Rapid and efficient site-specific mutagenesis.
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Cloning, Molecular↗
Biomedical subjects
W Goebel
Publications and source records attributed to W Goebel.
Characterization of a Listeria monocytogenes-specific protein capable of inducing delayed hypersensitivity in Listeria-immune mice.
Recovery of the host after infection by the intracellular pathogen Listeria monocytogenes is dependent on cell-mediated immunity. Little is known of the nature of listerial antigens that induce cell-mediated responses in the infected host. In this study we report on the identification and cloning of an Escherichia coli recombinant encoding a listerial antigen, designated ImaA, capable of eliciting a specific delayed-type hypersensitivity response in Listeria-immune mice. Nucleotide sequencing of the Listeria DNA insert in plasmid pLM10 showed that the ImaA gene product consisted of 170 amino acids with a molecular weight of 17,994. The predicted amino acid sequence suggests that the protein is localized to the bacterial plasma membrane or cell wall. The ImaA gene was unique to the pathogenic species L. monocytogenes and Listeria ivanovii; it was not present in any other species of the genus Listeria.
Amino Acid Sequence↗
Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes.
Listeriolysin is a virulence factor that appears to be necessary for the intracellular survival of Listeria monocytogenes. As shown in this investigation, listeriolysin is produced in only small amounts by clinical isolates of L. monocytogenes belonging to the serogroup 1/2a, but its synthesis can be induced by heat shock and to a lesser extent by oxidative stress. In addition to about 15 heat shock proteins that appear to be common to L. monocytogenes and Listeria species that are nonpathogenic for humans, at least five heat shock proteins are specifically coinduced with listeriolysin in all L. monocytogenes strains under heat shock conditions but not in the other Listeria species. One type of L. monocytogenes mutant blocked in the expression of listeriolysin failed to synthesize several of these specific heat shock proteins.
Bacterial Toxins↗
Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes, and basophils and from polymorphonuclear granulocytes.
We investigated the role of Escherichia coli 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 differed 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 effect was dependent on the concentration of bacteria used for preincubation as well as on the preincubation temperature. The various bacterial strains differed in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterial peptide FMLP, and peptidoglycan had no inhibitory effect or even increased subsequent leukotriene 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 B4 generation and reduced w-oxidation of leukotriene B4. Our data suggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after interaction with mannose-resistant E. coli.
Adhesins, Escherichia coli↗
The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes.
The gene of Listeria monocytogenes that encodes a major extracellular protein (p60) was cloned in Escherichia coli. The gene was designated iap, as p60 was previously shown to represent an invasion-associated protein (M. Kuhn and W. Goebel, Infect. Immun. 57:55-61, 1989). The recombinant E. coli clone expressed p60, as shown by immunoblotting. The complete nucleotide sequence of iap was determined. The deduced amino acid sequence of p60 (484 amino acids) contains a putative N-terminal signal sequence of 27 amino acids and an extended repeat region consisting of 19 threonine-asparagine units. Hybridization with the entire iap gene revealed the presence of homologous sequences in most other Listeria species. In contrast, a 400-base-pair internal iap probe which contained the whole repeat region hybridized only with genomic DNA from L. monocytogenes. Four oligonucleotides previously described as specific probes for the detection of L. monocytogenes (A. R. Datta, B. A. Wentz, D. Shook, and M. W. Trucksess, Appl. Environ. Microbiol. 54:2933-2937, 1988) were shown to be part of the iap gene.
Amino Acid Sequence↗
Translocation and compartmentalization of Escherichia coli hemolysin (HlyA).
Hemolysin plasmids were constructed with mutations in hlyB, hlyD, or both transport genes. The localization of hemolysin activity and HlyA protein in these mutants was analyzed by biochemical and immunological methods. It was found that mutants defective in hlyB accumulated internal hemolysin, part of which was associated with the inner membrane and was degraded in the late logarithmic growth phase. In an HlyB+ HlyD- mutant, hemolysin was predominantly localized in the membrane compartment. Labeling of these Escherichia coli cells with anti-HlyA antibody indicated that part of HlyA, presumably the C-terminal end but not the pore-forming domains, was already transported to the cellular surface. This finding suggests that HlyB is able to recognize the C-terminal signal of the HlyA protein and to initiate its translocation across the membranes.
