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S Normark

Publications and source records attributed to S Normark.

At least 55 records · Page 3Linked to original sources
PubMed1996

Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli.

Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins. We have found that polymerization of curlin to fimbriae-like structures (curli) on the surface of Escherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein. The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleator-presenting cell or by adjacent cells. The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation.

Amino Acid Sequence↗
PubMed1995

The PapG-adhesin at the tip of P-fimbriae provides Escherichia coli with a competitive edge in experimental bladder infections of cynomolgus monkeys.

Human urinary tract infection is an infectious disease that depends on a series of host-microbial interactions. The bacteria first colonize the colon and then the periurethral/vaginal areas; they ascend to and infect first the bladder and then the kidneys. Expression of Escherichia coli P-fimbriae constitutes the strongest correlation to renal pathogenicity, but is also related to first-time cystitis in children. The role of P-fimbriae in the preceding steps in the infectious process is unknown. To examine this, we constructed, from a P-fimbriated E. coli strain with a class II G-adhesin preferentially binding to globoside, one isogenic mutant lacking the G-adhesin and another isogenic mutant in which we replaced the papG class II allele with a class III adhesin preferentially binding to the Forssman antigen. We report here the comparison of the adhesin knockout mutant (DS17-8) and the class-switch mutant (DS17-1) with the wild-type (DS17) for in vivo colonization of the gut, vagina, and bladder of cynomolgus monkeys. It was recently shown that the class II tip G-adhesin is a prerequisite for acute pyelonephritis to occur in the monkey model in the absence of other kidney-specific adhesins or obstruction of the urinary flow. Here we show that it is not required for bladder infection but gives a competitive advantage in mixed infections. In the vagina and colon, the G-adhesin gives no competitive advantage.

Adhesins, Escherichia coli↗
PubMed1995

Role of asparagine 152 in catalysis of beta-lactam hydrolysis by Escherichia coli AmpC beta-lactamase studied by site-directed mutagenesis.

The role of asparagine 152 in the catalytic mechanism of Escherichia coli AmpC beta-lactamase has been investigated by site-directed mutagenesis. The residue has been replaced by aspartic acid, glutamic acid, histidine, and leucine. All the substitutions had similar effects on the activity toward substrates and inhibitors. The rate of substrate hydrolysis decreased by factors of 500-5000. The rates of both acylation (2-50-fold decrease) and deacylation (50-500-fold decrease) were affected, indicating a role for Asn152 in both processes. The wild-type AmpC beta-lactamase appears to exist as an equilibrium mixture of two forms, identified by their different kinetic properties. The Asn152 mutations affected the activity of the slow-reacting form much more than that of the fast-reacting form, but they did not appear to affect the interconversion of these two kinetic forms. Comparison of these observations with results obtained with mutation of the equivalent residues in other classes of penicillin-sensitive enzyme indicates that there are quite profound differences between the catalytic mechanisms of these enzymes despite a high degree of conservation of amino acids in the active center, and of the overall three-dimensional structure.

Anti-Bacterial Agents↗
PubMed1995

beta-Lactamase induction in gram-negative bacteria is intimately linked to peptidoglycan recycling.

A number of Gram-negative organisms normally express a chromosomally mediated class C beta-lactamase that is inducible by beta-lactam antibiotics. Data have recently emerged suggesting a close link between beta-lactamase induction and the recycling of released muramyl peptides from the bacterial peptidoglycan. Thus the AmpG transporter is responsible for the uptake into the cell of GlcNAc-anhMurNAc-tripeptide. A mutant unable to express AmpG is therefore unable to recycle the cell wall and is at the same time not possible to induce by a beta-lactam. Once inside the cytosol the above muramyl peptide and its derivative anhMurNAc-tripeptide is degraded by the cytosolic AmpD amidase that specifically releases the tripeptide from cytosolic muramyl peptides brought into the cell via AmpG. Mutants unable to produce AmpD are blocked in a cytosolic step for cell wall recycling and accumulate large amounts of cytosolic anhMurNAc-tripeptide. It is believed that cytosolic muramyl peptides can act as ligands for the beta-lactamase regulator AmpR to activate expression of beta-lactamase. AmpD mutants, therefore, constitutively overproduce the chromosomal beta-lactamase and are beta-lactam resistant. In wild-type strains beta-lactams that result in an increased cell wall breakdown will cause an increase in the cytosol of muramyl peptides leading to beta-lactamase induction. Mutants affected in the ampD gene arise readily during treatment with third-generation cephalosporins. Since these mutants lack a functional cell wall recycling system they may be at a disadvantage in the absence of selection. However, since muramyl peptides may act as cytotoxins, especially for respiratory epithelial cells, ampD mutants due to their large accumulation of anhMurNAc-tripeptide may be altered in their pathogenic properties as compared to wild-type cells possessing a normal cell wall recycling system.

