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Pathogenesis of Afa/Dr Diffusely Adhering Escherichia coli

Institut National de la Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie Paris XI, ChÂtenay-Malabry, France

SUMMARY

INTRODUCTION

GENETIC ORGANIZATION

Afa Adhesins

Dr Adhesins

Adhesin F1845

DIAGNOSIS

RECEPTORS FOR Afa/Dr ADHESINS

Type IV Collagen

DAF (CD55)

Receptor for human Afa/Dr adhesins.

DAF structure and functions.

CEACAMs as Receptors for Afa/Dr Adhesins

CEACAM1 structure and functions.

CEA structure and functions.

CEACAM6 structure and functions.

MECHANISMS OF PATHOGENICITY

UTIs

Internalization

Cell Signaling

Structural and Functional Lesions in the Intestinal Barrier

Inflammatory Responses

CONCLUDING REMARKS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Pathogenic Escherichia coli strains cause a spectrum of diseases in humans (103, 210, 219, 306, 369). Uropathogenic and diarrheagenic strains of E. coli are characterized by the expression of distinctive bacterial properties, products, or structures that are known as virulence factors because they help the organism overcome host defenses and colonize or invade the urinary or gastrointestinal tract (103, 210, 219, 306). These virulence factors allow pathogenic E. coli to interact with host molecules for colonization and usurping normal cell processes, including cytoskeletal dynamics and vesicle targeting for cellular structural and functional damage and host evasion (87, 233). In the case of uropathogenic strains of E. coli, some virulence factors specifically promote the development of pyelonephritis, whereas others promote cystitis or asymptomatic bacteremia (103, 210, 369). Consistent with the fecal-perineal-urethral hypothesis, acute pyelonephritis is recognized to be initiated by the dominance of uropathogenic strains in fecal flora (7). Moreover, although recurrent infections might occasionally be due to a persistent focus of infection (301, 302, 369), the majority have been thought to be reinfections caused by the initially infecting strain persisting in the fecal flora (7). Pathogenic enteric E. coli strains that cause human diarrhea can be divided into a least six groups based on their serotypes and the mechanism by which the disease is thought to be induced: enterotoxigenic E. coli (ETEC), attaching and effacing enteropathogenic E. coli (EPEC), enteroinvasive E. coli, enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), and diffusely adhering E. coli (DAEC) (219, 306).

DAEC strains have been identified from their diffuse adherence (DA) pattern on cultured epithelial HEp-2 as well as HeLa cells (307, 308, 365), and they appear to form a heterogeneous group (91, 308). The first class of DAEC strains includes E. coli strains that harbor Afa/Dr adhesins (Afa/Dr DAEC) (322). These E. coli strains have been found to be associated with urinary tract infections (UTIs) (pyelonephritis, cystitis, and asymptomatic bacteriuria) and with various enteric infections (11, 92, 103, 322). The genetic determinants responsible for the adherence of Afa/Dr DAEC to human epithelial cells have been identified in recent years by genetic and molecular methods. The data indicate that all Afa/Dr DAEC adhesins act as virulence factors. In addition, searches for DNA sequences that are present in Afa/Dr strain C1845, of intestinal origin (43), but absent from a nonpathogenic K-12 strain have revealed that several C1845-specific sequences are either homologous to putative virulence genes or show no homology with known sequences (48). E. coli C1845 harbors sequences encoding several iron transport systems found in other pathotypes of E. coli, including the yersiniabactin siderophore (irp2), the aerobactin siderophore (iuc), a catechole siderophore receptor (iroN), a heme transport system (shu), and a molybdenum transport system (modD). In addition, three C1845-specific sequences (MO30, S109, and S111) are highly prevalent (77 to 80%) among Afa/Dr strains but have low prevalence (12 to 23%) among non-Afa/Dr strains. Moreover, it is interesting that the Afa/Dr strain IH11128, recovered from a patient with a UTI (316), is genetically closely related to strain C1845 of intestinal origin (43). Finally, no genes encoding factors known to subvert host cell proteins, such as the type III secretion system or effector proteins expressed by EPEC (including intimin [Eae] and its receptor [Tir]) (155, 443), have been found in strain C1845. The phylogenic analysis of EAEC and DAEC strains has revealed five large clusters of strains (91). Strain C1845 and some other DAEC strains were present in the cluster DAEC1 and appear to be phylogenetically close to the EAEC strains. Moreover, Afa/Dr DAEC strains appear generally to express several characteristics that have been associated with extraintestinal E. coli strains, including the B2 phylogenetic group (188, 341), the O75 serotype (312), the production of aerobactin (71, 130, 441), the presence of iroN (358), and the presence of sequences from PAI_CFT073 (168), but not including the hlyA, hlyD, hp1 to hp4, papG, or papF sequences. As an exception, the pyelonephritogenic Afa/Dr DAEC strain EC7372, which harbors the Dr-II adhesin (340), expresses a functional hemolysin that is responsible for cell death by apoptosis or necrosis (165).

The second class of DAEC strains includes E. coli strains that express an adhesin involved in diffuse adherence (AIDA-I) (26-29), which is a potential cause of infantile diarrhea. These DAEC strains are likely to contain one or more homologues of the locus of the enterocyte effacement characteristic of EPEC, which may contribute to the pathogenic potential of these DAEC strains. These diarrheagenic E. coli strains have been shown to secrete similar patterns of proteins regulated by environmental parameters, namely, the medium, temperature, pH, and iron concentration (24). Proteins homologous to the EspA, EspB, and EspD proteins, which are necessary for signal transduction events inducing attaching and effacing (A/E) lesions, have been identified that induce the accumulation of actin and tyrosine-phosphorylated proteins at sites of bacterial attachment, leading to the formation of pedestals and/or extended surface structures phenotypically similar to the A/E lesions observed with enteropathogenic and some enterohemorrhagic E. coli strains carrying the LEE pathogenicity island (454).

GENETIC ORGANIZATION

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For extracellular colonization and internalization, microbial pathogens develop molecular interactions with the host cell surfaces. Bacterial pathogens, including pathogenic E. coli, have developed on their surfaces adhesins and invasins responsible for the recognition and binding of specific membrane-bound host molecules acting as receptors. In some cases, activation of complex signal transduction cascades associated with these host cell molecules follows the binding of adhesins and invasins within the active sites on these molecules. In many instances adhesins and invasins are located on the bacterial surface in extended hair-like appendages named pili or fimbriae or in amorphous outer membrane-associated structures termed afimbrial sheaths (404). The Afa/Dr family of adhesins contains representatives having fimbrial (37, 43, 90, 115, 316, 442), afimbrial (241, 243-245, 251, 455, 470), and nonfimbrial (340) architectures (Table 1).

