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AFA and F17 adhesins produced by pathogenicEscherichia coli strains in domestic animals

Chantal Le Bouguénec, yolande Bertin

To cite this version:Chantal Le Bouguénec, yolande Bertin. AFA and F17 adhesins produced by pathogenic Escherichiacoli strains in domestic animals. Veterinary Research, BioMed Central, 1999, 30 (2-3), pp.318-342.�hal-00902572�

Review article

AFA and F17 adhesins produced by pathogenicEscherichia coli strains in domestic animals

Chantal Le Bouguéneca Yolande Bertinb

a Laboratoire de pathogénie bactérienne des muqueuses, Institut Pasteur,28, rue du Dr-Roux, 75724 Paris cedex 15, France

h Laboratoire de microbiologie, Centre de recherche de Clermont-Fd-Theix, Inra,63122 Saint-Genès-Champanelle, France

* Correspondence and reprintsTel.: (33) 4 73 62 42 42; fax: (33) 4 73 62 45 91 ! c-mail: bertinC!clermont.inra.fr

(Received 22 October 1998; acceptcd I January 1999)

Abstraet-AFA and F17 are afimbrial and fimbrial adhesins, respectively, produced by pathogenicE.sf7)fnc/!;n coli strains in domestic animals. F17-related fimbriae are mainly detected on bovineand ovine E. coli associated with diarrhoea or septicaemia. The F17-G adhesin subunits recognize N-acetyl-D-glucosaminc (GIcNAc) receptors present on bovine intestinal cclls. Some F17 subtypesalso bind to GIcNAc receptors present on human uroepithelial and intestinal Caco-2 cells or to thelaminin contained in the basement of mammalian membranes. F]7 is often associated with othervirulence factors (aerobactin, serum resistance, CNF2 toxin, K99, CS31 A or AFA adhesins) onpathogenic E. coli. A cluster of only four genes is required to synthesize functional F17-related fim-brial structures. The hypothesis of multifunctional F17 fimbrial subunits is supported by the factthat: i) the N-terminal part of the adhesin subunit participates in receptor recognition, whereas the C-terminal part is required for biogenesis of the fimbrial filament; and ii) the interaction between struc-tural and adhesin subunits seems to be crucial for the initiation of monomer polymerization. Recently,determinants related to the crfh gene clusters from human pathogenic E. coli associated with intesti-nal and extra-iiitestinal infections were identified in strains isolated from calves and piglets withdiarrhoea and septicaemia. Two qfu-related gene clusters, designated ufa-7 and nfh-8, that encode afim-brial adhesins were cloned and characterized from bovine pathogenic E. coli. These animal ijfii geneclusters were plasmid and chromosome borne and were expressed by strains that produced othervirulence factors such as CNF toxins, F17, PAP and CS31 A adhesins. A high frequency ol’ qfii-8 anda low prevalence or (lfà-7 among bovine E. cnli isolates were suggested by preliminary epidemiologicalstudies. As with the human ufh gene clusters, the animal ones encode an adhesive structure composedof two proteins: AfaE which mediates adhesion to epithelial cells and AfaD which is an invasin.0 Inra/Elsevicr, Paris.

fimbrial adhesin / afimbrial adhesin / Escherichia coli / domestic animals / family of adhesins

Résumé-Adhésines AFA et FI7 produites par les souches d’Escherichia coli pathogènes chezles animaux. Les adhésines AFA et F17 de type respectivement afïmbriales et fimbriales sont pro-

duites à la surface de souches d’Escherichia coli pathogènes pour des animaux domestiques. Lesfimbriae apparentés à la famille F17 sont le plus souvent produits par des souches isolées de diarrhéeou de septicémie chez les bovins ou les ovins. L’adhésine F17 reconnaît un récepteur de type N-acétyl-D-glucosamine (GIcNAc) présent sur les cellules intestinales bovines. Quelques variants F17 7adhèrent également à des cellules epithéliales ou intestinales humaines et à la laminine des cellulesde mammifères. Les fimbriae F17 sont souvent associés à d’autres facteurs de virulence (aérobactine,résistance au sérum, toxine CNF2 (cytotoxic necrotizing factor 2) ou adhésine K99, CS31A ouAFA). Seulement quatre gènes sont nécessaires à la synthèse des fimbriae. La nature multifonc-tionnelle des sous-unités fimbriales est suggérée par le fait que : 1 ) la partie N-terminale de l’adhé-sine est impliquée dans la reconnaissance du récepteur alors que la partie C-terminale participe à labiogenèse du fimbriae ; et 2) l’interaction entre la sous-unité majeure et l’adhésine est essentielle pourl’initiation de la polymérisation du fimbriae. Récemment des déterminants similaires aux opérons qfàdes souches associées chez l’homme à des infections intestinales et extraintestinales, ont été identi-fiés dans des souches pathogènes pour les veaux et les porcelets. Deux opérons, designés afa-7 et afà-8, codant pour des adhésines afimbriales ont été clonés à partir de souches isolées chez des veauxatteints de septicémie. Ces opérons sont portés par le chromosome ou par un grand plasmide dans dessouches qui expriment d’autres facteurs de virulence telles les toxines CNF ou les adhésines F17, PAPou CS31 A. Des études épidémiologiques préliminaires ont suggéré une forte prévalence de afa-8 parmiles souches isolées chez les veaux. Comme les opérons afa des souches humaines, les opérons afa-7 et afa-8 codent pour une structure adhésive composée de deux protéines : AfaE qui permet l’adhé-sion aux cellules épithéliales et AfaD qui est une invasine. © Inra/Elsevier, Paris.

adhésines fimbriales / adhésines afimbriales / Escherichia coli / animaux domestiques / famillesd’adhésines

1. INTRODUCTION

Escherichia coli is a normal inhabitantof the intestinal tract of humans and ani-mals [59, 82]. However, pathogenic E. colistrains are also the causative agent of intesti-nal or extra-intestinal infections, such asurinary tract infection (UTI), meningitis orsepticaemia. The mechanisms involved inthe development of extra-intestinal infec-tions include the ability to colonize mucosalsurfaces, to traverse the epithelial cell layers,to resist the bactericidal effect of the com-

plement, to escape phagocytosis and to sur-vive and multiply in body fluids. Virulencefactors, such as cytotoxins, adhesins, aer-obactin, outer membrane proteins orlipopolysaccharides (LPS), are associatedwith the capacity of E. coli strains to causeextra-intestinal infections. A classification of

diarrhoeagenic E. coli has been established,based on clinical aspects of the disease andon the identification of the virulence fac-tors produced by the pathogenic strain [25,59].

In any case, the capacity of the bacteria toadhere to host cells is a prerequisite step forthe process of microbial colonization andinvasion. The binding of the pathogenic bac-teria to host receptors is usually mediatedby fimbrial or afimbrial adhesins [25, 59,821. Fimbriae (or pili) are supramolecularstructures produced at the bacterial cell sur-face. The bulk of the fimbriae is composedof the same repeated structural subunit anda specific adhesin subunit mediates the hostreceptor recognition. The adhesin subunitgenerally located at the top or within thefimbrial structure is presented away fromthe bacterial cell surface. The first E. coliadhesins studied were those associated withthe presence of fimbriae which can be visu-alized by electron microscopy. Later on,other adhesins were reported not to be asso-ciated with fimbriae. These non-fimbrial (orafimbrial) adhesins were found in humanand animal pathogenic isolates.

