Biomedical subjects
Publications and source records attributed to S Normark.
399 rural Tanzanian standard two pupils with a mean age of 10.9 years were dentally examined and interviewed. DMFS and defs were 0.7 and 0.5 respectively; gingival bleeding was present in 44% of the sites. Daily habits of the majority appeared satisfactory. Eight percent of the children displayed half of the caries amount.
The sequences of three Enterobacter cloacae ampC beta-lactamase genes have been determined. The deduced amino acid sequences are very similar: out of a total of 361 residues, only eight positions were found to be variable, and several mutations yielded residues with very similar properties. The kinetic properties of two of the enzymes were not significantly different. The three enzymes also exhibited a high degree of homology (greater than 70%) with the ampC beta-lactamases of Escherichia coli K12 and Citrobacter freundii, confirming the homogeneity of class-C beta-lactamases.
E. coli expressing the papA-I genes produce pili that mediate specific adhesion to mammalian cells. We show that the major pilus subunit gene, papA, is part of a polycistronic transcriptional unit subject to specific posttranscriptional processing. A primary transcript also encoding the papB regulatory gene product is endonucleolytically cleaved, resulting in the rapid decay of the papB-encoding 5' half of the mRNA, whereas the papA-encoding 3' half remains as a quite stable transcript. Processing and differential mRNA stability thereby result in accumulation of mRNAs encoding only the major pilus subunit. A sequence immediately downstream of the papA coding region may serve as a stability determinant for the papA transcript and concomitantly attenuate read-through transcription into the minor pilus subunit gene papH. This suggests that differential expression of genes within an operon may include endo- and exonucleolytic processing of the mRNA.
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Escherichia coli strains associated with extra-intestinal infections frequently express carbohydrate binding adhesins which are present as minor components of pili. The adhesin protein, PapG, and at least two other minor pilus subunits, PapE and PapF, are associated with the tips of Gal alpha (1-4)Gal-binding Pap pili or P fimbriae of uropathogenic E. coli. The structural and antigenic variation of these tip-associated proteins is discussed and evidence is presented showing that serologically identical pili may contain antigenically distinct adhesins each capable of binding to a specific receptor. One approach to the purification of these tip-associated proteins is presented and involves complex formation with the periplasmic transport protein PapD.
Different strains and isogenic variants of Neisseria gonorrhoeae were assayed for their ability to bind glycolipids extracted from various sources. Among a large number of reference glycolipids, binding was observed only to lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], isoglobotriaosylceramide [Gal(alpha 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriaosylceramide [GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer]. The latter two glycolipids bound gonococci with the highest affinity. Lactosylceramide and gangliotriaosylceramide were found in glycolipid preparations from ME180 cells, an epithelial cell line derived from a human cervical carcinoma, and thus are possible receptors for gonococci. The gonococcal surface component that bound the above glycolipids is a protein distinct from pilin and protein II.
Uropathogenic Escherichia coli frequently express P-pilus adhesins that recognize Gal alpha (1-4)Gal-containing glycoconjugates. The P-pilus adhesin of the E. coli isolate J96 is encoded by the pap gene cluster and has been shown to agglutinate P1-erythrocytes. We now describe a novel gene cluster from J96, prs, which is responsible for the agglutination of sheep erythrocytes. The structurally related gene clusters both expressed pili exhibiting the F13 antigen. Analysis of mutants of cloned prs sequences, together with trans-complementation of pap and prs genes, identified the sheep-specific adhesin as the 37-kD PrsG protein. The prsG gene occupies the equivalent position in prs as occupied by papG, which specifies the Gal alpha (1-4)Gal-specific adhesin of pap. PrsG was shown to be structurally distinct from PapG since PapG-specific antiserum did not cross-react with PrsG. Using a solid phase glycolipid receptor binding assay, PrsG was found to specify preferential binding to the Forssman antigen, a major constituent of sheep erythrocyte membranes. The binding epitope was identified as the GaINAc alpha (1-3)GaINAc moiety. This is the first direct evidence that serologically identical pili may present antigenically distinct adhesins, each capable of binding to a specific receptor.
Pyelonephritogenic Escherichia coli frequently expresses pili which bind to Gal alpha (1-4)Gal receptors present on the uroepithelium. Binding of these pili is mediated by a pilus-associated adhesin, PapG, and not by the major subunit which constitutes the bulk of the pilus structure. The adhesin and two pilinlike proteins, PapE and PapF, are present in only a few copies each at the pilus tip. Surface exposure of both PapF and PapG is required to achieve receptor-specific binding. The nucleotide sequences for the genes encoding the tip-associated proteins PapE, PapF, and PapG were determined for two E. coli clones expressing P pili of serotypes F11 and F7(2) and compared with the corresponding sequences established for proteins of F13 pili. Specific antisera were used to study the cross-reactivity between the F13 tip proteins and the equivalent proteins in F11 and F7(2) pili. We present data showing that, like the major pilus subunit, PapE varies its structure and antigenic properties among pili of different serotypes. In contrast, the PapF protein was highly conserved, and PapF-specific antisera raised against serotype F13 cross-reacted with the PapF proteins of both F11 and F7(2) serotypes. The PapG adhesin protein from F11 and F7(2) pili differed by only five amino acids out of 316 residues. However, the F13 adhesin showed only 45% amino acid homology with the other two variants.