View larger version (33K): [in this window] [in a new window]   FIG. 1. Genetic organization of Afa/Dr operons afa1 (243), afa3 (139, 251), afa7 and afa8 (140, 244), dra (316; accession number AF329316), and daa (43, 264, 265).

View larger version (33K): [in this window] [in a new window]   FIG. 2. Assembly of Dr adhesin via the chaperone-usher pathway.

The Afa-related operons in the A22 and A30 strains lack the Afa-I adhesin-encoding gene but do encode adhesins designated AfaE-II and AfaE-III (241). Le Bouguenec et al. (251) have reported that the cloned afa-3 gene clusters from strain A30 appeared to be carried by 9-kb plasmid regions, which displayed similar genetic organizations. The amino acid sequence of AfaE-III deduced from the nucleotide sequence of the afaE3 gene displays 98% identity to that of the Dr adhesin (157 out of 160 amino acids are identical) (340). Unlike Dr adhesin, in which receptor binding is inhibited by chloramphenicol (319), AfaE-III adhesin confers chloramphenicol-resistant adherence. The plasmid-borne afa-3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by both uropathogenic and diarrhea-associated strains of E. coli (137). The afa-3 gene cluster has been shown to contain six open reading frames, designated afaA to afaF (139). It is organized as two divergent transcriptional units. Five of the six Afa products showed marked homologies with proteins encoded by adhesion systems that have already been described. AfaE has been identified as the structural adhesin product, whereas based on homology with the pap operon, AfaB and AfaC have been identified as periplasmic chaperone and outer membrane anchor proteins, respectively. The AfaA and AfaF products have been shown to be homologous with the PapI-PapB transcriptional regulatory proteins. Upstream of the afa-3 gene cluster, a 1.2-kb region has been found to display 96% identity with the RepFIB sequence of one of the enterotoxigenic E. coli plasmids (P307), suggesting a common plasmid ancestor. This region contains an integrase-like gene (int). Sequence analysis has revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster. Two other IS1 elements have been detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments. This means that the afa-3 gene cluster is flanked by two IS1 elements in a direct orientation and two in opposite orientations. The afa-3 gene cluster, flanked by two directly oriented IS1 elements, has been shown to translocate from a recombinant plasmid into the E. coli chromosome. This translocation event occurred via IS1-specific recombination mediated by a RecA-independent mechanism. The afa-3 gene cluster is closely related to the daa operon, which codes for an adhesin, fimbrial adhesin F1845 (42, 43), that is closely related to the AfaE-III adhesin (137). Chimeras constructed between the afa-3 and daa operons demonstrate that the biogenesis of a fimbrial or an afimbrial adhesin is entirely determined by the amino acid sequences of the AfaE-III and F1845 adhesins (137). Determination of the atomic resolution structure for the AfaE-III subunit reveals that the adhesin assembles by donor strand complementation and for the architecture of capped surface fibers (8).

Two other Afa-related adhesins have been identified recently. The AfaE-VII and AfaE-VIII adhesins are encoded by the afa-7 and afa-8 gene clusters, respectively, and are expressed mostly by bovine isolates (244, 245). These animal afa gene clusters are expressed by strains that produce other virulence factors, such as the CNF toxins and the F17, PAP, and CS31A adhesins. It is noteworthy that although the AfaE-VIII adhesin has been detected in human E. coli (142), it has never been detected in diarrhea-associated human isolates (252). Like the afa-3 gene cluster, both the afa-7 and afa-8 gene clusters were found to encode the afimbrial adhesin AfaE and the invasin AfaD. The afa-8 operon is carried by a 61-kb genomic region with characteristics typical of a pathogenicity island, including a size in excess of 10 kb, the presence of an integrase-encoding gene, being inserted into a tRNA locus (PheR), and the presence of a small direct repeat at each extremity (245). The location of the afa-8 gene cluster on the plasmids or chromosomes of these isolates suggests that it could be carried by a mobile element, facilitating its dissemination among bovine-pathogenic E. coli strains (244). Sequences related to the afa-8 gene cluster have been identified in E. coli strains isolated from diseased calves, pigs, humans, and poultry, whereas no sequence related to the afa-7 gene cluster has been reported (140).

The EPEC O55 serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET5 comprises strains with different combinations of virulence genes, including those for localized adherence and DA. Interestingly, the ET5 DA strains possess an 11.6-kb chromosomal region including an operon that encodes a protein with 98% identity to AfaE-I, which is probably responsible for the DA (227).

The Dr-II adhesin has been identified from the pyelonephritogenic strain EC7372. This adhesin has a low level of sequence identity with other members of the Afa/Dr family (17 to 20% of the 160 amino acids are identical) (340). Dr-II is 96% identical to the nonfimbrial adhesin NFA-1, an adhesin associated with a UTI whose receptor has not been identified (4). It was noted that although nonfimbrial adhesins have not previously been considered to belong to the Afa/Dr family, in fact they have a very similar genetic organization. Strain EC7372 can be viewed as a prototype of a subclass of Afa/Dr DAEC isolates that have acquired a pathogenicity island similar to that described for the pyelonephritogenic strain CFT073, which carries both hly and pap operons (370) and which, unlike other Afa/Dr DAEC strains, triggers cell death by apoptosis or necrosis (165).

DIAGNOSIS

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Phenotypic and genotypic assays have been developed for detection of E. coli harboring Afa/Dr adhesins. On the basis that pathogenic E. coli strains attach to HeLa cells in different patterns (localized, diffuse, or aggregative), an adhesion assay has been proposed for the detection of the mannose-resistant diffuse adhesiveness of DAEC strains onto cultured epithelial Hep-2 or HeLa cells (307, 308, 365). The adhesion assay is not specific for Afa/Dr DAEC detection, since other pathogenic E. coli strains, including DA EPEC strains (26-29), have been reported to develop the diffuse phenotype of adhesion without the presence of Afa/Dr adhesins.

In order to detect the DAEC strains harboring the Afa/Dr adhesins, a hemagglutination inhibition assay with human erythrocytes and with human erythrocytes preincubated with anti-DAF monoclonal antibody IH4 has been further proposed on the basis that these E. coli strains show an MRHA phenotype (241, 243) and recognize as a receptor the human DAF in its complement control protein repeat 3 (CCP-3) domain (originally known as short consensus repeats) (318). Inhibition of MRHA presents several inconveniences, including the lack of viability of fresh human erythrocytes.