This article will specifically focus on thefimbrial and afimbrial adhesins, i.e. the F17 7

and AFA family of adhesins, which havebeen studied and associated with diarrhoeaor septicaemia in animals over the last fewyears.

2. FIMBRIAL ADHESINS

Fimbriae could be classified by theirreceptor binding specificities. For example,the type I fimbriae mediating mannose-sen-sitive haemagglutination (MSHA) are foundat the cell surface of both commensal and

pathogenic strains. In contrast, most fim-briae of pathogenic E. coli cause mannose-resistant haemagglutination (MRHA) [25,59].

Particular fimbriae are expressed by E.coli strains associated with human diarrhoea

[25, 59]. Briefly, the colonization of thehuman intestinal epithelium is mediated bythe colonization factor antigen (CFA)expressed by enterotoxigenic E. coli

(ETEC). The fimbrial adhesins designatedBfp (bundle-forming pilus) are produced byenteropathogenic E. coli (EPEC) strainsresponsible for infantile gastroenteritis. Fur-thermore, AAF/I (aggregative adherencefactor 1) mediates the aggregative adher-ence phenotype associated with severalenteroaggregative E. coli (EaggEC).

Fimbriae produced by E. coli strains asso-ciated with diarrhoea and/or septicaemia indomestic animals have also been intensivelystudied [59]. The K88 (F4) fimbriaeexpressed by ETEC strains bind to specificGal 1 -containing glycolipids present in theporcine small intestine mucus [23]. Therelease of heat-labile (LT) and/or heat-stable(ST) enterotoxins results in diarrhoea inneonatal and weaned piglets. The E. colistrains producing K99 (F5) adhesins bindto the small intestines of neonatal pigs,calves and lambs 18 The K99 fimbrialadhesion factor recognizes a sialic acidresidue of glycolipids in the intestinal mucuslayer of neonatal animals 1961. In addition,the 987P (F6) fimbriae are expressed byporcine ETEC [44] and F41 fimbriae by

bovine and porcine ETEC strains [ 19]. Fur-thermore, the non-enterotoxigenic E. colistrains isolated from domestic animals havealso been associated with diarrhoea and/or

septicaemia or bacteraemia. For example,the F165 fimbrial complex has been isolatedfrom piglets and calves with septicaemia orwith various diseases for which diarrhoeais the most prominent clinical sign [24]. TheF165! fimbriae included in the F165 com-plex mediates adhesion to the a-D-Gal(1,4)-(3-Gal receptors present on sheep erythro-cytes [42]. Throughout the last few years,the fimbriae included in the F17 family havebeen extensively detected among pathogenicE. coli strains isolated from domestic ani-mals.

2.1. F17-related fimbriae

2.1.1. F17-related fimbriae producedby E. coli strains

The family of the F17 fimbriae includesthe Fl7a, Fl7b, Fl7c and Fl7d fimbriaeexpressed by pathogenic E. coli isolatedfrom bovine with septicaemia and/or diar-rhoea, and from bacteriaemic lambs (table 1)16, 7, 61, 841. In addition, the G fimbriaealso included in the F17 family are producedby E. coli strains associated with human uri-nary tract infection (UTI) (table 1) [90]. Allthe F17 subtype fimbriae mediate adhesionof the bacteria to a N-acetyl-D-glucosamine(Glc-NAc)-containing receptor present onhost cells [6, 7, 34, 61, 84!.

The fimbriae appear as fine and flexible

protaneous filaments of 3-4 nm in diameteron the surface of bacterial cells cultured at37 °C [7, 61!. The F17 fimbriae are het-eropolymers composed of the F 17-A andF17-G subunits with an apparent molecu-lar mass of 20 and 36 kDa, respectively [6,7, 22, 62, 63 The bulk of the fimbriae areconstituted of the same repeated F17-A (orGafA for G fimbriae) structural subunit.F 17-G (or GatD) is the minor adhesin sub-unit conferring adhesive properties to the

fimbriae. The fimbriae included in the F17 7

family are serologic variants (or subtypes)based on differences in the amino acid com-

position of the F17-A or GafA structuralsubunits. Except for Fl7c-A and GafA sub-units which are identical, the amino acidsequences of the different F17-A structuralsubunits show 72-85 % identity (figure 1)[22, 71 ].

The Fl7a fimbrial subtype (formerlycalled FY) was first identified on the l laE. coli strain (019:K32:H9) isolated from adiarrhoeic calf in France [13, 34!. Thesefimbriae were further identified as Att25in Belgium [88] and characterized fromthe 25KH09 reference ETEC strain

(0101:K+:H-) at the National Institute forVeterinary Research (Brussels) [61 TheF 17a fimbrial subtype is chromosome-encoded, whereas the STa thermo-stabletoxin also produced by the 25KH9 refer-ence E. coli strain is plasmid-encoded [61]. ] .The F17b fimbriae subtype, formerly calledthe Vir antigen, was first described on the S5E. coli strain (O15:K+:H21) isolated fromblood of a lamb with fatal bacteriaemia inGreat Britain [73]. The genes encoding theFl7b fimbriae are located on the Vir trans-missible plasmid [73! that also controls thesynthesis of the cytotoxic necrotizing fac-tor type 2 (CNF2) [22, 83, 84]. The Fl7cfimbriae subtype (originally named 20Kfimbriae [7, 16]) was characterized from theE. coli strain 31 A at the ’Institut nationalde recherche agronomique’ of Clermont-Ferrand-Theix (France) [7]. The 31 A refer-ence strain (O153:K-:H-) isolated from thefaeces of a calf with diarrhoea produces nei-ther classical enterotoxins (STs or LTs) norCNF toxins but causes experimental septi-caemia in gnotobiotic calves [151. The Fl7dserological variant (formerly named Fill Ifimbriae) was first identified in Belgium atthe cell surface of the I I I KH86 E. coli strainisolated from the intestinal content of a diar-rhoeic calf [6]. In contrast to the other F17-related fimbriae, the apparent molecularweight of the F17d-A structural subunit isestimated to be 18 kDa by sulphate dode-

cyl sulphate-polyacrylamide gel elec-trophoresis (SDS-PAGE) [6, 8]. The G fim-briae were purified and characterized fromthe E. coli strain IH11165 (06 serogroup)present in the urine of a patient with acutepyelonephritis in Finland [90]. G fimbriaebind to erythrocytes treated with endo-p-galactosidase, a treatment that exposes ter-minal Glc-NAc residues on the erythrocytessurface [90]. The G fimbriae on the refer-ence strain are associated with an afimbrial

haemagglutinin able to recognize the bloodgroup M antigen [90].

