Biomedical subjects

J Hacker

Publications and source records attributed to J Hacker.

At least 73 records · Page 4Linked to original sources

PubMed1992

Genetics of Escherichia coli uropathogenicity: analysis of the O6:K15:H31 isolate 536.

E. coli strain 536 (O6:K15:H31) isolated from a case of acute pyelonephritis, expresses S-fimbrial adhesins, P-related fimbriae, common type I fimbriae, and hemolysins. The respective chromosomally encoded determinants were cloned by constructing a genomic library of this strain. Furthermore, the strain produces the iron uptake substance, enterocheline, damages HeLa cells, and behaves in a serum-resistant mode. Genetic analysis of spontaneously arising non-hemolytic variants revealed that some of the virulence genes were physically linked to large unstable DNA regions, termed "pathogenicity islands", which were mapped in the respective positions on the E. coli K-12 linkage map. By comparing the wild type strain and mutants in in vitro and in vivo assays, virulence features have been evaluated. In addition, a regulatory cross talk between adhesin determinants was found for the wild-type isolate. This particular mode of virulence regulation is missing in the mutant strain.

Chromosome Mapping↗

PubMed1992

Cloning and sequencing of a Shiga-like toxin II-related gene from Escherichia coli O157:H7 strain 7279.

Escherichia coli O157:H7 strains are newly recognized pathogens associated with haemorrhagic colitis and haemolytic-uremic syndrome. In addition to Shiga-like toxin types I and II known to be produced by E. coli O157 isolates, we have identified in E. coli O157:H7 strain 7279 a third toxin, designated SLT-IIvhc, that is neutralized neither by anti-SLT-I nor by anti-SLT-II antibodies. The genes for this toxin were isolated by using a PCR-mediated cell-free cloning technique. DNA sequence analysis revealed a high degree of homologies to SLT-II and several SLT-II variants. The predicted amino acid sequence of the A subunit of SLT-IIvhc differed from that of the O91:H7 toxin VT2ha and SLT-IIc from E. coli O157:H- strain E2511 by 2 and 3 amino acids, respectively. The amino acid sequence of its B subunit was identical to VT2ha and SLT-IIc but different from SLT-II and SLT-IIv. Immunological differences of SLT-IIvhc and SLT-II as well as their different toxicity to HeLa cells presumably resulted from the small deviations within the primary structure of the B subunit.

Amino Acid Sequence↗

PubMed1992

Adherence to and cytotoxicity of Escherichia coli for eucaryotic cell lines quantified by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide).

Adherence of Escherichia coli to human epithelial cells (HEp-2) was studied using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) which is cleaved by enzymes of eucaryotic or procaryotic cells to formazan. This method allows to quantify adherence of Escherichia coli to HEp-2 cells and offers the advantage of assaying a large number of eucaryotic cells without using specific antisera or radioactive material. Furthermore, toxic effects of isolated hemolysin cloned in Escherichia coli onto a renal tubular cell line (LLC-PK1) was investigated by this method, showing reduced cellular viability of tubular cells after an incubation period of 10 to 20 min. MTT is therefore considered to be useful to assay the adherence of Escherichia coli to eucaryotic cells and to quantify toxic effects in eucaryotic cells induced by bacterial virulence factors.

Bacterial Adhesion↗

PubMed1992

Restriction fragment length polymorphism and virulence pattern of the veterinary pathogen Escherichia coli O139:K82:H1.

Escherichia coli O139:K82:H1 strains originating from outbreaks and single cases of oedema disease in pigs were characterized by their genomic restriction fragment length polymorphism (RFLP), their virulence pattern, and by the occurrence as well as the genomic distribution of the determinants for hemolysin (hly) and verotoxins (shiga-like toxins; sltI, sltII). Whereas the RFLPs revealed considerable variation among the E. coli O139:K82:H1 isolates depending the origin and epidemic source of the strains, the virulence gene slt II was found to be present in nearly all strains in a particular chromosomal region. Similar to RFLPs, the plasmid profiles are useful for epidemiological analysis.

