TY - JOUR A1 - Bender, L. A1 - Ott, M. A1 - Debes, A. A1 - Rdest, U. A1 - Heesemann, J. A1 - Hacker, Jörg T1 - Distribution, expression and long range mapping of legiolysin gene (lly) specific DNA sequences in Legionellae N2 - The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal Uy-specific DNA probe was used in Southern hybridizations for the detection of Uy-specific DNA in the genomes of legioneUae and other gram-negative pathogenic bacteria. Under conditi9ns of high stringency, tlie Uy DNA probe specifically reacted with DNA fragments fr9m L. pneumophiüz isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bact~ria of other genera. The Uy genewas mapped by pulsed-field gel electrophoresis to the respective genomic Notl fragments of Legionelltz isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59744 ER - TY - JOUR A1 - Bender, L. A1 - Ott, M. A1 - Marre, R. A1 - Hacker, Jörg T1 - Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE) N2 - Various Legionella isolates from different sources and origins were analysed by orthogonal field alternation gel electrophoresis of Not I cleaved genomic DNA. The genome of L pneumophila Philadelphia I, the original isolate of the epidemics in 1976, exhibits only five Not I fragments. Two virulent derivatives. derived from L pneumophila Philadelphia I. which were obtained by prolonged passage on artificial cuhure media, did not differ from their isogenic virulent strain according the Not I fragment pattern. By summing the lengths of the Notl fragments, the genome size of L. pneumophila Philadelphia I was calculated as approximately 3.9 Mb. Environmental L pneumophila strains exhibited different Not I pattems, as did Legionella strains not belongi'ng to the species pneumophila. The usefulness of DNA long range mapping of Legionella ssp. with Notl for epidemiology and evaluation of their evolutionary rela· tionships is discussed. KW - Infektionsbiologie KW - Legionella ssp. KW - Genome analysis KW - Orthogonal field attenuation gel electrophoresis Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59657 ER - TY - JOUR A1 - Blum, G. A1 - Ott, M. A1 - Cross, A. A1 - Hacker, Jörg T1 - Virulence determinants of Escherichia coli O6 extraintestinal isolates analysed by Southern hybridizations and DNA long range mapping techniques N2 - A total of 16 Escherichia coli 06 strains isolated from cases of extraintestinal infections were analysed for the genetic presence and phenotypic expression of fimbrial adhesins ( P, S/FIC, type I), aerobactin and hemolysin. ln 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. ln 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 tagether with Southern hybridization represents a powerful tool for the genetic analysis of pathogenic bacteria. KW - Infektionsbiologie KW - E. coli serotype 06 KW - extraintestinal isolates KW - virulence factors KW - gene probes KW - DNA lang range mapping KW - epidemiology Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59717 ER - TY - JOUR A1 - Fischer, G. A1 - Bang, H. A1 - Ludwig, B. A1 - Mann, K. H. A1 - Hacker, Jörg T1 - Mip protein of Legionella pneumophila exhibits peptidyl-prolyl cis-trans-isomerase (PPIase) activity N2 - Legfonells pneumoph/la is an intracellular paraslte which ts able to survtve and multipJy in human monocytes and alveolar macrophages. The Mtp (macrophage lnfectiv1ty potentlator) protein has been shown to be an essential virulente factor. A search of translated nuclelt .acld data ba.ses has shown that the Mip proteJn from strain Wadsworth possesses reglons homologaus to those found in the FK.506-bindfng proteins (FKBPs) of several different eukaryotlc organisms. FKBPs are abte to bind to the fmmunosuppressant macrollde FK506 and possess peptidyf .. prolyl cisltrans Isomerase (PPiase) activlty. The gene coding for the Mlp proteln was cloned from the ehromo. some of L. pneumophila straln Philadelph·a I and sequenced. II was synthesl%ed in Escherichla coll ·K- 12 and alter purlfication it exhibited PPiase activity catalyslng the slow clsltrans lsomerization of prolyl peptlde bonds. ln ollgopeptides. Mip ls inhibi~ted by FK506 and fully reslstant to cyclosporln A, as was also found for the recently characterlzed FKBP-type PPiases of eukaryotes. However, the N-terminal extenslon of Mip and/or the substltutrons of the vari· ab1e amlno acrds ln the C-termlnal FKBP core Iead to variatlons,. when compared with eukaryotlc FKBPs, Jn substrate specfflclty wlth the Oligopeptide substrates of' type Suc-Aia-Xaa-Pro-Phe·4·nitroanUide. Never· theless, the Legionella Mip factor represents a bacte· rial gene product whtch shares some characteristics normally found in eukaryotic proteins. ln view of the activity of PPiases in protein-folding reactlonsf such prokaryotic FKBP analogues may represent a new class of bacterial. pathogenicity factors. KW - Infektionsbiologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59778 ER - TY - JOUR A1 - Hacker, Jörg A1 - Bender, L. A1 - Ott, M. A1 - Wingeder, J. A1 - Lund, B. A1 - Marre, R. A1 - Goebel, W. T1 - Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vivo and in vitro in various extraintestinal Escherichia coli isolates N2 - No abstract available KW - Infektionsbiologie KW - P-fimbriae KW - hemolysin KW - genomic deletions KW - extraintestinal E. coli KW - virulence modulation Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59608 ER - TY - JOUR A1 - Hacker, Jörg A1 - Hof, H. A1 - Emödy, L. A1 - Goebel, W. T1 - Influence of cloned Escherichia coli hemolysin genes, S fimbriae and serum resistance on pathogenicity in different animal models N2 - The virulence of the uropathogenic E. coli strain 536 (06: K 1 5: H31) which produces the S-fimbrial adhesin (Sfa•), is serum-resistant (Sre+) and hemolytic (Hiy+) and its derivatives were assessed in five different animal models. Cloned hemolysin (h/y) determinants from the Chromosomes of 06,018 and 075 E. colistrains and from the plasmid pHiy152 were introduced into the spontaneaus Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains {lnfect. Immun. 