Trans-complementation analysis showsthat the genetic determinants of F 17 a, Fl7band Fl7c fimbriae encode functionally inter-changeable systems confirming the concept

of a family of fimbriae [22, 71]. Further-more, a genotypic detection performed on alarge collection of F17-producing E. colistrains demonstrates that the F17 family isvery large and probably includes otherclosely related subtypes that have not yetbeen characterized [8].

2.1.2. F17-related fimbriae producedby Proteus mirabilis andHaemophilius influenzae strains

Fimbriae included in the F17 family arealso related to the UCA fimbriae expressedby human or canine uropathogenic Proteusmirabilis strains (table 1) [9, 17]. The pre-dicted amino acid sequence of the UcaA

structural subunit shows a 55-58 % iden-

tity to those of the different F17-A subtypes[9, 17, 71 ]. Furthermore, the codon usagefrequency of the gene encoding the F17a-A subunit is closely related to that of UCAand other P. mirabili.s fimbriae, suggestingthat UCA and F17 fimbrial genes may sharea common ancestor [t7]. A high sequencesimilarity (60 %) is also observed with theamino acid sequence of the Hif major fim-brial subunit (tablc 1) [32, 98]. The Hif (orHib) fimbriae are produced by a

Hacmophilivs iiifluefizae strain isolated fromthe throat of a child with meningitis andmediate adhesion of the bacteria to human

buccal cells [32, 39]. Moreover, the F17-Asubunits show an obvious homology withthose of the Haf fimbriae produced by a H.inf7uenzae biogroup aegyptius strain foundto be the etiologic agent of Brazilian pur-puric fever (BPF) and associated with pedi-atric purulent conjunctivitis and septicaemia[89]. The identity between the structuralsubunit genes shows that UCA, Hif or Haffimbriae are distant members of the F17 7

family. Phylogenetic analysis strongly sug-gests that the human P. mirabilis strain has

acquired the genes necessary for the UCAfimbrial biosynthesis recently in evolutionby a horizontal gene transfer, most probablyfrom E. coli [91. ] .

2.2. Prevalence of F17-related-

producing E. coli strains

Epidemiologic studies have mainly beenperformed by immunulogical detectionusing antibodies directed against the Fl7a orFl7c subtypes. Anti-Fl7c antibodies rec-ognize common determinants of the Fl7a,F17b, Fl7c, Fl7d and G native fimbriae [7 1.In contrast, the use of anti Fl7a antibodiesresults in a very weak reactivity with theFl7b, Fl7c or G subtypes 17, 22]. There-fore, differences in the specificity of F17 7detection can occur among the different

studies depending on the anti-F17 antibod-ies used. Recently, a multiplex PCR method

was developed to detect the genes encod-ing the different F 1 subtypes [8]. ] .F 17 are expressed by 15-46 % of bovine

E. coli associated with diarrhoea or septi-caemia in France and Belgium [7, 87, 88]. Incontrast, F17 fimbriac are detected among3 % of E. coli isolated from healthy calvessuggesting that F17 fimbriae are probablyinvolved in the infectious process [87]. Inaddition, 1 I-23 % of F17-positive isolatesare associated with diarrhoeic calves from

Japan, Algeria and India [40, 72, 95]. Only3.4 % of E. coli from diarrhoeic calves inScotland and England are F17-positive [74].However, 29 % of bacterial strains isolatedin Scotland from the intestinal content oflambs with a severe tubular disease

(nephropathy) [2] express F17 fimbriae (7!. ] .A clinicopathological survey of lambs withnephropathy demonstrates that about halfof the lambs investigated showed clinicalevidence of diarrhoea [2]. In addition, F17 7fimbriae are also produced by E. coli strainsisolated from kids and lambs with diarrhoeain Spain (8.6 %) [11 !. In humans, only 3 %of the E. coli strains isolated in France fromdiarrhoeic stools of hospitalized patientswere F17-positive, suggesting that F17 fim-briae do not play an important role in infan-tile and adult diarrhoea [7].

In addition to bacterial strains associatedwith diarrhoea or septicaemia, fimbriaeincluded in the F17-family are also

expressed by E. coli strains isolated fromthe milk of cows suffering from clinicalmastitis in the Netherlands (55 %) [64 Therole of F17 fimbriae in the infectious processof E. coli associated with mastitis infectionis unknown. However, in vitro adhesionexperiments have demonstrated that the F17-producing strains adhere to cultured mam-mae epithelial cells [64J.

2.3. Association of F17-related fimbriaewith virulence factors

F17 fimbriae are often associated withvirulence factors on pathogenic E. ooli. A

recent report reveals that 45 % of the F17-positive E. coli strains isolated from bovinewith diarrhoea are resistant to complementand produce aerobactin suggesting that thebacterial strains may cause septicaemia incalves [86]. Most of the bovine bacterialstrains expressing the F 17c subtype fim-briae also produce the afimbrial CS31Aadhesin that mediates adhesion to a N-

acetylneuraminic acid-containing receptorpresent in the human carcinoma cell lineCaco-2 [7, 8, 21, 36]. In addition, Fl7-related fimbriae are detected on 9 °l° of the

K99-positive bovine E. coli strains ] )4] andon 22 % of bovine E. coli strains associatedwith both the CNF2 toxin and AFA [56, 58,68J. This suggests a strategy used bypathogenic E. coli strains to adhere to dif-ferent host tissues and therefore increasetheir pathogenicity. The F17b subtype isprominent among bovine CNF2-producing gstrains with virulence properties associatedwith extra-intestinal infections (078serogroup, aerobactin and serum resistance)[8, 84]. Genes encoding the Fl7b fimbriaeand CNF2 toxin are located on the Vir trans-

missible plasmid [73, 841.

Pathogenic E. coli producing Fl7c sub-type fimbriae are prominent among bovineand ovine isolates (47 %) whereas Fl7a,F17b and Fl7d fimbriae subtypes aredetected on 30, 11 and I I % of the F 17-pos-itive bacterial strains, respectively [8!. Thehigh occurrence of Fl7c subtype fimbriaein France and Belgium [7 can be explainedby the use of antibiotics in animal care.Indeed, the bacterial strains producing theF17c subtype are strongly associated witha self-transmissible plasmid coding for vir-ulence factors (CS31 A adhesin and aer-obactin production) and for antibiotic resis-tance [7, 201. In addition, E. coli strainsproducing both CS31A and F 17c fimbrialsubtypes have a n2-inositol-positive pheno-type [7]. These strains may represent a newexample of the association between bacterialclones and plasmid-encoded virulence fac-tors [7].