Animals↗

PubMed1992

Inhibition of adhesion of S-fimbriated Escherichia coli to epithelial cells by meconium and feces of breast-fed and formula-fed newborns: mucins are the major inhibitory component.

We investigated the ability of meconium, feces from human milk-fed (HMF) newborns, and feces from formula-fed (FF) newborns to inhibit adhesion of S-fimbriated E. coli to human buccal epithelial cells. S-fimbriae are a common property of E. coli strains causing sepsis and meningitis in neonates. Meconium had the highest content of neuraminic acid and the strongest inhibitory effect on bacterial adhesion. HMF also exerted high inhibitory activity while FF was markedly less active: To achieve inhibitory effects comparable to HMF a sixfold amount of FF was required. Glycoproteins from excretions were separated by gel chromatography. Fractions obtained were analyzed for adhesion-inhibiting activity. In all excretions analyzed, the mucin-containing fraction could be identified as the major inhibitory component. Inhibition was probably mediated by specific interaction of this fraction with S-fimbriae, as shown by binding of isolated fimbriae on Western blots after electrophoretic separation of glycoproteins. In conclusion, our data support the view that the mucin-containing fraction from meconium and human milk exerts antibacterial functions by preventing adhesin-mediated binding of pathogenic bacteria to mucosal epithelia.

Adult↗

PubMed1992

Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity.

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macrophage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506-binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K-12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP-type PPIases of eukaryotes. However, the N-terminal extension of Mip and/or the substitutions of the variable amino acids in the C-terminal FKBP core leads to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc-Ala-Xaa-Pro-Phe-4-nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein-folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.

Amino Acid Isomerases↗

PubMed1992

Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of the protective function of mucins in the nonimmunoglobulin fraction.

We investigated the presence of factors in human milk that inhibit invasion of pathogenic bacteria. The effect of human milk fat globule membrane (HMFGM) components on adhesion of cloned S-fimbriated Escherichia coli to human buccal epithelial cells was analyzed. S fimbriae are a common feature of E. coli strains causing sepsis and meningitis in newborns and are bound to epithelia via sialyl-(alpha-2-3)galactoside structures. Human milk fat globules (HMFG) could be agglutinated by the above-mentioned bacteria. Agglutination could be inhibited by fetuin, human glycophorin, and alpha 1-acid glycoprotein. In addition, pretreatment of HMFG with Vibrio cholerae neuraminidase markedly reduced bacterium-induced agglutinations, indicating the involvement of neuraminic acid-containing glycoproteins. In contrast, lipid droplets of infant formula or artificial lipid emulsions (Intralipid) could not be agglutinated. HMFG were present in stools of breast-fed neonates as shown by indirect immunofluorescence staining with a monoclonal antibody directed against carbohydrate residues present on HMFGM. These HMFG could be agglutinated by bacteria. HMFG inhibited E. coli adhesion to buccal epithelial cells. To further characterize relevant E. coli binding structures, HMFGM components were separated by gel chromatography. The mucin fraction showed the most pronounced inhibitory effect on adhesion of S-fimbriated E. coli to human buccal epithelial cells. Our data suggest that HMFG inhibit bacterial adhesion in the entire intestine and thereby may provide protection against bacterial infection.

Bacterial Adhesion↗

PubMed1992

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.

Animals↗

PubMed1992

[Immunotherapy of urinary tract infections].

In the discussion on prevention of urinary tract infection, interest is now centered on virulence factors of microorganisms and immunological defense mechanisms of the host organism. In this article current research results and the clinical aspects of an immunological treatment method for urinary tract infection are summarized and discussed.

Animals↗

PubMed1991

Enhancement of tissue plasminogen activator-catalyzed plasminogen activation by Escherichia coli S fimbriae associated with neonatal septicaemia and meningitis.

