A Metabolite of Pseudomonas Triggers Prophage-Selective Lysogenic to Lytic Conversion in Staphylococcus aureus

Abstract

Bacteriophages have major impact on their microbial hosts and shape entire microbial communities. The majority of these phages are latent and reside as prophages integrated in the genomes of their microbial hosts. A variety of intricate regulatory systems determine the switch from a lysogenic to lytic life style, but so far strategies are lacking to selectively control prophage induction by small molecules. Here we show that Pseudomonas aeruginosa deploys a trigger factor to hijack the lysogenic to lytic switch of a polylysogenic Staphylococcus aureus strain causing the selective production of only one of its prophages. Fractionating extracts of P. aeruginosa identified the phenazine pyocyanin as a highly potent prophage inducer of S. aureus that, in contrast to mitomycin C, displayed prophage selectivity. Mutagenesis and biochemical investigations confirm the existence of a noncanonical mechanism beyond SOS-response that is controlled by the intracellular oxidation level and is prophage-selective. Our results demonstrate that human pathogens can produce metabolites triggering lysogenic to lytic conversion in a prophage-selective manner. We anticipate our discovery to be the starting point of unveiling metabolite-mediated microbe-prophage interactions and laying the foundations for a selective small molecule controlled manipulation of prophage activity. These could be for example applied to control microbial communities by their built-in destruction mechanism in a novel form of phage therapy or for the construction of small molecule-inducible switches in synthetic biology.

Figure 1

Activity-guided isolation and identification of the prophage inducer pyocyanin. (a) Screening scheme for the discovery of potential prophage inducers from microbial metabolites. (b) Two fractions of the extracts of P. aeruginosa increased phage production in S. aureus ATCC 6341. (c) The prophage inducer was identified as the phenazine pyocyanin, which compared to other phenazine compounds showed 2–3 orders of magnitude higher prophage induction. (d) Fold-induction measured by PFU counts relative to control of fractionated extracts of P. aeruginosa. In contrast to the wild type, a ΔphzM transposon mutant, which lacks the enzyme responsible for the N-methylation step in the pyocyanin biosynthesis, did not cause prophage induction. For parts b, c, and d three independent biological replicates were performed, and the mean PFU/mL values with the corresponding standard deviations (b, c) are reported.

Figure 2

Selective prophage induction of pyocyanin in the polylysogenic S. aureus ATCC 6341 strain. (a) Culture supernatants of S. aureus ATCC 6341 cultures treated with 25 μM pyocyanin (pyo) and 1.5 μM mitomycin C (mitC). Agarose gels show amplification prophage-like regions for mitomycin C treatment, whereas pyocyanin treatment only gave successful amplification of phiMBL3. Representatives of five independent biological replicates are shown for each treatment. Isolated genomic DNA was used as positive control (+). (b) Representative TEM microscopy images of propagated and isolated phage phiMBL3. (c) Volcano plots of p values versus the log 2 fold change in protein abundance between treated samples and DMSO control of cell lysates of S. aureus ATCC 6341 cultures treated with 25 μM pyocyanin (left) and 1.5 μM mitomycin C (right) (n = 3; FDR 0.05; s0 0.1). While only capsid protein of phiMBL3 was detected for pyocyanin treatment, also capisd proteins of the other phages were found for mitomycin C treatment.

Figure 3

Mechanism of pyocyanin-mediated selective prophage induction. (a) Transcriptional analysis showing upregulated oxidative stress response-related genes as the log 2 fold change for three different pyocyanin concentrations in comparison to DMSO control (n = 3). (b) Phage production dependent on increasing concentrations of the ROS scavenger N-acetylcysteine (NAC) as measured by plaque formation. (c) Prophage induction by plaque formation with wild type S. aureus ATCC 6341 and pyocyanin resistant mutants (pyo^R1-3) upon treatment with 25 μM pyocyanin (pyo) and 1.5 μM mitomycin C (mitC). For parts b and c three independent biological replicates were performed and the mean PFU/mL values with the corresponding standard deviations are shown. (d) Venn diagram of mutations shared between the three independently generated pyocyanin mutants. (e) Genome maps of the lytic–lysogenic genes of the prophages and pathogenicity islands in S. aureus ATCC 6341. Phage phiMBL3 harbors a truncated CI*-like repressor which lacks the C-terminal domain with cleavage site (purple triangle) and active site (pink arrows).

Figure 4

Species-selectivity of prophage induction. (a) Prophage induction relative to controls (fold induction) measured by plaque forming units for E. coli and P. aeruginosa strains upon treatment with different pyocyanin (pyo) concentrations and mitomycin C (mitC) using corresponding phage-sensitive indicator strains. (b) Percentage of colony forming units (CFU) of S. aureus ATCC 6341 and prophage-cured S. aureus RN4220 relative to DMSO control after treatment with different pyocyanin concentrations and mitomycin C. For each compound and concentration, three biological assay replicates were performed.