A new method of detection of Afa/Dr DAEC, named the DAF clustering assay (DCA), has recently been proposed by Goluszko et al. (149). This assay associates the diffuse adhesiveness of bacteria onto cultured epithelial HeLa cells and the previously reported human DAF receptor clustering around adhering bacteria (150, 166, 213, 252). Results show a high positive correlation of DCA with the hemagglutination inhibition assay described above and with a PCR protocol conducted with primers amplifying the afaB 750-bp sequences. However, the DCA did not allow the detection of all E. coli strains expressing Afa/Dr adhesins, since Afa/Dr DAEC strains that express the AfaE-VII and AfaE-VIII adhesins do not bind to human DAF (244). This is a particular inconvenience for the detection of Afa/Dr DAEC strains expressing the AfaE-VIII adhesin present in human extraintestinal clinical isolates (142, 245, 252).

DNA probes have been constructed for colony hybridization assays. The first DNA probe, named daaC, was generated by Stapleton et al. (413) and was a 300-bp PstI fragment of plasmid pSSS1 (daa operon), coding for part of an accessory protein of F1845 adhesin expressed by the diarrheagenic Afa/Dr DAEC C1845 strain (43). This DNA probe has been used in a large majority of the epidemiological studies of the association of Afa/Dr DAEC with diarrhea and urinary tract infections (1, 5, 68, 112, 127, 130, 131, 141, 143, 152, 208, 209, 256, 257, 293, 329, 344, 364, 366, 367, 400). Another constructed DNA probe, named drb (130), was a 260-bp PstI fragment of plasmid pIL14 (afa-1 operon) coding for the AfaE-I adhesin expressed by the uropathogenic Afa/Dr DAEC KS52 strain (243). This DNA probe has been used in epidemiological studies of the association of Afa/Dr DAEC with urinary tract infection (13, 130, 131, 250, 287, 466, 467). The results show a high positive correlation of the colony hybridization assay with the adhesion assay and the hemagglutination inhibition assay described above. However, this technique requires lengthy manipulations to prepare the DNA probes and is too time-consuming for testing of individual strains.

A more practical and faster method than the colony hybridization assay uses the PCR approach. Pham et al. (339) have developed primers designed to amplify a 750-bp fragment of the afaB gene, which encodes a periplasmic chaperone protein involved in the biogenesis of Afa/Dr adhesins. Two pertinent PCR assays that allow the detection of all of the Afa/Dr adhesins have been developed by Le Bouguenec's group (250, 252). The first PCR assay used the afa1 and afa2 primers, based on the partial sequence of the afa-1 gene operon, flanking a 750-bp DNA segment overlapping the afaB and afaC genes (250). After comparison of the nucleotide sequences of the afa-3, afa-7, and afa-8 operons, Le Bouguenec et al. (252), considering that the afa1 and afa2 primers did not detect all of the afa/dr gene clusters, constructed two new primers, afa-f and afa-r, which flanked a 672-bp DNA segment internal to the afaC gene. Strains positive in afa1-afa2 PCR expressed the afaE1, afaE2, afaE3, afaE5, afaE7, afaE8, draE and daaE genes clusters. Afa/Dr DAEC strains have been found equally in control and diseased patients.

Epidemiological studies conducted by use of colony blotting with the daaC DNA probe have demonstrated an age-related incidence of Afa/Dr DAEC in diarrhea in children, which apparently begins after age 2 or 3 (112, 127, 141, 143, 152, 167, 208, 209, 256, 344, 364). Moreover, E. coli strains expressing Afa/Dr adhesins have been found with similar frequencies in patients with diarrhea and control subjects (127, 167, 356). Recently Blanc-Potard et al. (48), using representational difference analysis, have revealed that three sequences (MO30, S109, and S111) were specifically present in the wild-type, diarrhea-associated C1845 strain (43). On the basis of these sequences, it should be of interest to develop a new PCR assay with primers specific for the detection of diarrhea-associated Afa/Dr DAEC strains.

RECEPTORS FOR Afa/Dr ADHESINS

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Type IV binding capacity appears to be important for urinary tract infection caused by Afa/Dr DAEC, since in the kidney the type IV collagen binding capacity of Dr adhesin leads to the formation of persistent mesangial deposits (285). Consistent with this previous observation, using an isogenic mutant in the DraE adhesin subunit that was unable to bind type IV collagen but retained binding to DAF, Selvaragan et al. (377) have shown that type IV collagen binding mediated by the DraE adhesin is a critical step for the development of persistent renal infection in a murine model of E. coli pyelonephritis. In contrast, the role of type IV collagen binding capacity in Afa/Dr DAEC-induced intestinal pathogenicity is questionable. Indeed, type IV collagen is never present at the apical domain of polarized epithelial cells, the site of Afa/Dr DAEC colonization, since it is mainly of mesenchymal origin (266). Together with fibronectin, laminin, tenascin, and heparan sulfate proteoglycans, type IV collagen is a component of the basement membrane, which is involved in complex interactions at the epithelial-mesenchymal interface. In particular, type IV collagen interacts with integrins expressed at the basal domain of polarized cells (23), to form a link between the basement membrane and epithelial cells (266). However, during inflammation, deregulated expression of membrane-bound molecules that are normally segregated in the basolateral domain of polarized intestinal cells occurs, and it is possible that in this context type IV collagen binding may contribute to the pathogenicity of Afa/Dr DAEC.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Membrane-associated receptors for Afa/Dr adhesins and invasins. DAF acts as a receptor for all of the human Afa/Dr adhesins (Afa/DrDAF) (318, 319). CEACAM1, CEA, and CEACAM6 act as receptors for AfaE-III, Dr, and F1845 adhesins (Afa/DrCEA) (33). Integrin 5ß1 acts as a receptor for internalization by Dr-positive IH11128 and AfaE-III-positive A30 strains (164, 343). Integrin 5ß1 recognition by AfaD-III invasin leads to cell entry (343).

Afa/Dr adhesins recognized the CCP-3 on DAF (318). Indeed, a single point substitution in CCP-3 (Ser165 to Leu, corresponding to the Dra-to-Drb allelic polymorphism) caused complete abolition of adhesin binding to DAF (318). Importantly, the Dr adhesin-binding and complement-regulating epitopes of DAF appear to be distinct and are approximately 20 Å apart (178). Indeed, it is residue Ser155, and not Ser165, in DAF CCP-3 that is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin, while residues Phe123 and Phe148 at the interface of CCP-2 and CCP-3, and also Phe154 in the CCP-3 cavity, are important in complement regulation. An atomic resolution model for functions of the AfaE-III adhesin reveals the pivotal role of CCP-2 and -3 in binding of adhesin onto DAF (8). Like DraE, AfaE-III binds to CCP-2 and -3, but CCP-3 contributes most to the free energy of binding. Interestingly, the binding regions for AfaE-III and the complement pathway convertases lie in close proximity to each other on DAF. This raises the possibility, previously invoked by Nowicki et al. (325), that binding of Afa/Dr adhesins might interfere with the complement-regulatory function of DAF, leading to immunopathological lesions.

