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W Goebel

Publications and source records attributed to W Goebel.

At least 199 records · Page 11Linked to original sources
PubMed1987

Marker exchange mutagenesis of the aerolysin determinant in Aeromonas hydrophila demonstrates the role of aerolysin in A. hydrophila-associated systemic infections.

We report here on the isolation of isogenic strains of Aeromonas hydrophila AB3 deleted for a segment of the aerolysin gene. All aer mutants obtained lacked the 49-kilodalton aerolysin gene product and were neither hemolytic for blood erythrocytes nor cytotoxic for Chinese hamster ovary tissue culture cells. One such mutant, AB3-5, was used in a mouse toxicity model to evaluate the role of aerolysin in the pathogenesis of A. hydrophila infections. The strain had a 50% lethal dose (LD50) of greater than 10(9) as compared with the parental strain which had an LD50 of 5 X 10(7). Reintegration of the deleted segment into AB3-5 resulted in an LD50 of 6 X 10(7) cells for this revertant. Furthermore, all mice injected with a sublethal dose of the parental strains developed necrotic lesions; this was never obtained with the aerolysin-deficient strain AB3-5. More importantly, specific neutralizing antibody to aerolysin was detected in mice surviving A. hydrophila infection, demonstrating that aerolysin is produced during the course of systemic A. hydrophila infections.

Aeromonas↗
PubMed1987

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.

DNA Transposable Elements↗
PubMed1987

Listeria monocytogenes ATCC 35152 and NCTC 7973 contain a nonhemolytic, nonvirulent variant.

Listeria monocytogenes NCTC 7973 and this same strain deposited as ATCC 35152 contain two phenotypes: hemolytic virulent colonies and nonvirulent colonies that show no zones of hemolysis when streaked on heart infusion agar containing 5% rabbit blood. Results of examinations of these virulent and nonvirulent strains by investigators at the Centers for Disease Control, Atlanta, Ga., the Pasteur Institute, Paris, France, and the University of Würzburg, Federal Republic of Germany, support the conclusion that the avirulent strain is a nonhemolytic mutant of the virulent strain and that hemolysin is a virulence factor for L. monocytogenes.

Animals↗
PubMed1986

Influence of cloned Escherichia coli hemolysin genes, S-fimbriae and serum resistance on pathogenicity in different animal models.

The virulence of the uropathogenic E. coli strain 536 (O6:K15:H31) which produces the S-fimbrial adhesin (Sfa+), is serum-resistant (Sre+) and hemolytic (Hly+) and its derivatives were assessed in five different animal models. Cloned hemolysin (hly) determinants from the chromosomes of O6, O18 and O75 E. coli strains and from the plasmid pHly152 were introduced into the spontaneous Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains (Infect. Immun. 42: 57-63) the O18-hly determinant but not the plasmid-encoded hly determinant of pHly152 transformed into 536-31 contribute to lethality in a mouse peritonitis model. Similar results were obtained with both Hly- host strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 15 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. In contrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. In a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a level comparable to that of the parental 536 strain.

Animals↗
PubMed1986

Characterization of Escherichia coli wild-type strains by means of agglutination with antisera raised against cloned P-, S-, and MS-fimbriae antigens, hemagglutination, serotyping and hemolysin production.

E. coli strains isolated from patients with urinary tract infections (UTI) very often possess mannose-sensitive (MS) and mannose-resistant (MR) adherence factors (fimbriae). According to their receptor specificity the mannose-resistant adhesins can be divided into several types, P, S, M and X. We have cloned the determinants of three groups of UTI E. coli adhesins, MS, P and S, and prepared specific antisera against the fimbriae antigens. 189 hemagglutination (HA+)-positive strains, 96 fecal isolates and 93 strains isolated from UTI have been tested with these specific antisera and further characterized by receptor specific HA, HA patterns and further of the "common O serogroups" 01, 02, 04, 06, 07, 08, 018, 025, 075, most prevalent in UTI, and hemolysin production. 68 (73%) of the UTI strains and 50 (52%) of the fecal isolates showed P-receptor specificity; 16 (17%) of the uropathogenic bacteria and 33 (34%) of the fecal strains exhibited S, M or X-fimbriae antigens. 24% of the P-hemagglutinating (P+) strains reacted with P (F8)-specific antiserum. In contrast, more than three quarter of the S+-strains were agglutinated by S-specific antiserum. HA-pattern VI and 018 antigen were found to be associated with P-fimbriae strains, whereas HA-pattern V and VII and the O antigens 02 (M-type), 06 and 018 (S-type) occurred most frequently in P--strains. A high percentage of P-fimbriated strains showed mannose-sensitive hemagglutination and hemolysin production.