The biogenesis of Escherichia coli Pap pili, encoded by the pap gene cluster, was studied. A novel gene, papH, was identified and found to encode a weakly expressed pilin-like protein. PapH was dispensable for digalactoside-specific binding and for formation of Pap pili. However, in papH deletion mutants 50%-70% of total pilus antigen was found free of the cells. We present evidence showing coregulation of papH and the adjacent gene, papA, which encodes the major pilin subunit. A decrease in the PapA to PapH ratio resulted in a large fraction of cells producing shortened pili, whereas overproduction of PapA relative to PapH resulted in cells with lengthened pili. The data show that PapH has roles in anchoring the pilus to the cell and in modulating pilus length.
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The adherence process in pathogenesis involves the attachment of bacteria to structures present on eukaryotic cell surfaces. To investigate components necessary for this interaction, we have characterized the binding of N. gonorrhoeae to eukaryotic glycolipids immobilized on thin layer chromatograms. The gonococci specifically bind to a subset of glycolipids consisting of lactosylceramide, gangliotriosylceramide, and gangliotetraosylceramide. This binding was identified in both piliated and nonpiliated cells, and is postulated to be mediated by a nonpilin lectin-like adhesin protein.
Uropathogenic Escherichia coli adhere to uroepithelial cells by their digalactoside alpha-D-galactopyranosyl-(1----4)-beta-D-galactopyranose [alpha-D-Galp-(1----4)-beta-D-Galp or Gal alpha (1----4)Gal]-binding pili, which are composed of repeating identical subunits. The major subunit (PapA) of these pili is not required for binding, but the papF and papG gene products are essential for adhesion. Transcomplementation analysis between the pap gene cluster and a related gene cluster encoding a different binding specificity showed that PapG and not PapF is the Gal alpha (1----4)Gal-specific adhesin. Antibodies against PapG were obtained upon immunizing with whole Pap pili, showing that the adhesin is a pilus component. Antisera specific for different Pap proteins were used to demonstrate that a pilin protein, either PapA or PapE, together with both PapG and PapF, must be exposed on the cell surface to allow E. coli to bind. The DNA sequence of the papG gene is presented, and the deduced primary structure showed similarities both to the B-chain sequence of the digalactoside-binding Shigella toxin and to established amino acid sequences of pilins.
The papC gene of uropathogenic Escherichia coli is required for the formation of digalactoside-binding Pap pili. papC forms part of an operon wherein the regulatory gene papB, the major pilin gene papA, a minor pilin-like gene papH, and papC are co-transcribed. Furthermore, the extent of PapC synthesis was found to affect the number of pili expressed on the cell surface. The DNA sequence of the papC gene is presented and its deduced amino acid sequence is compared to that of the FaeD protein encoded by the K88 pili gene cluster. The PapC protein was localized to the E. coli outer membrane where it may form a trans-membrane channel through which pilin subunits are surface localized.
In Gram-negative bacteria, beta-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic beta-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the beta-lactamases have been studied and their significance in creating beta-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of beta-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC beta-lactamase, or a plasmid-coded TEM-type beta-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter, 'target access index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.
Expression of the chromosomal beta-lactamase from the ampC gene in inducible in both Enterobacter cloacae and Citrobacter freundii. Cloning of ampC as well as its regulatory gene, ampR, from E. cloacae P99 revealed a gene organization indentical to that of C. freundii in the corresponding region. Although almost no similarities could be found between the restriction maps of ampC and ampR in the two species, the genes cross-hybridize. Also, both ampR gene products have a size of about 31,000. The regulatory features of E. cloacae beta-lactamase induction are very similar to those in C. freundii, i.e., beta-lactamase synthesis is repressed by AmpR in the absence, and stimulated in the presence, of inducer. The AmpR function can be transcomplemented between the two species, but there are quantitative regulatory aberrations in such hybrids, in contrast to the total complementation obtained within each system. These results suggest that the mechanism of beta-lactamase induction is the same in E. cloacae, C. freundii, and other gram-negative bacteria with inducible chromosomal beta-lactamase expression.
In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression.
The Citrobacter freundii OS60 ampC beta-lactamase gene was sequenced and found to encode a 380-amino-acid-long precursor with a 19-residue signal peptide. The mature protein has a predicted molecular mass of 39781 Da. The first 60 residues of the purified enzyme, as determined by sequential Edman degradation, are identical to the amino acid sequence inferred from the gene sequence. Also, the amino acid composition determined for the purified beta-lactamase and that given by the gene sequence are in good agreement. 77% of the amino acid positions hold identical residues in the C. freundii and Escherichia coli K12 chromosomal AmpC beta-lactamases. This clearly puts the C. freundii enzyme into the class C of beta-lactamases. Of the 68 amino-terminal residues determined for the Enterobacter cloacae P99 beta-lactamase, 44 are identical to the corresponding residues of the C. freundii enzyme. All three enzymes, as well as that of Pseudomonas aeruginosa 18S/H are highly similar around the active-site serine at position 64 of the mature protein.
The papE, papF, and papG genes of uropathogenic Escherichia coli are dispensable for the synthesis and assembly of pili associated with pyelonephritis, called Pap pili. Phenotypically, papF and papG mediate digalactoside [alpha-D-Galp-(1----4)-beta-D-Galp)-specific adhesion. Although whole bacterial cells of a papE mutant bind to this receptor, purified pili from such a mutant do not. This is in contrast to pili purified from the wild type, which bind specifically. The DNA sequences of the papE and papF genes are presented, together with the deduced primary structure of the gene products. Both proteins have most of the features characteristic of Escherichia coli type 1 and Pap pilins. The PapE protein can be detected in the purified wild-type pilus by NaDodSO4/polyacrylamide gel electrophoresis followed by silver staining or by autoradiography of gels to which radioiodinated pili have been applied. In rabbits immunized with purified Pap pili, antibodies specific for both PapE and PapF are produced. We propose that PapE and PapF are minor pilins in the Pap pilus.