ABSTRACT
We have used RT-PCR and GFP-mediated fluorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells. Our data show that most of the PrfA-regulated virulence genes are more efficiently expressed, as measured by transcript levels, when L. monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells. The promoters for hly and plcA are predominantly activated within the phagosomal compartment, while those for actA and inlC are predominantly activated in the host cell cytosol. Expression of actA and plcB precedes that of inlC after infection of epithelial cells and macrophages. Little transcription of inlA or inlB is observed in epithelial cells and there is only slightly more in macrophages. In both cell types the level of transcription of the inlAB operon is lower than is seen under extracellular growth conditions in rich media, which is compatible with the assumption that InlA and InlB are not required during intracellular growth of the bacteria. Activation of the PrfA-independent iap promoter is also low during intracellular growth, although the gene product (p60) is required for cell viability. The levels of the PrfA-dependent virulence gene transcripts do not correlate with the amount of prfA transcript present, which is low under all intracellular conditions analysed, suggesting that the prfA transcript is either highly unstable in bacteria that are growing intracellularly, or that the small amount of PrfA produced is highly activated by additional component(s).
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Genes, Bacterial , Listeria monocytogenes/genetics , Trans-Activators/metabolism , Animals , Caco-2 Cells , Cell Compartmentation , Cell Line , Cells, Cultured , Fluorescence , Green Fluorescent Proteins , Humans , Listeria monocytogenes/growth & development , Listeria monocytogenes/pathogenicity , Luminescent Proteins/genetics , Macrophages/cytology , Mammals , Mice , Mice, Inbred BALB C , Peptide Termination Factors , Plasmids , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , VirulenceABSTRACT
The interaction of the purified PrfA transcription factor with the regulatory sequences located upstream of the PrfA-dependent listeriolysin (hly) and internalin (inlA) genes was studied in the presence and in the absence of Paf (PrfA-activating factor)-containing extracts. It is shown that PrfA protein is able to bind, independently of additional factors, to a 109bp DNA fragment including the entire hly promoter sequence with the anticipated PrfA binding site ('PrfA-box'). PrfA alone, but not in combination with Paf, can also bind to a shorter target sequence of 28 bp comprising essentially the PrfA-box of the hly promoter. The addition of a Paf-containing extract does not lead to significant protein binding to these two hly target sequences in the absence of PrfA but converts the complex (CIII) consisting of PrfA and the 109 bp hly DNA fragment to a slower migrating PrfA-Paf-DNA complex (CI). Incubation of cell-free extracts of wild-type Listeria monocytogenes with the 109 bp DNA fragment leads to the formation of CI. The addition of polyclonal PrfA antibodies causes a supershift of CIII. Purified PrfA and PrfA-Paf also bind to a DNA fragment containing the PrfA-dependent promoter P2 of inlA, albeit at a lower rate when compared with the corresponding hly sequence. In contrast to the hly target DNA, the inlA promoter sequence efficiently binds Paf alone, and this Paf binding reduces that of PrfA and PrfA-Paf to the inlA target DNA. DNase I footprint experiments show that purified PrfA protects sequences of dyad symmetry previously proposed as PrfA binding sites in the hly and in the inlA promoter regions.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins , Listeria monocytogenes/genetics , Promoter Regions, Genetic , Trans-Activators/metabolism , Bacterial Proteins/genetics , Binding Sites , Centrifugation, Density Gradient , DNA Probes , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Genes, Bacterial , Heat-Shock Proteins/genetics , Hemolysin Proteins/genetics , Immunoblotting , Listeria monocytogenes/pathogenicity , Peptide Termination Factors , Protein Binding , Regulatory Sequences, Nucleic Acid , Trans-Activators/genetics , Transcription Factors/genetics , Virulence/geneticsABSTRACT
Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes. L. monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin. Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L. monocytogenes strain. After delivery by L. monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).