The major structural subunits of Dr adhesin (DraE) and F1845 adhesin (DaaE) bind to the DAF receptor in a specific manner resembling the distinct phenotypes of the corresponding Dr adhesin (448). Individual amino acid changes at positions 10, 63, 65, 75, 77, 79, and 131 of the mature DraE sequence significantly reduce the ability of the DraE adhesin to bind DAF. Considering that more than half of the mutants obtained had substitutions within amino acids 63 to 81, this suggests that these proximal residues may cluster to form a binding domain for DAF (449).

As described above for binding of DraE adhesin to type IV collagen, binding of DraE adhesin to DAF is sensitive to chloramphenicol, as demonstrated by the ability of chloramphenicol to inhibit the MRHA of erythrocytes (319, 321). In contrast, the MRHA produced by AfaE-I, AfaE-III, and F1845 adhesins is not sensitive to chloramphenicol (251, 319). Swanson et al. (424) reported that the domains responsible for the chloramphenicol-sensitive hemagglutination of Dr adhesin reside within the amino-terminal portion of the fimbrial subunit. Examination of the X-ray structure of a DraE-chloramphenicol complex has recently revealed the precise atomic basis for the sensitivity of DraE-DAF binding to chloramphenicol (338). The chloramphenicol-DraE complex structure reveals that chloramphenicol binds in a surface pocket between the N-terminal portion of strand B and the C-terminal portion of strand E and lies within the recently identified DAF-binding site (8). Moreover, in contrast to other chloramphenicol-proteins complexes, chloramphenicol binding to DraE is mediated via recognition of the chlorine "tail" rather than by the intercalation of the benzene rings into the hydrophobic pocket. Carnoy and Moseley demonstrated that the single Ile111Thr mutation in DraE completely abolishes chloramphenicol binding (73). The X-ray structure of a DraE-chloramphenicol complex reveals that the chloramphenicol binding site is in close proximity to two of the three sequence differences between DraE and AfaE-III (338), providing an explanation for the previously reported lack of activity of chloramphenicol against AfaE-III binding to DAF (251, 319).

Among E. coli strains isolated from gestational pyelonephritis patients and used to investigate the expression of Dr adhesin, several Dra-positive strains did not fulfill the specific criteria for Dr adhesins (339). Indeed, the binding sites of several of these E. coli strains were located within the CCP-3 domain of DAF but outside the region blocked by a monoclonal anti-CCP-3 antibody, and in other cases they were located on the CCP-4 domain. This reveals heterogeneity in the binding sites of E. coli expressing Afa/Dr adhesins that may reflect the ability of these adhesins to evolve so as to recognize alternative peptide epitopes on DAF in order to achieve efficient colonization.

As described above for Afa/Dr DAEC, DAF is hijacked by coxsackieviruses and enteroviruses as part of their pathogenicity mechanisms. DAF is a major cell attachment receptor for coxsackieviruses B-1, -3, and -5 (3, 30, 32, 277, 380, 383), but cell infection requires an association with the coxsackievirus and adenovirus receptor (CAR) (31, 75, 76, 332, 381, 382, 384). Enterovirus 70 utilizes DAF as an attachment protein (221), recognizing CCP-1 as a binding site (184, 220), but some enteroviruses that bind to DAF also bind to cells of human and murine origins in a DAF-independent manner, suggesting that they use a multiplicity of receptors to achieve infection of the host (153, 154). Finally, human DAF is the receptor that mediates attachment and infection by several echoviruses (30, 84, 153, 184, 249, 347-349). Interestingly, echovirus and coxsackie B viruses all display highly specific recognition of human DAF, since all failed to recognize rat, mouse, or pig DAF (412), like Afa/Dr DAEC (197).

DAF structure and functions. DAF is a CRP (58, 239, 273, 294). These structurally related regulatory proteins are all encoded in the regulators of C activation gene cluster on chromosome 1q32. The regulators of C activation gene family encodes four membrane-bound proteins: C receptor 1 (CD35), C receptor 2 (CD21), membrane cofactor protein (CD46), and DAF (74, 263, 270, 271, 346, 354). Human DAF is a cell-associated protein with an M_r of 55,000 to 70,000, depending on its glycosylation level. Large quantities of membrane-associated DAF have been found on the epithelial surfaces of oral and gastrointestinal mucosae, renal tubules, ureter and bladder, and cervical and uterine mucosa (86, 199, 281).

Under physiological conditions, DAF plays a central role in preventing the amplification of the complement cascade on host cell surfaces (133, 280). DAF interacts directly with membrane-bound C3b or C4b and prevents the subsequent uptake of C2 and factor B.

The DAF domains involved in complement regulation have been characterized. Biophysical explorations of the structural biology of CRPs have shown that the five human proteins responsible for regulating the early events of complement are homologous and consist mainly of building blocks containing CCPs. The structures of the individual CCPs exhibit wide variations on a common theme, while the extent and nature of their intermodular connections are diverse. Some neighboring modules within a protein stabilize each other, and some cooperate to form specific binding surfaces (232). Molecular cloning of human DAF from HeLa cells has revealed two classes of DAF mRNA (69). The major spliced DAF mRNA (90%) encodes membrane-bound DAF, whereas the minor unspliced DAF mRNA (10%) may encode secreted DAF. The two DAF proteins have divergent C-terminal domains with differing hydrophobicities, and the deduced DAF sequence contains four repeating units homologous to a consensus repeat found in the CRP family. Membrane-bound DAF is attached to the cell surface membrane by a glycosylphosphatidylinositol (GPI) anchor (70, 95), followed by a serine-threonine-proline-rich region and by the repeating units of the CCPs, consisting of 60 to 70 amino acids arranged in tandem (74, 354). A model of the regulatory region of human DAF has revealed that the four CCPs are arranged in a helical fashion (238). Removal of CCP-1 had no effect on DAF function, but individual deletion of CCP-2, CCP-3, or CCP-4 totally abolished DAF function (59, 88). Molecular modeling of the protein has predicted that a positively charged surface area on CCP-2 and -3 and nearby exposed hydrophobic residues on CCP-3 may act as ligand-binding sites and that L147F148 in a hydrophobic area of CCP-3 is essential in the regulation of both regular and alternative pathway C3 convertase, whereas KKK125 to -127 in the positively charged pocket between CCP-2 and -3 is necessary for the regulatory activity of DAF on the alternative pathway C3 convertase but plays a lesser role in its activity on the regular pathway enzyme (58). The N-linked glycan of DAF is not involved in its regulatory function (88). Because of the increased lateral mobility due to the GPI anchor, this gives a functional advantage in contacting ligand C3b or C4b on the cell surface. However, a transmembrane (TM) version of DAF (DAF-TM) is effective in protecting CHO transfectants against cytotoxicity (268). Finally, deletion of the serine-threonine-proline-rich region totally abolished DAF function, since this region serves as a crucial but nonspecific spacer required to project the DAF functional domains above the plasma membrane (88).