The F17-producing E. coli strains iso-lated from bovine intestinal contents aresometimes referred to as ETEC strains but

only when F41 and/or K99 fimbrial adhesinsare also produced at the bacterial cell surface[86]. Although some bovine F17-positivestrains producing STa and/or STb entero-toxins have been isolated in India [40), a arecent study clearly demonstrated thatK99/F41-negative isolates producing F17 7fimbriae do not correspond to the definitionof ETEC strains [86]. When associated withthe K99 adhesin, Fl7a-positive isolates rep-resent 10 % of bovine ETEC strains isolatedin France and 30 % of those isolated in Bel-

gium in surveys performed in the early1980s [14, 88]. Experimental studies haverevealed that colostral antibodies againstboth K99 and F41 adhesins or against F17adhesins alone do not provide protection forcalves inoculated with ETEC strains pro-ducing K99, F17 and F41 adhesins [12]. Aprotective effect is obtained only when anti-bodies directed against the three adhesinsare associated in the colostrum of vacci-nated cows [ 12J. The ETEC strains carry-ing both K99 and F17 fimbriae adhere morestrongly to bovine enterocytes than to ETECthat only express K99 1351. In contrast toETEC that cause diarrhoea in lambs during gthe first days of life, non-ETEC strainsexpressing Fl7 fimbriae are isolated fromcalves between days 4 and 21 [14, 87, 102].

2.4. Adhesive properties

It is well documented that F17-relatedfimbriae mediate in vitro adhesion to N-

acetyl-D-glucosamine (GIcNAc)-contain-ing receptors present on bovine erythrocytesand intestinal cells [7, 34, 61]. The recog-nition of host receptors is mediated by theF 17-G protein exported at the bacterial cellsurface and located within the F17 fimbrialstructures [22, 63, 71, 91]. Analysis ofamino acid comparisons reveals that all thesubstitutions are located within the N-ter-minal part of the F17-G adhesin subunit. In

contrast, an absolute conservation isobserved on the C-terminal part of the pro-tein (figure 2) [22, 71]. Lintermans et al.speculate that Fl7a fimbriae may initiatethe binding of bacteria to calf intestinal villiby hydrophobic interactions between theintestinal mucus and the Fl7a-A structuralsubunit that is characterized by a highhydrophobicity [63]. This interaction is thenfollowed by a specific Glc-Nac-dependentadhesion to the epithelial carbohydratereceptors mediated by the Fl7a-G subunit[63].

The F17-fimbrial adhesin subtypes canbe subdivided on the basis of their receptorspecificities. Different studies demonstrate

that: i) Fl7a-producing E. coli strains bindto a specific receptor of calf brush borderenterocytes but not to human receptors onhuman Caco-2 cells; ii) G fimbriae bind tohuman Caco-2 cells in culture but recogni-tion of bovine enterocyte receptors appearsto be influenced by the cell surface

hydrophobicity; iii) Fl7c adhesin recog-nizes receptors on both the Caco-2 cells andbovine enterocyte brush borders; and (iv)only the Fl7a subtype fimbriae adhere tohuman uroepithelial cells [7, 63, 71, 84!.

Mannose-resistant haemagglutination(MRHA) inhibition experiments show thatGIcNAc binds to F17 agglutinins with dif-ferent affinities in the preferential order:

Fl7c>G>Fl7b>Fl7a>Fl7d(tableln[7]. The high inhibitory potency of bovinesubmaxillary gland mucin (BSM) (contain-ing 25 % sialic acid) and the influence offree N-acetylneuraminic acid, cerebrosidesand glycophorin A on the binding of Fl7band G fimbrial lectins to receptors present onbovine erythrocytes suggests that sialic acidmay be involved in the binding of bothlectins (table II) [7]. Porcine intestinal mucin(PGM) (containing 0.9 % of sialic acid) alsoinduces a strong MRHA inhibition with theFl7c, G and Fl7d subtypes (table 11) [7].The differences in the inhibitory potencies ofBSM and PGM probably reflect variousreceptor specificities of the GlcNAc-bindingfimbrial lectins.

Mucin is a high molecular weight glyco-protein constituting the mucus layer whichcovers the surface of the gastrointestinaltract !77j. The mucus layer may protect thehost cells against the infectious process bypreventing bacterial attachment. However,the host receptors recognized by the F17 7adhesins are the O-glycosidically linkedoligosaccharides of bovine intestinal mucinwhere the sequence GleNac(31-3Ga1(31 isinternally located [75]. In fact, the minimalGlcNac(31-3Ga1(31 sequence of the GlcNAc-containing receptors strongly binds to theFl7a lectin when located in a terminal orinternal position in carbohydrate moieties

[75]. The receptors recognized by the F17 7lectins are present in both the mucus layerand the membrane of the epithelial brush-border. Several interactions established byF17 fimbriae are involved in the infection

process: F17 adhesins first mediate the ini-tial binding to the mucus layer and thenfacilitate the subsequent development of thesecond stage binding to the surface of intesti-nal villi [75, 93]. Furthermore, a decreasein adhesion of F17-producing strains isobserved with older-calf ileal mucus sug-gesting that the density of receptors recog-nized by F17 adhesins are dependant on theage of the calf and the intestinal segment[751.

In addition, the G fimbriae mediate theadhesion of the bacterial strains to thelaminin contained in the basement of mam-malian membranes [92]. Laminin is a multi-domain glycoprotein produced by a varietyof cell types, including epithelial andendothelial cells [70]. Terminal Glc-NAcresidues in short side chains of the corestructures of laminin are recognized by F17 7adhesins [92[. It is well documented thatlaminin are recognized by other fimbrialadhesins: the mannose and sialyloligosac-charide chains of laminin are specific bind-ing sites for E. coli strains producing type Iand S fimbriae, respectively [52, 99]. Inaddition to the recognition of the GlcNAc

receptors present in intestinal mucosal cells,the adhesion of F17 fimbriae to laminin mayincrease the bacterial colonization at dam-

aged intestinal tissue sites and may, there-fore, promote the translocation of the bac-

terial strains to extra-intestinal sites [92].

2.5. Genetic organization

The organization of several fimbrial geneclusters has been intensively studied. Forexample, the biosynthesis of functional K99,type I or P fimbriae produced by E. colistrains requires the transcription of complexgene clusters of about 10 kb in size includ-

ing eight to eleven genes each [25, 59]. Onthe basis of functional properties, the geneclusters can be divided into different groupsof genes encoding: i) the proteins that reg-ulate the expression of fimbrial genes clus-ters; ii) the structural, adhesin and minorsubunits included in the heteropolymericfimbrial structure exported at the cell sur-face; and iii) the periplasmic chaperon andthe outer membrane ’usher’. The periplasmicchaperon binds to subunit proteins and pre-vents improper polymerization, aggregationand proteolytic activity in the periplasm[48]. The chaperon-subunit complex is thentargeted to the ’usher’ that acts as a molec-ular doorkeeper which receives the com-plexes and incorporates the subunits intothe growing fimbriae 148 1.

