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.

Fas-mediated cytotoxicity remains intact in perforin and granzyme B antisense transfectants of a human NK-like cell line.

Cell-mediated cytotoxicity (CMC) has traditionally been thought to involve the release of granule components, including perforin and granzymes, from the effector cell (EC) onto the target cell (TC) membrane. Recently, a granule-independent cytolytic mechanism involving the interaction of Fas antigen (CD95) with Fas ligand has been described. We have generated antisense perforin (YT-xP1) and granzyme B (YT-xGrB) transfectants of the human NK-like cell line YT-INDY.

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.

Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability.

In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte.

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.

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.

Synthesis and secretion of bacterial antigens by attenuated Salmonella via the Escherichia coli hemolysin secretion system.

We describe a plasmid system which allows the secretion of foreign antigens in attenuated Salmonella aroA strains by the secretion apparatus of E. coli hemolysin. The gene (or gene fragment) encoding the antigen is inserted in frame into a residual position of the hlyA gene, encoding the HlyA secretion signal (HlyAs).

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.

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.

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.

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.

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.

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.

Ultrastructural study of Listeria monocytogenes entry into cultured human colonic epithelial cells.

Evidence that Listeria monocytogenes enters Caco-2 cells through the apical surface is presented. Attachment of bacteria to host cells seems to induce modifications of microvilli which are either in direct contact with the bacterial surface or in close vicinity, resulting in the formation of lamellipodia involved in the cellular uptake of the bacteria. Such modifications are not induced by L.

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.

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.

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.

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.

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.

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.

Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation.

Coexpression of pairs of nonhaemolytic HlyA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one HlyA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region.

The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity.

Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RIII and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L.

Listeria monocytogenes--a model system for studying the pathomechanisms of an intracellular microorganism.

Virulence of Listeria monocytogenes is determined by a cluster of five genes in the order plcA, hly, mpl, actA and plcB, which are coordinately regulated by a transcriptional activator, termed PrfA. The gene for PrfA is located in front of plcA. Mutations within each of these genes reduce the virulence considerably and render the mutants unable to properly multiply and/or spread within the infected host cells.