His6-OPH and Its Stabilized Forms Combating Quorum Sensing Molecules of Gram-Negative Bacteria in Combination with Antibiotics

Abstract

Background: In most species of gram-negative pathogenic and conditionally pathogenic bacteria, quorum sensing-dependent systems have been discovered, in which various N-acyl-L-homoserine lactones function as signaling molecules inducing the development of resistance towards the effects of antibiotics. Objectives: The purpose of this study was to investigate the possibility of stabilizing lactonase activity of His6-OPH by forming enzyme polyelectrolyte non-covalent complexes (EPNCs) containing both this enzyme and polyanionic polymer. It also targeted at studying the efficacy of these EPNCs in combination with a variety of antibiotics (ampicillin, gentamicin, kanamycin, and rifampicin) against gram-negative bacteria. Cheap non-toxic biodegradable polyamino acids (poly-L-glutamic acid and poly-L-aspartic acid) were selected as polyanionic polymers to produce EPNCs. Methods: Recombinant Escherichia coli strain SG13009 [pREP4], transformed by plasmid encoding His6-OPH, was used for His6-OPH production. The His6-OPH lactonase activity was determined according to known methods, using a pH-sensitive indicator (cresol red) and a colorimetric method. The minimum inhibitory concentration (MIC) of antibiotics was determined with the suspension of bacteria cells, with antibiotic added in a concentration range from 0 to 500 mg/L at 37°C during 16 hours. Results: All the tested enzyme preparations were equally efficient in catalyzing the hydrolysis of N-acyl-homoserine lactones of various structures containing and lacking a 3-oxo group in the acyl radical. It has been established that the presence of enzyme preparations facilitates the reduction of MIC of antibiotics (ampicillin, gentamicin sulfate, kanamycin sulfate, and rifampicin) on the growth of highly concentrated (106 cells/mL) cell populations of Pseudomonas aeruginosa B-6643 and Escherichia coli B-6645. Polyelectrolyte complexes of this enzyme have an increased efficiency at a lower pH (6.5) of the medium in comparison to its highly purified form. It is possible to reduce the minimum inhibitory concentration of antibiotics by 10% to 850% in combined use of antibiotics and His6-OPH enzyme preparations. Conclusions: The discussed approaches for the use of original biological products based on His6-OPH opens up opportunities for creating new ways of effectively controlling bacterial diseases in animals.