The outer membrane protein P5, expressed by Haemophilus influenzae, a commensal of the human respiratory mucosa, recognizes CEACAM1 (189, 450). A major outer membrane protein of Moraxella catarrhalis strains, belonging to the ubiquitous surface protein family, also interacts with CEACAMs as receptors (189). CEACAM1, CEA, and CEACAM6 have been shown to bind some uncharacterized E. coli strains and some Salmonella species (253-255, 363). CEACAMs play an important role in the pathogenicity of Neisseria gonorrhoeae, since opacity (Opa) proteins mediate the adherence and signaling required to allow this bacterium to penetrate into human tissues (96, 97, 180, 182, 183, 282, 283, 310, 452). As described above for Afa/Dr adhesins, groups displaying distinct specificities of Opa interaction with CEACAMs have been identified (159). CEACAM1, CEACAM3, CEA, and CEACAM6 all act as Opa_CEA receptors, whereas CEACAM4, CEACAM7, and CEACAM8 do not (44, 49-52, 79, 158, 298, 345). Opa₅₂ binds CEACAM1, CEACAM3, CEA, and CEACAM6; Opa₅₃ is CEACAM1 specific; Opa₅₄ binds CEACAM1 and CEA; and Opa₅₅ is CEA specific.

CEACAMs belong to the immunoglobulin (Ig) superfamily of adhesion molecules (163, 172, 327, 431). The members of the CEACAM gene family are clustered on chromosome 19q13.2. CEACAMs share a conserved N-terminal Ig variable (Ig_v)-like domain that is followed by 0 to 6 Ig constant (Ig_c)-like domains. The CEACAMs, consistent with the recently redefined nomenclature (22), now comprise seven members, i.e., CEACAM1 (biliary glycoprotein, CD66a), CEACAM3 (CEA gene family member 1 [CGM1], CD66d), CEACAM4 (CGM7), CEA (carcinoembryonic antigen, CD66e), CEACAM6 (nonspecific cross-reacting antigen, CD66c), CEACAM7 (CGM2), and CEACAM8 (CGM6, CD66b). CEACAM receptors are differentially expressed by various epithelial, endothelial, and hematopoietic cells in vivo (22, 163). CEACAM1, CEACAM3, and CEACAM4 are inserted into the cellular membrane via a carboxy-terminal transmembrane and cytoplasmic domain, whereas CEA, CEACAM6, CEACAM7, and CEACAM8 have a GPI anchor instead. The level of glycosylation of CEACAM receptors may vary, depending on their cell type and differentiation state, and multiple glycoforms of the same protein have been isolated. CEACAMs generally function as intercellular adhesion molecules (25). Moreover, the observation that CEACAM1, CEA, CEACAM6, and CEACAM7 are all located on the apical glycocalyx of normal colonic epithelium suggests that they could play a role in innate immunity (118).

CEACAM1 structure and functions. CEACAM1 contains the conserved N-terminal Ig_v-like domain of CEACAMs, which is followed by three Ig_c-like domains (22, 396, 431). CEACAM1 is inserted into the cellular membrane via a carboxy-terminal transmembrane and cytoplasmic domain. Differential splicing of CEACAM1 finally yields eight transmembrane isoforms, including CEACAM1-4L, CEACAM1-3L, CEACAM1-4S, and CEACAM1-3S, with different numbers of extracellular domains, and either a long or a truncated cytoplasmic domain. CEACAM1 has been shown to be expressed on leukocytes, including granulocytes, activated T cells, B cells, and CD16^– CD56⁺ natural killer cells (163), and has also been observed in endothelial cells, in the apical poles of enterocytes and colonic cells, and in the epithelia of esophageal and Brunner's glands, bile ducts and gallbladder, pancreatic ducts, proximal tubules of the kidney, prostate, endometrium, and mammary ducts (195, 350).

CEACAM1 functions as a cell-cell adhesion molecule that mediates homophilic cell adhesion (429, 457, 458). CEACAM1 contributes to contact inhibition of cell proliferation in confluent cells but allows proliferation when expressed at different isoform ratios (117, 129, 394). CEACAM1 expression has been reported to be generally downregulated in carcinomas of the colon and liver of human, rat, and mouse origins (235, 311, 372), and in human colon and prostate cancer downregulation is associated with the loss of cell polarity (66) and results in enhanced tumor cell growth and tumorigenicity (15). CEACAM1 directly associates with the cytoskeleton proteins actin and tropomyosin (61, 374). CEACAM1-L is located at cell-cell boundaries, and its association with the actin cytoskeleton is regulated by the Rho family of GTPases (359). Consistent with this, CEACAM1 colocalizes with paxillin at the plasma membrane, and CEACAM1-paxillin complexes have been isolated in granulocytes, the colonic cell line HT29, and human umbilical vein endothelial cells (111). In polarized Madin-Darby canine kidney (MDCK) epithelial cells, activation of Cdc42 and Rac1, or of their downstream effector PAK1, targeted CEACAM1 to sites of cell-cell contacts. The transmembrane domain of CEACAM1 was responsible for the Cdc42-induced targeting at cell-cell contacts (128).

