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. 2022 Jun 29;10(3):e0118222.
doi: 10.1128/spectrum.01182-22. Epub 2022 May 16.

PrrT/A, a Pseudomonas aeruginosa Bacterial Encoded Toxin-Antitoxin System Involved in Prophage Regulation and Biofilm Formation

Esther Shmidov  1   2 , Ilana Lebenthal-Loinger  1 , Shira Roth  2   3 , Sarit Karako-Lampert  4 , Itzhak Zander  1   2 , Sivan Shoshani  1   2 , Amos Danielli  2   3 , Ehud Banin  1   2
Affiliations

PrrT/A, a Pseudomonas aeruginosa Bacterial Encoded Toxin-Antitoxin System Involved in Prophage Regulation and Biofilm Formation

Esther Shmidov et al. Microbiol Spectr. .

Abstract

Toxin-antitoxin (TA) systems are genetic modules that consist of a stable protein-toxin and an unstable antitoxin that neutralizes the toxic effect. In type II TA systems, the antitoxin is a protein that inhibits the toxin by direct binding. Type II TA systems, whose roles and functions are under intensive study, are highly distributed among bacterial chromosomes. Here, we identified and characterized a novel type II TA system PrrT/A encoded in the chromosome of the clinical isolate 39016 of the opportunistic pathogen Pseudomonas aeruginosa. We have shown that the PrrT/A system exhibits classical type II TA characteristics and novel regulatory properties. Following deletion of the prrA antitoxin, we discovered that the system is involved in a range of processes including (i) biofilm and motility, (ii) reduced prophage induction and bacteriophage production, and (iii) increased fitness for aminoglycosides. Taken together, these results highlight the importance of this toxin-antitoxin system to key physiological traits in P. aeruginosa. IMPORTANCE The functions attributed to bacterial TA systems are controversial and remain largely unknown. Our study suggests new insights into the potential functions of bacterial TA systems. We reveal that a chromosome-encoded TA system can regulate biofilm and motility, antibiotic resistance, prophage gene expression, and phage production. The latter presents a thus far unreported function of bacterial TA systems. In addition, with the emergence of antimicrobial-resistant bacteria, especially with the rising of P. aeruginosa resistant strains, the investigation of TA systems is critical as it may account for potential new targets against the resistant strains.

Keywords: Pseudomonas aeruginosa; bacteriophages; biofilm; biofilms; prophages; toxin-antitoxin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
The prrT/prrA gene pair act as a type II TA system. (A) Deletion of prrA in 39016 strain revealed a decrease in the growth rate; the growth curve of the mutants in comparison to the WT, and complementation by arabinose induced prrA expression in the mutant. (B) prrT expression is toxic only for the ΔprrA strain; the growth curve of prrT induced OE in the WT and mutant strains, and the inducer was added immediately after the dilution. (C) prrT/prrA gene pair expressed as a polycistron; the following sets of primers were used for the operon verification: (a) 500 bp upstream to prrT, (b) prrT_F, (c) prrT_R, (d) prrA_F, (e) prrA_R, and (f) 500 bp downstream to prrA. The cDNA results represent the transcripts, while gDNA results represent the bacterial genome as a positive control. (D) PrrT/PrrA form a protein complex together; BACTH assay on indicative LB plates containing IPTG and x-gal. The above (A) and (B) graphs are the averages of three independent experiments consisting of five replicates each. Error bars represent the standard deviations.
FIG 2
FIG 2
The prrT/A promoter activity is affected by PrrA, PrrT, and NOR stressor. (A) PrrA represses the promoter while PrrT expression elevates the activity; fluorescence measurement of the WT and the ΔprrTA strain with single complementation, and PrrA and PrrT were induced in the ΔprrTA strain at time zero with 10 mM arabinose. (B) NOR treatment elevated the promoter activity exclusively in strains with an intact prrA; fluorescence measurement of the WT and mutant strains with or without 0.1 mM NOR treatment at time zero. (C) PrrA directly binds to the prrT/A promoter; competition sample with biotinylated DNA and an unlabeled competitor DNA (lane 1), biotinylated DNA with the addition of decreasing amount of the PrrA protein (lanes 2–7). For the A and B graphs, m-Cherry fluorescence measurements were taken in the early stationary phase. The above A and B graphs are the average of three independent experiments consisting of five replicates each. Error bars represent the standard deviation. According to t test: *, P < 0.05; **, P < 0.01.
FIG 3
FIG 3
prrA deletion influenced swarming motility and biofilm formation. (A) prrA deletion significantly increased biofilm formation; CV stained 24 h biofilm of the strains. (B) The deletion of prrA significantly impacts the amrZ gene; RT- PCR analysis comparing the expression levels of amrZ the IN prrA mutant strain compared to the WT and complementation strains. (C) prrA deletion reduced swarming motility; the WT, mutants, and complementation strains were grown for 48 h. The A graph is the average of three independent experiments with five replicates each. The B graph is the average of three independent experiments with three replicates each. The C pictures represent three independent experiments conducted with three replicates each. Error bar represents standard error. **, P < 0.01 with WT strain as a reference, according to t test.
FIG 4
FIG 4
The prrT/prrA system is involved in prophage regulation and impacts phage production. (A) The prrA deletion resulted in a significant downregulation for most prophage encoded genes; heat-map was constructed with the RNA-seq results of the WT and ΔprrA strain, 1 h post prophage induction. The following prophage region (coordinates predicted by PHASTER [60]) genes were analyzed; PR1, PR2, PR3, PR4, and PR5. (B) The deletion of prrA resulted in decreased PR5 phage production; PA14 strain was used as a host, and phages were induced and extracted from WT, mutants, and the complementation strain. (C) prrA gene confers partial defense against phage infection; phages extracted from 39016 were used to infect the strains PA14/pUCP18 (PA14_VEC) and PAO1/pUCP18 prrA (PA14_prrA). The plaque-forming units presented in the above B and C graphs are the average of three independent experiments consisting of three replicates each. Error bars represent the standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001 when the WT and VEC is the reference strain, according to t test.
FIG 5
FIG 5
The MexXY system is upregulated in the prrA mutant. Log2 fold change of the mutant strain relative to the WT strain. Values were calculated with the transcriptomics levels. Error bars represent standard error.
FIG 6
FIG 6
The prrA mutants show enhanced fitness in the subinhibitory treatment of aminoglycosides. (A) MIC experiment with Kan antibiotic, with concentrations ranging from 0 to 300 μg/mL. The MIC for Kan in both strains is 198 μg/mL, and the subinhibitory concentration is 131 μg/mL. (B) MIC experiment with Strep antibiotic, with concentrations ranging from 0 to 800 μg/mL. The MIC for Kan in both strains is 400 μg/mL, and the subinhibitory concentration is 200 μg/mL. (C) The growth curve of the different strains with the treatment of Kan in the concentration of 100 μg/mL. (D) The growth curve of the different strains with the treatment of Strep in the concentration of 400 μg/mL. (E) Competition assay for WT and prrA mutant coculture. The y axis represents the percentage of prrA strain in the different treatments. The above graphs are the average of three independent experiments with three replicates each. Error bars represent the standard deviations.
FIG 7
FIG 7
Model for the PrrTA system and its involvement in bacterial processes.
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