF fimbriae. In the absence of dispersin, the fimbriae cling to the surface of the bacterial cell, although their number and diameter do not appear changed. This would imply that the surface of the outer membrane under the dispersin coat is largely hydrophobic; such hydrophobicity could be mediated by outer membrane proteins, or perhaps by other hydrophobic surface structures previously described in EAEC (30).

Notably, despite the fact that aap is coregulated with AAF adhesins and that dispersin has C-terminal characteristics consistent with secretion by the usher-chaperone pathway, we found that mutants in either the usher or the chaperone secreted normal levels of dispersin. Thus, the mechanism by which dispersin is translocated across the outer membrane is as yet unknown. We found it interesting that AAF mutants bound less dispersin protein in the presence of Triton X-100. Immunofluorescence microscopy experiments suggest that some dispersin is bound within AAF-mediated aggregates (not shown), providing one possible explanation for this observation.

The requirement for AggR in aap expression is highly significant. AggR has been shown to be required for expression of AAF biogenesis genes (31), yet there are many strains that carry aggR and do not have an identified AAF adhesin (6). Our data thus suggest that AggR acts as a more versatile regulator of virulence genes in EAEC. Indeed, recent data from our laboratory suggest that AggR activates additional unlinked genes on the pAA plasmid and the EAEC chromosome (J. Nishi et al., unpublished results; and E. Dudley et al., unpublished results). Additional characterization of the AggR regulon and its role in EAEC pathogenesis is underway. Coexpression of AAF and dispersin in a common AggR regulon is consistent with an interactive role of the two factors in colonization, but it is interesting that the bacterium would use the same regulator to express both an adhesin and a negative modulator of adhesion.

EAEC is an emerging pathogen, but vaccine development is in its earliest stages. Several obstacles must be overcome before a candidate vaccine can be constructed. Chief among these obstacles is the antigenic heterogeneity of EAEC strains. In addition, given that EAEC infection is a serious health concern predominantly in developing countries, there is little economic incentive toward the development of a vaccine. Therefore, the best approach to EAEC vaccine development would be to identify a highly conserved, highly prevalent, surface-exposed and immunogenic protein that could be expressed in an attenuated vector vaccine. The ideal vector would be one engineered to provide protection against another, perhaps more serious threat (such as shigellosis or typhoid fever). Dispersin fulfills many of these criteria, and experiments are underway to test its feasibility as an EAEC immunogen.

This work was supported by NIH grant AI-33096 to J.P. Nataro. We thank James Kaper for critical review of the manuscript and Alya Raphael for excellent technical assistance.

Ian R. Henderson’s present address is: Division of Immunity and Infection, University of Birmingham Medical School, Edgbaston, Birmingham, United Kingdom.

Conflict of interest: No conflict of interest has been declared.

Nonstandard abbreviations used: enteroaggregative Escherichia coli (EAEC); aggregative adherence (AA); aggregative adherence fimbriae (AAF); trichloroacetic acid (TCA).

