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Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate
Published on: February 22, 2020
Inhibition of Pseudomonas aeruginosa Alginate Synthesis by Ebselen Oxide and Its Analogues
Soo-Kyoung Kim1, Huy X Ngo2, Emily K Dennis2
1Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, College Park, Maryland 20742, United States.
Abstract:
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is frequently found in the airways of cystic fibrosis (CF) patients due to the dehydrated mucus that collapses the underlying cilia and prevents mucociliary clearance. During this life-long chronic infection, P. aeruginosa cell accumulates mutations that lead to inactivation of the mucA gene that results in the constitutive expression of algD-algA operon and the production of alginate exopolysaccharide. The viscous alginate polysaccharide further occludes the airways of CF patients and serves as a protective matrix to shield P. aeruginosa from host immune cells and antibiotic therapy. Development of inhibitors of alginate production by P. aeruginosa would reduce the negative impact from this viscous polysaccharide. In addition to transcriptional regulation, alginate biosynthesis requires allosteric activation by bis (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding to an Alg44 protein. Previously, we found that ebselen (Eb) and ebselen oxide (EbO) inhibited diguanylate cyclase from synthesizing c-di-GMP. In this study, we show that EbO, Eb, ebsulfur (EbS), and their analogues inhibit alginate production. Eb and EbS can covalently modify the cysteine 98 (C98) residue of Alg44 and prevent its ability to bind c-di-GMP. However, P. aeruginosa with Alg44 C98 substituted with alanine or serine was still inhibited for alginate production by Eb and EbS. Our results indicate that EbO, Eb, and EbS are lead compounds for reducing alginate production by P. aeruginosa. Future development of these inhibitors could provide a potential treatment for CF patients infected with mucoid P. aeruginosa.
Insights
Ebselen and its derivatives inhibit alginate production in Pseudomonas aeruginosa, a key factor in cystic fibrosis lung infections. These compounds offer a potential therapeutic strategy to reduce airway occlusion and enhance antibiotic efficacy in CF patients.
Area of Science:
- Microbiology
- Drug Discovery
- Biochemistry
Background:
- Pseudomonas aeruginosa is a major pathogen in cystic fibrosis (CF) patients, causing chronic infections due to its ability to produce alginate.
- Alginate production is crucial for P. aeruginosa virulence, forming a protective matrix that hinders host immunity and antibiotic treatment.
- Inhibiting alginate biosynthesis is a promising therapeutic strategy for managing CF lung infections.
Purpose of the Study:
- To investigate the potential of ebselen (Eb), ebselen oxide (EbO), and ebsulfur (EbS) as inhibitors of alginate production in P. aeruginosa.
- To elucidate the mechanism by which these compounds affect alginate biosynthesis, particularly their interaction with the Alg44 protein and cyclic diguanylate monophosphate (c-di-GMP).
Main Methods:
- Treatment of P. aeruginosa with EbO, Eb, EbS, and their analogues to assess alginate production.
- Investigating the interaction of Eb and EbS with the Alg44 protein, focusing on cysteine 98 (C98).
- Utilizing site-directed mutagenesis to substitute Alg44 C98 with alanine or serine to evaluate the role of this residue in inhibition.
Main Results:
- EbO, Eb, and EbS effectively inhibit alginate production in P. aeruginosa.
- Eb and EbS covalently modify Alg44 at C98, disrupting c-di-GMP binding and consequently inhibiting alginate synthesis.
- Inhibition of alginate production by Eb and EbS was observed even when Alg44 C98 was mutated, suggesting additional mechanisms of action.
Conclusions:
- EbO, Eb, and EbS are promising lead compounds for developing novel therapeutics against mucoid P. aeruginosa infections.
- These compounds can reduce alginate production, thereby mitigating airway occlusion and improving treatment outcomes for CF patients.
- Further development of these ebselen derivatives could lead to effective treatments for chronic P. aeruginosa infections in cystic fibrosis.
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