Search PubMed⌕ Search

Biomedical subjects

W Goebel

Publications and source records attributed to W Goebel.

At least 109 records · Page 6Linked to original sources
PubMed1996

Conversion of bacterial gene products to secretion-competent fusion proteins.

We describe an efficient and easy procedure that allows the generation, detection and secretion of foreign proteins by the secretion apparatus of E. coli hemolysin. The gene (or gene fragment) encoding the foreign protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyAs). Generally, the expressed fusion is efficiently secreted into the culture supernatant of the producing strain. The new approach allows the direct generation of fusion proteins from genomic DNA fragments. The successful use of this method is demonstrated by cloning of random chromosomal DNA fragments from Salmonella typhimurium.

Bacterial Proteins↗
PubMed1995

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli.

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the overexpressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.

Amino Acid Sequence↗
PubMed1995

Salmonella strain secreting active listeriolysin changes its intracellular localization.

We describe the construction of an attenuated Salmonella dublin aroA strain which secretes via the Escherichia coli hemolysin secretion machinery an active hybrid cytolysin consisting of listeriolysin from Listeria monocytogenes and the C-terminal secretion signal of E. coli hemolysin. This hemolytic S. dublin strain is partially released into the cytoplasm of the host cell following uptake by J774 macrophage cells, whereas the nonhemolytic control S. dublin aroA strain remains in the phagosome.

Animals↗
PubMed1995

Listeria monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated Salmonella typhimurium.

The extracellular protein p60 is a major virulence factor of the intracellular bacterium Listeria monocytogenes. Its roles in pathogen survival in vivo and host cell invasion in vitro were studied. To this end, Salmonella typhimurium SL7207 was used as carrier for secreted p60-HlyA fusion protein by Escherichia coli HlyB and HlyD transport proteins. C57BL/6 mice infected intravenously with this strain suffered from increased bacterial numbers in livers and spleens compared with the p60-nonexpressing control strain, but only transiently. In vitro experiments showed that p60 promotes invasion of recombinant S. typhimurium SL7207 p60 into hepatocytes and resting macrophages independent from complement. Moreover, the uptake of wild-type L. monocytogenes EGD and L. monocytogenes BUG 8, an internalin-deficient strain, into hepatocytes was partially blocked by anti-p60 antibodies. The impaired invasion of dissociated bacterial chains of L. monocytogenes RIII, a p60 expression mutant, into hepatocytes and macrophages was partially restored by addition of p60- or p60-HlyA-enriched bacterial supernatants. These data suggest that the L. monocytogenes surface-associated proteins, p60 and internalin, act in concert to achieve optimal uptake into nonprofessional phagocytes and macrophages. Together, these experiments reveal a substantial impact of p60 on cell invasion and virulence and thus emphasize the importance of the intracellular habitat for survival of L. monocytogenes in the host.

Animals↗
PubMed1995

Color Doppler imaging: a new technique to assess orbital blood flow in patients with diabetic retinopathy.

PURPOSE: Color Doppler imaging is a new noninvasive technique that enables measuring blood flow velocity in small orbital vessels, arteries as well as veins. Because hemodynamic changes are seen in patients with diabetic retinopathy by other techniques, the authors compared 61 eyes with proliferative, 59 eyes with nonproliferative, and 26 eyes with preproliferative diabetic fundus changes with a matched control group of 70 patients without diabetes (128 eyes). METHODS: The central retinal artery (CRA), short posterior ciliary artery (PCA), and ophthalmic artery (OA) of all patients were examined, and the systolic, diastolic, and mean velocities were measured for each vessel. RESULTS: Differences between the groups were most prominent in the CRA. The perfusion velocity was significantly lower (P < 0.001) in proliferative eyes (Vsystolic 5.7 +/- 1.8 cm/sec) than in the control group (Vsystolic 9.4 +/- 1.2 cm/sec) or in nonproliferative eyes (Vsystolic 8.4 +/- 1.8 cm/sec). In the preproliferative group, there was greater variability in velocity distribution. Consequently, no statistically significant difference could be deduced, either in the group with background retinopathy or in the group with proliferative diabetes. In the OA and PCA, neither group showed significant differences from normal. CONCLUSIONS: Measurements indicate a correlation between severity of diabetic retinopathy and decreased flow velocity in the CRA.