1. Nataro, JP, Steiner, T, Guerrant, RL. Enteroaggregative Escherichia coli. Emerg Infect Dis 1998. 4:251-261.
   View this article via: PubMed Google Scholar
2. Nataro, JP, Kaper, JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998. 11:142-201.
   View this article via: PubMed Google Scholar
3. Okeke, IN, Nataro, JP. Enteroaggregative Escherichia coli. Lancet Infect Dis 2001. 1:304-313.
   View this article via: PubMed CrossRef Google Scholar
4. Nataro, JP, Hicks, S, Phillips, AD, Vial, PA, Sears, CL. T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis. Infect Immun 1996. 64:4761-4768.
   View this article via: PubMed Google Scholar
5. Eslava, CE, et al. Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli. Infect Immun 1998. 66:3155-3163.
   View this article via: PubMed Google Scholar
6. Czeczulin, JR, et al. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun 1999. 67:2692-2699.
   View this article via: PubMed Google Scholar
7. Tzipori, S, et al. Studies with enteroaggregative Escherichia coli in the gnotobiotic piglet gastroenteritis model. Infect Immun 1992. 60:5302-5306.
   View this article via: PubMed Google Scholar
8. Nataro, JP, et al. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr Infect Dis J 1987. 6:829-831.
   View this article via: PubMed Google Scholar
9. Nataro, JP, et al. Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes. Infect Immun 1992. 60:2297-2304.
   View this article via: PubMed Google Scholar
10. Czeczulin, JR, et al. Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli. Infect Immun 1997. 65:4135-4145.
    View this article via: PubMed Google Scholar
11. Savarino, SJ, Fox, P, Yikang, D, Nataro, JP. Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis. J Bacteriol 1994. 176:4949-4957.
    View this article via: PubMed Google Scholar
12. Elias (Jr), WP, Czeczulin, JR, Henderson, IR, Trabulsi, LR, Nataro, JP. Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli. J Bacteriol 1999. 181:1779-1785.
    View this article via: PubMed Google Scholar
13. Ausubel, F.M., et al. 1999. Current protocols in molecular biology. John Wiley & Sons. New York, New York, USA. 3.0.1–3.19.8.
14. Penfold, RJ, Pemberton, JM. An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 1992. 118:145-146.
    View this article via: PubMed CrossRef Google Scholar
15. Sheikh, J, Hicks, S, Dall’Agnol, M, Phillips, AD, Nataro, JP. Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 2001. 41:983-997.
    View this article via: PubMed CrossRef Google Scholar
16. Harlow, E., and Lane, D. 1999. Using antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York, USA. 726 pp.
17. Cravioto, A, Gross, RJ, Scotland, SM, Rowe, B. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr Microbiol 1979. 3:95-99.
    View this article via: CrossRef Google Scholar
18. Hasman, H, Chakraborty, T, Klemm, P. Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation. J Bacteriol 1999. 181:4834-4841.
    View this article via: PubMed Google Scholar
19. Lehker, MW, Sweeney, D. Trichomonad invasion of the mucous layer requires adhesins, mucinases, and motility. Sex Transm Infect 1999. 75:231-238.
    View this article via: PubMed Google Scholar
20. Hicks, S, Candy, DC, Phillips, AD. Adhesion of enteroaggregative Escherichia coli to formalin-fixed intestinal and ureteric epithelia from children. J Med Microbiol 1996. 44:362-371.
    View this article via: PubMed Google Scholar
21. Levine, MM, et al. Coli surface antigens 1 and 3 of colonization factor antigen II-positive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans. Infect Immun 1984. 44:409-420.
    View this article via: PubMed Google Scholar
22. Savarino, SJ, Fox, P, Deng, Y, Nataro, JP. Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis. J Bacteriol 1994. 176:4949-4957.
    View this article via: PubMed Google Scholar
23. Schoolnik, G. 1994. Purification of somatic pili. In Bacterial pathogenesis. Volume 236. V. Clark and P. Bavoil, editors. Academic Press Inc. San Diego, California, USA. 271–282.
24. Nataro, J, Hicks, S, Phillips, A, Vial, P, Sears, C. T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis. Infect Immun 1996. 64:4761-4768.
    View this article via: PubMed Google Scholar
25. Hicks, S, Candy, D, Phillips, A. Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro. Infect Immun 1996. 64:4751-4760.
    View this article via: PubMed Google Scholar
26. Soto, GE, et al. Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus. EMBO J 1998. 17:6155-6167.
    View this article via: PubMed CrossRef Google Scholar
27. Knutton, S, et al. The type IV bundle-forming pilus of enteropathogenic Escherichia coli undergoes dramatic alterations in structure associated with bacterial adherence, aggregation and dispersal. Mol Microbiol 1999. 33:499-509.
    View this article via: PubMed CrossRef Google Scholar
28. Benitez, JA, et al. Adherence of Vibrio cholerae to cultured differentiated human intestinal cells: an in vitro colonization model. Infect Immun 1997. 65:3474-3477.
    View this article via: PubMed Google Scholar
29. Atuma, C, Strugala, V, Allen, A, Holm, L. The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am J Physiol Gastrointest Liver Physiol 2001. 280:G922-G929.
    View this article via: PubMed Google Scholar
30. Wai, SN, Takade, A, Amako, K. The hydrophobic surface protein layer of enteroaggregative Escherichia coli strains. FEMS Microbiol Lett 1996. 135:17-22.
    View this article via: PubMed CrossRef Google Scholar
31. Nataro, JP, Yikang, D, Yingkang, D, Walker, K. AggR, a transcriptional activator of aggregative adherence fimbria I expression in enteroaggregative Escherichia coli. J Bacteriol 1994. 176:4691-4699.
    View this article via: PubMed Google Scholar
32. Nataro, JP, et al. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J Infect Dis 1995. 171:465-468.
    View this article via: PubMed Google Scholar
33. Guzman, LM, Belin, D, Carson, MJ, Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 1995. 177:4121-4130.
    View this article via: PubMed Google Scholar