Enzyme-Linked Immunosorbent Assay↗
Production, purification and characterization of hemolysins from Listeria ivanovii and Listeria monocytogenes Sv4b.
In culture supernatants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as listeriolysin O (LLO). In the case of L. ivanovii a second major supernatant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supernatants of L. ivanovii a sphingomyelinase and a lecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-terminal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.
Amino Acid Sequence↗
Mutations affecting hemolysin production in Listeria monocytogenes located outside the listeriolysin gene.
We have investigated the molecular basis of spontaneous mutations leading to non-hemolytic and avirulent variants of the Listeria monocytogenes serotype 1/2a strain NCTC 7973 using Southern hybridization to DNA fragments that harbor the listeriolysin gene (hlyA) and adjacent regions cloned from a L. monocytogenes serotype 1/2a strain. The analysis of such non-hemolytic variants revealed the presence of a deletion of 300 base pairs, located 1.6 kb upstream of an otherwise intact listeriolysin gene. The importance of regions upstream of the hlyA gene in controlling the expression of the listeriolysin gene was further emphasized by the detection of a transposon-derived nonhemolytic mutant in which the transposon had inserted approximately 200 bp upstream of the listeriolysin gene. We conclude that at least two elements, contained within a region encompassing 1.6 kb of sequences upstream of the hlyA gene, may be required for expression of the listeriolysin gene.
Bacterial Toxins↗
Synthesis of listeriolysin in Listeria monocytogenes under heat shock conditions.
Listeriolysin, produced by all virulent Listeria monocytogenes isolates, is an essential virulence factor which appears to be necessary for the intracellular survival of these bacteria. It has been postulated that the intracellular environment imposes stress conditions similar to heat shock on invading bacteria. We show here that listeriolysin was still very efficiently synthesized in one Listeria monocytogenes strain even intracellularly and induced under heat shock conditions in another L. monocytogenes strain. Listeriolysin appears to be the only major extracellular protein synthesized under heat shock conditions; all other heat shock proteins remain cell associated.
Bacterial Toxins↗
Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells.
Mutants of Listeria monocytogenes were recently isolated which are impaired in the synthesis of a major extracellular protein (p60). As shown in this investigation, the p60 mutants have lost the capability of invading nonprofessional phagocytic 3T6 mouse fibroblast cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant proteins of these mutants indicated that no other extracellular protein was altered in these mutants. The p60 mutants formed long cell chains which disaggregated to normal-sized single bacteria upon treatment with partially purified p60. These disaggregated bacterial cells were able to invade 3T6 cells. Physical disruption of the cell chains by ultrasonication produced similar single cells which were, however, noninvasive. Treatment of these ultrasonicated mutant cells with wild-type p60 restored their ability to invade 3T6 cells.
Animals↗
Pore formation by the Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates.
Lipid bilayer experiments were performed in the presence of hemolysin of Escherichia coli. The toxin had a rather low activity in membranes formed of pure lipids, such as phosphatidylcholine or phosphatidylserine. In membranes from asolectin, a crude lipid mixture from soybean, hemolysin was able to increase the conductance by many orders of magnitude in a steep concentration-dependent fashion, which suggested that several hemolysin molecules could be involved in the conductive unit. Furthermore, the much higher toxin activity in asolectin membranes would be consistent with the assumption that this lipid contains a receptor needed for membrane activity of the toxin. The results of single-channel records showed that the membrane activity of hemolysin is due to the formation of ion-permeable channels with a single-channel conductance of about 500 pS in 0.15 M KCl. The hemolysin channel seemed to be formed by a toxin oligomer which showed an association-dissociation reaction and had a mean lifetime of about 2 s at small transmembrane voltages. The conductance of the hemolysin channels was only moderately dependent on the salt concentration in the aqueous phase. Zero-current membrane potential experiments showed that the hemolysin channel is cation selective. The mobility sequence of the cations in the channel was similar to their mobility sequence in the aqueous phase, which was consistent with the assumption that the hemolysin channel is wide and that the interior field strength is not very high. From the single-channel conductance, a lower limit of about 1.0 nm for the effective channel diameter could be estimated.