Subject(s)
Genetic Vectors , Listeria monocytogenes/genetics , Macrophages/metabolism , Plasmids/genetics , Vaccines, DNA , Animals , Bacterial Vaccines , Bacteriolysis , Bacteriophages/enzymology , Bacteriophages/genetics , Cytomegalovirus/genetics , Cytosol/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , Genes, Reporter , Listeria monocytogenes/virology , Mice , Mice, Inbred BALB C , Promoter Regions, Genetic , Tumor Cells, Cultured , Vaccines, AttenuatedABSTRACT
As previously reported, Listeria monocytogenes infection of P388D1 macrophages results in a rapid induction of NF-kappaB DNA-binding activity. Here we show that this induction of NF-kappaB activity occurs in a biphasic mode: first, a transient, IkappaBalpha degradation-dependent phase of activity, also induced by the nonvirulent species Listeria innocua, which is mediated by binding of the bacteria to the macrophage, or by adding Listeria-derived lipoteichoic acid to the macrophage; the second persistent phase of activation is only markedly induced when the bacteria enter the cytoplasm of the host cell and express the virulence genes plcA and plcB, encoding two phospholipases. We suggest that products of the enzymatic activity of phospholipases directly interfere with host cell signal transduction pathways, thus leading to persistent NF-kappaB activation via persistent IkappaBbeta degradation.
Subject(s)
DNA-Binding Proteins/metabolism , I-kappa B Proteins , Leukemia P388/physiopathology , Lipopolysaccharides/pharmacology , Listeria monocytogenes , Listeriosis/metabolism , Macrophages/microbiology , NF-kappa B/metabolism , Phospholipases/pharmacology , Teichoic Acids/pharmacology , Animals , Humans , Macrophages/metabolism , Mice , NF-KappaB Inhibitor alphaABSTRACT
The E. coli hemolysin (HlyA) secretion apparatus represents a type I secretion system that is fully functional in Salmonella. The system which consists of the two specific membrane proteins HlyB and HlyD and the outer membrane protein TolC, recognizes on HlyA a C-terminally located signal sequence of about 60 amino acids. Fusion proteins to which this signal sequence is covalently linked at the C-terminus are also recognized by this secretion apparatus. The efficiency of secretion is dependent on the rate of folding of the reporter protein. Secretion-competent regions of a given reporter protein that is not secretable as entire protein can be screened by a recently constructed transposon TnhlyAs which allows the insertion of the secretion signal into any region of the reporter protein. The genetic information for antigens of any source ranging in size between 10 and 1000 amino acids can be easily inserted into a recently constructed secretion vector which will allow the secretion of the fused antigen(s) in attenuated Salmonella typhimurium strains and in other attenuated Enterobacteriaceae. By manipulation of the Hly secretion system the antigen can be either completely secreted into the environment, fixed on the outer membrane or arrested in the cytoplasm of the used carrier strain. By the use of appropriate attenuated Salmonella strains the antigen is delivered in isolated compartments or to the cytosolic compartment. The extracellular delivery of such antigens is also possible with the help of appropriate carrier strains. The immunological consequences of the different display of the processed antigen will be discussed in the paper by Hess et al in this volume. With a similar antigen delivery system the easy identification and molecular characterization of unknown antigens recognized by the immune system in an infection is also feasible.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/biosynthesis , Bacterial Vaccines , Escherichia coli Proteins , Escherichia coli/genetics , Hemolysin Proteins/biosynthesis , Salmonella typhimurium/immunology , Salmonella/immunology , Vaccines, Attenuated , Vaccines, Synthetic , Animals , Antigens, Bacterial/biosynthesis , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Enterobacteriaceae/immunology , Genes, Reporter , Genetic Vectors , Humans , Open Reading Frames , Plasmids , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunologyABSTRACT
The interaction of Listeria monocytogenes with human umbilical vein endothelial cells was studied. We show that L. monocytogenes invades human umbilical vein endothelial cells independently of internalin A, internalin B, internalin C, and ActA. L. monocytogenes replicates efficiently inside the cells and moves intracellularly by the induction of actin polymerization. We further show that L. monocytogenes-infection of human umbilical vein endothelial cells induces interleukin-6 and interleukin-8 expression during the first 6 h of infection. The expression of MCP-1 and the adhesion molecules VCAM-1 and ICAM-1 was not altered under the experimental conditions used here.
Subject(s)
Cell Movement/physiology , Endothelium, Vascular/microbiology , Listeria monocytogenes/growth & development , Listeriosis/microbiology , Actins/physiology , Bacterial Proteins/genetics , Endothelium, Vascular/chemistry , Endothelium, Vascular/immunology , Gene Expression Regulation, Bacterial , Humans , Interleukin-6/genetics , Interleukin-8/genetics , Listeria monocytogenes/genetics , Listeriosis/immunology , Mutagenesis , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , Umbilical Veins/cytologyABSTRACT
We describe the development of plasmid vectors carrying the expression sites, an hlyA cassette and the secretion genes of Escherichia coli hemolysin. These allow the synthesis and secretion of heterologous microbial antigens in E. coli and attenuated Salmonella aroA strains. Genes or gene fragments encoding microbial antigens are inserted in-frame into a residual part of the hlyA gene which essentially encodes the HlyA secretion signal (HlyAs). In general, the fused genes, carrying the hlyAs sequence at the 3' terminus, are efficiently expressed, and the synthesized antigens are secreted into the culture supernatant of the producing strain. Attenuated Salmonella strains synthesizing either HlyAs-fused listeriolysin or p60 of Listeria monocytogenes were constructed by this procedure and shown to provide protective immunity against L. monocytogenes in mice. The most effective protection was obtained when these microbial antigens were secreted by the attenuated Salmonella strains. We further present new approaches which may allow the application of this antigen-delivery system to any microbial antigen.
