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A Cyclic Disulfide Diastereomer From Bioactive Fraction of Bruguiera gymnorhiza Shows Anti-Pseudomonas aeruginosa Activity.

ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen that commonly causes hospital-acquired infection and is of great concern in immunocompromised patients. The quorum sensing (QS) mechanism of P. aeruginosa is well studied and known to be responsible for pathogenicity and virulence. The QS inhibitor derived from the natural product can be an important therapeutic agent for pathogen control. The present study reports the role of Bruguiera gymnorhiza purified fraction (BG138) in inhibiting virulence factor production, biofilm formation, quorum sensing molecules, and expression of QS-related genes of P. aeruginosa. Structural characterization of BG138 by high resolution mass spectrometry, Fourier transform infrared spectroscopy, 1D (¹H and ¹³C NMR) and 2D NMR reveals that the fraction is a mixture of already known cyclic disulfide diastereomer, namely, brugierol and isobrugierol. The minimum inhibitory concentration (MIC) of BG138 against P. aeruginosa was 32 μg/ml. Biofilm formation was significantly reduced at sub-MIC concentrations of BG138. Scanning electron microscopy analysis reports the concentration-dependent biofilm inhibition and morphological changes of P. aeruginosa. Flow cytometry-based cell viability assay showed that P. aeruginosa cells exhibit increased propidium iodide uptake on treatment with 32 and 64 μg/ml of BG138. At sub-MIC concentrations, BG138 exhibited significant inhibition of virulence factors and reduced swimming and swarming motility of P. aeruginosa. Furthermore, the effect of BG138 on the expression of QS-related genes was investigated by qRT-PCR. Taken together, our study reports the isolation and structural characterization of bioactive fraction BG138 from B. gymnorhiza and its anti-biofilm, anti-virulence, anti-quorum sensing, and cell-damaging activities against P. aeruginosa.

SUBMITTER: Dahibhate NL

PROVIDER: S-EPMC9201687 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED:Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. IMPORTANCE:The human pathogen Pseudomonas aeruginosa causes life-threatening infections in immunodepressed and cystic fibrosis patients. The emergence of P. aeruginosa strains resistant to all of the available antibacterial agents calls for the urgent development of new antibiotics active against this bacterium. The pathogenic power of P. aeruginosa is mediated by an arsenal of extracellular virulence factors, most of which are stabilized by disulfide bonds. Thus, targeting the machinery that introduces disulfide bonds appears to be a promising strategy to combat P. aeruginosa. Here, we unraveled the oxidative protein folding system of P. aeruginosa in full detail. The system uniquely consists of two membrane proteins that generate disulfide bonds de novo to deliver them to P. aeruginosa DsbA1 (PaDsbA1), a soluble oxidoreductase. PaDsbA1 in turn donates disulfide bonds to secreted proteins, including virulence factors. Disruption of the disulfide bond formation machinery dramatically decreases P. aeruginosa virulence, confirming that disulfide formation systems are valid targets for the design of antimicrobial drugs.

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A Cyclic Disulfide Diastereomer From Bioactive Fraction of Bruguiera gymnorhiza Shows Anti-Pseudomonas aeruginosa Activity.

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