PubMed Health⌕ Search

Biomedical subjects

J Hacker

Publications and source records attributed to J Hacker.

At least 181 records · Page 10Linked to original sources
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↗
PubMed1991

Analysis of Legionella pneumophila serogroup 6 strains isolated from a hospital warm water supply over a three-year period by using genomic long-range mapping techniques and monoclonal antibodies.

Over a period of 3 years, Legionella pneumophila serogroup 6 strains were isolated from warm water outlets and dental units in the Dental Faculty and from the Surgery and Internal Medicine Clinics at the University of Dresden, Dresden, Germany. In the bacteriological unit of the above-mentioned facility, L. pneumophila serogroups 3 and 12 were grown from warm water specimens. The medical facilities are located in separate buildings connected with a ring pipe warm water system. All L. pneumophila serogroup 6 strains isolated from the warm water supply reacted with a serogroup-specific monoclonal antibody, but not with two other monoclonal antibodies which are subgroup specific, reacting with other serogroup 6 strains. The NotI genomic profiles obtained by pulsed-field gel electrophoresis of 25 serogroup 6 strains isolated from the Dental Faculty over a 3-year period, 1 isolate from the Internal Medicine Clinic, and 4 strains from the Surgery Clinic were identical. Furthermore, all these strains hybridized with a 300-kb NotI fragment when a legiolysin (lly)-specific DNA probe was used. The NotI pattern, however, differed from those of six serogroup 6 strains of other origins, one serogroup 12 strain from the bacteriological unit, and another six unrelated strains of serogroups other than serogroup 6. L. pneumophila serogroup 6 strains which can be divided into only two subgroups by the use of monoclonal antibodies are differentiated in at least six NotI cleavage types obtained by pulsed-field electrophoresis.

Antibodies, Monoclonal↗
PubMed1991

Cloning, genetic analysis, and nucleotide sequence of a determinant coding for a 19-kilodalton peptidoglycan-associated protein (Ppl) of Legionella pneumophila.

A genomic library of Legionella pneumophila, the causative agent of Legionnaires disease in humans, was constructed in Escherichia coli K-12, and the recombinant clones were screened by immuno-colony blots with an antiserum raised against heat-killed L. pneumophila. Twenty-three clones coding for a Legionella-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found to be associated with the peptidoglycan layer both in L. pneumophila and in the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions with a monospecific polyclonal 19-kDa protein-specific antiserum. The protein was termed peptidoglycan-associated protein of L. pneumophila (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb ClaI fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18.9 and 16.8 kDa, respectively, were located on the ClaI fragment. Exonuclease III digestion studies confirmed that pplA is the gene coding for the peptidoglycan-associated 19-kDa protein of L. pneumophila. The amino acid sequence of PplA exhibits a high degree of homology to the sequences of the Pal lipoproteins of E. coli K-12 and Haemophilus influenzae.

Amino Acid Sequence↗
PubMed1991

Virulence patterns and long-range genetic mapping of extraintestinal Escherichia coli K1, K5, and K100 isolates: use of pulsed-field gel electrophoresis.

A total of 127 extraintestinal Escherichia coli strains of the capsule serotypes K1, K5, and K100 from human and animal sources were analyzed for DNA sequences specific for the genes for various adhesins (P fimbriae [pap] and P-related sequences [prs], S fimbriae [sfa]/F1C fimbriae [foc], and type I fimbriae [fim]), aerobactin (aer), and hemolysin (hly). The expression of corresponding virulence factors was also tested. Twenty-four selected strains were analyzed by long-range DNA mapping to evaluate their genetic relationships. DNA sequences for the adhesins were often found in strains not expressing them, while strains with hemolysin and aerobactin genes usually did express them. Different isolates of the same serotype often expressed different virulence patterns. The use of virulence-associated gene probes for Southern hybridization with genomic DNA fragments separated by pulsed-field gel electrophoresis revealed that a highly heterogeneous restriction fragment length and hybridization pattern existed even within strains of the same serotype. Long-range DNA mapping is therefore useful for the evaluation of genetic relatedness among individual isolates and facilitates the performance of precise molecular epidemiology.

Adhesins, Escherichia coli↗
PubMed1991

Distribution, expression, and long-range mapping of legiolysin gene (lly)-specific DNA sequences in legionellae.

The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal lly-specific DNA probe was used in Southern hybridizations for the detection of lly-specific DNA in the genomes of legionellae and other gram-negative pathogenic bacteria. Under conditions of high stringency, the lly DNA probe specifically reacted with DNA fragments from L. pneumophila isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bacteria of other genera. The lly gene was mapped by pulsed-field gel electrophoresis to the respective genomic NotI fragments of Legionella isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates.

