Abstract
Listeria monocytogenes is a Gram-positive pathogen able to cause severe human infections. Its major virulence regulator is the transcriptional activator PrfA, a member of the Crp/Fnr family of transcriptional regulators. To establish a successful L. monocytogenes infection, the PrfA protein needs to be in an active conformation, either by binding the cognate inducer glutathione (GSH) or by possessing amino acid substitutions rendering the protein constitutively active (PrfA*). By a yet unknown mechanism, phosphotransferase system (PTS) sugars repress the activity of PrfA. We therefore took a transposon-based approach to identify the mechanism by which PTS sugars repress PrfA activity. For this, we screened a transposon mutant bank to identify clones able to grow in the presence of glucose-6-phosphate as the sole carbon source. Surprisingly, most of the isolated transposon mutants also carried amino acid substitutions in PrfA. In transposon-free strains, the PrfA amino acid substitution mutants displayed growth, virulence factor expression, infectivity, and DNA binding, agreeing with previously identified PrfA* mutants. Hence, the initial growth phenotype observed in the isolated clone was due to the amino acid substitution in PrfA and unrelated to the loci inactivated by the transposon mutant. Finally, we provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.IMPORTANCE The Gram-positive bacterium Listeria monocytogenes is a human pathogen affecting mainly the elderly, immunocompromised people, and pregnant women. It can lead to meningoencephalitis, septicemia, and abortion. The major virulence regulator in L. monocytogenes is the PrfA protein, a transcriptional activator. Using a growth-based selection strategy, we identified mutations in the PrfA protein leading to constitutively active virulence factor expression. We provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.
Highlights
Listeria monocytogenes is a Gram-positive pathogen able to cause severe human infections
We found that L. monocytogenes PrfA* mutants can grow in defined medium (DM) supplemented with G-6-P and that this growth phenotype is strictly dependent on high expression of Hpt
In line with these findings, we tested if a constitutively active form of PrfA, PrfA*, could grow in defined medium (DM) with sugar phosphate as the sole carbon source. To test this hypothesis and evaluate it as a selection strategy for identifying genes involved in phosphotransferase system (PTS) sugar-mediated repression of PrfA activity, four strains were plated on DM supplemented with G-6-P
Summary
Listeria monocytogenes is a Gram-positive pathogen able to cause severe human infections. We took a transposon-based approach to identify the mechanism by which PTS sugars repress PrfA activity. In transposon-free strains, the PrfA amino acid substitution mutants displayed growth, virulence factor expression, infectivity, and DNA binding, agreeing with previously identified PrfA* mutants. The major virulence regulator in L. monocytogenes is the PrfA protein, a transcriptional activator. Using a growth-based selection strategy, we identified mutations in the PrfA protein leading to constitutively active virulence factor expression. The major regulator of virulence factors in L. monocytogenes is the transcription activator PrfA, a member of the Crp/Fnr family of regulators. Unlike glucose and cellobiose, are taken up by the Hpt transporter and not by the PTS, it has been suggested that an active PTS represses PrfA activity, the mechanism remains unclear. PrfA becomes active once inside the mammalian host, where the sugar sources are available in the form of sugar phosphate
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