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Institute
- Institut für Molekulare Infektionsbiologie (301) (remove)
Sonstige beteiligte Institutionen
- Genelux Corporation, San Diego Science Center, 3030 Bunker Hill Street, Suite 310, San Diego, California 92109, USA (1)
- Institut für Molekulare Infektionsbiologie (MIB) der Universität Würzburg (1)
- MRB Forschungszentrum für Magnet-Resonanz-Bayern e.V., Am Hubland, D-97074 Würzburg (1)
- Research Center for Infectious Diseases (ZINF), University of Wuerzburg, Wuerzburg, Germany, (1)
- Research Center of Infectious Diseases (ZINF) of the University of Wurzburg, Germany (1)
Die MHK-Werte von Desacetylcefotaxim gegen verschiedene, z. T. ampicillinresistente Stämme von Escherichia coH, die mit Hilfe einer Agardilutionsmethode erhoben wurden, waren höher als die von Cefotaxim und Ceftriaxon, jedoch niedriger als die von Cefoxitin. In einem Modell der systemischen Infektion der Maus mit einem plasmidtragenden, betalactamaseproduzierenden Stamm von E. coli führte die Therapie mit Desacetylcefotaxim zu einer starken Reduktion der Keime pro Leber. Im Vergleich zur Therapie mit Cefotaxim trat die protektive Wirkung aber verlangsamt ein. Desacetylcefotaxim ist also kein unnützes Abbauprodukt von Cefotaxim.
Role of alpha-Hemolysin for the in vitro Phagocytosis and intracellular killing of Escherichia coli
(1989)
The_role of a-hemolysin for the elimination of Eschericbia coli by phagocyres in vitro was investigated using sets of isogenic strains which included wild-type a -hemolyric srrains, derived strains with a reduced production of a-hemolysin and derived nonhemolytic strains. Phagocyrosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition rechniques. a-hemolytic strains were phagocytosed and killed ro a Jesser extent than isogenic strains with a reduced production of o:hemoJysin and isogenic nonhemolytic strains. The results obrained with granulocyres were similar to rhose obtained with monocyres although the elimination of bacteria by monocytes was less than that by granulocytes. These resulcs strongJy suggest that production of ahemolysin is a means by which E. coli counteracrs the activity of phagocytes by injuring these cells with the toxin.
DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pi/) exist in a single copy on the chromosome of E. coli 018:K5 strain 2980. In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined. The pil genes were mapped close to the gene clusters thr and Jeu controlling the biosynthesis of threonine and leucine, respectively. The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E. coli chromosomallinkage map.
The avirulent Salmonella typhimurium F885 was transformed with a plasmid carrying the cloned S fimbriae genes of a uropathogenic Escherichia coli. The resulting transformant (F885-1) produced efficiently E. coli S fimbriae and was used for live oral vaccination of rats. For comparison rats were immunized subcutaneously with isolated S fimbriae. Both routes of vaccination resulted in a significant lgG antibody response to S fimbriae. In addition live oral vaccination induced a serum lgA response against S fimbriae. After transurethral infection of rats with a S fimbriae producing E. coli a 10-fold reduction of bacterial counts in the kidney was observed in rats orally vaccinated with F885-1 as compared to unvaccinated controls. This study suggests that the avirulent Salmonella F885 may be used as a fimbrial antigen carrier for oral vaccination against renal infections.
Genetically cloned E. co/i strains expressing cloned virulence factors were studied with regard to their capability to induce inflammatory mediator release from various target cells. Among the strains were E. co/i strains with mannose-resistant haemagglutination (MRH +) and mannose-resistant adhesins, e.g. E. coli 536/21 pANN 80 I /4, E. coli 536/21 pANN 921 and E. coli 536/21 pANN 801-1. In comparison, E. coli 536/21, E. coli 536/21 pGB 30 int and E. coli Kl2, without and with mannosesensitive haemagglutination (MSH±), and adhesins were studied. The properties of the various strains for human PMN with regard to adherence and phagocytosis, chemiluminescence, 5-lipoxygenase activation of arachidonic acid, leukotriene formation, granular enzyme release and release of histamine from rat mast cells were analysed. It is evident that the various 'biochemical processes of cell activation are dissociated events. The highest chemiluminescence response is obtained with strains expressing MSH+, P-M RH+ or S-M RH+; the presence of S-adhesins suppressed the response. Highest leukotriene formation is obtained with E. coli 536/21 pANN 801-4, while E. coli with MSH was inactive. The concomitant presence of haemolysin secretion enhanced mediator release significantly. Our data suggest a potent role for mannose-resistant haemagglutination (MRH), adhesins and haemolysin as virulence factors in inducing the release of inflammatory mediators.
