No abstract

The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth () operon of , catalyze the dehydrogenation of the cholesterol metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA), with a 3-carbon side chain, and subsequent hydration of the product 3-oxo-4,17-pregnadiene-20-carboxyl-CoA (3-OPDC-CoA) to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). The gene downstream of , i.e.,, was expressed in recombinant RHA1 in combination with other genes within the operon. His-tagged Ltp2 copurified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2, which contains a domain of unknown function (DUF35). Ltp2 in association with ChsH1-ChsH2 or just the DUF35 domain of ChsH2 was shown to catalyze the retroaldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Steady-state kinetic analysis using the Ltp2-DUF35 complex showed that the aldolase had optimal activity at pH 7.5, with a of 6.54 ± 0.90 μM and a of 159 ± 8.50 s ChsH1-ChsH2 could hydrate only about 30% of 3-OPDC-CoA, but this unfavorable equilibrium could be overcome when the aldolase was present to remove the hydrated product, providing a rationale for the close association of the aldolase with the hydratase. Homologs of ChsH1, ChsH2, and Ltp2 are found in steroid-degrading Gram-positive and Gram-negative bacteria, suggesting that side chains of diverse steroids may be cleaved by aldolases in the bacteria. The C-C bond cleavage of the D-ring side chain of cholesterol was shown to be catalyzed by an aldolase. The aldolase associates with the hydratase that catalyzes the preceding reaction in the cholesterol side chain degradation pathway. These enzymes are encoded by genes within the intracellular growth () operon of , and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice.

No abstract

The glycoside hydrolase, PslG, attacks and degrades the dominant Psl polysaccharide in the exopolymeric substance (EPS) matrix of Pseudomonas aeruginosa biofilms and is a promising therapy to potentiate the effect of antibiotics. However, the need for coadministration with an antibiotic and the potential susceptibility of PslG to proteolysis highlights the need for an effective delivery system. Here, we compared liposomes versus lipid liquid crystal nanoparticles (LCNPs) loaded with PslG and tobramycin as potential formulation approaches to (1) protect PslG from proteolysis, (2) trigger the enzyme's release in the presence of bacteria, and (3) improve the total antimicrobial effect in vitro and in vivo in a Caenorhabditis elegans infection model. LCNPs were an effective formulation strategy for PslG and tobramycin that better protected the enzyme against proteolysis, triggered and sustained the release of PslG, improved the antimicrobial effect by 10−100-fold, and increased the survival of C. elegans infected with P. aeruginosa. Digestible LCNPs had the advantage of triggering the enzyme's release in the presence of bacteria. However, compared to nondigestible LCNPs, negligible differences arose between the LCNPs' ability to protect PslG from proteolysis and potentiate the antimicrobial activity in combination with tobramycin. In C. elegans, the improved antimicrobial efficacy was comparable to tobramycin-LCNPs, although the PslG + tobramycin-LCNPs achieved a greater than 10-fold reduction in bacteria compared to the unformulated combination. Herewith, LCNPs are showcased as a promising protective delivery system for novel biofilm dispersing enzymes combined with antibiotics, enabling infection-directed therapy and improved performance.

The bacterium Pseudomonas aeruginosa can colonize the airways of patients with chronic lung disease. Within the lung, P. aeruginosa forms biofilms that can enhance resistance to antibiotics and immune defenses. P. aeruginosa biofilm formation is dependent on the secretion of matrix exopolysaccharides, including Pel and Psl.