Other cell functions mediated by CEACAM1 have also recently attracted interest. In human T and natural killer (NK) cells (291) and small intestinal intraepithelial lymphocytes (292), CEACAM1 phosphorylation undergoes a rapid increase following stimulation with the chemoattractant formyl-Met-Leu-Phe peptide (395). Ligation of CEACAM1 strongly increases adhesion to fibrinogen by Fc receptor- and ß₂ integrin-dependent mechanisms (419). Interestingly, coligation of CEACAM1 plus CEACAM6 and CEACAM8 has also been reported to cause increased ß₂ integrin-mediated adhesion and receptor clustering, whereas ligation of CEACAM6 or CEACAM8 separately did not cause neutrophil activation. CEACAM1 acts as a novel class of immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing regulatory molecules on T cells that are active during the early phases of the immune response in mice (215, 216, 303). The cytoplasmic domain of CEACAM1 contains two tyrosine residues in amino acid motifs interacting with pp60c-src, which are located in ITIM consensus sequences (62). Phosphorylation of CEACAM1 tyrosine by an associated tyrosine kinase may have a functional role (395). Lyn and Hck account for much of the tyrosine kinase activity associated with CEACAM1 (395), which activates extracellular signal-regulated kinases 1 and 2 (392). The structural features surrounding the tyrosine residues in the cytoplasmic domain of CEACAM1 share similarities with the consensus sequence of the ITIM, the docking site for SHIP, SHP-1, and SHP-2 molecules. When phosphorylated, these residues associate with the protein-tyrosine phosphatases SHP-1 and SHP-2, and the C-terminal amino acids of CEACAM1 are critical for these interactions (196). The intracytoplasmic domain, which contains two ITIM-like domains, is required for activation of a fraction of T cells in the lamina propria that express CEACAM1 by interleukin-7 (IL-7) and IL-15 cytokines, indicating that CEACAM1 amplifies T-cell activation and thus could facilitate cross talk between epithelial cells and T lymphocytes in the intestinal immune response (102).

The particular role of CEACAM1 in Neisseria pathogenicity has been documented. By its functional ITIM, CEACAM1 plays pivotal role in Opa_CEA-mediated signaling (283, 284). CEACAM1 functions as a microbial receptor in human granulocytes and epithelial cells, since Opa_CEA proteins bind to the N-terminal domain of CEACAM1 on the nonglycosylated surface of the molecule (451, 453). Interestingly, the N-terminal domain is implicated in homophilic adhesion by CEACAM1 (458). No pathogen-directed reorganization of the actin cytoskeleton is required for invasion of the epithelial cell lines via CEACAM1 to occur (44). Neisseria infection induces the expression of CEACAM1, CEACAM1-3L, and CECAM1-4L splice variants through activation of an NF-B heterodimer consisting of p50 and p65. Subsequently, increased Opa₅₂-dependent binding of gonococci by these cells develops (297, 299). The ability of N. gonorrhoeae to upregulate its epithelial receptor CEACAM1 via NF-B reveals an important pathogen-elicited mechanism that allows efficient bacterial colonization to occur during the initial infection process. In addition, the regulation of CEACAM1 expression by NF-B also implies that this receptor plays a broader role in the general inflammatory response to infection (299). N. gonorrhoeae evades host immunity by switching off T lymphocytes (56). In N. gonorrhoeae, the Opa₅₂ protein is able to bind the CEACAM1 expressed by primary CD4⁺ T lymphocytes and to suppress their activation and proliferation after the Opa gonococcal protein associates with the tyrosine phosphatases SHP-1 and SHP-2 in the ITIM of CEACAM1 (55, 80). In addition, Opa_CEA interaction with CEACAM1 leads to inhibition of the activation and proliferation of Neisseria-infected CD4⁺ T lymphocytes (315). It remains to be determined whether or not the recognition of CEACAM1 by Afa/Dr_CEA adhesins is followed by the signaling events and the cellular responses observed for Opa_CEA.

CEA structure and functions. CEA is a well-established tumor-associated marker (398). CEA shares the conserved N-terminal Ig_v-like domain of CEACAMs, which is followed by six Ig_c-like domains (22, 431). CEA is expressed by M cells (19), enterocytes (34), and colonic cells (456) and is an integral component of the apical glycocalyx (173). CEA has been shown to act, in vitro at least, as a homotypic intercellular adhesion molecule. CEA is known to mediate Ca²⁺-independent, homotypic aggregation of cultured human colon adenocarcinoma cells. CEA is produced in excess in virtually all human colon carcinomas and in a high proportion of carcinomas at many other sites (372). The engagement of neutrophil CEA with anti-CEA Ig results in activation-associated phenomena, including shape change and activation of ß₂-integrin (418). CEA can also inhibit the differentiation of several other cell types and thus contributes to tumorigenesis, an activity that requires CEA-CEA interactions (78). This differentiation-blocking activity resides in its GPI anchor (375). Deregulated expression of CEA could directly contribute to colon tumorigenesis by inhibiting terminal differentiation and anoikis (201). CEA may act as a chemoattractant in colorectal cells, a function related to type IV collagen and laminin (230). In fully differentiated polarized epithelial cells, CEA is apically expressed. It has not been shown whether CEA mediates functions in normal cells. CEA is anchored in the cell membrane via a GPI anchor, and like other GPI-anchored proteins (82, 386, 387, 415, 417), CEA can signal.

The role of CEA in signaling events following its recognition as a receptor by microbial pathogens is poorly documented. Opa_CEA-mediated stimulation of CEA leads to activation of the small GTPases Rac1 and Cdc42 (44) and downregulation of the tyrosine phosphatase SHP-1 (181). It remains to be analyzed what the signaling events that follow the recognition of CEA by Afa/Dr_CEA adhesins are. Moreover, it could be of interest to examine whether the CEA, which is a GPI-anchored protein, triggers the same signaling events observed following recognition of DAF. Finally, analyzing the cellular responses that occur after the recognition of CEA by Afa/Dr_CEA adhesins is of interest, considering that the functions of CEA are poorly documented.

CEACAM6 structure and functions. CEACAM6 shares the conserved N-terminal Ig_v-like domain of CEACAMs, which is followed by two Ig_c-like domains (22, 397, 431). CEACAM6 is anchored in the cell membrane via a GPI anchor. Intriguingly, unlike GPI-anchored proteins, CEACAM6 could not be dislodged from the cell membrane by phosphatidylinositol-specific phospholipase C. CEACAM6, due to its GPI anchor, is apically expressed in polarized epithelial cells. Like CEA, the GPI-anchored CEACAM6 can signal (82, 386, 387, 415, 417). Consistent with this, CEACAM6-cross-linking increased c-Src activation and induced tyrosine phosphorylation of p125FAK focal adhesion kinase, for which caveolin-1 was required (106). Moreover, CEACAM6 cross-linking initiates c-Src-dependent cross talk between CEACAM6 and _vß3 integrin, leading to increased extracellular matrix component adhesion (107).