Adhesiveness↗
PubMed1986

Production of listeriolysin by beta-hemolytic strains of Listeria monocytogenes.

Listeriolysin was isolated from target rabbit erythrocyte membranes after lysis of the cells with partially purified toxin derived from a culture supernatant of Listeria ivanovii. The membrane form of the toxin exhibited properties similar to those previously found for streptolysin O. Detergent-solubilized, delipidated listeriolysin was found to comprise a heterogeneous population of partially and fully circularized, amphiphilic oligomers whose embedment within the lipid bilayer generated large transmembrane pores. The molecular weight of the toxin monomer was estimated to be 55,000 to 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological cross-reactions between the toxin and streptolysin O were demonstrable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. An immunoblot assay for detecting listeriolysin in agar-incorporated, lysed erythrocyte membranes was developed, and 28 defined, clinical isolates of Listeria monocytogenes were examined for toxin production. These isolates caused beta-hemolysis on the agar plates and had previously been regarded as listeriolysin producers. However, we found that only two isolates produced genuine listeriolysin, since the sensitive immunoblot assay entirely failed to detect the toxin in all other cases. We excluded that this finding derived from proteolytic degradation of membrane-bound toxin. Thus, the great majority of human pathogenic Listeria strains appear to produce one or several hemolysins that are immunologically and, by inference, molecularly distinct from the streptolysin O-related listeriolysin. We propose that the streptolysin O-related toxin be designated alpha-listeriolysin and that the other hemolysin(s) be termed beta-listeriolysin.

Animals↗
PubMed1986

Analysis of the genetic determinants coding for the S-fimbrial adhesin (sfa) in different Escherichia coli strains causing meningitis or urinary tract infections.

Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Escherichia coli isolates. Fimbriae from the resulting Sfa+ E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including O83:K1 isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with F1C fimbriae. Furthermore the sfa+ recombinant DNAs and some cloned sfa-flanking regions were used as probes in Southern experiments. Chromosomal DNAs isolated from O18:K1 and O83:K1 meningitis strains with and without S fimbriae and from uropathogenic O6:K+ strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. No sfa-specific sequences were observed on the chromosome of E. coli K-12 strains and an O7:K1 isolate. With the exception of small alterations in the sfa-coding region the genetic determinants for S fimbriae were identical in uropathogenic O6:K+ and meningitis O18:K1 and O83:K1 strains. The sfa determinant was also detected on the chromosome of K1 isolates with an Sfa-negative phenotype, and specific cross-hybridization signals were visible after blotting against F1C-specific DNA. In addition homology among the different strains was observed in the sfa-flanking regions.

Adhesins, Escherichia coli↗
PubMed1986

Contribution of cloned virulence factors from uropathogenic Escherichia coli strains to nephropathogenicity in an experimental rat pyelonephritis model.

Escherichia coli 536 (O6:K15:H31), which was isolated from a case of urinary tract infection, determines high nephropathogenicity in a rat pyelonephritis system as measured by renal bacterial counts 7 days after infection. The loss of S fimbrial adhesin formation (Sfa-) (mannose-resistant hemagglutination [Mrh-] and fimbria production [Fim-]), serum resistance (Sre-), and hemolysin production (Hly-) in the mutant 536-21 led to a dramatic reduction of bacterial counts from almost 10(5) to only 40 cells per g of kidney. The reintroduction of the cloned S fimbrial adhesin determinant (sfa) increases the virulence of the avirulent mutant strain by a factor of 20; almost the same effect was observed after restoration of serum resistance by integration of an sfa+ recombinant cosmid into the chromosome. Additional reintroduction of the Hly+ phenotype by transformation of two hly determinants increased the virulence of the strains. Hemolysin production determined increased renal elimination of leukocytes and erythrocytes. Thus all three determinants investigated, S fimbriae, serum resistance, and hemolysin, contribute to the multifactorial phenomenon of E. coli nephropathogenicity.

Adhesins, Escherichia coli↗
PubMed1986

Role of Escherichia coli alpha-hemolysin and bacterial adherence in infection: requirement for release of inflammatory mediators from granulocytes and mast cells.

