Res. MicrobioL 1990, 141, 785-786
© INSTITUTPASTEUR/ELsEVIER Paris 1990
DIFFUSE ADHERENCE OF ENTEROPATHOGENIC ESCHERICHIA
COLI STRAINS I. Benz and M.A. Schmidt Zentrum fiir Molekulare Biologie Heidelberg, 6900 Heidelberg (Germany)
Summary. For the identification and characterization of the factor(s) responsible for the diffuse adherence (DA) pattern of enteropathogenic Escherichia coli strains, E. coli strain 2787 isolated from a case of infantile diarrhoea was employed. A plasmidderived 11-kb fragment was cloned into pBR322. The recombinant plasmid pIB6 was shown to confer the diffuse adherence phenotype on different E. coli K12 strains as well as pIB4, a plasmid with a 9.2-kb insert. The DNA fragment necessary for the expression of the DA phenotype could be reduced to 6.0 kb. Antiserum obtained against pIB4-encoded proteins recognized a surface-associated protein of about 100 kDa in Western blotting. The isolated 100-kDa protein was found to bind to HeLa cells, The a r iserum against C600(pIB4) inhibits adherence ofE. coli 2787 and C600(pIB6) to HeLa cells. For this reason, the protein is called adhesin involved in diffuse adherence (AIDA-I). KEY-WORDS: Escherichia coil, Enteropathogenicity, Adherence, Plasmid; Diarrhoea, Infants, AIDA-I.
Introduction. Enteropathogenic Escherichia coil (EPEC) strains are a major cause of neonatal and infantile gastroenteritis throughout the world. The pathogenic mechanisms involved in the development of EPEC diarrhoea, however, remain unclear. Adherence of EPEC strains to the small bowel mucosa, as demonstrated by histopathological studies, seems to be of prime importance for infection. Using the adherence of EPEC to HeLa and HEp2 cells as a model system, different attachment patterns are observed: localized adherence (LA), where bacteria attach to and form microcolonies in defined regions, and diffuse adherence (DA), where bacteria adhere evenly to the whole cell surface. The genes coding for the factors responsible for LA have been located on a 60-megadalton plasmid (pMAR2). A fragment of pMAR2 hybridizes to DNA from other EPEC strains expressing LA. However, homology with DA-EPEC strains was not observed (Nataro et aL, 1985). Thus, the factors mediating the LA and DA phenotype are genetically different. Recently, a chromosomal DNA fragment coding for a fimbrial adhesin mediating DA was isolated (Bilge et al., 1989). Here we present the cloning and initial characterization of plasmid-encoded factors responsible for the DA pattern exhibited by the EPEC strain 2787 (Benz and Schmidt, 1989).
786
/. B E N Z A N D M . A . S C H M I D T
Results.
EPEC strain 2787 isolated from a case of infantile diarrhoea was employed for our studies. This strain harbours three plasmids, one of about 3 kb and two of about 100 kb. To clone the gone(s) responsible for the DA pattern exhibited by this strain, plasmid DNA was partially digested with EcoRI and the resulting fragments were ligated to pBR322. After transformation of E. coli C600 ampr transformants were screened for their ability to adhere to HeLa cells. One clone expressing DA was found to contain a plasmid with an 11-kb insert in pBR322 (pIB6). Southern blot analysis showed homology between pIB6 ant~ one of the large plasmids, thus indicating that this plasmid is responsible for the DA phenotype, pIB6 conferred the ability to adhere to HeLa cells to other E. coil K12 Strains. Parts of the l l - k b insert of pIB6 could be deleted without loss of the adherence property. The fragment length still encoding