Minor Subunit Proteins Research Articles

Functional analysis of the minor subunits of S fimbrial adhesin (SfaI) in pathogenic Escherichia coli

S fimbrial adhesins I and II (SfaI and II), produced by extraintestinal Escherichia coli pathogens that cause urinary tract infections (UTI) and newborn meningitis (NBM), respectively, mediate bacterial adherence to sialic acid-containing glycoprotein receptors present on host epithelial cells and extracellular matrix. The S fimbrial adhesin complexes consist of four proteins: SfaI-A, the major subunit protein and the minor subunit proteins SfaI-G, SfaI-S and SfaI-H. Sialic acid-specific binding is mediated by the minor subunit protein SfaI-S. In order to determine whether the minor subunit proteins SfaI-G, -S and -H play a role in the modulation of adherence and the degree of fimbriation, a trans-complementation system was developed. A non-adhesive E. coli K-12 derivative, harbouring the sfaI-A gene but lacking sfaI-G, -S and -H, was transformed with sfaI-G, -S or -H. Only SfaI-S was able to increase the degree of fimbriation and to confer adhesion properties on the recombinant E. coli K-12 strains. Amino acid residues in SfaI-S that are involved in modulation of fimbriation as well as in receptor recognition were localized by random and site-directed mutagenesis.

Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate

S fimbrial adhesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newborn meningitis. We recently described the cloning and molecular characterization of a determinant, termed sfaI, from the chromosome of an E. coli urinary tract infection strain. Here we present data concerning a S fimbria-specific gene cluster, designated sfaII, of an E. coli newborn meningitis strain. Like the SfaI complex, SfaII consists of the major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding the subunit proteins of SfaII were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with those of the SfaI complex subunits. Although the sequences of the two major SfaA subunits differed markedly, the sequences of the minor subunits showed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of the meninigitis isolate. These data were confirmed by the isolation and characterization of the SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of SfaI and SfaII revealed that differences between the two Sfa complexes with respect to their capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other than SfaS.

Role of fimbrial adhesins in the pathogenesis of Escherichia coli infections.

Escherichia coli strains are able to cause intestinal (enteritis, diarrhoeal diseases) and extraintestinal (urinary tract infections, sepsis, meningitis) infections. Most pathogenic E. coli strains produce specific fimbrial adhesins, which represent essential colonization factors: intestinal E. coli strains very often carry transferable plasmids with gene clusters specific for fimbrial adhesins, like K88 and K99, or colonization factor antigens (CFA) I and II. In contrast, the fimbrial gene clusters of extraintestinal E. coli strains, such as P, S, or F1C fimbriae, are located on the chromosomes. The fimbrial adhesin complexes consist of major and minor subunit proteins. Their binding specificity can generally be assayed in hemagglutination tests. In the case of fimbrial adhesins of intestinal E. coli strains, the major subunit proteins preferentially represent the hemagglutinating adhesins, whereas minor subunit proteins are the hemagglutinins of extraintestinal E. coli strains. Recently "alternative" adhesin proteins were identified, which have the capacity to bind to eukaryotic structures different from the receptors of the erythrocytes. Fimbrial adhesins are not constitutively expressed but are stringently regulated on the molecular level. Extraintestinal E. coli wild-type strains normally carry three or more fimbrial adhesin determinants, which have the capacity to influence the expression of one another (cross talk). Furthermore the fimbrial gene clusters undergo phase variation, which seems to be important for their contribution to pathogenesis of E. coli.

Functional analysis of the fsoC gene product of the F71 (fso) fimbrial gene cluster

Contrary to what would be expected from data in the literature, mutations in the fsoC gene of the F7(1) (fso) P-fimbrial gene cluster do not completely block fimbrial biogenesis. fsoC mutants still express small amounts of fimbriae of normal length, which carry the non-adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG. The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins. The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogenesis.

Biochemical Relationship among Three F-Type Pyocins, Pyocin F1, F2, and F3, and Phage KF1

The immunological and the biochemical relationships between F-type pyocins and phage KF1, which is cross-reactive with anti-F-type pyocin sera, were studied. The primary structures of six subunit proteins of each pyocin were also compared among three F-type pyocins, pyocin F1, F2, and F3. Both anti-pyocin F1 and anti-pyocin F3 sera gave precipitin bands with phage KF1 by Ouchterlony's test, and were found to bind to minor subunit proteins P3 and P6 of the phage, which were separated by SDS-polyacrylamide gel electrophoresis. Electron microscopic studies showed that these sera bound to some loci of the rod part of the phage tail. These results showed that the subunit proteins P3 and P6 carried antigenically common parts to F-type pyocin and that P3 and/or P6 were components of the rod part of the phage KF1. The subunit proteins P3 and P6 of the phage were of the same mobilities in SDS-polyacrylamide gel electrophoresis as those of band 1 and band 5 of F-type pyocins, respectively. Tryptic peptide mapping after iodination with 125I also showed a partial homology between P3 and band 1, and between P6 and band 5. The tryptic peptide mapping of the six subunit proteins of each F-type pyocin showed that the primary structure of subunit protein band 1 was almost the same among these pyocins, and subunit protein bands 2, 3, 5, and 6 showed were similar. Only subunit protein band 4 was different among these pyocins. The difference in the action spectra of pyocin F1, F2, and F3 is probably due to the difference in the primary structure of subunit protein band 4, which is a component of the fiber part.

Isolation and characterization of a new bacteriophage, KF1, immunologically cross-reactive with F-type pyocins.

Pseudomonas aeruginosa strain NIH S produced a bacteriophage, KF1, immunologically cross-reactive with F-type pyocins. Phage KF1 was neutralized by both anti-pyocin F1 and anti-pyocin F3 sera, although the efficiency was very low. About eleven polypeptides were detected by SDS-polyacrylamide gel electrophoresis of the phage. Most of the subunit proteins were different from those of F-type pyocins, but the molecular weights of minor subunit proteins P3 and P6 seemed to be the same as those of band 1 and band 5 of F-type pyocins, respectively. The head of the phage appeared to have an icosahedral structure, approximately 63 nm in diameter, with a long (190 nm, 11 nm wide and about 45 striations) flexuous tail connected to a fiber structure (about 53 nm in length). The density in CsCl and the sedimentation coefficient of the phage were 1.54 g/ml and 392S, respectively. Some other biochemical properties were described. The nucleic acid of the phage was linear, double stranded DNA of molecular weight 4 x 10(7). The density of the DNA in CsCl was 1.719 g/ml, the melting temperature was 95.4 degrees C. The guanine plus cytosine content was calculated to be 60 to 64%.