Identification of Salmonella typhi promoters activated by invasion of eukaryotic cells.
The interaction of pathogenic microorganisms with host tissues, and the underlying genetic events which regulate these interactions, are difficult to analyse where no suitable animal model exists. The approach described here, for obtaining information on the genes involved in these interactions, employs an infection system based on the invasion of Henle cells by Salmonella typhi to select promoter-containing DNA sequences able to activate gene expression inside eukaryotic cells. Several DNA fragments exhibiting different promoter strengths and extent of selective activation within eukaryotic cells were identified. Three were selected and characterized according to the expression level of the reporter gene, the polynucleotide sequence, the transcription start, and the dependence upon regulatory proteins. All fragments gave much stronger expression of the reporter gene when the recombinant S. typhi carrier strains invaded cells compared with the expression measured in growth medium. One promoter-containing region exhibited sequence homology to sigma 54-dependent promoters, whereas another appears to be dependent on the stationary-phase RNA polymerase subunit sigma s. S. typhi containing the S1 subunit gene of pertussis toxin cloned under the control of these promoters, selectively expressed the S1 subunit following infection of different phagocytic and non-phagocytic cell lines of human or murine origin. Deletion and point mutant derivatives of two promoters enabled the identification of the main motif required for promoter activity. This method may be helpful for the analysis of pathogenesis in organisms previously difficult to study because of the lack of a convenient animal model, and could provide insights into the chronology and topology of gene expression during infection, including a possible genetic basis for tissue tropism.
Staendner
L H
LH
Division of Microbiology, GBF-National Research Centre for Biotechnology, Braunschweig, Germany.
Rohde
M
M
Timmis
K N
KN
Guzmán
C A
CA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004269:DNA, Bacterial; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D011401:Promoter Regions, Genetic; D012485:Salmonella typhi / Q000235:genetics*; D014158:Transcription, Genetic; D010566:Virulence Factors, Bordetella / Q000235:genetics
D004269:DNA, Bacterial; D010566:Virulence Factors, Bordetella; D015500:Chloramphenicol O-Acetyltransferase; D037342:Pertussis Toxin