the factor(s) necessary for diffuse adherence could be reduced to about 6.0 kb. The plasmid with the smallest insert mediating adherence is called pIB264. Antiserum was raised in rabbits against whole E. coli C600 cells harbouring the pIB4 plasmid (mediates DA phenotype, 9.2 kb insert). Preadsorption with C600(pBR322) resulted in pIB4-specific antiserum which reacted with a 100-kDa protein of a total cell extract of C600(pIB4). The surface-associated 100-kDa protein could be isolated by mild heat treatment (20 min, 60°C). Two of the deletion mutants not showing the DA phenotype contain a membrane-associated protein of about 80-kDa cross-reacting with the pIB4-specific antiserum. Partially purified 100-kDa protein was incubated with fixed HeLa cells. Bound protein was detected by ELISA employing specific antiserum to pIB4-encoded proteins. The results show that the 100-kDa protein binds to HeLa cells and thus represents the adhesin involved in diffuse adherence (AIDA-I) of EPEC strains. Antiserum obtained against strain C600(pIB4) recognized a 100-kDa protein in other EPEC strains exhibiting a diffuse adherence pattern. This indicates that the 100-kDa protein is scrologically conserved. In Southern blotting experiments, however, DNA homology between different DA-EPEC strains could not be demonstrated. Adherence of bacteria to HeLa cells was inhibited by antiserum and Fab fragments, respectively. The extent of inhibition was determined by measurement of [~galactosidase activity of the attached bacteria. It was shown that antiserum against C600(pIB4) inhibits the attachment of 2787 as well as of C600(pIB6). Attachment was inhibited even better by Fab fragments than by antiserum.
References.
BENZ, I. & SCHMIDT,M.A. (1989), Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli. Infect. Immun., 57, 1506-1511. BILGE,S.S., CLAUSEN,C.R., LAu, W. & MOSELEY,S.L. (1989), Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coil to HEp-2 cells. J. Bact., 171, 4281-4289. NATARO, J.P., SCAL~rSKY,I.C.A., KAPER, J.B., LEVIN~, M.M. & TRABULSl,L.R. (1985), Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenie Escherichia coll. Infect. lmmun., 48, 378-383.
AIDA = adhesininvolvedin diffuseadherence. DA = diffuse adherence.
EPEC = enteropathogenicEscherichia coll. LA localizedadherence.
© INSTITUTPASTEUR/ELsEVIER Paris 1990
DIFFUSE ADHERENCE OF ENTEROPATHOGENIC ESCHERICHIA
COLI STRAINS I. Benz and M.A. Schmidt Zentrum fiir Molekulare Biologie Heidelberg, 6900 Heidelberg (Germany)
Summary. For the identification and characterization of the factor(s) responsible for the diffuse adherence (DA) pattern of enteropathogenic Escherichia coli strains, E. coli strain 2787 isolated from a case of infantile diarrhoea was employed. A plasmidderived 11-kb fragment was cloned into pBR322. The recombinant plasmid pIB6 was shown to confer the diffuse adherence phenotype on different E. coli K12 strains as well as pIB4, a plasmid with a 9.2-kb insert. The DNA fragment necessary for the expression of the DA phenotype could be reduced to 6.0 kb. Antiserum obtained against pIB4-encoded proteins recognized a surface-associated protein of about 100 kDa in Western blotting. The isolated 100-kDa protein was found to bind to HeLa cells, The a r iserum against C600(pIB4) inhibits adherence ofE. coli 2787 and C600(pIB6) to HeLa cells. For this reason, the protein is called adhesin involved in diffuse adherence (AIDA-I). KEY-WORDS: Escherichia coil, Enteropathogenicity, Adherence, Plasmid; Diarrhoea, Infants, AIDA-I.
