Biomedical subjects

M Leimeister-Wächter

Publications and source records attributed to M Leimeister-Wächter.

At least 19 recordsLinked to original sources

PubMed1993

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. Under growth-limiting conditions PrfA-dependent proteins are preferentially synthesised. These studies indicate the existence of additional PrfA-regulated proteins in L. monocytogenes. The synthesis of catalase, superoxide dismutase, LmaA and p60 is not under the control of PrfA. These proteins seem to be also associated with virulence of L. monocytogenes. P60-related proteins are found as major extracellular proteins in all Listeria species but only p60 of L. monocytogenes is able to restore the failure of R-mutants (exhibiting a drastically reduced synthesis of p60) to adhere to 3T6 mouse fibroblasts. Adherence of L. monocytogenes to the epithelial Caco-2 cells seem to be independent of p60. The p60 protein of L. monocytogenes differs characteristically from the p60-related proteins of the nonvirulent Listeria species.

Amino Acid Sequence↗

PubMed1993

Detection of a prfA-independent promoter responsible for listeriolysin gene expression in mutant Listeria monocytogenes strains lacking the PrfA regulator.

Expression of listeriolysin, a major virulence factor of pathogenic Listeria monocytogenes, is positively regulated by the pleiotropic virulence regulator PrfA. In this study, we demonstrate that L. monocytogenes strains lacking the prfA regulator gene produce listeriolysin in small, albeit detectable, amounts when analyzed in a hemolysin assay and by immunoblots with listeriolysin-specific monoclonal antibodies. Transcriptional analysis revealed the existence of a PrfA-independent promoter that was responsible for the hemolytic activity expressed by these strains.

Bacterial Proteins↗

PubMed1992

A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.

The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.

Actin Cytoskeleton↗

PubMed1992

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.

Base Sequence↗

PubMed1992

Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene.

The prfA gene of Listeria monocytogenes encodes a protein that activates transcription of the listeriolysin gene (lisA). In order to explore the role of the prfA gene product in the pathogenesis of listerial infection, we constructed a site-directed insertion mutation in prfA by the chromosomal integration of a novel suicide vector containing a portion of the prfA coding region. This mutation not only transcriptionally silenced the listeriolysin (lisA) gene but also abrogated production of specific RNA transcripts corresponding to the phosphatidylinositol-specific phospholipase C (pic) and metalloprotease (mpl) genes, two further virulence gene products expressed only by pathogenic Listeria strains. The strain was also found to be avirulent when tested in a mouse model of listerial infection. The concomitant loss of multiple characteristics such as production of LisA, Pic, Mpl, and loss of virulence in a mouse infection model is the result of a mutation in a single gene and demonstrates that the prfA gene product is a positive regulator of multiple virulence determinants in L. monocytogenes.

Animals↗

PubMed1992

The expression of virulence genes in Listeria monocytogenes is thermoregulated.

The expression of listeriolysin, a major virulence factor of the gram-positive facultative intracellular pathogen Listeria monocytogenes, is positively regulated by a transcriptional activator, the prfA gene product. We had previously shown that mutations within the prfA gene lead to loss of listeriolysin production. In this communication, the regulation of expression of listeriolysin by a specific environmental condition, namely, temperature, was studied in wild-type strains of Listeria monocytogenes. We found that expression of the hemolysis phenotype was thermoregulated. A lisA::lacZ fusion was constructed, and its expression in the wild-type strain was studied at various growth temperatures. The results showed that the fusion beta-galactosidase activity was expressed only when cultures were grown at temperatures above 30 degrees C. This activity could be either specifically repressed or induced, depending on growth temperature. No change in activity was detected in a strain harboring a control beta-galactosidase fusion at the various growth temperatures tested. Northern (RNA) blot analysis of lisA-specific RNA transcripts showed that thermoregulation is manifested at the level of transcription. We also found that the transcription of other PrfA-regulated virulence genes in L. monocytogenes was similarly affected by growth temperature. Hence, as in other facultative intracellular pathogens, Shigella and Yersinia spp., temperature is an important cue in the induction of expression of virulence genes in L. monocytogenes. Our studies revealed that a higher level of regulation is imposed on the PrfA-mediated activation of virulence genes in pathogenic L. monocytogenes.

Bacterial Toxins↗

PubMed1991

Studies on the pathogenicity of Listeria monocytogenes.

The characterization of mutants of Listeria monocytogenes with reduced virulence properties is described. Reduction in the amount of the extracellular protein p60 (encoded by the ipa gene) leads to cell filaments with impaired invasiveness. Mutants which cannot synthesize listeriolysin are still invasive but unable to survive within phagocytic cells. One type of listeriolysin-negative mutants is defective in the synthesis of a positive regulatory element PrfA which coordinately regulates the listeriolysin gene (lisA) together with several other genes, including those for a phosphatidylinositol-specific phospholipase and a metalloprotease.

