Genetic manipulation of major P-fimbrial subunits and consequences for formation of fimbriae

Abstract

was studied. Deletion of two regions that code for hypervariable parts of the P fimbrillin resulted in strong reduction or total absence of fimbria production. Replacement of deleted amino acids by other amino acid residues restored the formation of fimbriae. The hypervariable regions may be important for biogenesis of fimbriae by imposing correct spacing between conserved regions of the protein.The potential for substituting amino acids in the P-fimbrial subunit opens interesting possibilities for use of fimbriae as carriers of foreign antigenic determinants. An antigenic determinant of foot-and-mouth disease virus (FMDV) was incorporated in the F11 fimbrial subunit. Hybrid fimbriae, recognized by an FMDV-specific neutralizing monoclonal antibody directed against FMDV, were formed.Fimbriae are long filamentous appendages that occur on many bacteria and consist of about 1,000 protein subunits (for a review, see reference 14). Many uropathogenic Escherichia coli strains expose on their cell surfaces P fimbriae that mediate adherence of the pathogen to the uroepithelium (11,31,32). P fimbriae recognize the a-D-galactose-(1,4)-P-D-galactose moiety of antigens of the P blood group (13,18). Among P fimbriae, the serotypes F7 through F13 have been distinguished (27). From uropathogenic E. coli, the genes responsible for synthesis of various serologically different P fimbriae, i.e., F71, F72, F8, F9, Fll, and F13, have been cloned (5,6,10,12,28,36,37). It has been shown that these fimbrial gene clusters are very similar in general organization (25,36,38), which suggests that the biogenesis of P fimbriae may follow a general concept.The composition of P fimbriae is very complex. The major subunit, or P fimbrillin, is predominant and determines the antigenic properties (35). Several minor components, among them the adhesin protein, are also present in the fimbrial structure (19,20,29 MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. E. coli K-12 HB101 (2), a strain deficient in the production of type 1 fimbriae, was used as the host strain in all experiments unless otherwise indicated. JM101 was used as the host strain for M13mp8 derivatives (24). HB2154 (3) was used as the host strain for M13mpl8 (26) derivatives in the localized mutagenesis experiments. The mutant plasmids described were derived from pPIL110-75, carrying thefso gene cluster, and pPIL291-15, carrying the fel (F-eleven) gene cluster (6,29,36

Pap pili as a vector system for surface exposition of an immunoglobulin G-binding domain of protein A of Staphylococcus aureus in Escherichia coli

“…Figure 3 indicates that HB1O1(pPAP5) reacted with Ra-pilin and not with the other antibodies; therefore, the test In summary, we show that adventitiously coding sequences can be functionally incorporated in the PapA subunit of pili and can become exposed at the surfaces of the bacteria, presumably as a part of a genuine pilus structure. This conclusion is consistent with the observations of Van Die et al (30), who incorporated an 8-amino-acid foot-and-mouth disease virus epitope in the Fll major pilus subunit and could observe the presence of the epitope at the cell surface, incorporated in a pilus structure. Our observations show that a stretch of at least 58 amino acids can be inserted into PapA and can be functionally exposed at the surface of the bacterium.…”

F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits

“…Analysis of plasmid-encoded proteins in minicells was performed as described previously (39 performed as described previously (40). The monoclonal antibody M9-6, a FocA-specific antibody, was used as an FlC-specific antibody (26).…”

Structure and Function of Hib Pili fromHaemophilus influenzaeType b

“…1. Alignment of HifA and PapA after accounting for data from previous studies (10,12,13,17,20,22,26). HifA from two strains of H. influenzae, Eagan and M43pϩ, have 82% identity and 95% similarity or 76% identity and 91% similarity, excluding and including, respectively, the highly variable regions of HifA.…”

“…This shift reduced the sequence alignments from 23% identical and 61% similar to 22% identical and 57% similar, excluding the HifA inserts. Data from previous alignments of the major structural pilins of class I pili were used to indicate regions of conservation and of variability for predicted secondary structures in HifA (4, 13) and PapA (26). These data were also used as guidelines in our realignment of the HifA/Eagan and PapA/J96 pilin sequences (Fig.…”

“…With biochemical, mutational, immunologic, and evolutionary data (4,8,10,12,13,17,20,26), we have produced an alignment in which the absence of crossreactivity of antibodies to HifA amino acids 62 to 72 and 97 to 102 (20) is correlated with insertion regions of HifA compared with PapA, and the extended hypervariable region defined by Krasan et al (17) corresponds to the largest insertion region. This region is expected to be surface exposed.…”