Adult↗
PubMed1994

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD.

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HlyD (region B), located between amino acids Leu127 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HlyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.

Amino Acid Sequence↗
PubMed1994

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA.

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hylA from the 5' end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E. coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

Antibodies, Bacterial↗
PubMed1994

Host cell responses to Listeria monocytogenes infection include differential transcription of host stress genes involved in signal transduction.

We examined the effect of Listeria monocytogenes infection of J774 macrophage-like mouse cells on induction of several stress genes, including genes for heat shock proteins (HSPs) and a protein-tyrosine phosphatase (PTP), to understand the host response in various steps of the bacterial invasion process. Exposure to wild-type L. monocytogenes strain EGD elicited an early induction of HSP70 mRNA with a corresponding early appearance of HSP70 protein. Cytochalasin D pretreatment prevented the induction of HSP70 mRNA in L. monocytogenes-infected macrophages. After a 2-hr infection with L. monocytogenes, PTP and to a lesser extent HSP90 mRNA levels were elevated. A listeriolysin-negative mutant of L. monocytogenes strain EGD and a noninvasive species of Listeria, Listeria innocua, did not induce PTP or HSP90 mRNA in infected macrophages. Mutations in other virulence genes did not affect transcription of PTP or HSP90. Expression of HSP60 mRNA remained constant over the time course studied in wild-type or mutant strains. These results suggest that phagocytosis of L. monocytogenes triggers transcription of HSP70 mRNA in macrophages; however, escape from the phagosome appears to be necessary for induction of PTP and HSP90 mRNA. Since both PTP and HSP90 may have links with signal transduction pathways in eukaryotic cells, the induction of these mRNAs suggests a role for L. monocytogenes in influencing the signal transduction routes of the host cell.

Animals↗
PubMed1994

A cytolysin encoded by Salmonella is required for survival within macrophages.

A Salmonella gene encoding a cytolysin has been identified by screening for hemolysis on blood agar. DNA sequence analyses together with genetic mapping in Salmonella suggest that it is unrelated to other toxins or hemolysins. The gene (slyA) is present in every strain of Salmonella examined, in Shigella, and in enteroinvasive Escherichia coli but not in other Enterobacteriaceae. SlyA (salmolysin) purified from a derivative of the original clone has hemolytic and cytolytic activity and has a molecular weight predicted by the DNA sequence. The median lethal dose and infection kinetics in mice suggest that the toxin is required for virulence and facilitates Salmonella survival within mouse peritoneal macrophages.

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of prfA and PrfA-regulated virulence genes in Listeria monocytogenes.

The ActA protein, the lecithinase PlcB and listeriolysin are the major PrfA-dependent proteins synthesized when brain-heart infusion (BHI)-cultured Listeria monocytogenes is shifted to minimum essential medium (MEM) in the presence of the transcriptional inhibitor rifampicin. Enhanced synthesis of all three proteins under these conditions depends, however, on a short incubation (about 5 min) of the bacteria in MEM without rifampicin, suggesting that induction of these proteins in MEM requires de novo transcription. The enhanced synthesis of these three proteins is observed in the L. monocytogenes wild-type strains EGD and NCTC 7973, both of which belong to the serotype 1/2 a. A significant induction of the bicistronic mRNA for ActA and PlcB is observed in both strains shortly after shifting the bacteria from BHI to MEM. This mRNA as well as the monocistronic listeriolysin (hly)-specific mRNA is highly stable in L. monocytogenes NCTC 7973 shifted to MEM. In contrast to the actA-plcB mRNA, no enhanced transcription in MEM is observed for the regulatory prfA gene or for the PrfA-controlled virulence genes hlyA and plcA in strain NCTC 7973. However, transcription of these genes is induced in strain EGD. Transcriptional induction of the mpl gene is observed in neither strain NCTC 7973 nor in strain EGD. The life-time of the prfA, plcA, and mpl transcripts is short. ActA was also found to be the most abundant newly synthesized surface protein when the two wild-type strains of L. monocytogenes replicated within the phagocytic cell line J774. ActA synthesis seemed to be induced in the cytoplasm since the non-haemolytic mutant M3 did not induce ActA when taken up by J774 cells.

Animals↗
PubMed1994

The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators.