Cations, Monovalent↗
A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage.
A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular level. Nucleotide sequence determination revealed the presence of two open reading frames. Both open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.
Amino Acid Sequence↗
Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins.
Extra- and intracellular Escherichia coli hemolysin expressed by two cloned hly determinants, both under the control of the activator element hlyR, were analyzed. One determinant carried all four hly genes (hlyC, hlyA, hlyB, and hlyD), whereas the other carried only the two genes (hlyC and hlyA) required for synthesis of active hemolysin but not those essential for its secretion. It was shown that the total amounts of HlyA protein and of hemolytic activity are similar in both cases in logarithmically growing cultures. The E. coli strain carrying the complete hly determinant released most hemolysin into the media and accumulated very little HlyA intracellularly. The active extracellular hemolysin (HlyA*) was inactivated in the stationary phase without degradation of the HlyA protein. In contrast, the hemolysin which accumulated intracellularly in the E. coli strain carrying hlyA and hlyC only was proteolytically degraded at the end of the logarithmic growth phase. Immunogold labeling indicates that active intracellular HlyA bound preferentially to the inner membrane, whereas that part of the extracellular HlyA which remained cell-bound was located exclusively at the cell surface. It was shown by fluorescence-activated cell sorter analysis that active extra- and intracellular HlyA* bound with similar efficiency to erythrocytes, whereas hemolytically inactive HlyA protein did not bind to these target cells.
Biological Transport↗
Purification and characterization of cytolysins from Listeria monocytogenes serovar 4b and Listeria ivanovii.
Several exoproteins from Listeria monocytogenes serovar 4b (NCTC 10527) and Listeria ivanovii (ATCC) 19119, SLCC 2379), respectively, have been purified to homogeneity by thiol-disulfide exchange chromatography and gel filtration. Both strains produce a haemolytic/cytolytic protein of Mr 58 kDa, which has all the properties of a SH-activated cytolysin, the prototype of which is streptolysin O (SLO), and this protein has therefore been termed listeriolysin O (LLO). In addition a protein of Mr 24 kDa from culture supernatants of L. ivanovii co-purified with LLO. The N-terminal aminoacid sequences of both proteins from L. ivanovii have been determined. By mutagenesis with transposons of Gram-positive origin (Tn916 and Tn1545), which have been introduced via conjugation into L. ivanovii, several phenotypic mutants (altered haemolysis on sheep blood agar or lecithinase-negative) were obtained. Results on the properties of these mutants will be presented.
Amino Acid Sequence↗
Role of cloned virulence factors (mannose-resistant haemagglutination, mannose-resistant adhesions) from uropathogenic Escherichia coli strains in the release of inflammatory mediators from neutrophils and mast cells.
Genetically cloned E. coli 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. coli strains with mannose-resistant haemagglutination (MRH+) and mannose-resistant adhesins, e.g. E. coli 536/21 pANN 801/4, 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 K12, without and with mannose-sensitive 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-MRH+ or S-MRH+; 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.
Adhesins, Escherichia coli↗
[Erythrocyte aggregation and stasis: effect of intravascular sedimentation on microvascular perfusion in hypotension and treatment with defibrinogenization (Arwin)].
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Ancrod↗
[Rheology of giant capillary occlusion in secondary Raynaud's phenomenon within the scope of systemic sclerosis].
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Blood Flow Velocity↗
Isolation and characterization of genes coding for proteins involved in the cytolysis by Listeria ivanovii.
We established a library of chromosomal DNA of Listeria ivanovii in the pTZ19R plasmid system, using Escherichia coli DH5 alpha as the host. One recombinant clone reacted strongly with a polyclonal antiserum raised against the listeriolysin O and a second exoprotein (24kDa) of L. ivanovii, which is most probably also involved in cytolytic processes. The recombinant E. coli clone may contain part of the listeriolysin O gene of L. ivanovii.
Blotting, Western↗