The S flmbrial adhesln (Sfa) enables Esch richla colito attach to slalfc acld-containing receptor molecules of eukaryotJc cells. As prevlously reported, the genetlc determinant coding for the Sfa of an E. co/1 06 strain was cloned, the gene codlng for the major fimbrfal subunit was ldentlfled and sequenced and th.e S speclflc adhesin was detected. Here we present evidence that ln addltlon to the major subunit proteln SfaA three other minor subunit proteins, SfaG (17 kD), SfaS (14kD) and SfaH (31 kD) can be isolated from the S..speclfic flmbrial adhesln complex. The genes coding for these minor subunits were ldenblied, mutagenlzed separately and sequenced. Using haemagglutlnatton tests. electron-microscopy and quantitative ELISA assays with monoclonal anti-SfaA and anti-SfaS antlbodles the functlons of the minor subunlts were determined. lt was determlned that SfaS ls ldentlcal to the S-specific adhesln; whlch also plays a role ln deterrninatlon of the degree of fimbri· ation ofthe cell. The mlnor subunit SfaH also had some Jnfluence on the Ievei of fimbrlation of the cell. while StaG ls necessary for full expression of S·specific binding. lt was further shown that the amino-terminal proteln sequence of the isolated SfaS profein was identJcal to the proteln sequence calculated from the DNA sequence of the sfaS gene locus.
Evidence for T cell recognition in mice of a purified lipophosphoglycan from Leishmania major
(1989)
We have previously reported that a Leishmania major lipophosphoglycan (LPG), given with killed Corynebacterium parvum as an adjuvant, can vaccinate mice against cutaneous leishmaniasis. In order to analyze whetber T cells are able to recognize this important parasite antigen, we have studied both humoral and cellular immune responses to L. major LPG that bad been isolated from promastigotes by sequential solvent extraction and bydrophobic chromatography. The data sbow that immunization of mice with highly purified LPG induced an increase in frequency of L. major-reactive T cells and the production of immunoglobulin G antibodies to LPG. Furthermore, genetically resistant mice infected with L. major were able to develop a specific delayed-type hypersensitivity response in the ear to L. major LPG. These findings strongly suggest that T cells can recognize and respond to glycolipid antigens, in this case a bost-protective Leishmania LPG, even though such antigens appear not to be potent T-cell stimulators in mice.
Leishmanien besitzen eine Vielzahl von Mechanismen, die humorale und zelluläre Immunabwehr effektiv zu unterlaufen. Diese hängen eng mit der Expression von hauptsächlich zwei Glykokonjugaten auf der Parasitenoberfläche zusammen, dem gp63 und dem Lipophosphoglykan. Die Parasiten sind einerseits schlechte Aktivatoren des alternativen Komplementweges und umgehen damit ihre eigene extrazelluläre Lyse. Oberflächengebundene Komplementfaktoren fördern andererseits die Aufnahme der Leishmanien durch Makrophagen. Solange diese nicht durch T-Zellen aktiviert sind, dienen sie den Parasiten als "Refugium". Dies gilt insbesondere, als Leishmanien in der Lage sind, 1. den "oxidative burst" zu hemmen; 2. toxische Sauerstoffmetaboliten zu entgiften; 3. abbauende lysosomale Enzyme zu hemmen und 4. das saure Milieu in den Lysosomen für ihren eigenen Metabolismus auszunutzen. Schließlich unterlaufen Leishmanien die zelluläre Immunabwehr des Wirts, indem sie die Aktivierung von T-Lymphozyten hemmen und die Expansion von T-Zell-Sub-populationen bewirken, die für ihr eigenes Überleben nützlich sind.