Subject(s)
Antigens, Bacterial/administration & dosage , Bacterial Proteins/administration & dosage , Bacterial Proteins/physiology , Bacterial Toxins , Escherichia coli Proteins , Heat-Shock Proteins/administration & dosage , Hemolysin Proteins/physiology , Animals , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Antigens, Bacterial/therapeutic use , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Bacterial Vaccines/therapeutic use , Genetic Vectors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Hemolysin Proteins/genetics , Listeriosis/prevention & control , Mice , Plasmids/genetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/therapeutic use , Salmonella/genetics , Vaccination , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/therapeutic useABSTRACT
The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility-shift assays (EMSAs) using cell-free extracts from the two L. monocytogenes strains EGD and NCTC 7973. PrfA-specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels. Both strains exhibited increased PrfA-specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain-heart infusion (BHI). The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA-free extracts from various Listeria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA-activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA-free extracts. Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S. Specific binding of PrfA-containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced. The iron effect seems to be mediated by Paf rather than by PrfA itself.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins , DNA, Bacterial/metabolism , Heat-Shock Proteins/genetics , Iron/pharmacology , Membrane Proteins/genetics , Promoter Regions, Genetic , Trans-Activators/metabolism , Bacterial Proteins/isolation & purification , Culture Media/pharmacology , Escherichia coli/metabolism , Gene Expression , Hemolysin Proteins , Humans , Listeria monocytogenes , Peptide Termination Factors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Trans-Activators/genetics , Trans-Activators/isolation & purificationABSTRACT
Listeria monocytogenes isolates belonging to serogroup 4 (subtypes 4a, 4ab, 4b, 4c, 4d, and 4e) exhibit different levels of virulence in mice. Molecular studies indicate that in comparison with the control strain EGD (serotype 1/2a), these strains differ in the expression of the PrfA-regulated virulence genes, including prfA itself. Strains of serotypes 4c, 4d, 4e, and especially 4a show a low level of invasiveness in Caco-2 cells, which correlates in part with the low level of expression of the inlA gene. All serotypes reach the cytoplasm, at the latest, 2 h postinfection and become surrounded by polymerized actin within the next hour, but actin tail formation by serotype 4a, 4c, 4d, and 4e strains is drastically reduced. The actA genes in these serogroup 4 strains are expressed in minimum essential medium and within the phagocytic cell line J774. However, the amounts and (in part) the sizes of the ActA proteins in these strains differ under these conditions. The reduced actin tail formation by serotype 4a, 4c, 4d, and 4e strains may be due to the low level of in vivo expression of ActA. In addition, the loss of one repeat unit in the ActA proteins of serotype 4a and 4e strains may also contribute to the less efficient actin tail formation observed with these strains.
Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Listeria monocytogenes/genetics , Trans-Activators/genetics , Animals , Bacterial Proteins/biosynthesis , Base Sequence , Cell Line , Culture Media , Female , Listeria monocytogenes/classification , Listeria monocytogenes/pathogenicity , Mice , Molecular Sequence Data , Peptide Termination Factors , Serotyping , Transcription, Genetic , VirulenceABSTRACT
A mutant of Listeria monocytogenes EGD was constructed that carries an extended deletion removing the entire PrfA-regulated gene cluster from plcA to plcB and a second deletion inactivating the inlA gene. Upon supplementation of this mutant with multiple gene copies of prfA, a protein of 30 kDa was detected in the supernatant of the mutant strain. The gene encoding this protein was obtained by direct and inverse polymerase chain reaction using oligonucleotide primers that were deduced from partial amino acid sequences of the purified 30 kDa protein. The amino acid sequence of the gene product revealed a protein of 297 amino acids that carried eight repeat units with high homology to those of the two known internalin proteins A and B. This secretory protein, termed internalin C, is much smaller than InlA or InlB and its complete sequence is related to the two known internalins. The gene InlC is transcribed into a monocistronic mRNA from a single promoter which shows a typical consensus sequence for PrfA-binding at the position -40. In contrast to the transcription of the InlAB operon, which is downregulated after shift of an L. monocytogenes EGD culture from brain-heart infusion into minimum essential medium (MEM), transcription of inlC is induced in MEM like most of the other known PrfA-regulated virulence genes. In addition, InlC is strongly transcribed in the cytoplasm of phagocytic J774 cells whereas inlA is poorly transcribed under these conditions, suggesting that internalin C may play a role in a late stage of L. monocytogenes infection rather than in the uptake of L. monocytogenes by non-professional phagocytic cells. An InlC deletion mutant shows reduced virulence when tested in an intravenous mouse model, but intracellular replication of the mutant in Caco-2 and J774 cells appears to be comparable with that of the wild-type strain.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/physiology , Genes, Bacterial/genetics , Listeria monocytogenes/genetics , Trans-Activators/physiology , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Base Sequence , Caco-2 Cells , Cloning, Molecular , Culture Media , Gene Expression Regulation, Bacterial/physiology , Humans , Listeria monocytogenes/growth & development , Listeria monocytogenes/pathogenicity , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Molecular Weight , Peptide Termination Factors , RNA, Bacterial/analysis , RNA, Messenger/analysis , Sequence Analysis , Sequence Homology, Amino Acid , Species Specificity , Trans-Activators/genetics , Transcription, Genetic , VirulenceABSTRACT
The two Listeria monocytogenes strains EGD and NCTC 7973 display a different regulation pattern of their PrfA-dependent genes. All PrfA-dependent genes from L. monocytogenes NCTC 7973 are much more efficiently transcribed in brain-heart infusion medium than those from strain EGD. Transcription of these genes in EGD is, however, induced after shift into Minimal Essential Medium (MEM) to a level that is comparable to that of strain NCTC 7973. Expression of the internalin gene (inlA) is also influenced by PrfA, but only one (P2) out of three mapped promoters is strictly dependent on PrfA. In contrast to the other PrfA-regulated genes, transcription of inlA even from the P2 promoter is reduced in both strains after shift to MEM. The prfA deletion mutant SLCC 53 complemented with multiple copies of prfA synthesizes large amounts of monocistronic prfA transcript, but there is no concomitant increase in the transcripts of the PrfA-dependent genes. However, upon a shift into MEM, transcription of the PrfA-dependent genes (with the exception of the inlA gene) is highly induced even in the absence of de novo protein synthesis. The PrfA proteins of the two studied L. monocytogenes strains differ in their ability to activate PrfA-dependent genes. This difference is probably the result of amino acid exchange(s) in the C-terminal part of these proteins. Strain EGD supplemented with multiple copies of prfA-7973 shows a similar regulation of the PrfA-dependent genes as strain NCTC 7973, whereas multiple copies of prfA-EGD introduced into strain EGD hardly change the rate of transcription of the PrfA-dependent genes. Our data thus suggest that PrfA-mediated gene expression depends not only on the amount of the PrfA protein and the 'quality' of the putative PrfA-binding sites, but also on the 'quality' of the PrfA protein which seems to be influenced by its amino acid composition and by environmental parameters.
Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Listeria monocytogenes/genetics , Trans-Activators/genetics , Bacterial Proteins/metabolism , Gene Deletion , Gene Dosage , Genetic Complementation Test , Listeria monocytogenes/metabolism , Listeria monocytogenes/pathogenicity , Peptide Termination Factors , Trans-Activators/metabolism , Virulence/geneticsABSTRACT
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.
Subject(s)
Bacterial Toxins , Heat-Shock Proteins/physiology , Hemolysin Proteins/physiology , Salmonella/physiology , Animals , Base Sequence , Cell Line , Macrophages/microbiology , Mice , Molecular Sequence DataABSTRACT
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). Generally, the fused gene is efficiently expressed and the synthesized antigen is in part secreted into the culture supernatant and in part exposed on the surface of the producing Salmonella strain. The successful use of this approach is demonstrated with two antigens of Salmonella typhimurium, PagC and SlyA, both of which are potent virulence factors but produced only in small amounts under in vitro culture conditions and two virulence proteins of Listeria monocytogenes, p60 and listeriolysin. Interestingly the listeriolysin fusion protein proved to be cytolytically active and allowed, when expressed in Salmonella, the escape of these bacteria into the cytoplasm of infected macrophages.
