Dataset Information

An antipathogenic compound that targets the OxyR peroxide sensor in Pseudomonas aeruginosa.

ABSTRACT: Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance.Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy.Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa.Methodology. Computer-based virtual screening under consideration of the 'eNTRy' rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR.Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H₂O₂ treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus.Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.

SUBMITTER: Oh HY

PROVIDER: S-EPMC8289212 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The adaptive response to hydrogen peroxide (H(2)O(2)) in Pseudomonas aeruginosa involves the major catalase, KatA, and OxyR. However, neither the molecular basis nor the relationship between the aforementioned proteins has been established. Here, we demonstrate that the transcriptional activation of the katA promoter (katAp) in response to H(2)O(2) was abrogated in the P. aeruginosa PA14 oxyR null mutant. Promoter deletion analyses revealed that H(2)O(2)-mediated induction was dependent on a region of DNA -76 to -36 upstream of the H(2)O(2)-responsive transcriptional start site. This region harbored the potential operator sites (OxyR-responsive element [ORE]) of the Escherichia coli OxyR binding consensus. Deletion of the entire ORE not only abolished H(2)O(2)-mediated induction but also elevated the basal transcription, suggesting the involvement of OxyR and the ORE in both transcriptional activation and repression. OxyR bound to the ORE both in vivo and in vitro, demonstrating that OxyR directly regulates the katAp. Three distinct mobility species of oxidized OxyR were observed in response to 1 mM H(2)O(2), as assessed by free thiol trapping using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. These oxidized species were not observed for the double mutants with mutations in the conserved cysteine (Cys) residues (C199 and C208). The uninduced transcription of katAp was elevated in an oxyR mutant with a mutation of Cys to serine at 199 (C199S) and even higher in the oxyR mutant with a mutation of Cys to alanine at 199 (C199A) but not in oxyR mutants with mutations in C208 (C208S and C208A). In both the C199S and the C208S mutant, however, katAp transcription was still induced by H(2)O(2) treatment, unlike in the oxyR null mutant and the C199A mutant. The double mutants with mutations in both Cys residues (C199S C208S and C199A C208S) did not differ from the C199A mutant. Taken together, our results suggest that P. aeruginosa OxyR is a bona fide transcriptional regulator of the katA gene, sensing H(2)O(2) based on the conserved Cys residues, involving more than one oxidation as well as activation state in vivo.

Project description:Background: Quorum quenching, the interference of a Quorum sensing (QS) system that contributes to the pathogenesis through triggering the production of various virulence determinants, is among the newly suggested antivirulence strategies. Purpose: This study aimed at screening of N-Acyl homoserine lactonase activity from local bacterial isolate, and investigating its effect on Pseudomonas aeruginosa (P. aeruginosa) virulence and biofilm formation. Materials and methods: Soil bacteria were screened for aiiA gene coding for lactonase enzyme by Polymerase Chain reaction and sequencing of aiiA gene homologs. Lactonase activity and spectrum were assessed in the cell-free lysate by well diffusion assay using Agrobacterium tumafaciens KYC55. A bacterial isolate showing the highest N-acyl-homoserine lactones degradation percentage was identified by gene amplification and sequencing of the 16S rRNA gene and its aiiA gene homolog. High performance liquid chromatography was used to confirm N-acyl-homoserine lactone degradation. The effect of cell-free lysate on the biofilm formation ability and cytotoxicity of P. aeruginosa PAO1 and P. aeruginosa clinical isolates from different clinical sources were assessed by static microtiter plate and viability assay, respectively Results: Lactonase gene and activity were identified in three Bacillus spp. isolates. They showed broad catalytic activities against tested N-acyl-homoserine lactones. However, The lactonase activity in the cell- free lysate of isolate 30b showed the highest significant degradation percentage on all tested signals; N-butanoyl-L-homoserine lactone (71%), N-hexanoyl-l-homoserine lactone (100%), N-decanoyl-homoserine lactone (100%), N-(3-oxohexanoyl)-L-homoserine lactone (37.5%), N-(oxodecanoyl)-L-homoserine lactone (100%), and N-(3-oxododecanoyl)-L-homoserine lactone (100%). Alignment of the amino acid sequences of AiiA protein of isolate 30b showed 96% identity with Bacillus cereus (B. cereus) homologous lactonases in the GenBank database, and the isolate was designated as B. cereus isolate 30b. Cell-free lysate of B. cereus isolate 30b reduced biofilm formation significantly in 93% of P. aeruginosa isolates. The highest mean percentage of reduction in the biofilm was 86%. Moreover, the viability percentage of human lung carcinoma A549 cells infected by P. aeruginosa and treated with cell-free lysate of B. cereus isolate 30b increased up to 15%. Conclusion: The results of this study highlight the potential of lactonases as a promising strategy to combat Pseudomonas aeruginosa virulence.

Project description:Pseudomonas aeruginosa possesses an extensive armament of genes involved in oxidative stress defense, including katB-ankB, ahpB, and ahpC-ahpF. Transcription of these genes was regulated in response to H(2)O(2), paraquat, or organic peroxides. Expression of katB-lacZ and the observed KatB catalase levels in P. aeruginosa PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also, ahpB-lacZ and ahpC-lacZ expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 microM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a DeltaoxyR mutant, indicating that OxyR was essential for this response. The transcriptional start sites of katB-ankB, ahpB, and ahpC-ahpF were mapped, putative OxyR-binding sites were identified upstream of the -35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The oxyR mutant was hypersusceptible to oxidative stress-generating agents, including H(2)O(2) and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The oxyR phenotype was fully complemented by a plasmid containing the oxyR gene, while any of the katB, ahpB, or ahpCF genes alone resulted in only marginal complementation. Increased katB-lacZ expression and higher KatB catalase levels were detected in a DeltaahpCF background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In P. aeruginosa, oxyR is located upstream of recG, encoding a putative DNA repair enzyme. oxyR-lacZ and recG-lacZ reporter activities and oxyR-recG mRNA analysis showed that oxyR and recG are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of oxyR. Mutants affected in recG but not oxyR were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the oxyR-recG locus is essential for oxidative stress defense and for DNA repair.

Dataset Information

An antipathogenic compound that targets the OxyR peroxide sensor in Pseudomonas aeruginosa.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets