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 - Rdest, Ursula A1 - Wintermeyer, E. A1 - Ludwig, B. T1 - Legiolysin, a New Hemolysin from L. pneumophila N2 - Legionella pneumophila generares exotoxins, cytolysins, proteases oc hemolysins that darnage host cells llke erythrocytes or rissue cu lrure cells. The gene for a new L. pneumophila hemolysin withour a proteolytic activiry was idemified, cloned in E. coli and sequenced. The gene producr was analysed by SDS-Polyacrylamide-gel-electrophoresis. N2 - Legionella pneum.ophila bildet Exoroxine, Zytolysine, Proteasen oder Hämolysine, die Wirtszellen wie Erythrozyten oder Animalzellen schädigen. Das Gen für ein neues L. pneumophila Hämolysin ohne proteolytische Aktivität wurde identifiziert, in E. coli kloniert und sequenziert. Das Genprodukt wurde durch SDS-Gelelcktropborese analysiert. KW - Hämolysin KW - Legionella pneumophila Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-73070 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 - TY - JOUR A1 - Wintermeyer, E. A1 - Rdest, U. A1 - Ludwig, B. A1 - Debes, A. A1 - Hacker, Jörg T1 - Characterization of legiolysin (lly); responsible for hemolytic activity, colour production and fluorescence of Legionella pneumophila N2 - No abstract available KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59706 ER - TY - JOUR A1 - Ludwig, B. A1 - Schmid, A. A1 - Marre, R. A1 - Hacker, Jörg T1 - Cloning, genetic analysis and nucleotide sequence of a determinant coding for a 19 kd peptidoglycan-associated protein (Ppl) of Legionella pneumophila N2 - A genomic library of Legionello pneumophihz, the causative agent of Legionnaires disease in humans, was constructed in Escherichill coli K-12, and the recombinant clones were screened by immuno-colony blots with im antiserum raised against heat-killed L. pneumophilo. Twenty-three clones coding for a LegioneUa-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found tobe associated with the peptidoglycan layer bothin L. pneumophilo andin the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions witb a monospecific polyclonal 19-kDa protein-specific antiserum. Tbe protein was termed peptidoglycan-associated protein of L. pneumophilo (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb Clol 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 Clol fragment. Exonuclease 111 digestion studies confirmed tbat pplA is the gene coding for the peptidoglycan.;.associated 19-kDa protein of L. pneumophilo. The amino acid sequence of PpiA exhibits a high degree of homology to the sequences of the Pal Iipoproteins of E. coli K-12 and liaemophilus injluenvze. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59721 ER - TY - JOUR A1 - Schmoll, T. A1 - Morschhäuser, J. A1 - Ott, M. A1 - Ludwig, B. A1 - Van Die, I. A1 - Hacker, Jörg T1 - Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesin determinant: nucleotide sequence of the genes sfaB, C, D, E, F. N2 - The S fimbrial adhesin (sfa) determinant of E. co/i comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfaC 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 sturlies indicated that the genes sfaB and sfaC play a role in regulation of the sfa determinant. ln addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfaA and sfaG responsible for S subunit proteins, were determined. lt 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), F1 C fimbriae (foc) and type I fimbriae ( fim). The evolutionary relationship of fimbrial adhesin determinants is discussed. KW - Infektionsbiologie KW - Escherichia coli KW - S fimbrial adhesin (Sfa) KW - genetic organization KW - gene regulation KW - nucleotide sequence Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59661 ER -