Introduction. Enteropathogenic Escherichia coil (EPEC) strains are a major cause of neonatal and infantile gastroenteritis throughout the world. The pathogenic mechanisms involved in the development of EPEC diarrhoea, however, remain unclear. Adherence of EPEC strains to the small bowel mucosa, as demonstrated by histopathological studies, seems to be of prime importance for infection. Using the adherence of EPEC to HeLa and HEp2 cells as a model system, different attachment patterns are observed: localized adherence (LA), where bacteria attach to and form microcolonies in defined regions, and diffuse adherence (DA), where bacteria adhere evenly to the whole cell surface. The genes coding for the factors responsible for LA have been located on a 60-megadalton plasmid (pMAR2). A fragment of pMAR2 hybridizes to DNA from other EPEC strains expressing LA. However, homology with DA-EPEC strains was not observed (Nataro et aL, 1985). Thus, the factors mediating the LA and DA phenotype are genetically different. Recently, a chromosomal DNA fragment coding for a fimbrial adhesin mediating DA was isolated (Bilge et al., 1989). Here we present the cloning and initial characterization of plasmid-encoded factors responsible for the DA pattern exhibited by the EPEC strain 2787 (Benz and Schmidt, 1989).
786
/. B E N Z A N D M . A . S C H M I D T
Results.
EPEC strain 2787 isolated from a case of infantile diarrhoea was employed for our studies. This strain harbours three plasmids, one of about 3 kb and two of about 100 kb. To clone the gone(s) responsible for the DA pattern exhibited by this strain, plasmid DNA was partially digested with EcoRI and the resulting fragments were ligated to pBR322. After transformation of E. coli C600 ampr transformants were screened for their ability to adhere to HeLa cells. One clone expressing DA was found to contain a plasmid with an 11-kb insert in pBR322 (pIB6). Southern blot analysis showed homology between pIB6 ant~ one of the large plasmids, thus indicating that this plasmid is responsible for the DA phenotype, pIB6 conferred the ability to adhere to HeLa cells to other E. coil K12 Strains. Parts of the l l - k b insert of pIB6 could be deleted without loss of the adherence property. The fragment length still encoding the factor(s) necessary for diffuse adherence could be reduced to about 6.0 kb. The plasmid with the smallest insert mediating adherence is called pIB264. Antiserum was raised in rabbits against whole E. coli C600 cells harbouring the pIB4 plasmid (mediates DA phenotype, 9.2 kb insert). Preadsorption with C600(pBR322) resulted in pIB4-specific antiserum which reacted with a 100-kDa protein of a total cell extract of C600(pIB4). The surface-associated 100-kDa protein could be isolated by mild heat treatment (20 min, 60°C). Two of the deletion mutants not showing the DA phenotype contain a membrane-associated protein of about 80-kDa cross-reacting with the pIB4-specific antiserum. Partially purified 100-kDa protein was incubated with fixed HeLa cells. Bound protein was detected by ELISA employing specific antiserum to pIB4-encoded proteins. The results show that the 100-kDa protein binds to HeLa cells and thus represents the adhesin involved in diffuse adherence (AIDA-I) of EPEC strains. Antiserum obtained against strain C600(pIB4) recognized a 100-kDa protein in other EPEC strains exhibiting a diffuse adherence pattern. This indicates that the 100-kDa protein is scrologically conserved. In Southern blotting experiments, however, DNA homology between different DA-EPEC strains could not be demonstrated. Adherence of bacteria to HeLa cells was inhibited by antiserum and Fab fragments, respectively. The extent of inhibition was determined by measurement of [~galactosidase activity of the attached bacteria. It was shown that antiserum against C600(pIB4) inhibits the attachment of 2787 as well as of C600(pIB6). Attachment was inhibited even better by Fab fragments than by antiserum.
References.
BENZ, I. & SCHMIDT,M.A. (1989), Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli. Infect. Immun., 57, 1506-1511. BILGE,S.S., CLAUSEN,C.R., LAu, W. & MOSELEY,S.L. (1989), Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coil to HEp-2 cells. J. Bact., 171, 4281-4289. NATARO, J.P., SCAL~rSKY,I.C.A., KAPER, J.B., LEVIN~, M.M. & TRABULSl,L.R. (1985), Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenie Escherichia coll. Infect. lmmun., 48, 378-383.
AIDA = adhesininvolvedin diffuseadherence. DA = diffuse adherence.
EPEC = enteropathogenicEscherichia coll. LA localizedadherence.