Enzyme Induction↗
PubMed1995

Human milk kappa-casein and inhibition of Helicobacter pylori adhesion to human gastric mucosa.

Readily digested caseins, which account for almost half of the protein content in human milk, are important as nutritional protein for breast-fed infants. It has also been advocated that part of the antimicrobial activity of human milk resides in the caseins, most likely the glycosyated K-casein. Top explore this possibility, we purified K-casein from human milk to homogeneity by a two-step size-exclusion chromatography procedure. Purified human K-casein, in contrast to K-casein purified from bovine milk, effectively inhibited the cell lineage-specific adhesion of fluoroisothiocyanate-labeled Helicobacter pylori to human gastric surface mucous cells. The inhibitory activity was abolished by metaperiodate oxidation and considerably reduced by preincubation with alpha-L-fucosidase but not with alpha-N-acetylneuraminidase or endo-beta-galactosidase. These results strongly support the view that fucose containing carbohydrate moieties of human K-casein are important for inhibition of H. pylori adhesion and, thus, infection. They also suggest that breastfeeding may protect from infection by H. pylori during early life and that species-specific glycosylation patterns, as illustrated by human bovine K-casein, partly determine both the narrow host spectrum of this human gastric pathogen and the capacity to resist infection.

Animals↗
PubMed1995

Pilus biogenesis gene, pilC, of Neisseria gonorrhoeae: pilC1 and pilC2 are each part of a larger duplication of the gonococcal genome and share upstream and downstream homologous sequences with opa and pil loci.

Pili of Neisseria gonorrhoeae mediate attachment of the bacteria to target cells and undergo both phase and antigenic variation. PilC is a 110 kDa minor pilus-associated protein involved in pilus biogenesis and attachment. The expression of PilC is turned on and off at high frequency and is controlled by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Most strains of N. gonorrhoeae carry two copies of pilC. The DNA sequence of pilC1 of strain MS11 is presented and compared to the sequence of the 3' end of pilC2. These two genes are highly homologous, but not identical. The putative transcriptional terminator of pilC1 contains a pair of inverted uptake sequences for gonococcal DNA (5'-GCCGTCTGAA-3'). An 88 bp sequence located upstream of the pilC1 gene has also been reported to precede several opa genes of N. gonorrhoeae. Shorter regions positioned both downstream and upstream of pilC1 can also be found in silent pil loci as well as close to opa genes. The pilC genes are part of a duplication of a larger DNA region extending more than 2 kb downstream of the coding region.

Amino Acid Sequence↗
PubMed1995

Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma s in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

Bacterial Proteins↗
PubMed1995

AmpD, essential for both beta-lactamase regulation and cell wall recycling, is a novel cytosolic N-acetylmuramyl-L-alanine amidase.

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of beta-lactamase expression. It is shown here that the AmpD protein is a novel N-acetylmuramyl-L-alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptidoglycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a 'recycling tag' allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for beta-lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan.

Amino Acid Sequence↗
PubMed1994

The Gal(alpha 1-4)Gal-specific tip adhesin of Escherichia coli P-fimbriae is needed for pyelonephritis to occur in the normal urinary tract.

