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Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

ABSTRACT: The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.

SUBMITTER: Bakker D

PROVIDER: S-EPMC207580 | biostudies-other | 1992 Oct

REPOSITORIES: biostudies-other

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Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

Bakker D D Willemsen P T PT Willems R H RH Huisman T T TT Mooi F R FR Oudega B B Stegehuis F F de Graaf F K FK

Journal of bacteriology 19921001 20

The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K8 ...[more]

PMID: 1400188

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Project description:Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Project description:Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.

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Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

Publications

Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

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