2.5.1. Genetic organization of the f17 7gene clusters

A more simple genetic organization isobserved for the different F17 fimbriae sincethe gene clusters required to synthesize func-tional fimbriae are 5 kb in size and include

only four genes figure 3) [8, 22, 62, 63,71]. The analysis of the f] 7a gene clusterreveals the presence ofjJ7a-A,jJ7a-D,f17a-C and fl7a-G encoding a structural subunit,a periplasmic chaperon, an outer membraneusher and an adhesin subunit, respectively(figure 3) [63]. Few bovine E. coli strains

can express two distinct F17 structural sub-units (Fl7c-A/Fl7d-A or Fl7b-A/Fl7d-A)encoded by two complete and separate geneclusters [8]. Moreover, in some cases, thetwo distinct structural subunits are producedat the bacterial cell surface [8]. It seems thatthe two F17-related gene clusters haveevolved from each other in these bacterialstrains by duplication [8]. The strains pro-ducing both Fl7c /Fl7d or Fl7b /Fl7d fim-briae subtypes possess two separate andcomplete gene clusters but the fl7d-A struc-tural subunit gene is associated with the

fl7c-G or,fl7b-G adhesin gene, respectively[8]. This finding suggests that the relatedgene clusters encoding the different F]7 7fimbriae subtypes are probably organizedin mosaic operons constituted of F17-relatedstructural and adhesin gene subunits [8].

Interestingly, a cluster of five genes isrequired for the expression of the two mostdistantly related members of the F17-family:the Hif and Haf fimbriae produced by H.influenzae strains associated with meningi-tis and Brazilian purpuric fever (BPF),respectively [89, 98] (figure 3). In additionto the four proteins with functions similarto those required for F17 fimbrial biogene-sis, the HifD minor subunit (predicted to bea lipoprotein) may take part in the forma-tion of the adhesin-usher complex (HifE-HifC) implicated in the Hif fimbrial poly-merization [98]. The lipoprotein propertiesof HifD probably serve to anchor the proteinto the bacterial membrane [98]. HifDremains membrane associated during thefimbrial polymerization and the incorpora-tion of HifD in the growing fibrillum wouldprobably terminate the polymerization [98].Moreover, H. influenzae strains associatedwith BFP carry two distinct fimbrial geneclusters (hafl and haf2) with more than99 % identity, similarly to some F17-pro-ducing E. coli strains [89]. Read et al. [89]strongly suggest that: i) the duplication ofthe two gene clusters probably occurred bya recent intragenic recombination rather thanby the acquisition of DNA from anotherstrain through natural transformation; and

ii) these strains with two functional sets ofpilus genes may produce significantlygreater amounts of fimbriae on the cell sur-face.

2.5.2. Hypothesis of multifunctional F 17 7fimbrial proteins

The comparison of amino acid sequencesof the F17 adhesin subunits, reveals that thesubstitutions are located on the N-terminalhalves of the respective proteins, whereasthe C-terminal parts are identical !gure 2)[22, 71, 91]. Genetic analysis of G fimbriaebiosynthesis is performed by means of dele-tion mutagenesis of the gajd adhesin sub-unit gene [91]. A deletion in the N-terminalhalf of GafD results in the biosynthesis ofmorphologically intact G fimbriae but theadhesion to the Glc-Nac-containing receptoris not retained [91 In contrast, deletions inthe C-terminal half of the protein abolishboth Glc-NAc recognition and expressionof G fimbriae [91 J. The absolute conserva-tion of the C-terminal part of the differentF17-G proteins strongly suggests a strictstructural or functional role of the F 17 7adhesin subunits. Saarela et al. [91 ] proposethat the N-terminal part of GafD participatesin receptor recognition, whereas the C-terminal part is required for the biogenesis ofthe fimbrial filament or for the integrationof GafD in the fimbrial filament. This agrees

well with a secondary structure predictionanalysis on the adhesin and structural sub-units (including Fl7a-A and Fl7a-G pro-teins) showing a clear structural relatednessbetween the structural subunit and the C-terminal half of the adhesin [33]. The C-ter-minal-part of the adhesin, which folds as astructural subunit, can serve as an assemblymodule required for adhesin presentation atthe fibrillum tip [33] (a model for the assem-bly of F17 fimbrial structures is proposed infigure 4). Moreover, the PapG adhesin isdescribed as a two-domain protein in whichthe highly conserved C-terminal part isrequired for forming a complex with thePapD chaperon [43]. The interaction of PapDwith PapG is required for the incorporationof the adhesin subunit into the fimbrial struc-ture and for the enhancement of the cleavageof the PapG signal sequence [43].

In addition, the interactions between theGafA structural subunit and the GafDadhesin subunit seem to be crucial for theinitiation of fimbrial monomer polymeriza-tion [91]. When compared to Hif fimbrialbiogenesis, the additional HifD protein isimplicated in the initiation of fimbrial poly-merization by anchoring the adhesin-ushercomplex to the bacterial membrane [98].Moreover, during P fimbrial biogenesis, thetwo distinct proteins PapK and PapF arerequired as polymerization initiators of Pfimbrial monomers and for the correct pre-sentation of the adhesin subunit within thefimbriae [46]. Genes encoding equivalentproteins are not present on the cluster ofgenes required to functionally express F17 7fimbriae. A multifunctional nature of fim-brial proteins could explain why only fourgenes are necessary for the F 17 fimbrial bio-

genesis [91]. ] .

3. AFIMBRIAL ADHESINS

The first report of determinants encod-

ing an adhesin that was not associated withthe presence of visible fimbriae on the bac-terial surface was that of an afa gene cluster

encoding an adhesive structure designatedAFA, for afimbrial adhesin in 1984 [53].The cjfa gene cluster was shown to belocated on a 6.7-kb fragment cloned fromthe genomic DNA of a pyelonephritic E.coli strain (KS52). The cloned fragmentexpressed polypeptides with molecularmasses of 13, 30, 100, 18.5 and 16 kDa andwas encoded by five genes, afaA to afaE,respectively, organized in an operon (fig-ure 5). Among the crfa genes, only alaS,afaC and afaE were demonstrated to berequired for MRHA expression and adher-ence to uroepithelial cells [53, 54J. The utaEgene has been identified as the structuraladhesin gene which mediates specific bind-ing to uroepithelial cells as well as to HEp-2 and HeLa cells with a so-called diffuseadherence pattern [100!.

Hybridization experiments and westernblot (immunoblot) analyses demonstratedthe existence of gene clusters structurallyrelated to the first afa operon to be describedbut encoding antigenically distinct adhesins.These experiments demonstrated that all the

afa gene clusters harbour a highly conserved4.1-kb DNA segment (carrying the afaB,afaC and nfaD genes) and revealed hetero-geneity for the afae sequences (figure 5).Thus, it was proposed that at least four dif-ferent afa operons exist, am- 1, ufa-2, afa-3and afa-4, which encode variable adhesinsfirst designated as AFA-1, AFA-11, AFA-III and AFA-IV, respectively [54]. Severalyears following these descriptions, the reportthat the afimbrial adhesive structure was

composed of two proteins, AfaE and AfaD,allowed the renaming of the adhesinsencoded by the various afaE genes as AfaE-I to AfaE-IV [28]. Recently, the AfaE-Vtype adhesin was described in human iso-lates associated with both intestinal or extra-intestinal disorders [27, 103]. Finally, thepresence of afà-rclated sequences wasdetected among isolates from both diseased

piglets and calves !41, 68J and two ala-related operons, designated afa-7 and atil-8,encoding the AfaE-VII and AfaE-VIII typeadhesins were characterized from bovine

pathogenic E. coli [56J (table IIO.