Nonobstructive acute pyelonephritis in humans is most often caused by P-fimbriated Escherichia coli. P-fimbriae are heteropolymeric fibers carrying a Gal(alpha 1-4)Gal-specific PapG adhesin at its distal end. The pyelonephritic strain DS17 expresses P-fimbriae from a single gene cluster. A mutant strain, DS17-8, which expresses P-fimbriae tacking the PapG adhesin, was constructed by allelic replacement introducing a 1-bp deletion early in the papG gene. In cynomolgus monkeys, DS17 and DS17-8 were equally able to cause bladder infection, whereas only the wild-type strain DS17 could cause pyelonephritis as monitored by bacteriological, functional, and histopathological criteria. Since DS17, but not DS17-8, adheres to renal tissue, these data underscore the critical role of microbial adherence to host tissues in infectious disease and strongly suggest that the PapG tip adhesin of P-fimbriae is essential in the pathogenesis of human kidney infection.

Adhesins, Escherichia coli↗
PubMed1994

Bacterial cell wall recycling provides cytosolic muropeptides as effectors for beta-lactamase induction.

A mechanism for bacteria to monitor the status of their vital cell wall peptidoglycan is suggested by the convergence of two phenomena: peptidoglycan recycling and beta-lactamase induction. ampG and ampD, genes essential for beta-lactamase regulation, are here shown to be required for recycling as well. Cells lacking either AmpG or AmpD lose up to 40% of their peptidoglycan per generation, whereas Escherichia coli normally suffers minimal losses and instead recycles 40 or 50% of the tripeptide, L-alanyl-D-glutamyl-meso-diaminopimelic acid, from its peptidoglycan each generation. The ampG mutant releases peptidoglycan-derived material into the medium. In contrast, the ampD mutant accumulates a novel cell wall muropeptide, 1,6-anhydro N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (anhMurNAc-tripeptide), in its cytoplasm. This work suggests that AmpG is the permease for a large muropeptide and AmpD is a novel cytosolic N-acetylmuramyl-L-alanine amidase that cleaves anhMurNAc-tripeptide to release tripeptide, which is then recycled. These results also suggest that the phenomenon of beta-lactamase induction is regulated by the level of muropeptide(s) in the cytoplasm, since an ampD mutation that results in beta-lactamase expression even in the absence of a beta-lactamase inducer coincides with accumulation of anhMurNAc-tripeptide. The transcriptional regulator AmpR is presumably converted into an activator for beta-lactamase production by sensing the higher level of muropeptide(s). This may be an example of a general mechanism for signaling the progress of external events such as cell wall maturation, cell division or cell wall damage.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1994

The role of tyrosine 150 in catalysis of beta-lactam hydrolysis by AmpC beta-lactamase from Escherichia coli investigated by site-directed mutagenesis.

The kinetics of beta-lactam hydrolysis by wild-type AmpC beta-lactamase from Escherichia coli and three mutant proteins created by substitution of tyrosine 150 have been examined. The catalytic efficiency was decreased 10- to 1000-fold according to the substrate and mutant being studied. The effect of the mutation was much stronger with rapidly hydrolyzed substrates (e.g., cephalothin) than it was with slowly hydrolyzed substrates (e.g., ceftriaxone). With the latter substrates, the mutagenesis had a much stronger effect on apparent affinity than it did on rates of catalysis. Indeed, the enzyme appeared to be more reactive toward certain of the slowly hydrolyzed substances (e.g., methicillin, aztreonam, and ceftriaxone). These observations were not compatible with an obligatory role of tyrosine 150 in catalysis. The analysis of the effects of the mutation on activity was complicated by the observation of at least two, kinetically distinct, forms of the enzymes. It appeared that mutation of tyrosine 150 influenced the kinetic properties of one state and that this residue is involved in the partitioning of the enzyme between the different reactive states.

Acylation↗
PubMed1994

Catalytic mechanism of active-site serine beta-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad.

The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.

Amino Acid Sequence↗
PubMed1994

Role of residue Lys315 in the mechanism of action of the Enterobacter cloacae 908R beta-lactamase.

