Toxin-antitoxin (TA) systems are prevalent and transcribed in clinical isolates of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus

Abstract

The percentage of bacterial infections refractory to standard antibiotic treatments is steadily increasing. Among the most problematic hospital and community-acquired pathogens are methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA). One novel strategy proposed for treating infections of multidrug-resistant bacteria is the activation of latent toxins of toxin-antitoxin (TA) protein complexes residing within bacteria; however, the prevalence and identity of TA systems in clinical isolates of MRSA and PA has not been defined. We isolated DNA from 78 MRSA and 42 PA clinical isolates and used PCR to probe for the presence of various TA loci. Our results showed that the genes for homologs of the mazEF TA system in MRSA and the relBE and higBA TA systems in PA were present in 100% of the respective strains. Additionally, reverse transcriptase PCR analysis revealed that these transcripts are produced in the clinical isolates. These results indicate that TA genes are prevalent and transcribed within MRSA and PA and suggest that activation of the toxin proteins could be an effective antibacterial strategy for these pathogens.

Figure 1. MLVA of the MRSAand PAclinical isolates

The 91% or 97% clonal cutoff value and 75% similarity cutoff value are indicated by solid and dashed vertical lines, respectively for MRSA (A) and PA (B). The clusters generated are shown in corresponding solid and dashed brackets alongside the dendrogram.

Figure 1. MLVA of the MRSAand PAclinical isolates

The 91% or 97% clonal cutoff value and 75% similarity cutoff value are indicated by solid and dashed vertical lines, respectively for MRSA (A) and PA (B). The clusters generated are shown in corresponding solid and dashed brackets alongside the dendrogram.

Figure 2. Locations of primary homology for primers used in PCR screen, RT-PCR and flanking region PCR

(A) Primer sequences were based off the S. aureus COL genome. The same internal primers were used to amplify a region of mazEF_Sa for both the PCR-based screen and RT-PCR. Flanking mazEF_Sa are the genes rbsU and alr. Primers were designed to amplify the sequences from rbsU to mazF_Sa and from mazE_Sa to alr. (B–D) Primer sequences were based off the P. aeruginosa PAO1 genome. (B) The same internal primers were used to amplify a region of parDE_Pa for both the PCR-based screen and RT-PCR. Flanking parDE_Pa are genes encoding an integrase, a tRNA and a transferase. Primers were designed to amplify the sequence between the integrase gene and parD_Pa and between parE_Pa and the transferase gene. (C) Separate sets of internal primers were used to amplify regions of relBE_Pa for the PCR-based screen and for RT-PCR. Flanking relBE_Pa are genes encoding a transcriptional regulator and a hypothetical protein. Primers were designed to amplify the sequence from the transcriptional regulator gene to relB_Pa and from relE_Pa to the hypothetical protein gene. (D) Separate sets of internal primers were used to amplify regions of higBA_Pa for the PCR-based screen and for RT-PCR. Flanking higBA_Pa are genes encoding a hypothetical protein and a Ton-B dependent receptor. Primers were designed to amplify the sequence from the hypothetical protein gene to higA_Pa and from higB_Pa to the Ton-B dependent receptor gene. (E) In P. aeruginosa PA7, higBA_Pa is flanked by genes encoding a restriction endonuclease and a hypothetical protein. Primers were designed to amplify the sequence from the restriction endonuclease gene to higA_Pa and from higB_Pa to the hypothetical protein gene.

Figure 3. RT-PCR analysis of MRSA and P. aeruginosa clinical isolates

RT-PCR with primers complementary to the genes encoding each TA system indicates that transcripts are produced in the clinical isolates of (A) MRSA and (B) P. aeruginosa, as shown in the (+) RT row. Controls for DNA contamination, in which the reverse transcriptase is omitted from the reaction mix, yield no product, as shown in the (−) RT row. Clinical isolates analyzed are indicated by the strain number above each column.