We investigated the role of bacterial mannose-resistant fimbriation of S fimbriae (Fim), mannose-resistant hemagglutination (S-Mrh), and hemolysin (Hly) production by an Escherichia coli parent and genetically cloned strains as regards their effect on histamine release from rat mast cells and generation of the chemiluminescence response, leukotriene, and enzyme release from human polymorphonuclear granulocytes. These mediators are involved in the induction of inflammatory disease processes and lead, e.g., to the enhancement of vascular permeability, chemotaxis, aggregation of granulocytes (leukotriene B4), lysosomal enzyme release, and smooth-muscle contraction (leukotrienes C4, D4, and E4). The content of azurophilic and specific granules in polymorphonuclear granulocytes consists of highly reactive enzymes which amplify inflammatory reactions. Washed bacteria (E. coli 764 Hly+/-, E. coli 21085 Hly+/- Fim+/- Mrh+/-), as well as their culture supernatants, were analyzed at various times during their growth cycle. No differences exist between parent and cloned or mutant strains with respect to their outer membrane proteins and lipopolysaccharide pattern. Washed bacteria [E. coli 764 and 21085(pANN202-312)] which produced hemolysin, unlike Hly- strains, induced high levels of histamine release from rat mast cells and led to a significant chemiluminescence response and enzyme and leukotriene release from human polymorphonuclear granulocytes. Bacterial culture supernatants from Hly+ and secreting strains showed similar results with the exception of E. coli 21085(pANN202-312), which is a hemolysin-producing but not a secretory strain. Our data suggest a potent role for hemolysin as a stimulus for noncytotoxic mediator release from various cells. Furthermore, we showed that the presence of Fim and S Mrh potentiates mediator release. The simultaneous presence of Mrh and Fim [E. coli 535/21(pANN801-4)] increased mediator release compared with Mrh+ Fim- strains [E. coli 536/21(pANN801-1)]. E. coli 536/21 (Msh- Mrh- Fim- Hly-) did not induce mediator release.

Arachidonic Acid↗
PubMed1986

Cloning, expression, and mapping of the Aeromonas hydrophila aerolysin gene determinant in Escherichia coli K-12.

DNA sequences corresponding to the aerolysin gene (aer) of Aeromonas hydrophila AH2 DNA were identified by screening a cosmid gene library for hemolytic and cytotoxic activities. A plasmid containing a 5.8-kilobase EcoRI fragment of A. hydrophila DNA was required for full expression of the hemolytic and cytotoxic phenotype in Escherichia coli K-12. Deletion analysis and transposon mutagenesis allowed us to localize the gene product to 1.4 kilobases of Aeromonas DNA and define flanking DNA regions affecting aerolysin production. The reduced hemolytic activity with plasmids lacking these flanking regions is associated with a temporal delay in the appearance of hemolytic activity and is not a result of a loss of transport functions. The aerolysin gene product was detected as a 54,000-dalton protein in E. coli maxicells harboring aer plasmids and by immunoblotting E. coli whole cells carrying aer plasmids. We suggest that the gene coding aerolysin be designated aerA and that regions downstream and upstream of aerA which modulate its expression and activity be designated aerB and aerC, respectively.

Aeromonas↗
PubMed1986

Large, unstable inserts in the chromosome affect virulence properties of uropathogenic Escherichia coli O6 strain 536.

The hemolytic, uropathogenic Escherichia coli 536 (O6:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequency of 10(-3) to 10(-4). These inserts were not found in the chromosome of two nonhemolytic E. coli strains, whereas the chromosomal sequences adjacent to these inserts appeared to be again homologous in the uropathogenic and two other E. coli strains. Insert I was 75 kilobases in size and was flanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation. For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp flanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome. Both inserts contained a functional hemolysin determinant. However, the loss of the inserts not only affected the hemolytic phenotype but led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type fimbriae (sfa). It is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes. Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems.

Adhesins, Escherichia coli↗
PubMed1985

Characterization of the 7S RNA and its gene from halobacteria.

The 7S RNA is an abundant nonribosomal RNA in H. halobium and other halobacteria. A specific 7S RNA gene probe shows high homology to genomic DNA of all halobacteria tested but not to those of several other archaebacteria, eubacteria and eukaryotes. All halobacterial genomes seem to carry a single copy of the 7S RNA gene. The coding region of the 7S RNA gene is highly G+C rich whereas the 5'- and 3'-noncoding regions possess a rather low G+C content. An extended double stranded structure for the 7S RNA is deduced from its nucleotide sequence. The 7S RNA of H. halobium (304 nucleotides) resembles in size and structure the 7S-L RNA from mammalian cells and shares with it a sequence homology of about 50% when arranged in a colinear fashion. The similarities in sequence are found particularly at the 3'- and 5'-termini. No similarity was detected between the 7S RNA from H. halobium and the nonribosomal 6S RNA from Escherichia coli.

Animals↗
PubMed1985

Common structural features of the genes for two stable RNAs from Halobacterium halobium.

The genes coding for the 5S rRNA and another stable RNA, termed 7S RNA, in Halobacterium halobium were isolated from a genomic library of this archaebacterium and their nucleotide sequences determined. Both genes are colinear with their transcripts (5S rRNA and 7S RNA), but 5S rRNA and possibly also 7S RNA isolated from other halobacteria carry additional nucleotides within the RNA transcript. Both genes are located in the G + C rich chromosomal fraction I of H. halobium. Comparison of the 3' non-coding regions of both genes shows a 20 bp sequence of high homology immediately at the 3' ends which is almost symmetrically flanked by two stem-loop structures, one being situated close to the 3' end but within the coding region and the other downstream of the common 20 bp sequence.