The two pathogenic Listeria species, L. ivanovii and L. monocytogenes, can be differentiated biochemically and show different host ranges. Virulence of L. monocytogenes is dependent on the integrity of prfA which positively and co-ordinately regulates transcription of several virulence genes. Until now, a prfA homologue had not been identified in L. ivanovii. We have now cloned a chromosomal region from L. ivanovii comprising two genes with high homology to the plcA and prfA genes from L. monocytogenes. Distal from prfA, an open reading frame highly homologous to a phosphoribosyl pyrophosphate synthetase gene (prs) was newly identified, defining the border of the virulence gene cluster. Transcription of the gene for ivanolysin O and expression of other genes of the virulence gene cluster in L. ivanovii were dependent on PrfA. The pattern of PrfA-dependent proteins (PdPs) expressed in L. ivanovii was similar, but not identical to that of L. monocytogenes. The PrfA proteins, as predicted from nucleotide sequences of both pathogenic Listeria species, are very similar and show significant homology to the Crp-Fnr family of global transcription regulators.

Amino Acid Sequence↗
PubMed1994

Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins.

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.

Amino Acid Sequence↗
PubMed1994

Pore formation by the Escherichia coli alpha-hemolysin: role for mediator release from human inflammatory cells.

The Escherichia coli alpha-hemolysin represents a potent stimulus for inflammatory mediator release (O2-, beta-glucuronidase release, and leukotriene generation) from human polymorphonuclear granulocytes, for histamine release from a suspension of human lymphocyte/monocyte basophil cells (LMB), and for serotonin release and 12-hydroxyeicosatetraenoic acid generation from human platelets. In contrast, the E. coli alpha-hemolysin leads to a downregulation of cytokine release (interleukin-1 beta [IL-1 beta], IL-6, and tumor necrosis factor alpha) from human LMB. Recently, it became apparent that the E. coli alpha-hemolysin is composed of several functional structures. We analyzed the role of pore formation, pore stability, and calcium-dependent membrane binding for inflammatory mediator release by using washed bacteria as well as culture supernatants of isogenic recombinant E. coli strains expressing no hemolysin (Hly-), the wild-type hemolysin (Hly+), or hemolysin molecules deficient or modulated in defined functions (pore formation, calcium-dependent membrane binding, or pore stability). In human granulocytes and platelets, mutant hemolysin with enhanced pore stability did not lead to a further increase in induction; mutant hemolysin deficient in pore-forming activity or calcium-dependent membrane binding no longer induced leukotriene B4 generation or beta-glucuronidase release compared with the wild-type hemolysin. Similar results were obtained with regard to histamine release from human LMB. The induction of cytokine release from human LMB differed depending on the type of mutant E. coli alpha-hemolysin. The wild-type hemolysin, the mutant hemolysin with enhanced pore-forming activity, and, to a lesser degree, the mutant hemolysin deficient in pore-forming activity decreased cytokine release (IL-1 beta, IL-6, IL-8, and tumor necrosis factor) compared with untreated cells. In contrast, the mutant hemolysin deficient in calcium-dependent membrane binding led to an increase of up to 50% in cytokine release compared with that by unstimulated cells. Our results indicate that simultaneous expression of the pore-forming and calcium-dependent membrane-binding activities of the hemolysin molecule was necessary to obtain the full cellular inflammatory response pattern observed with the wild-type hemolysin.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua.

Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. The species secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by the plcA gene. A plcA gene from L. monocytogenes was cloned downstream of a gram-positive promoter in the plasmid pWS2-2. To determine what effect plcA would have on intracellular survival when introduced into Listeria innocua, a species that does not growth intracellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/pWS2-2 was confirmed on a brain heart infusion-phosphatidylinositol agar plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured in the macrophage-like cell line J774 by Giemsa staining and viable count determinations at specific time points following infection. The J774 cells infected with wild-type L. innocua showed a falling viable count through 8 h postinfection. Although J774 cells infected with L. innocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h postinfection before a subsequent decline again at 16 h postinfection. Giemsa staining revealed fewer than 6 bacteria in individual macrophage cells at 2 h postinfection, and yet approximately 15% of the J774 cells had 6 to 12 bacteria localized to one area of the macrophage cell after 6 h; moreover, electron micrographs showed that the L. innocua/pWS2-2 cells were replicating inside the phagosome of the host cell. Furthermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the compartments were acidified. These results demonstrate that L. innocua cells transformed with the plasmid-borne plcA gene, and expressing functional PI-PLC, are able to grow intracellularly in what appear to be phagolysosomes, although between 3 and 6 h is needed for this to manifest itself. Intracellular growth specifically in L. innocua may be a secondary function associated with the plcA gene product. The addition of this one gene, plcA, to a species of Listeria that in the wild-type state does not replicate intracellularly apparently can now allow some of the bacteria to transiently multiply inside the phagosomes of host macrophage cells.