Ten monoclonal antibodies (McAbs) raised to Schistosoma japonicum eggs could be assigned using several serological and immunochemical techniques to 3 groups. The McAbs, termed A, B and C-McAbs, apparently recognize carbohydrate epitopes that can be located on the same antigen molecule. The antibodies, generally of IgM isotype, are idiotypically related. They are distinct from another IgM McAb (Group D-McAb) the carbohydrate target epitope of which can also be associated with the epitopes of A. B and C-McAbs. The McAbs produce large vacuolated bleb reactions in the circumoval precipitin test (COPT) and target epitopes have different representations in various life cycle stages such as immature and mature eggs, male and female worms (including S. mansoni). Antigens affinity purified on columns containing A, B, C and D-McAbs stimulate proliferation of T cells from egg-sensitized mice and elicit DTH reactions in such mice. This raises the possibility that the target antigens of these carbohydrate-reactive monoclonal antibodies are immunopathologic and involved in egg-induced granuloma formation.
We have previously shown that during an infection with Leishmania major, susceptible BALB/c mice, as opposed to mice of a resistant strain (C57BLl6), are primed by lipopolysaccharide for the production of high levels of tumor necrosis factor-\(\alpha\) (TNF-\(\alpha\)) which is known to be a potent maerophage M\(\Phi\) stimulator in other parasitic diseases. In the present study we investigated whether TNF-\(\alpha\) activates M\(\Phi\) for killing of L. major parasites. In the absence of interferon-y (IFN-\(\gamma\)) or lipopolysaccharide, TNF-\(\alpha\) (0.025-25000 U/ml) failed to activate peritoneal exudate M\(\Phi\) from BALB/c mice for killling of L. major amastigotes. In the presence of suboptimal doses of IFN-\(\gamma\) (5 or 10 Vlml), however, TNF-\(\alpha\) mediated a rapid elimination of intracellular parasites, which was highly significant compared to IFN-\(\gamma\) alone. Tbe combination of TNF with interleukin 4, in contrast, was inactive in this respect and allowed survival of intracellular parasites. From these data we conelude that the presence of IFN-\(\gamma\) is crucial for TNF-\(\alpha\)-mediated killing of L. major parasites by M\(\Phi\). Disease progression in susceptible mice therefore seems to be a consequence of a deficiency of IFN-\(\gamma\) and a predominance of interleukin 4 rather than the result of an excess amount of TNF-\(\alpha\).
We have assessed the role of tumour necrosis factor-a (TNF) during cutaneous leishmaniasis and demonstrated that significant levels of TNF were released by spleen cells from infected mice after in cirro restimulation with Leishmania major promastigotes. Spleen cells from both genetically resistant and genetically susceptible mice were equally capable of producing TNF. After challenge with bacterial endotoxin, TNF activity could also be demonstrated in the serum of L. mujor-infected mice and the titres correlated with the course of cutaneous disease in susceptible and resistant mice. TNF did not exert a direct leishmanicidal effect in uitro. Furthermore, our study indicated that macrophages are the source of L. major-induced TNF activity and that its elicitation is dependent on the presence of T cells. These findings suggest that TNF acts in concert with other cytokines produced during L. major infection and that its role depends on the composition of T cell subsets and cytokines present.
S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the inftuence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme ß-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-Iac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain S36WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain S36WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate bad a strong inftuence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.
We investigated the roJe of Escherichia coU expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains dUfered in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory eft'ect was dependent on the concentration of bacteria used for preincubation as weil as on the preincubation temperature. The various bacterial strains dift'ered in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterfal peptide FMLP, and peptidoglycan bad no inhibitory eft'ect or even increased subsequent IeukotrieDe formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene 84 generation and reduced w-oxidation of leukotriene 84 • Our data soggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after Interaction with mannose-resistant E. coli.
Escherichia coU K-12 strains producing S-fimbrial adhesins, FlC fimbriae, and mutagenized fimbriae were tested in a binding assay with a renal tubular cell line. S-fimbrial adhesins and FlC fimbriae mediated bindlog 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 Inhibitionprofile of FlC fimbriae resembled that of S fimbriae.
Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE)
(1990)
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.
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.