The effect of Escherichia coli strains isolated from blood and cerebrospinal fluid of septic infants on plasminogen activation was studied. These strains typically carry a filamentous surface protein, S fimbria, that has formerly been shown to bind to endothelial cells and interact with plasminogen. The bacteria effectively promoted plasminogen activation by tissue plasminogen activator (t-PA) which was inhibited by epsilon-aminocaproic acid. A recombinant strain expressing S fimbriae accelerated t-PA-catalyzed plasminogen activation to a similar extent as did the wild-type strains whereas the nonfimbriate recipient strain had no effect. After incubation with t-PA and plasminogen, the S-fimbriate strain displayed bacterium-bound plasmin activity whereas the nonfimbriate strain did not. Bacterium-associated plasmin generation was also observed with a strain expressing mutagenized S fimbriae that lack the cell-binding subunit SfaS but not with a strain lacking the major subunit SfaA. Both t-PA and plasminogen bound to purified S fimbriae in a lysine-dependent manner and purified S fimbriae accelerated t-PA-catalyzed plasminogen activation. The results indicate that E. coli S fimbriae form a complex with t-PA and plasminogen which enhances the rate of plasminogen activation and generates bacterium-bound plasmin. This may promote bacterial invasion and persistence in tissues and contribute to the systemic activation of fibrinolysis in septicaemia.

Amino Acid Sequence↗

PubMed1991

Analysis of the variability of S-fimbriae expression in an Escherichia coli pathogen.

The uropathogenic Escherichia coli wild-type strain 536 produces S-fimbriae, P-related fimbriae and type I fimbriae. Using immuno-colony dot and ELISA techniques, variants were detected showing an increased degree of S-fimbrial production. It was demonstrated by immunofluorescence microscopy that in normal (wild-type) and hyper-S-fimbriated E. coli populations non-fimbriated cells also exist, and that the percentage of S-fimbriated and non-fimbriated bacteria was roughly identical in either population. Hyper-S-fimbriated variants could be stably maintained. The transition from wild-type to hyper-S-fimbriation, which occurs spontaneously, is markedly higher than vice versa. Southern blot analysis of the S fimbrial adhesin (sfa) determinants of normal and hyper-fimbriated strains revealed no marked difference in the gene structure.

Adhesins, Escherichia coli↗

PubMed1991

Intracellular survival and expression of virulence determinants of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as well as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human embryo lung fibroblastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further studies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella.

Bacterial Toxins↗

PubMed1991

Phenotype versus genotype of the 19 kD peptido-glycan associated protein of Legionella (PpIA), among Legionellae and other gram-negative bacteria.

The protein PpIA (19 kD) cloned from a genomic library of Legionella pneumophila, Philadelphia 1, represents a peptido-glycan associated outer membrane protein in recombinant E. coli K-12 and L. pneumophila. It exhibits distinct sequence homology to lipoproteins of Haemophilus influenzae and E. coli. A ppIA specific DNA probe generated by PCR was used in Southern hybridizations of chromosomal DNA of Legionella strains and other Gram-negative pathogens. Under conditions of high stringency, hybridization could only be observed in L. pneumophila isolates, but all other Legionella strains tested displayed hybridization under lower stringency. No signals appeared after hybridization of chromosomal DNA from a variety of other bacteria. Using anti-PpIA monospecific polyclonal antibodies in Western blots, it was demonstrated that PpIA related proteins of nearly the same size are found in all L. pneumophila isolates and in a variety of, but not all, the Legionella species analysed here.

Bacterial Outer Membrane Proteins↗

PubMed1991

Virulence determinants of Escherichia coli O6 extraintestinal isolates analysed by Southern hybridizations and DNA long range mapping techniques.

