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  • 1
    Electronic Resource
    Electronic Resource
    Extracellular PagC-HlyAS fusion protein for the generation and identification of Salmonella-specific antibodies (1996)
    Springer
    Applied microbiology and biotechnology 45 (1996), S. 629-637 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E. coli hemolysin protein (HlyAS) to the mature PagC peptide. This PagC-HlyAS fusion protein was expressed and efficiently secreted into the culture supernatant by E. coli upon complementation with the hemolysin secretion proteins HlyB and HlyD. Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested. A monoclonal antibody against the PagC-HlyAS fusion protein recognized only PagC in membrane fractions. The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains. Sera from Salmonella-infected human patients and from a rabbit infected with S. typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050740
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  • 2
    Electronic Resource
    Electronic Resource
    The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 144-158 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsListeria monocytogenes inlGHE locus ; inlC2DE locus ; internalin multigene family ; leucine-rich repeat (LRR) proteins ; Listeria virulence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-β-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615–1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3′-terminal end of inlC2 and the 5′-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA- independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380050880
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  • 3
    Electronic Resource
    Electronic Resource
    The Listeria monocytogenesiap gene as an indicator gene for the study of PrfA-dependent regulation (1997)
    Springer
    Molecular genetics and genomics 256 (1997), S. 54-62 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsListeria monocytogenes ; p60 protein ; PrfA protein ; Regulation of gene expression ; Cell division
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The iap gene of Listeria monocytogenes encodes the extracellular protein p60, which possesses a murein hydrolase activity necessary for septum separation. We constructed L. monocytogenes EGD strains harbouring plasmids that carry the iap gene under the control of the PrfA-regulated promoters of the L. monocytogenes genes hly, mpl, and actA. After insertional inactivation of the chromosomal iap gene in L. monocytogenes EGD, p60 synthesis was strictly dependent on PrfA. Elevated temperature (40 °C) enhanced synthesis of p60 in L. monocytogenes when the iap gene was under the control of the hly promoter; this appeared to be associated with increased synthesis of PrfA at this temperature. Synthesis of p60 in L. monocytogenes was significantly lower when the iap gene was placed under the control of the actA or the mpl promoter. Transcription of the iap gene was repressed in L. monocytogenes in the presence of PrfA when iap expression was under the control of the prfA promoter P2. Under the control of the hly promoter the gene produced low levels of secreted p60 in the presence of low amounts of PrfA, and this in turn led to the generation of long listerial cell filaments consisting of bacteria that had failed to separate. Overexpression of p60 in the presence of high levels of PrfA caused formation of single cells, which showed reduced viability depending on the level of secreted p60. These data suggest that the iap gene may be a valuable tool for monitoring virulence gene regulation by PrfA under in vivo conditions, without disturbing the integrity of the infected host cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380050545
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  • 4
    Electronic Resource
    Electronic Resource
    Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila (2000)
    Springer
    Molecular genetics and genomics 262 (2000), S. 1060-1069 
    Details
    ISSN: 1617-4623
    Keywords: Key words Green fluorescent protein (GFP) ; Legionella pneumophila ; Intracellular survival ; Macrophage infectivity potentiator (Mip) protein ; Acanthamoeba castellanii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (macrophage infectivity potentiator) promoter (P mip ) and the sod (superoxide dismutase) promoter (P sod ) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was asssociated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P mip -gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P mip -gfp and P sod -gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by, L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00008649
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  • 5
    Electronic Resource
    Electronic Resource
    Differential expression of Listeria monocytogenes virulence genes in mammalian host cells (1999)
    Springer
    Molecular genetics and genomics 261 (1999), S. 323-336 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsListeria monocytogenes ; Green fluorescent protein ; Reverse-transcriptase polymerase chain reaction ; Virulence gene expression ; Mammalian cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00008633
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  • 6
    Electronic Resource
    Electronic Resource
    Catalase-negativeListeria monocytogenes causing lethal sepsis and meningitis in an adult hematologic patient (1996)
    Springer
    European journal of clinical microbiology & infectious diseases 15 (1996), S. 965-967 
    Details
    ISSN: 1435-4373
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01690521
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