The role of the highly conserved Lys315 residue in the catalytic mechanism of a class C beta-lactamase has been probed by site-directed mutagenesis. Lys315 has been replaced by a histidine in the Enterobacter cloacae 908R beta-lactamase, thus introducing a tritratable group to probe the role of the positive charge, and by a glutamine. The effects of these mutations have been studied on the kinetics of penicillin G and cephalothin turnover and on the pre-steady-state kinetics with carbenicillin at different pH. Results showed that substrate binding was not impaired by the mutations, so that an interaction with the substrate-free carboxylate in the Henri-Michaelis complex could be ruled out. Lys315 must have a catalytic role as shown by the decreased acylation and deacylation rates observed with the mutant enzymes. The mutants exhibited a lower activity at acidic pH, and this observation could be correlated with a decreased affinity for (3-aminophenyl)boronate, a compound devoid of free carboxylate which binds to the active site and forms an adduct mimicking the tetrahedral intermediate. This suggested that Lys315 was somehow involved in accelerating the nucleophilic substitutions along the reaction pathway. The study was extended to modified substrates where the free carboxylate had been esterified. Neither acylation nor deacylation seemed severely impaired with these compounds, showing that the interaction between the enzyme and the substrate-free carboxylate did not play a major role in catalysis.

Amino Acid Sequence↗
PubMed1994

Roles of pilin and PilC in adhesion of Neisseria meningitidis to human epithelial and endothelial cells.

Pili and pilin antigenic variation play important roles in adhesion of Neisseria meningitidis (MC) to human epithelial and endothelial cells. We recently identified one pilin variant that confers high adhesiveness of MC to human epithelial cells in culture. However, other factor(s) also play a role in MC adhesiveness, since some nonadhesive variants of MC strain 8013 are piliated and produce the same pilin variant as adhesive derivatives. PilC1 and PilC2, high molecular weight outer membrane proteins in Neisseria gonorrhoeae, are proposed to play roles in pilus assembly. Strain 8013 also contains pilC1 and pilC2; their products function in a similar if not identical manner in pilus biogenesis. PilC1 has an additional function in that it also modulates adhesiveness of strain 8013.

Bacterial Adhesion↗
PubMed1994

Helicobacter pylori: molecular basis for host recognition and bacterial adherence.

The bacterium Helicobacter pylori is tropic for epithelial cells and the mucus layer in the stomach lining, and is associated with the development of gastritis, ulcers and possibly also gastric malignancies. Adherence to the gastric epithelial cells is mediated by fucosylated blood-group antigens associated with blood-group O phenotype, which could explain the higher prevalence of ulcerative disease in individuals with this blood group.

ABO Blood-Group System↗
PubMed1994

Sequence changes in the pilus subunit lead to tropism variation of Neisseria gonorrhoeae to human tissue.

Pili of Neisseria gonorrhoeae are correlated with increased bacterial attachment to epithelial cells and undergo both phase and antigenic variation. Phase variation of gonococcal pili can be brought about by recombination events in the pilin structural gene, pilE, or by the on/off switch in expression of PilC, a pilus biogenesis protein for which two loci exist. We have studied the binding to epithelial cell lines and to fixed tissue sections of N. gonorrhoeae MS11 derivatives and mutants carrying structurally defined PilE and PilC proteins. In situ binding studies of N. gonorrhoeae to formalin-fixed tissue sections resulted in a binding pattern similar to that obtained using viable epithelial cell lines of different origin. Piliated gonococcal clones, containing different pilE sequences, varied dramatically from one another in their efficiencies at binding to corneal and conjunctival tissue, but bound equally well to cervical and endometrial tissues. Further, the binding data suggested that PilC expression by itself, i.e. without pili, cannot confer bacterial binding and that expression of either PilC1 or PilC2 does not confer different binding properties to the bacterial cells. Possible receptors for piliated gonococci were expressed in human tissues, such as cervix, endometrium, cornea, intestine, stomach, mid-brain and meninges, but not in human kidney. Pretreatment of the target tissues with Proteinase K decreased the gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect attachment of gonococci. These data argue that pilus-dependent attachment of N. gonorrhoeae to human tissue may be mediated by a eukaryotic receptor having protein characteristics, and that the pilus subunit sequence may play an important role in the interaction with human cornea.

Amino Acid Sequence↗