A total of 16 Escherichia coli O6 strains isolated from cases of extraintestinal infections were analysed for the genetic presence and phenotypic expression of fimbrial adhesins (P, S/FIC, type 1), aerobactin and hemolysin. In addition restriction fragment length polymorphisms (RFLPs) of Xbal-cleaved genomic DNA of seven selected strains, separated by orthogonal field alternation gel electrophoresis (OFAGE) were determined and virulence-associated DNA probes were used for Southern hybridization studies of the Xbal-cleaved genomic DNAs. The virulence characteristics and hybridization patterns obtained differed between the various isolates. In three isolates hemolysin genes and P fimbrial determinants were located on the same Xbal fragments. Furthermore, multiple copies of FIC determinants (foc) could be detected in two strains. Our data show that the new technique of pulse field electrophoresis together with Southern hybridization represents a powerful tool for the genetic analysis of pathogenic bacteria.

Adhesins, Escherichia coli↗

PubMed1991

Legiolysin, a new hemolysin from L. pneumophila.

Legionella pneumophila generates exotoxins, cytolysins, proteases or hemolysins that damage host cells like erythrocytes or tissue culture cells. The gene for a new L. pneumophila hemolysin without a proteolytic activity was identified, cloned in E. coli and sequenced. The gene product was analysed by SDS-Polyacrylamide-gel-electrophoresis.

Animals↗

PubMed1991

Binding of cloned S-fimbriated E. coli to human buccal epithelial cells--different inhibition of binding by neonatal saliva and adult saliva.

Investigations were carried out on the adhesion of cloned S-fimbriated E. coli, labelled with fluoresceinisothiocyanate (FITC) to human buccal epithelial cells. Fluorescence microscopic analysis revealed binding of bacteria to 75-95% of epithelial cells. Inhibition experiments with fetuin, alpha 1-acid glycoprotein and N-acetyl neuraminic acid confirmed the specificity of bacterial binding to sialoglycoproteins. Further studies using saliva as an inhibitor resulted in a 4-5 times stronger binding inhibition by newborn saliva in comparison to adult saliva coinciding with a 4-5 times higher content of total N-acetyl neuraminic acid in samples of newborn saliva. In Western blot analysis sialoglycoprotein bands with a molecular weight greater than 200 kD reacting with wheat germ agglutinin (WGA), were only identified in samples of newborn saliva. These bands are classified as mucins on account of molecular weight and staining. These data suggest that saliva mucins could represent a major defense mechanism against bacterial infections at a stage of ontogeny where the secretory IgA-system is not yet developed.

Adult↗

PubMed1991

Temperature-dependent expression of flagella in Legionella.

Legionella pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa flagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserum also reacted with flagellin subunits of L. micdadei, L. hackelia [serogroup (SG) 1 and SG2] and L. longbeachae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent of strain L. pneumophila Philadelphia I was shifted from 30 degrees C to either 37 or 41 degrees C, a decrease in the percentage of flagellated bacteria within the population was observed.

Bacterial Outer Membrane Proteins↗

PubMed1991

Characterization of legiolysin (lly), responsible for haemolytic activity, colour production and fluorescence of Legionella pneumophila.

A genomic library of Legionella pneumophila, the causative agent of Legionnaires' disease in humans was constructed in Escherichia coli K12 and the recombinant clones were tested for haemolysis and other phenotypic properties. Seven clones were identified which were able to confer haemolysis of human, sheep, and canine erythrocytes but which were unable to mediate proteolytic activities or cytotoxic effects on CHO- or Vero cells. Clones that exhibited this haemolytic property were also able to produce a brown colour and a yellow-green fluorescence activity detected on M9 plates containing tyrosine. The genetic determinant encoding these properties, termed legiolysin (lly) was mapped by Tn1000 mutagenesis and by subcloning experiments. Southern hybridization with an lly-specific gene probe showed that this determinant is part of the genome of L. pneumophila but is not identical to a protease gene of L. pneumophila which also mediates haemolysis. Minicell analysis of lly-specific plasmids exhibited a protein of 39 kDa. Polyclonal antibodies generated against a LacZ-Lly hybrid protein also recognized a 39 kDa protein produced either by the recombinant legiolysin-positive E. coli K12 clones or by L. pneumophila wild-type strains.

Animals↗