CEACAM6 is coexpressed with CEA in normal colorectal epithelia and is deregulated in colorectal cancers, where it could play a role in tumorigenesis (201). CEACAM6 revealed a broader expression zone in proliferating cells in hyperplasic polyps and adenomas than in the normal mucosa. Anoikis is the apoptotic response induced in normal intestinal cells by inadequate or inappropriate adhesion to substrate. Deregulated overexpression of CEA/CEACAM6 inhibits anoikis (330). Furthermore, increased CEACAM6 expression and CEACAM6 cross-linking both induced a significant increase in cellular resistance to anoikis, and CEACAM6 gene silencing reversed this acquired resistance (108). It has been observed that Akt, which is known to mediate cell survival, is activated in colonic T84 cells expressing CEA and CEACAM6 and infected with Afa/Dr DAEC (F. Betis, A. L. Servin, and P. Hofman, unpublished data).

CEACAM6 is involved in the invasion of epithelial cell lines by Neisseria. As for CEACAM1, no pathogen-directed reorganization of the actin cytoskeleton is required for Opa_CEA-expressing bacteria (44). Moreover, the CEACAM6-mediated uptake of Neisseria is not blocked by dominant-negative versions of the small GTPase Rac (371). This mechanism of cell entry resembles the mechanism by which Afa/Dr DAEC is internalized following recognition of CEA and/or CEACAM6, which does not require mobilization of the actin cytoskeleton and which is not inhibited by substances that block the signaling molecules involved in F-actin rearrangements (218). It remains to be determined whether Neisseria uses lipid rafts, like Afa/Dr DAEC (148, 164, 218), to invade the cells following recognition of the GPI-anchored CEACAM.

MECHANISMS OF PATHOGENICITY

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DAF has been shown to regulate complement activation on glomerular epithelial cells (351), and expression of DAF was increased on the glomerulus of patients with diffuse proliferative glomerulonephritis (12). Experimental studies have shown that urinary complement components have a role in mediating tubulointerstitial damage, which is known to be closely correlated with the progression of chronic renal diseases. Both GPI-anchored and transmembrane-anchored DAF proteins, each of which can be derived from two different genes (Daf1 and Daf2), are produced in mice, and nephrotoxic serum nephritis develops in both wild-type mice and Daf1 gene-floxed mice (259). Increased susceptibility to antiglomerular basement membrane glomerulonephritis has been reported in DAF-deficient mice (401). The possible role of virulence factors of Dr-positive E. coli in the persistence of bacteria in renal tissue and in the pathogenesis of chronic pyelonephritis has been investigated. Goluszsko et al. (146) examined the hypothesis that E. coli renal interstitial binding mediated by the Dr adhesin is important for the development of chronic ascending pyelonephritis in mice. Dr⁺ E. coli colonized the renal interstitium, since a substantial amount of fimbrial antigen was detected in the injured parenchymal regions, and significant histological changes corresponding to tubulointerstitial nephritis, including interstitial inflammation, fibrosis, and tubular atrophy, were found in the kidney tissue of Dr⁺-infected mice but not in that of Dr^–-infected mice. Considering that the Dr adhesin mediates interaction with DAF and type IV collagen (321, 459, 460), whether these phenotypes are necessary for the development of tubulointerstitial nephritis in mice has been investigated. Meittinen et al. (285) observed that in kidney, the type IV collagen binding capacity of Dr adhesin results in the formation of mesangial deposits that persist but does not induce histological damage, indicating that additional factors provided by the bacteria and/or the host are needed for glomerular damage to occur. Selvarangan et al. (377) recently demonstrated that the type IV, collagen-binding phenotype is crucial for E. coli virulence in the mouse model of chronic pyelonephritis. Indeed, an isogenic DraE adhesin subunit mutant that was unable to bind type IV collagen but retained binding to DAF was eliminated from the mouse renal tissues, while the parent strain caused persistent renal infection. In addition, transcomplementation with the intact Dr operon restored type IV collagen-binding activity, basement membrane interstitial tropism, and the ability to cause persistent renal infection. The role of DAF in the development of chronic ascending pyelonephritis in Dr-positive E. coli-infected mice is currently not established, and a recent report is not in favor of a role of mouse DAF. Indeed, Hudault et al. (197) showed that like the echovirus and coxsackie B viruses, which bind specifically to human DAF but fail to recognize rat and mouse DAF (412), Dr and F1845 adhesins fail to recognize mouse, rat, or pig DAF. This could result because although mouse DAF contains four CCPs similar to those found in human and guinea pig DAF (174), the base sequences of mouse and human DAF show 63.7% identity and the deduced degree of amino acid sequence identity between mouse and human DAF is only about 47% (134, 410). In addition, considering that in rodents Crry is a membrane-associated complement-regulating protein (126, 258, 290, 352) expressed on glomerular mesangial, endothelial, and epithelial cells and that like DAF, Crry protects against complement injury (231, 289, 304, 313, 314, 368), Hudault et al. (197), examining the role of Crry in Afa/Dr binding, showed that Crry does not act as a receptor for human Afa/Dr adhesins. In conclusion, from these reports, it is probable that the recognition of DAF by Dr adhesin is not necessary for the induction of tubulointerstitial nephritis in Dr-positive E. coli-infected mice, whereas the recognition of type IV collagen is a critical step for the development of persistent renal infection in mice.

UTIs are associated with approximately 27% of premature births. E. coli Dr family adhesins have been found to be frequently expressed in strains associated with pyelonephritis in pregnant females (176, 320, 339, 340). Within the uterus, DAF has been found in the endometrial glands, spiral arterioles, and myometrial arteries protecting tissues against complement-induced damage, and the DAF density in the endometrium may affect sensitivity to complement activation (222, 223). The presence of DAF in the endometrium and interindividual differences in DAF density in the endometrium may affect sensitivity to the attachment of Dr-bearing E. coli (223, 225). Moreover, DAF has been found to be overexpressed in endometrial biopsies from patients without malignancy at the proliferative phase (326).

