Pseudomonas aeruginosa ttcA encoding tRNA-thiolating protein requires an iron-sulfur cluster to participate in hydrogen peroxide-mediated stress protection and pathogenicity

Abstract

During the translation process, transfer RNA (tRNA) carries amino acids to ribosomes for protein synthesis. Each codon of mRNA is recognized by a specific tRNA, and enzyme-catalysed modifications to tRNA regulate translation. TtcA is a unique tRNA-thiolating enzyme that requires an iron-sulfur ([Fe-S]) cluster to catalyse thiolation of tRNA. In this study, the physiological functions of a putative ttcA in Pseudomonas aeruginosa, an opportunistic human pathogen that causes serious problems in hospitals, were characterized. A P. aeruginosa ttcA-deleted mutant was constructed, and mutant cells were rendered hypersensitive to oxidative stress, such as hydrogen peroxide (H2O2) treatment. Catalase activity was lower in the ttcA mutant, suggesting that this gene plays a role in protecting against oxidative stress. Moreover, the ttcA mutant demonstrated attenuated virulence in a Drosophila melanogaster host model. Site-directed mutagenesis analysis revealed that the conserved cysteine motifs involved in [Fe-S] cluster ligation were required for TtcA function. Furthermore, ttcA expression increased upon H2O2 exposure, implying that enzyme levels are induced under stress conditions. Overall, the data suggest that P. aeruginosa ttcA plays a critical role in protecting against oxidative stress via catalase activity and is required for successful bacterial infection of the host.

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“…The virulence of P . aeruginosa and mutants were evaluated using the Drosophila melanogaster model as previously described [ 7 , 58 ]. Essentially, the late-exponential-phase cultures (OD 600nm of 0.8) of P .…”

Top-Down Characterization of an Antimicrobial Sanitizer, Leading From Quenchers of Efficacy to Mode of Action

“…Oxidative stress in general is well known to target surface-exposed FeS clusters in proteins ( Imlay et al, 2019 ). FeS proteins are highly reactive with FAC ( Albrich et al, 1981 ) and NaOCl may, potentially via ROS generation in cells, damage the clusters in bacterial FeS proteins ( Romsang et al, 2018 ). This study shows that EW impairs activity of the FeS cluster protein aconitase, a known target of pro-oxidant agents ( Lalève et al, 2016 ).…”

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“…Conversely, the Fe-S tRNA-modifying enzyme, TtcA, is associated with the peroxide stress response in P. aeruginosa. Inactivation of ttcA results in hypersensitivity towards hydrogen peroxide and hypochlorite treatments [ 68 ]. TtcA catalyzes the thiolation of C32 on tRNA Arg1 (s 2 C32), a rare modification that restricts codon recognition and improves aminoacylation [ 69 ].…”

“…Interestingly, ttcA mRNA expression levels increase 13-fold upon hydrogen peroxide challenge through a transcriptional response involving the OxyR regulator. Moreover, the activity of TtcA is associated with the accumulation of KatA [ 68 ]. Although hydrogen peroxide challenge directly inactivates TtcA, it is possible that higher expression levels of this enzyme is a compensatory mechanism.…”