42: 57-63) the 018-hly determinant but not the plasmid-encoded hly determinant of pHiy 1 52 transformed into 536-31 contribute to lethality in a mouse peritonitis modal. Similar results were obtained with both Hlyhost strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 1 5 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. ln centrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. ln a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a Ievel comparable to that of the parental 536 strain. KW - Infektionsbiologie KW - E. coli hemolysin KW - S-fimbriae KW - serum resistance KW - E. coli virulence KW - animal models KW - gene cloning Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59423 ER - TY - JOUR A1 - Hacker, Jörg A1 - Hughes, C. A1 - Hof, H. A1 - Goebel, W. T1 - Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice N2 - After intraperitoneal injection of mice with Escherichia coli strains isolated from patients with urinary tract infections, the mortality due to hemolytic (Hly+) and nonhemolytic (Hiy-) isolates was 77 and 40%, respectively. Deletion of the chromosomal hemolysin (h/y) determinant in an E. co/i 06:K15:H31 urinary tract infection strain led to a significant reduction in toxicity for mice, and its reintroduction on a recombinant plasmid partially restored the original toxicity. Although introduction of the cloned plasmid pHiy152-encoded hly determinant into the Hly- E. coli 06 mutant strain increased toxicity by only a marginal degree, transformation with the cloned chromosomal hly determinants from two E. coli strains of serotypes 018ac:K5:H- and 075:K95:H? resulted in markedly greater toxicity, even exceeding that of the original Hly+ E. coli 06 wild-type strain. KW - Infektionsbiologie Y1 - 1983 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59330 ER - TY - JOUR A1 - Hacker, Jörg A1 - Kestler, H. A1 - Hoschützky, H. A1 - Jann, K. A1 - Lottspeich, F. A1 - Korhonen, T. K. T1 - Cloning and characterization of the S fimbrial adhesin (SfaII) complex of an Escherichia coli O18:K1 meningitis isolate N2 - S fimbrial adbesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newbom meningitis. We recently described tbe cloning and molecular cbaracterization of a determinant, termed sftJI, from the chromosome of an E. coli urinary tract infection strain. Herewe present data conceming a S fimbria-specific gene duster, designated sfall, of an E. coli newbom meningitis strain. Like tbe Sfal complex, Sfall consists of tbe major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding tbe subunit proteins of Sfall were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with tbose of the Sfal complex subunits. Altbough the sequences ofthe two major SfaA subunits ditf'ered markedly, tbe sequences ofthe minor subunits sbowed 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 tbe meninigitis isolate. These data were confirmed by tbe isolation and characterization of tbe SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of Sfal and Sfall revealed that difl'erences between the two Sfa complexes with respect to tbeir capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other tban SfaS. KW - Infektionsbiologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59853 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, M. A1 - Hof, H. T1 - Effects of low, subinhibitory concentrations of antibiotics on expression of a virulence gene cluster of pathogenic E. coli by using a wild-type gene fusion N2 - No abstract available KW - Infektionsbiologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59874 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, M. A1 - Ludwig, B. A1 - Rdest, U. T1 - Intracellular survival and expression of virulence determinants of Legionella pneumophila N2 - Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as weil 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 fibro:blastic 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 sturlies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella. Zusammenfassung: Intrazelluläres Überleben N2 - Intrazelluläres Überleben und Expression der Virulenzdeterminanten vonLegionella pneumophila. L. pneumophila, der Erreger der Legionärskrankheit, hat die Fähigkeit, sowohl in Makrophagen als auch in Protozoen zu leben und sich dort zu vermehret;t. Legionellen inhibieren die Fusion von Phagosom und Lysosom und hemmen die Ansäuerung des Phagosoms. Mit Hilfe von zwei unterschiedlichen Zellkultur-Systemen konnte gezeigt werden, daß Legionella-Stämme ihre Virulenz nach Laborpassage verlieren. Um die Mechanismen zu studieren, die für das intrazelluläre Überleben von Legionellen verantwortlich sind, haben wir eine Genbank des Legionella pneumophila-Stammes Philadelphia I in Escherichia coli K-12angelegt. Mit Hilfe der Cosmid-Klonierungstechnik war es möglich, fünf putative Virulenzfaktoren zu klonieren. Zwei von diesen Faktoren haben hämolytische Eigenschaften und drei sind Membran-assoziierte Proteine mit Molekulargewichten von 19, 26 und 60 kilodalton. Eines der hämolytischen Proteine, das Legiolysin, lysiert spezifisch humane Erythrozyten. Das zweite Hämolysin zeigt zusätzlich proteolytische Eigenschaften und schädigt sowohl Vero- als auch CHO-Zellen. Weitere Studien sind notwendig, um die Rolle der klonierten Proteine in der Pathogenese von Legionella exakt zu bestimmen. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59681 ER -