Using the experimental model of chronic pyelonephritis developed with E. coli bearing Dr adhesin, Kaul et al. (224) observed that nearly 90% of pregnant mice infected with Dr-positive E. coli delivered preterm, compared to 10% of mice infected with Dr-negative E. coli. Urogenital tract colonization by Dr-positive E. coli is accompanied by a defense mechanism involving nitric oxide (NO), which is known to induce antimicrobial activity (120). NO is generated in the uterus, and one of its functions is to inhibit uterine contractility (465). Current data support the suggestion that gestation, parturition, steroid hormones, and prostaglandins all modulate both the generation and the effects of NO on the uterus (439). Moreover, the NO synthases (NOS) that produce NO are induced by lipopolysaccharide (LPS) and/or cytokines. One theory is that NO plays a role in uterine quiescence during pregnancy and that any change in this system at term or preterm could play a role in inhibiting labor and delivery (309). An increase in rat uterine NOS activity has been found in pregnancy and declines at term, suggesting that NO functions in an autocrine and/or paracrine manner (355). Indeed, two isoforms of NOS have been found, an endothelial constitutive form located in vascular endothelium and an inducible form that is expressed in the myometrium of the pregnant rat uterus but not in that of the virgin rat and the expression of which declines at term when labor occurs. A localized increase in type II NOS expression and NO production occurs in response to intrauterine infection (121, 122). Moreover, the invasion of human endometrial adenocarcinoma Ishikawa cells by Dr-positive E. coli was reduced by elevated NO production and increased by NO inhibition. In addition, elevated NO production significantly reduced DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner (123). In addition, changes in NO and LPS responsiveness were significantly associated with the increased sensitivity of C3H/HeJ mice to experimental Dr-induced pyelonephritis. Infection of LPS responder (C3H/HeN) and nonresponder (C3H/HeJ) mice with E. coli strain O75 (bearing Dr fimbriae) and an O75 strain (bearing P fimbriae) has shown that the E. coli infection rate in Dr-infected C3H/HeN mice treated with the inhibitor of nitric oxide, L-NAME, was approximately 100-fold greater than that in the P-infected group (323). E. coli infection is followed by complications in pregnancy, and death occurs in pregnant mice within 24 to 48 h following infection. This death rate was increased twofold by treatment with the NO blocker L-NAME. In contrast, no deaths occurred in nonpregnant animals with or without L-NAME treatment, suggesting that infectious complications of pregnancy may be related to gestation-dependent sensitivity to the pathogenic microorganism and to the host's NO status (317). Overall, these findings add to our understanding of the NO-dependent mechanism of defense and have provided reliable insights into how this system works as an epithelial defense against urogenital tract infection. As already discussed for Dr-induced pyelonephritis, the role of mouse CRPs in Dr-induced urogenital tract infection is intriguing, since mouse DAF and Cryy do not act as receptors for human Afa/Dr adhesins (197).

One study indicates that CEACAM1 could be implicated in the human implantation process (16, 17). Data from immunohistochemistry studies and flow cytometry and Western blotting of isolated trophoblast populations show that CEACAM1 is present in epithelial cells of the pregnant endometrium as well as in small endometrial vessels, whereas it is absent from decidual cells. In the fetus, CEACAM1 is strongly expressed by the extravillous, intermediate trophoblast at the implantation site, as well as by extravillous trophoblastic cells. Expression is also observed in placental villous core vessels but is absent from both villous cyto- and syncytiotrophoblasts throughout pregnancy. A subfamily of Afa/Dr adhesins, including Dr, AfaE-III, and F1845, bind to CEACAM1, CEA, or CEACAM6 (33). Human CEACAM1, CEA, and CEACAM6 are not expressed in mice. However, it has been reported that murine CEACAM1 and CEACAM2 can serve as receptors for mouse hepatitis virus, a murine coronavirus (234, 288, 392, 428, 432, 438). The possibility that murine CEACAM1 acts as a receptor for human Dr adhesin in the infectious mouse model has been investigated by using BHK cells transfected with mouse CEACAM1a, and the results show that it does not act as a receptor (S. Hudault and A. L. Servin, unpublished data).

According to Nowicki's group, the DraE adhesin harbored by Dr-positive strains and encoded by the draE gene is sufficient to promote internalization, even though this strain expresses a DraD invasin (469). Indeed, purified Dr fimbriae applied to polystyrene beads were capable of triggering receptor clustering and the accumulation of actin at the adhesion sites on cells where beads were engulfed and ultimately internalized by the cells (150). In addition, the internalization of Dr-positive E. coli was inhibited by anti-Dr fimbria IgG and anti-CCP-3 of DAF, and the draE, draC, and draB insertional mutants and adherent draD mutant were unable to enter epithelial cells, whereas complementation of the dra mutation restored their invasiveness (147). Consistent with a role of DraE adhesin in internalization, Selvaragan et al. (376) have demonstrated the role of extracellular domains and the GPI anchor of DAF in the internalization process of Dr-positive E. coli. Binding to the CCP-3 domain and replacement of the GPI anchor of DAF were critical for internalization to occur. Internalization of Dr-positive E. coli is associated with the recruitment of _5ß1 integrin around the adhering bacteria (164, 218). Interestingly, it has been reported that ß₁ integrin plays a critical role in echovirus-1 binding preceding the DAF-dependent entry (99, 421). Dr-mediated internalization is inhibited by nocodazole (148, 164), indicating that the microtubules play a role in the entry process, as has been observed for a few pathogens, including Campylobacter jejuni (328). In enterocyte-like epithelial cells, an Afa/Dr diffusely adhering E. coli strain bearing the Dr adhesin entered basolaterally but not apically (164). In addition, it has been observed that surviving Dr-positive bacteria residing within the host cell have no effect on the functional differentiation of these cells (164).

According to Le Bouguenec's group, the AfaD protein harbored by an Afa-III-positive strain and encoded by the afaD gene acts as an invasin (137). The AfaD invasin is structurally and functionally conserved among Afa-expressing human strains, independently of the AfaE subtype and clinical origin of the E. coli isolate (138). The AfaD protein, like the AfaE protein, was exposed at the bacterial cell surface, but unlike AfaE, it was able to detach itself from the surface of bacterium to become internalized (137, 138, 157). Moreover, recent data suggest that the AfaE-III adhesin assembles into a flexible fiber that provides the link between the bacterial membrane usher and the invasin at the tip (8). Recombinant E. coli producing the AfaD or AfaE-III protein demonstrated that AfaE-III allows the E. coli to bind to cells and that AfaD mediates the internalization of the adherent bacteria (213). Moreover, colloidal gold tagging of AfaE-III and AfaD proteins has shown that AfaE-III-gold complexes are simply bound to the cell surface, whereas AfaD-gold complexes actually enter the cells. The role of AfaD in cell entry has been confirmed by the observation that coating of polycarbonate beads with AfaD protein enables the beads to enter the cell (137). As observed for Dr-positive bacteria (164), the entry of recombinant AfaD-coated beads into both cervical HeLa and undifferentiated intestinal Caco-2 cells was dependent on the accessibility of ß₁ integrins (343) (Fig. 3). It has been suggested that AfaD could be the prototype of a family of invasins encoded by adhesion-associated operons in pathogenic E. coli (138). This hypothesis is based on two observations. First, the AggB protein from enteroaggregative E. coli has also been found to be an AfaD-related invasin (138). Second, despite their differences, the recombinant AfaD-III and AfaD-VIII proteins both bind to ß₁ integrins (343).

To reconcile the two mechanisms propose