Animals↗
PubMed1991

Pulsed field electrophoresis of genomic restriction fragments for the detection of nosocomial Legionella pneumophila in hospital water supplies.

Ten Legionella pneumophila strains isolated from different sources were analyzed according to their restriction fragment patterns obtained by cleavage of genomic DNA with NotI and SfiI and separation by pulsed field electrophoresis. Three L. pneumophila isolates from a nosocomial outbreak in Lübeck (Germany) and three other L. pneumophila strains independently isolated from a water tap located in the care unit where the patients were hospitalized exhibited identical restriction fragment profiles. Therefore, we concluded that these environmental specimens were the source of the Legionnaires disease. Another two isolates from patients and two strains from the environment, all unrelated to the outbreak described, showed different cleavage patterns.

Adult↗
PubMed1990

Genome analysis of Legionella ssp. by orthogonal field alternation gel electrophoresis (OFAGE).

Various Legionella isolates from different sources and origins were analysed by orthogonal field alternation gel electrophoresis of NotI cleaved genomic DNA. The genome of L. pneumophila Philadelphia I, the original isolate of the epidemics in 1976, exhibits only five NotI fragments. Two virulent derivatives, derived from L. pneumophila Philadelphia I, which were obtained by prolonged passage on artificial culture media, did not differ from their isogenic virulent strain according the NotI fragment pattern. By summing the lengths of the NotI fragments, the genome size of L. pneumophila Philadelphia I was calculated as approximately 3.9 Mb. Environmental L. pneumophila strains exhibited different NotI patterns, as did Legionella strains not belonging to the species pneumophila. The usefulness of DNA long range mapping of Legionella ssp. with NotI for epidemiology and evaluation of their evolutionary relationships is discussed.

Biological Evolution↗
PubMed1990

Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vitro and in vivo in various extraintestinal Escherichia coli isolates.

Fimbrial adhesins and hemolysins contribute to pathogenicity of extraintestinal Escherichia coli isolates causing urinary tract infections (UTI), sepsis and new born meningitis (NBM). Using gene cloning techniques and pulse field electrophoresis in combination with Southern hybridizations it was demonstrated that the genetic determinants coding for P and 'P-related' fimbrial adhesins and hemolysins are closely linked on the chromosomes of different pathogenic E. coli wild-type isolates. For two UTI strains, 536 (O6:K15) and J96 (O4:K6), a co-deletion of the linked gene clusters coding for hemolysin and fimbriae was observed. The deleted DNA regions which also comprise flanking DNA sequences were termed 'pathogenicity DNA islands'. Such 'pathogenicity DNA islands' were also detected in the genome of O18:K1 isolates of OMP type 6 but were absent on the chromosomes of O18:K1 strains of OMP type 9. A mutant strain, 536-22 was selected from rat kidneys after intraurethral infection of animals with the wild-type parental strain 536. This particular isolate also shows deletions of 'pathogenicity islands' leading to a non-pathogenic phenotype. It is therefore concluded that excisions of 'pathogenicity islands' from chromosomes of pathogenic E. coli strains are not restricted to the laboratory but also occur in vivo. The generation of deletions may represent a general mechanism of bacterial virulence modulation.

Adhesins, Escherichia coli↗
PubMed1990

Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesion determinant: nucleotide sequence of the genes sfa B, C, D, E, F.

The S fimbrial adhesin (sfa) determinant of E. coli comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfa C situated proximal to the main structural gene sfaA is described. Sfa-LacZ fusions show that the two genes are transcribed in opposite directions. The isolation of mutants in the proximal region of the sfa gene cluster, the construction of sfa-phoA gene fusions and subsequent transcomplementation studies indicated that the genes sfa B and sfa C play a role in regulation of the sfa determinant. In addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfa A and sfa G responsible for S subunit proteins, were determined. It is suggested that these genes are involved in transport and assembly of fimbrial subunits. Thus the entire genetic organization of the sfa determinant is presented and compared with the gene clusters coding for P fimbriae (pap), F1C fimbriae (foc) and type I fimbriae (fim). The evolutionary relationship of fimbrial adhesion determinants is discussed.

Adhesins, Escherichia coli↗
PubMed1990

Genetically engineered S and F1C fimbriae differ in their contribution to adherence of Escherichia coli to cultured renal tubular cells.

Escherichia coli K-12 strains producing S-fimbrial adhesins, F1C fimbriae, and mutagenized fimbriae were tested in a binding assay with a renal tubular cell line. S-fimbrial adhesins and F1C fimbriae mediated binding to tubular cells. The SfaA, SfaG, and SfaS subunits of S fimbriae contributed to attachment. Site-specific mutations in the sfaS gene reduced binding. The inhibition profile of F1C fimbriae resembled that of S fimbriae.

Adhesins, Escherichia coli↗