Amino Acid Sequence↗
PubMed1994

Mammalian cells transfected with the listeriolysin gene exhibit enhanced proliferation and focus formation.

Mouse 3T6 and 3T3 fibroblasts and rat epithelial L2 cells were transfected with recombinant plasmids containing the listeriolysin gene (hly) of Listeria monocytogenes. This bacterial gene (with and without the 5' signal sequence) was cloned under the control of a murine metallothionein promoter, resulting in elevated transcription of both forms of the hly gene after induction with ZnSO4. However, the gene product could be observed only when the listeriolysin gene lacking the 5' signal sequence was used. Intact listeriolysin could not be detected in the cytoplasm or in the supernatant of the hly-transfected cells. 3T6 and L2 cells transfected with the intact hly gene exhibited significantly increased cell proliferation and increased formation of actin microfilaments upon induction of hly expression with ZnSO4. Both cell types are not contact inhibited and formed large piles of spherical cells after transfection with hly. In contrast, contact-inhibited 3T3 cells transfected with the hly gene showed increased proliferation but no formation of such cell aggregates. When 3T6 fibroblasts were transfected with the hly gene without the 5' signal sequence, inhibition of growth, lack of cell layer confluency, and altered (spherical) cell morphology were observed.

3T3 Cells↗
PubMed1994

Induction of cytokines in phagocytic mammalian cells infected with virulent and avirulent Listeria strains.

The present paper analyzes the cytokine response of mouse macrophages during infection by Listeria monocytogenes. The use of different mutants of L. monocytogenes impaired in various steps of the infection process allowed us to dissect the cytokine response. Cytokine mRNA expression was detected by PCR-assisted amplification of RNA extracted from macrophages after infection with different Listeria strains. An increase in the amount of mRNA for tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6 was detected in P388D1 macrophages infected with L. monocytogenes at 4 h postinfection. Interestingly, only hemolytic strains of L. monocytogenes were able to induce IL-1 alpha, IL-6, and TNF-alpha mRNA. This indicated that the induction of these cytokine mRNAs requires entry of the listeriae into the host cell cytoplasm. In contrast, IL-1 beta was also induced by infection with nonhemolytic mutants of L. monocytogenes which remain entrapped within the phagosome. The levels of TNF, IL-1 alpha, and IL-6 found in the supernatants of Listeria-infected P388D1 macrophages generally correlated well with the induction of the respective mRNAs, but it became obvious that cytokine activity is also regulated through posttranscriptional mechanisms. In vitro induction of the cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha was also observed by infection of bone-marrow-derived macrophages with L. monocytogenes.

Animals↗
PubMed1994

Listeria monocytogenes infection enhances transcription factor NF-kappa B in P388D1 macrophage-like cells.

In the present study, we investigated the effect of Listeria monocytogenes infection on the cellular level of the transcription factors NF-kappa B, AP-1, and NF-IL6 in the macrophage-like cell line P388D1 by using electrophoretic mobility shift assays. Infection with L. monocytogenes enhanced the formation of two NF-kappa B-like DNA-protein complexes, C1 and C2, whereas the concentration of AP-1 and NF-IL6 complexes remained unaffected. In supershift assays using NF-kappa B-specific antibodies, complex C2 was identified to be a p50 homodimer (KBF1) and complex C1 was identified as a p50/p65 heterodimer. Both complexes were formed within 10 min after addition of the bacteria. Since the synthesis of tumor necrosis factor alpha and interleukin-1 occurs at later times, these cytokines cannot be the mediators of enhanced NF-kappa B formation. Infection experiments with different nonhemolytic mutants of L. monocytogenes and the use of the phagocytosis inhibitor cytochalasin B suggest that events prior to invasion and escape of the bacteria from the phagosome into the cytoplasm enhance the nuclear transport of p50/p65 NF-kappa B components.

Animals↗