Cloning and expression of colonization factor antigen I (CFA/I) epitopes of enterotoxigenic Escherichia coli (ETEC) in Salmonella flagellin.
Oligonucleotides coding for linear epitopes of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) were cloned and expressed in a deleted form of the Salmonella muenchen flagellin fliC (H1-d) gene. Four synthetic oligonucleotide pairs coding for regions corresponding to amino acids 1 to 15 (region I), amino acids 11 to 25 (region II), amino acids 32 to 45 (region III) and amino acids 88 to 102 (region IV) were synthesized and cloned in the Salmonella flagellin-coding gene. All four hybrid flagellins were exported to the bacterial surface where they produced flagella, but only three constructs were fully motile. Sera recovered from mice immunized with intraperitoneal injections of purified flagella containing region II (FlaII) or region IV (FlaIV) showed high titres against dissociated solid-phase-bound CFA/I subunits. Hybrid flagellins containing region I (FlaI) or region III (FlaIII) elicited a weak immune response as measured in enzyme-linked immunosorbent assay (ELISA) with dissociated CFA/I subunits. None of the sera prepared with purified hybrid flagella were able to agglutinate or inhibit haemagglutination promoted by CFA/I-positive strains. Moreover, inhibition ELISA tests indicated that antisera directed against region I, II, III or IV cloned in flagellin were not able to recognize surface-exposed regions on the intact CFA/I fimbriae.
Luna
M G
MG
Laboratório de Fisiologia Celular, Instituto de Biofísica Carlos Chagas Filho, UFRJ-CCS, Cidade Universitária, Brazil.
Martins
M M
MM
Newton
S M
SM
Costa
S O
SO
Almeida
D F
DF
Ferreira
L C
LC
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000276:immunology*; D005408:Flagellin / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006385:Hemagglutination Inhibition Tests; D006386:Hemagglutination Tests; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D012475:Salmonella / Q000235:genetics
D001426:Bacterial Proteins; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C022924:colonization factor antigens; D005408:Flagellin; D036781:Fimbriae Proteins