Tet-ON and Tet-OFF regulation in hypervirulent Klebsiella pneumoniae
Luisa Lutz1,2, Ipek Ekemen1,3, Diana Rasp1,3
1Institute of Laboratory Medicine and Infectious Diseases, Paracelsus Medical Private University, Nuremberg General Hospital, Nuremberg, Germany.
Abstract:
Klebsiella pneumoniae is an opportunistic gram-negative bacterial pathogen responsible for a variety of infections in hospitals and in the community. Hypervirulent K. pneumoniae (hvKp) strains are characterized by their ability to cause liver abscesses and bloodstream infections. This study describes plasmid-based tetracycline-dependent regulation systems for induction or repression of target genes in hvKp. These plasmids encode a wild-type tetracycline repressor (TetR) or a reverse variant (revTetR), and carry tet-sensitive promoters, for anhydrotetracycline-dependent Tet-ON or Tet-OFF regulation, respectively. The dynamic range, determined by gfp under tet control, was up to 19.3-fold with wt-TetR and 5.5-fold with revTetR. As analyzed for up to 48 h, the reporter-gene responses in both systems were controllable in an inducer concentration-dependent manner. When the virulence-associated factor peg-344 was induced by the Tet-ON system, two different hvKp strains exerted slightly increased pathogenicity in a Galleria mellonella infection model. These tet vectors can be useful tools for characterizing potential virulence factors and other genes of interest in an important pathogen.
Importance:
Hypervirulent Klebsiella pneumoniae (hvKp) strains are a serious threat in healthcare environments and beyond. Even in healthy individuals, infections often result in liver abscesses, and the bacteria can spread to multiple infection sites. We here describe two kinds of gene expression control in hvKp, dependent on anhydrotetracycline (ATc). The Tet-ON system uses wild-type tetracycline repressor (TetR), and addition of ATc leads to target gene induction. In the Tet-OFF mode, reverse TetR is co-repressed by ATc, resulting in target gene repression. We have quantified both systems by reporter gene assays and also subjected the hypervirulence marker gene peg-344 to Tet-ON control. When induced, two different hvKp strains exerted slightly increased pathogenicity in a Galleria mellonella (greater wax moth) infection model. These systems can be useful tools for characterizing potential virulence factors and other genes of interest in an important pathogen.
Insights
Researchers developed novel tetracycline-regulated gene expression systems for hypervirulent Klebsiella pneumoniae (hvKp). These Tet-ON and Tet-OFF systems enable precise control of gene expression, aiding in the study of hvKp virulence factors.
Area of Science:
- Microbiology and Molecular Biology
- Bacterial Pathogenesis
- Genetic Engineering
Background:
- Hypervirulent Klebsiella pneumoniae (hvKp) poses a significant threat, causing severe infections like liver abscesses and bloodstream infections.
- Understanding hvKp virulence mechanisms requires precise tools to manipulate gene expression.
- Existing methods may lack the specificity or dynamic range needed for detailed studies.
Purpose of the Study:
- To develop and characterize novel tetracycline-dependent gene expression systems (Tet-ON and Tet-OFF) for hypervirulent Klebsiella pneumoniae.
- To assess the efficacy of these systems in controlling reporter gene expression and a key virulence factor (peg-344).
- To evaluate the impact of induced virulence factor expression on bacterial pathogenicity in a model organism.
Main Methods:
- Construction of plasmid-based systems encoding wild-type TetR (Tet-ON) or reverse TetR (revTetR) with tet-sensitive promoters.
- Quantification of gene expression dynamics using GFP reporter assays under anhydrotetracycline (ATc) induction.
- Assessment of pathogenicity by inducing the peg-344 gene in hvKp strains within a Galleria mellonella infection model.
Main Results:
- The Tet-ON system achieved up to a 19.3-fold dynamic range, while the Tet-OFF system showed a 5.5-fold range.
- Gene expression was controllable in an inducer concentration-dependent manner for up to 48 hours.
- Induction of the peg-344 virulence factor led to slightly increased pathogenicity in Galleria mellonella for two hvKp strains.
Conclusions:
- Plasmid-based Tet-ON and Tet-OFF systems provide effective and controllable gene expression regulation in hypervirulent Klebsiella pneumoniae.
- These systems are valuable tools for dissecting the roles of specific genes, including virulence factors, in hvKp.
- The developed tetracycline-inducible vectors facilitate further research into the pathogenesis of this important opportunistic pathogen.
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