The existence of non-fimbrial adhesinsencoded by different gene clusters was thendescribed at the cell surface of E. coli strains

belonging to various pathotypes (table IIO.CS6 is a colonization factor (CF) of humanenterotoxigenic E. coli (ETEC) strains thatwas characterized in 1985. It is one of theseven most commonly found CFs on humanETEC strains. The structure of CS6 remainsundefined. It is composed of the CssA andCssB subunits which may constitute eithera single fine fibrillar structure, or individ-ual fine fibrillae, or an afimbrial adhesivesheath [101 The M-specific agglutinin wasidentified in 1986 from a strain isolated fromthe urine of a patient with acute pyelonephri-tis !47, 90, 97]. This adhesin specificallyrecognizes the blood group M substanceglycophorin AM. Epidemiological studiesshowed that the production of the M-agglu-tinin is very infrequent in the strains iso-lated in pyelonephritis [3]. The NFAadhesins (NFA-I to NFA-VI) were charac-terized from human uropathogenic strains[ 1, 5 1 ]. The AIDA-1 adhesin was identifiedin a diarrhoea-associated E. coli strain. This100-kDa afimbrial adhesin is plasmidencoded and mediates, like the AfaE

adhesins, a diffuse adherence phenotype ofthe bacteria on HeLa and HEp-2 cells [5].

The CS3lA adhesin has been identified

among bovine and human enterotoxigenic orsepticaemic E. coli [36]. The CS31A antigenthat has been described as a capsule-likestructure around the bacteria, is composed ofthe ClpG protein. The CS3 A adhesin medi-ates adhesion to a N-acetylneuramidic acid-containing receptor present in the humancarcinoma cell line Caco-2 [36]. It is mainlyproduced by bovine pathogenic strains alsoproducing the Fl7c subtype (see above).

Among the afimbrial adhesins, thoseencoded by the ala gene clusters have beenmore-extensively studied.

3.1. The afa family of gene clusters

The (!fa family includes closely relatedgene clusters that are expressed byuropathogenic and diarrhoea-associatedE. coli (table I!. The drn determinantsencoding the Dr adhesins produced byuropathogenic strains [78, 85J as well as thedan determinants encoding the fimbrialF1845 adhesin produced by a strain isolated

from stools of a child with persistent diar-rhoea [10] have been demonstrated to belongto the afa family [26, 28]. All these oper-ons shared a very similar genetic organiza-tion and were closely related at the DNAlevel [26,58]. Consequently, probes andPCR assays used to detect the presence of

af« gene clusters in a strain also detect thepresence of dra and daa operons (figure 5).The afa/daa-specific DNA probes that con-sist of sequences internal to the highly con-served region (nfaB, qfaC and ntaD genes)were first defined [3, 10]. Then, a specificPCR assay using primers chosen from thesequence of the DNA probes was developed[57] figure 5).

High degrees of similarities were foundbetween the nucleotide sequence of the afa-3 gene cluster and the partial sequence ofthe non-fimbrial NFA-I-encoding operon[ 1, 26]. Whereas the structural gene encod-ing the NFA-1 adhesin had no homologywith any of the genes encoding the AfaEadhesins (nor was the NFA-I related toAfaE-III at the peptide level (table Il!), theAfaB chaperon exhibited 95.9 % identitywith the homologous NfaE protein. More-over, the non-coding region located

upstream of the adhesin-encoding genes(ufaE and nfaA) displayed strong similarities(79.5 % identity over 138 nucleotides) [26].Such conservation at the DNA level sug-gests that both the nfa and ufa determinantsare closely related. Recently, the descrip-tion of NFA-related adhesins, designatedNFAIlI, NFA 116 and Dr-II [85, 103!(table IV) encoded by strains that gave pos-itive amplification using the ajh specificPCR strongly enhanced this hypothesis.

The organization of the genes encodingthe M-specific haemagglutinin blood groupwas studied with a cloned 6.5-kb DNA frag-ment from the E. coli strain IH 11165 [90].This DNA segment was shown to containfive genes (bmaA to bmaE) which code forpolypeptides of 12.5, 30, 80, 18.5 and21 kDa, respectively. Although the geneticorganization of the M-agglutinin gene clus-ter resembles those of other afimbrial

adhesins, no relation could be deduced sinceonly the brnaE gene which was demon-strated to encode the M-agglutinin subunithas been sequenced. Until the cloning andsequence analysis of the afa-8 gene clusterwas obtained, no significant sequencehomology between the M-agglutinin sub-unit and other adhesin subunits was evi-denced. The observation that the AfaE-VIIIafimbrial adhesin encoded by the afa-8 genecluster exhibits strong homology with theM-agglutinin (97 % identity) [56] (table IV)suggests that the bma determinants belong tothe same phylogenetic family of gene clus-ters as the ak, and 4a operons.

3.2. Genetic and structural

organization of the afa geneclusters

3.2.1. afa Gene clusters from humanpathogenic E. coli strains

The information deduced from the

sequence analysis of the ccfa-3 gene cluster[26], encoding the AfaE-111 adhesin, hasshown that the cifa gene clusters are com-posed of six genes (afaA to afaF) organizedin two divergent transcriptional units cor-responding to C!f’aA-qfàE and qfiif, respec-tively (figure -!). Five of the six Afa productsshowed marked homologies with proteinsencoded by previously described adhesionsystems which allowed the attribution of a

putative function in the biogenesis of theafimbrial adhesive sheath to each of them.The biogenesis of the AFA adhesive struc-ture, like that of a fimbrial one [45 requiresproteins with specialized functions such asa periplasmic chaperon (AfaB protein), anouter membrane anchor protein (AfaC), andtranscriptional regulators (AfaA and AfaF)!26]. The afca and qfaF genes were foundto encode products that belong to the familyof the Papl-PapB regulators commonlyinvolved in the regulation of E. coli fim-brial adhesin expression [26]. The nfaE geneencoded the structural adhesin. No function

could be attributed to the AfaD productencoded by a gene known to be non-essen-tial for the expression of MRHA and for theadherence to epithelial cells [55]. The role ofAfaD in the biogenesis of the afimbrialstructure was then assessed by immunogoldand immunofluorescence experiments. Sim-ilar to AfaE, AfaD was found to be a sur-face-exposed protein as well as an adhesin;both AfaD and AfaE are concomitantlyexpressed by the bacterial cell [28 Theroles of the two components of the AFAafimbrial sheath during the association ofafa-expressing strains with epithelial cellswas investigated by ultrastructural andgenetic analyses. These experimentsrevealed that bacteria producing both AfaDand AfaE could be internalized into HeLacells. Then, the examination of mutants andpolystyrene beads coated with either AfaDor AfaE demonstrated that AfaE and AfaDare involved in initial binding to HeLa cells sand in the internalization process, respec-tively [49]. These data demonstrate that theafa gene cluster is unique among bacteria, asit alone encodes both the adhesion to andthe invasion of epithelial cells.