Subject(s)
Antigens, Bacterial/biosynthesis , Bacterial Vaccines/biosynthesis , Escherichia coli Proteins , Escherichia coli/physiology , Hemolysin Proteins/biosynthesis , Salmonella/immunology , Vaccines, Synthetic , Antigens, Bacterial/immunology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Base Sequence , DNA Primers , Genes, Bacterial , Hemolysin Proteins/genetics , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Salmonella/genetics , Salmonella/ultrastructureABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Bacterial Toxins , Cyclic AMP Receptor Protein/genetics , Escherichia coli Proteins , Genes, Bacterial , Iron-Sulfur Proteins , Listeria/genetics , Multigene Family , Trans-Activators/genetics , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Carrier Proteins , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Hemolysin Proteins , Listeria/pathogenicity , Listeria monocytogenes/genetics , Listeria monocytogenes/pathogenicity , Molecular Sequence Data , Peptide Termination Factors , Sequence Alignment , Sequence Homology, Amino Acid , Virulence/geneticsABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Bacterial Toxins , Gene Expression Regulation, Bacterial , Listeria monocytogenes/pathogenicity , Trans-Activators/genetics , Transcription, Genetic , Animals , Base Sequence , Genes/genetics , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Hemolysin Proteins , Hemolysis , Listeria monocytogenes/genetics , Macrophages/microbiology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/genetics , Mice , Molecular Sequence Data , Peptide Termination Factors , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoric Diester Hydrolases/biosynthesis , Phosphoric Diester Hydrolases/genetics , RNA Precursors/biosynthesis , Rifampin/pharmacology , Transcription, Genetic/drug effects , Type C Phospholipases/biosynthesis , Type C Phospholipases/genetics , Virulence/geneticsABSTRACT
The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA-controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37 degrees C to the logarithmic growth phase. Stress conditions such as heat-shock or stationary culture conditions lead to the induction of additional PrfA-dependent proteins (PdPs): ActA (92 kDa), a 38 kDa protein of unknown function and a 34 kDa protein which probably represents PlcA. Under nutrient-stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Toxins , Gene Expression Regulation, Bacterial , Genes, Bacterial , Listeria monocytogenes/metabolism , Stress, Physiological/metabolism , Trans-Activators/physiology , Animals , Bacterial Adhesion/genetics , Bacterial Proteins/genetics , Cell Line , Culture Media/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Hemolysin Proteins , Listeria monocytogenes/genetics , Mice , Peptide Termination Factors , Virulence/geneticsABSTRACT
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.
Subject(s)
Listeria monocytogenes/pathogenicity , Amino Acid Sequence , Animals , Bacterial Adhesion/genetics , Bacterial Adhesion/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Gene Expression Regulation, Bacterial , Genes, Bacterial , Humans , Listeria monocytogenes/genetics , Listeria monocytogenes/physiology , Mice , Models, Biological , Molecular Sequence Data , Mutagenesis , Sequence Homology, Amino Acid , Virulence/genetics , Virulence/physiologyABSTRACT
Antibodies directed against the major secreted protein of Listeria monocytogenes, termed p60, were found more frequently than antilisteriolysin antibodies in sera of listeriosis patients. Anti-p60 antibodies were also identified in all tested sera from healthy individuals. To test whether p60 provides protection against L. monocytogenes, we constructed an attenuated Salmonella typhimurium aroA strain which secretes p60 via the Escherichia coli hemolysin secretion pathway. Application of this Salmonella strain to BALB/c mice prior to an L. monocytogenes infection induced p60 antibodies in these mice and led to a significantly reduced number of viable bacteria in the spleen compared with that in control animals which were primed with the S. typhimurium aroA strain alone.
Subject(s)
Antibodies, Bacterial/analysis , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Carrier Proteins/immunology , Listeria monocytogenes/immunology , Recombinant Fusion Proteins/immunology , Salmonella typhimurium/immunology , Animals , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Base Sequence , Carrier Proteins/genetics , Female , Hemolysin Proteins , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence DataABSTRACT
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.
Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Toxins , Listeria monocytogenes/pathogenicity , Bacterial Proteins/chemistry , Conjugation, Genetic , Cross Reactions , Extracellular Space/chemistry , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Hemolysin Proteins/biosynthesis , Hemolysin Proteins/genetics , Humans , Listeria monocytogenes/genetics , Mutation , VirulenceABSTRACT
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.