Antigens, Bacterial↗

PubMed1991

Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is co-ordinately expressed with listeriolysin in Listeria monocytogenes.

A phosphatidylinositol-specific phospholipase C (PI-PLC) that is unique to the pathogenic Listeria species L. monocytogenes and L. ivanovii has been detected. Deletion analysis performed with Escherichia coli recombinants expressing PI-PLC activity together with maxicell analysis showed that a 34 kDa polypeptide was responsible for this activity. Nucleotide sequencing revealed that the gene encoding this polypeptide comprises 317 amino acid residues with a 22-amino-acid signal peptide. This gene, designated pic for phosphatidylinositol-specific phospholipase C, is located back to back with the listeriolysin gene on the chromosome of L. monocytogenes where these genes are transcribed by divergent non-overlapping promoters. Expression of the pic gene is dependent on the product of the prfA gene, which also regulates expression of the listeriolysin gene in L. monocytogenes.

Amino Acid Sequence↗

PubMed1991

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.

Base Sequence↗

PubMed1991

Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene.

The entire nucleotide sequence of an open reading frame located immediately downstream of the listeriolysin gene from a virulent Listeria monocytogenes serotype 1/2a strain was determined. The product of the open reading frame was 510 amino acids with a predicted molecular weight of 57,400. The deduced amino acid sequence of this open reading frame is highly similar to that of a family of secreted metalloproteases produced by various members of the genus Bacillus, of which thermolysin is the prototype. Immunoblots performed with specific antisera raised against thermolysin from Bacillus stearothermophilus allowed the detection of a 60-kDa polypeptide, corresponding to the pro-form of the protease, in culture supernatants of L. monocytogenes strains. In maxicell experiments, Escherichia coli recombinants harboring this open reading frame also specifically directed production of a 60-kDa protein. Protease activity was low to undetectable in both Listeria strains and E. coli recombinants. This is due to lack of processing of the inactive pro-form of the protease to its mature active form in both species. We have designated this gene mpl for metalloprotease of L. monocytogenes. The gene was present only in pathogenic L. monocytogenes strains, in which it was physically linked to the listeriolysin gene.

Amino Acid Sequence↗

PubMed1990

Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes.

We have isolated, by molecular cloning and genetic complementation of a listeriolysin-negative mutant, a gene required for the expression of this virulence factor in Listeria monocytogenes. The mutant strain SLCC53, which was nonhemolytic and avirulent, harbored a deletion of 450 base pairs located approximately 1500 base pairs upstream of the listeriolysin gene. No transcripts corresponding to the listeriolysin gene were detected in the mutant. DNA sequencing of this region from the hemolytic strain EGD revealed that the region deleted in the mutant would abrogate expression of a 27-kDa polypeptide. Introduction of a recombinant plasmid expressing this 27-kDa polypeptide restored hemolytic activity to the mutant and increased the hemolytic activity of the wild-type L. monocytogenes strain EGD. We have designated the gene encoding the 27-kDa polypeptide prfA, for positive regulatory factor of listeriolysin (lisA) expression. The prfA gene regulates transcription of the lisA gene positively.

Amino Acid Sequence↗

PubMed1990

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↗

PubMed1990

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↗

PubMed1989

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↗

PubMed1989

Specific gene probe for detection of biotyped and serotyped Listeria strains.

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.

Bacterial Typing Techniques↗

PubMed1989

Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri.

The listeriolysin gene from a weakly hemolytic but virulent strain of Listeria monocytogenes serotype 1/2a was cloned in Escherichia coli K-12. Recombinants were identified on the basis of their cross-reactivities to hyperimmune antisera raised against streptolysin O and listeriolysin. Low levels of hemolytic activity were detected in crude lysates of strains harboring the listeriolysin gene. In DNA hybridization studies with five DNA probes that encoded the listeriolysin gene and surrounding sequences, highly homologous listeriolysin genes were found to be present in the species L. monocytogenes, Listeria ivanovii, and Listeria seeligeri. Immunoblotting performed with affinity-purified antibody to listeriolysin allowed the detection of this protein in supernatants of all three species. This study demonstrates for the first time that listeriolysin is produced by L. seeligeri and documents the genetic homology between the various listeriolysins produced by Listeria spp. Sequences unique to the species L. monocytogenes were found to be located downstream of the listeriolysin gene. Furthermore, the restriction fragment length polymorphisms detected with probes flanking the hlyA gene may be useful epidemiological markers in identifying and distinguishing virulent Listeria strains from each other.

Bacterial Toxins↗

PubMed1989

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↗

PubMed1989

Gene probes for the detection of Listeria spp.

Four cloned genes of Listeria monocytogenes coding for listeriolysin O, beta-haemolysin, CAMP-factor and a DTH-inducing protein (DTH-18) were used as gene probes in DNA-DNA hybridization with different listerial references strains. The fragment encoding for DTH-18 is the only probe reacting specifically with pathogenic strains of L. monocytogenes and Listeria ivanovii.

Bacterial Proteins↗