The sequence of the a ’fcie structuraladhesin-encoding gene is highly heteroge-neous among the qfa gene clusters, leadingto the production of antigenically distinctadhesins. The determinants encoding theadhesin subunit were cloned from various

afa-expressing strains. The sequences of thegenes encoding the AfaE-I, AfaE-11, AfaE-III and AfaE-V type adhesins as well asthose encoding the Dr (Dr-I and Dr-II),F1845 and other adhesin variants called

Drbl2l, Drb122, NFAIII, NFAH6 6adhesins have been reported [10, 26, 27, 53,54, 58, 78, 85, 103]. The homologiesbetween the known AfaE subtypes are pre-sented in table IV. A large number of AfaEadhesins still, however, remain to be iden-tified. PCR assays have been developed fortyping of the gene encoding the AfaE-I,AfaE-II, AfaE-III, AfaE-V and F1845adhesin variants [27, 1031 (figure 5). TheAfaE-III adhesin encoded by the afa-3 gene

cluster is closely related to the F1845adhesin encoded by the daa operon (53.6 %identity); however, unlike the AfaE-IIIadhesin, F 1845 is a fimbrial adhesin. Cre-ation of recombinant chimeras between the

afa-3 and the daa gene clusters haverevealed that the production of an afimbrialAfaE-1I1 or a fimbrial F1845 adhesin is

solely dependent on the nucleotide sequenceof the adhesin-encoding gene [28 Thesefindings suggest that the respective archi-tectural differences among the adhesivematerials are determined by differences inthe packing of AfaE-III or F1845 subunitsinto three-dimensional macromolecularstructures. Epidemiological studies haverevealed that the fimbrial state is exceptionalamong adhesive sheaths encoded by the afirfamily of gene clusters [103]. ] .

3.2.2. afa Gene clusters from animalpathogenic E. coli strains

Pathogenic E. coli isolates of porcine andbovine origin possess adhesin-encoding geneclusters (pap, .sfa, afa) related to extra-intestinal infections in human beings. Thepresence of afa-related sequences wasdetected among isolates from both diseased

piglets and calves !41, 68]. However, underhigh stringency hybridization conditions orin the afa-specific PCR derived from thesequence analysis of the conserved region ofthe human afcr operons, the afa animalstrains were negative. This suggests that theafa operons of animal pathogenic E. coliare structurally different from the operons ofhuman E. coli isolates 168]. Additionally,hybridization experiments suggested thatthe 4a animal operons are various [691.Recently, two different afa-related geneclusters, designated q/a-7 and qfh-8 respec-tively, were cloned from E. coli strains iso-lated from calves with diarrhoea and septi-caemia 156]. They encode afimbrialadhesins. The genetic organization of afa-7and q/a-8 was found to be similar to that ofthe ufa gene clusters from human strains.Partial sequence determination of these gene

clusters indicated that the C-terminus of theAfaC proteins from o/a-7, ufcr-8 and afa-3are highly conserved (82 Glo identity over79 amino acids between AfaC peptides fromqfh-7 and a/a-3; 67 % identity over79 amino acids between AfaC from afa-7and afa-8). The AfaD peptides from the ani-mal gene clusters display only 46 % identitywith each other and with the AfaD invasinfrom ufa-3. The AfaD products encoded byala-7 and afa-8 complement the invasionof HeLa cell deficiency of an afa-3-express-ing strain with a mutated afaD gene, sug-gesting that they are also invasins [56!. TheAfaE-VII product displayed a weak simi-larity (26 °lo identity) with the fimbrialAAF/I adhesin encoded by enteroaggrega-tive E. coli implicated as an agent of pedi-atric diarrhoea in the developing world [76]. ] .AfaE-VII encodes a mature protein of151 amino acids that includes two cysteineresidues located 33 amino acids from eachother as in the previously studied AfaEadhesins. In contrast, the afaE gene fromqfa-8 encodes an adhesin that exhibits verystrong homology with the M-agglutinin [90],an afimbrial adhesin described in

uropathogenic human E. coli (97 %n iden-tity).

3.2.3. Dissemination of the afa-relatedgene clusters

The location of gene clusters of the afafatnily is dual. By subtyping, the a/a-3, qfii-5, ufa-8 and daa gene clusters were foundboth on large plasmids and on the chromo-some [56, 58, 103]. Some ufcr-8 determi-nants were located on the Vir plasmid whichalso carry sequences encoding CNF2 156]. ].All the other identified subtypes were foundonly on the chromosome [10, 53, 54, 56,103]. The plasmid-borne qfh-3 gene clus-ters were found to be tlankcd by two IS Ielements in direct orientation and two in

opposite orientations [26]. The afa-3 genecluster, llanked by two directly oriented IS1 Ielements, was shown to translocate from arccombinant plasmid to the E. coli chromo-

some. This movement occurred via ISl-spe-cific recombination mediated by a recA-independent mechanism [26]. Whereas sucha situation has already been reported fordrug resistance genes and for genes encod-ing toxins, this was the first evidence thata pathogenic determinant responsible foradhesion properties is capable of transloca-tion from one replicon to another, allowingus to speculate on the possible disseminationof such determinants among pathogenicE. coli.

3.3. Prevalence of A FA -producingstrains

3.3.1. Prevalence among human

pathogenic E. coli strains

Several studies suggest that am-positivestrains play an important role in urinary tractinfection pathogenesis. The Dr blood antigencomponent of the decay-accelerating factorthat is the receptor of the AfaE adhesinsfrom human operons [79, 80 is widely dis-tributed along the urinary tract, underlyingthe importance of the AFA-producing strainsin the ascending colonization of the urinarytract. The AFA strains occur frequentlyamong E. coli isolates from pregnant women

[81 J and children [3, 4] with UTI and arealso associated with recurring UTI [103!.Moreover, development of an animal modelfor experimental chronic pyelonephritis hasprovided evidence for the importance ofexpression of the <!Y< gene cluster, an nfa-related operon, in establishing persistentcolonization of the urinary tract [38 j.

Human diarrhoeagenic E. coli comprisesa heterogeneous group of pathogenic bac-teria classified into six different pathotypesbased on clinical aspects of the disease andidentification of virulence factors producedby the isolates [76J. The frequency of eachpathotype depends on the geographic areaand varies among epidemiological studies.Diffusely adherent E. coli (DAEC) are oneof these categories. The only known viru-

lence factor of DAEC strains is their dif-fuse pattern of adherence to cultured epithe-lial cells. Two families of gene clusters

encoding diffuse adherence have been iden-tified in DAEC strains: afaldaa [10, 58] andthe AIDA (adhesin involved in diffuseadherence)-encoding genes [5]. Recent epi-demiological studies have implicated DAECstrains carrying afa genes as a cause of acuteand persistent diarrhoea in 2-6-year-oldchildren in developing countries [30, 37,60]. In New Caledonia, AFA strains werefound to be the first bacterial pathogensassociated with diarrhoea in children (cor-responding to 22 % of the bacterial

pathogens isolated from stools) (Y. Ger-mani, pers. comm.). In contrast, several epi-demiological studies have demonstrated thatthe AIDA-encoding genes are rare (0-1 %)in DAEC [30, 60].

