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Published on: May 8, 2013
The soluble pyocins S2 and S4 from Pseudomonas aeruginosa bind to the same FpvAI receptor
Ameer Elfarash1, Qing Wei, Pierre Cornelis
1Department of Bioengineering Sciences, Research Group of Microbiology, VIB Department of Structural Biology, Vrije Universiteit Brussel Pleinlaan 2, B-1050, Brussels, Belgium.
Abstract:
Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa cells by gaining entry via a specific receptor, which, in the case of pyocin S2, is the siderophore pyoverdine receptor FpvAI, and in the case of pyocin S3, FpvAII. The nucleic acid sequence at the positions 4327697-4327359 of P. aeruginosa PAO1 genome was not annotated, but it was predicted to encode the immunity gene of the flanking pyocin S4 gene (PA3866) based on our analysis of the genome sequence. Using RT-PCR, the expression of the immunity gene was detected, confirming the existence of an immunity gene overlapping the S4 pyocin gene. The PA3866 coding for pyocin S4 and the downstream gene coding for the immunity protein were cloned and expressed in Escherichia coli and the His-tagged S4 pyocin was obtained in pure form. Forty-three P. aeruginosa strains were typed via PCR to identify their ferripyoverdine receptor gene (fpvAI-III) and were tested for their sensitivity to pyocin S4. All S4-sensitive strains had the type I ferripyoverdine receptor fpvA gene. Some S4-resistant type I fpvA-positive strains were detected, but all of them had the S4 immunity gene, and, following the deletion of the immunity gene, became S4-sensitive. The fpvAI receptor gene was deleted in a S4-sensitive strain, and, as expected, the mutant became resistant to S4. The N-terminal receptor binding domain (RBD) of pyocin S2, which also uses the FpvAI receptor to enter the cell, was cloned in the pET-15b vector, and expressed in E. coli. When the purified RBD was mixed with pyocin S4 at different ratios, an inhibition of killing was observed, indicating that S2 RBD competes with the pyocin S4 for the binding to the FpvAI receptor. The S2 RBD was also shown to enhance the expression of the pvdA pyoverdine gene, suggesting that it, like pyoverdine, works via the known siderophore-mediated signalization pathway.
Insights
Pseudomonas aeruginosa pyocins S4 and S2 utilize the ferripyoverdine receptor FpvAI for entry. Pyocin S4
Area of Science:
- Microbiology
- Bacteriology
- Molecular Biology
Background:
- Soluble (S-type) pyocins are bacteriocins produced by Pseudomonas aeruginosa.
- These pyocins target and kill non-immune P. aeruginosa strains.
- Entry into target cells is mediated by specific receptors, such as the ferripyoverdine receptor FpvAI for pyocin S2.
Purpose of the Study:
- To investigate the receptor usage and immunity mechanisms of pyocin S4.
- To explore the interaction between pyocin S4 and the ferripyoverdine receptor FpvAI.
- To understand the potential for cross-inhibition between different pyocins.
Main Methods:
- Genome sequence analysis to identify pyocin S4 and its immunity gene.
- RT-PCR to confirm gene expression.
- Cloning and expression of pyocin S4 and its immunity protein in Escherichia coli.
- PCR typing of P. aeruginosa strains for ferripyoverdine receptor genes.
- Sensitivity testing of strains to pyocin S4.
- Gene deletion experiments to assess immunity and receptor function.
- Cloning and expression of the pyocin S2 receptor binding domain (RBD).
- In vitro competition assays between pyocin S4 and S2 RBD.
Main Results:
- An unannotated nucleic acid sequence was identified as the immunity gene for pyocin S4.
- The pyocin S4 gene and its overlapping immunity gene were successfully cloned and expressed.
- All pyocin S4-sensitive strains possessed the type I ferripyoverdine receptor (fpvA) gene.
- Strains resistant to pyocin S4 carried the S4 immunity gene, which, upon deletion, restored sensitivity.
- Deletion of the fpvAI receptor gene rendered a sensitive strain resistant to pyocin S4.
- Pyocin S2 RBD inhibited pyocin S4 killing activity, indicating competition for FpvAI receptor binding.
- Pyocin S2 RBD enhanced pyoverdine gene expression, suggesting a role in siderophore-mediated signaling.
Conclusions:
- Pyocin S4 utilizes the FpvAI receptor for cell entry, similar to pyocin S2.
- The identified immunity gene is crucial for protecting P. aeruginosa from pyocin S4.
- Competition for the FpvAI receptor can occur between different pyocins.
- Pyocin S2 RBD may modulate siderophore-mediated signaling pathways.
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