Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences

Pallesen, Lars; Poulsen, Lars; Christiansen, Gunna; Klemm, Per

doi:10.1099/13500872-141-11-2839

Cited by 62 publications

(63 citation statements)

References 30 publications

(21 reference statements)

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“…This is consistent with what is found with other bacterial adhesins (4). FimH has been used to express foreign antigens by inserting heterologous gene segments into the fimH gene (33) and used on its own as an effective immunogen in preventing experimental urinary tract infections in mice (20).…”

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confidence: 67%

Genetic Characterization of Escherichia coli Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype

et al. 2000

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Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31°C but not at 42°C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42°C but sensitive after growth at 31°C. The ts mutant thus binds macrophages with one receptor specificity at 31°C and another at 42°C.Type 1 pili are filamentous proteinaceous appendages produced by several members of the Enterobacteriaceae. In Escherichia coli, type 1 pili have been studied extensively with regard to their genetics, biosynthesis, and ability to bind mannose-containing receptor molecules on a variety of eucaryotic cells (reviewed in reference 29). Although the pili are made principally of a single protein monomer, the product of the fimA gene, several minor protein components are also incorporated (12, 34). These are most often found at the ends of pili and are organized into fibrillar structures (15). One of the minor components, the product of the fimH gene (FimH), binds directly to the receptor (18). Whereas the specificity of the interaction of the fimH product can be influenced by other fimbrial components (23), several studies have linked certain naturally occurring fimH allelic types to the specificity of receptor binding (43, 46) and the strength of receptor binding (45). Additional experiments have suggested that some fimH allelic differences can contribute to tissue tropism (35,43,44). Gene fusion experiments have indicated that FimH binding capacity resides in the amino one-third to one-half of the pr...

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supporting

confidence: 67%

Genetic Characterization of Escherichia coli Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype

et al. 2000

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“…Subsequently, a 52 aa peptide mimicking the preS2 region of the hepatitis B surface antigen and the previously mentioned CTB epitope were inserted into both positions. In all cases, the insert positions proved to be compatible with integration of the heterologous sequences with regard to surface display and full or at least partial conservation of the mannose-binding function of the chimeric FimH proteins (Pallesen et al, 1995). Furthermore, both the CTB-and the preS2 Fimbrial surface display systems in bacteria segment were displayed on the surface of the chimeric FimH proteins in conformations which were immunologically similar to the conformations in the parental proteins, as evidenced by immunofluorescence microscopy and immunoelectron microscopy (Pallesen et al, 1995).…”

Section: Heterologous Antigen Display In the Major Structural Proteinmentioning

confidence: 86%

“…In all cases, the insert positions proved to be compatible with integration of the heterologous sequences with regard to surface display and full or at least partial conservation of the mannose-binding function of the chimeric FimH proteins (Pallesen et al, 1995). Furthermore, both the CTB-and the preS2 Fimbrial surface display systems in bacteria segment were displayed on the surface of the chimeric FimH proteins in conformations which were immunologically similar to the conformations in the parental proteins, as evidenced by immunofluorescence microscopy and immunoelectron microscopy (Pallesen et al, 1995). Recent research has established that the FimH adhesin is a key player in E. coli-mediated urinary tract infections (UTI) (Connell et al, 1996 ;Sokurenko et al, 1998).…”

Section: Heterologous Antigen Display In the Major Structural Proteinmentioning

confidence: 86%

Fimbrial surface display systems in bacteria: from vaccines to random libraries

Klemm

Schembri

2000

Microbiology

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“…Electron microscopy and immunoelectron microscopy were carried out essentially as described previously Pallesen et al, 1995). In short…”

Section: Western Blot (Immunoblot)mentioning

confidence: 99%

Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

et al. 1997

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The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions in the fim gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fim in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested with respect to host background in three different Escherichia coli strains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenous fim gene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized cholera toxin B chain, confirming the utility of the employed strategy.

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Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences

Cited by 62 publications

References 30 publications

Genetic Characterization of Escherichia coli Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype

Genetic Characterization of Escherichia coli Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype

Fimbrial surface display systems in bacteria: from vaccines to random libraries

Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

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