Type-specific PCR were used to investi-gate the distribution of AfaE subtypes inuropathogenic and diarrhoeagenic afa-expressing E. coli. The data indicated thatthe adhesin subtype did not determine thepathotype (M.I. Garcia, pers. comm.). AnE. coli isolate carrying an afa gene clusterencoding an AfaE-1 adhesin has recentlybeen reported to be the causative agent ofboth diarrhoea and cystitis in the same child[31]. afa-1, afa-3 and afa-5 gene clusterspredominate in both uropathogenic and diar-rhoeagenic strains (M.I. Garcia, pers. comm.and [103]).

3.3.2. Prevalence among bovine and

porcine pathogenic E. coli strains

A preliminary study of the frequency ofafa-7 and afa-8 gene clusters among thebovine and porcine E. coli isolates previ-ously reported to carry afa-relatedsequences, suggests a high incidence of theafa-8 gene cluster and a low prevalence ofafa-7 among these strains [56]. afa-8 hasbeen identified in bovine necrotoxigenicstrains (producing a cytotoxic necrotizingfactor (CNF1 or CNF2)) associated withintestinal and extra-intestinal infections in

farm animals. Most of these strains also

hybridized with the F17 and/or PAP andCS31A probes [56]. The sequences of theafaD and afaE genes are highly conservedamong these afa-8-positive strains [56]. Theqf!-8 gene cluster has also been detected inCNF-negative bovine isolates and in Fl 65-positive bovine and porcine E. coli isolatesassociated with intestinal infections (J. Fair-brother, pers. comm.). ).

3.4. Adhesion properties

3.4.1. AfaE adhesins produced by humanpathogenic E. coli strains

The AfaE adhesins mediate a mannose-resistant agglutination of human erythro-cytes (MRHA) expressing the Dr bloodgroup antigen. The AfaE receptor has beenmore precisely identified as the short con-sensus repeat-3 (SCR-3) domain of thedecay-accelerating factor (DAF or CD55)[80], one of the cell membrane proteins thatregulate the complement cascade and protecteucaryotic cells from being lysed by anautologous complement. DAF is a 70-kDaglycoprotein widely distributed on

haematopoietic cells, intestinal and urinaryepithelia and endothelial cells [65-67 Thedensity of DAF molecules, however, isdependent on the cellular type and DAFmolecules show tissue and host tropisms.AfaE adhesins from human pathogenicstrains mediate a specific attachment touroepithelial cells and to HEp-2, HeLa,Caco-2 and T84 epithelial cells with a so-called diffuse adherence pattern [49, 50].

3.4.2. AfaE adhesins produced by animalpathogenic E. coli strains

The AfaE adhesin produced by the afa-7operon mediates a mannose-resistant agglu-tination of human, bovine and porcine ery-throcytes [56]. Preliminary data suggest thatAfaE-VII also recognizes DAF moleculesas a receptor (L. Lalioui, pers. comm.).

AfaE-VII mediates a specific attachment tohuman and bovine uroepithelial cells(Urotsa, and Mabin-Darby bovine kidneycell lines, respectively), and HeLa, Caco-2cell lines [56].

The afa-8 gene clusters mediate a man-nose-resistant agglutination of human ery-throcytes [56]. AfaE-VIII adhesins are inca-pable of haemagglutinating bovine, porcine,sheep, cat, dog and horse erythrocytes (J.Fairbrother, pers. comm.). Since the AfaE-VIII adhesins displayed a high degree ofhomology (97 % identity) with the M-agglu-tinin, the blood group M substance gly-cophorin AM which is the receptor of theM-agglutinin [47] was supposed to also bethe receptor of AfaE-VllI. The AfaE-VIIIadhesins mediate attachment to human andcanine uroepithelial cells (Urotsa and Mabin-Darby canine kidney cell lines, respectively)[56]. The receptor of AfaE-VIII on epithelialcells remains to be identified.

3.5. Interaction of afa-expressing E. coliwith epithelial cells

The afa gene clusters have the uniquefeature of encoding both an adhesin and aninvasin that seem to be involved in a two-

stage process. AfaE is an adhesin that isabsolutely required for adhesion to eukary-otic cells whereas AfaD is an invasin pro-moting internalization of adherent bacteriainto these cells [28, 49] (figure 6).

During bacterium-epithelial cell inter-actions, it has been observed that adhesionof bacteria to cells is accompanied by a mas-sive recruitment of DAF molecules at theadhesion site, with DAF molecules beingparticularly concentrated in the membraneextensions of the epithelial cells that tendto enwrap the adherent bacteria figure 6)[50]. The DAF molecule is a glycosyl phos-phatidylinositol (GPI)-anchored protein thathas been reported to function in signal trans-duction in leucocytes [94]. Identification ofthe putative signal transduced to epithelialcells following AfaE-DAF interactions

should elucidate the pathological processinvolved in diseases caused by the afa-expressing E. coli strains.

The afaD genes are structurally and func-tionally conserved among human afa-expressing E. coli isolates [29]. The AfaDvariants produced by the afa-7 and afa-8gene clusters and the AfaD homologue(AggB) produced by enteroaggregative E.coli (which are associated with persistentdiarrhoea in humans) were also found to beinvasins [29, 56]. Strains carrying an afagene cluster are associated with acute and

persistent forms of diarrhoea and urinarytract infections in human beings and thehuman AFA strain internalization occurs inboth intestinal and urothelial cell lines [50]. ] .The role, if any, of the invasion of epithelialcells in the pathogenesis of these diseasesremains to be established. Epithelial cellpenetration possibly provides a protectedniche for the organism so that it can surviveand persist within the host.

4. CONCLUSION

In conclusion, the F17-related fimbrialadhesins and AFA-related afimbrial adhesins

produced by pathogenic E. coli strains havebeen extensively detected among domesticanimals with diarrhoea and/or septicaemia.In addition, afimbrial adhesins are producedby pathogenic E. coli associated with vari-ous pathologies in humans. High percent-ages of identity are observed between F 17 7fimbrial proteins not only produced by dif-ferent bacterial species (E. coli, P. mirabilis,H. influenzae), but also by bacteria fromdifferent infectious sites (intestinal tract,blood, urinary tract or throat) and differenthosts (human, bovine, ovine or canine). TheF17-family seems to be very large and prob-ably includes other uncharacterized sub-types.

The study of the afa-related family ofgene clusters demonstrated that most of theafimbrial adhesin-encoding operons belongto the same phylogenetic family of deter-

minants. The afh gene cluster is unusual inthat a single operon encodes both adhesionof the bacteria to the epithelial cells and theinvasion of these cells. Consequently, theafa gene clusters are good models for study-ing bacterium-cell interaction mechanisms.The AFA family seems to be large and prob-ably includes other uncharacterized vari-ants. In human pathogenic E. coli associ-ated with intestinal and extra-intestinal

infections, at least three subtypes of afa geneclusters (ufcr-1, afa-3, and afa-5) are pre-dominant. In contrast, only one qla genecluster, the afa-8 operon, seems to be pre-dominant in bovine pathogenic E. coli.

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