Base Sequence↗
PubMed1985

Analysis of the flanking regions from different haemolysin determinants of Escherichia coli.

The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element. Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E. coli strains. Chromosomal hly determinants lack both flanking sequences (IS2 and RS) in the immediate vicinity of the hly genes. The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152. We show that these sequences, which contain one promoter (left promoter, phlyL) in all hly determinants tested, vary considerably although common sequence elements can still be identified. In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes. The A + T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A + T) but even the structural hly genes show a considerably higher A + T content (about 60 mol%) than the E. coli chromosome on average (50 mol% A + T) suggesting that the hly determinant may not have originated in E. coli.

Amino Acid Sequence↗
PubMed1985

Cloning and characterization of genes involved in production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from a uropathogenic O6:K15:H31 Escherichia coli strain.

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) exhibits a mannose-resistant hemagglutination phenotype (Mrh) with bovine erythrocytes and delayed Mrh with human and guinea pig erythrocytes. Neuraminidase treatment of the erythrocytes abolishes mannose resistant hemagglutination, which is typical for X fimbriae. E. coli strain 536 synthesizes two different fimbriae (Fim phenotype) protein subunits, 16.5 and 22 kilodaltons in size. In addition the strain shows mannose-sensitive hemagglutination and common type I (F1) fimbriae. The cosmid clone E. coli K-12(pANN801) and another nine independently isolated Mrh+ cosmid clones derived from a cosmid gene bank of strain 536 express the 16.5-kilodalton protein band, but not the 22-kilodalton protein, indicating an association of the Mrh+ property with the "16.5-kilodalton fimbriae." All cosmid clones were fimbriated, and they reacted with antiserum produced against Mrh+ fimbriae of the E. coli strain HB101(pANN801) and lacked mannose-sensitive hemagglutination (F1) fimbriae. From the Mrh fim cosmid DNA pANN801, several subclones coding for hemagglutination and X fimbriae were constructed. Subclones that express both hemagglutination and fimbriae and subclones that only code for the hemagglutination antigen were isolated; subclones that only produce fimbriae were not detected. By transposon Tn5 mutagenesis we demonstrated that about 6.5 kilobases of DNA is required for the Mrh+ Fim+ phenotype, and the 1.5- to 2-kilobase DNA region coding for the structural protein of the fimbriae has been mapped adjacent to the region responsible for the Mrh+ phenotype. Two different regions can thus be distinguished in the adhesion determinant, one coding for hemagglutination and the other coding for fimbria formation. Transformation of plasmid DNA from these subclones into a Mrh- Fim- mutant of E. coli 536 and into a galE (rough) strain of Salmonella typhimurium yielded transformants that expressed both hemagglutination and fimbria production.

Animals↗
PubMed1985

Bacterial adherence and hemolysin production from Escherichia coli induces histamine and leukotriene release from various cells.

We investigated the role of bacterial adherence and hemolysin production from Escherichia coli parent and genetically cloned strains as to their effects on histamine release from rat mast cells and leukotriene generation from human polymorphonuclear granulocytes. These mediators were involved in the induction of inflammatory disease processes and led, for example, to enhancement of vascular permeability, chemotaxis (leukotriene B4 [LTB4]), chemoaggregation, lysosomal enzyme release, and smooth muscle contraction, (LTC4, LTD4, and LTE4). Washed bacteria (E. coli K-12 MS+ Hly +/-; E. coli 536 MS+ MR +/-) as well as their culture supernatants were analyzed. Washed E. coli K-12 (Hly+), unlike Hly- strains, induced high amounts of histamine release from rat mast cells and chemotactic activity from human polymorphonuclear granulocytes. Significant leukotriene release was obtained with washed E. coli K-12 Hly+ strains and their bacterial culture supernatants. Leukotriene induction was dependent on the amount of hemolysin activity present in the supernatant. However, additional soluble factors should also be considered. The presence of hemolysin appeared to accelerate and enhance the rate of phagocytosis of bacteria by neutrophils. When E. coli 536 (MS+ MR +/- Hly +/-) strains were analyzed, the simultaneous presence of MR+ pili and hemolysin production led to an increase in histamine release as compared with MR- Hly+ strains. The genetically cloned MR+ Hly+ E. coli 536 strain induced higher amounts of leukotrienes as compared with the wild-type strain. Our data suggest a potent role for adhesins and hemolysin as virulence factors in inducing the release of inflammatory mediators.

Adhesiveness↗