Crescente, Vincenzo (2015) Azoreductases : genes and proteins in ' Pseudomonas aeruginosa '. (PhD thesis), Kingston University, .
Abstract
Pseudomonas aeruginosa is one of the primary causes of opportunistic infections in humans and it is associated with both acute and chronic infections in immunocompromised individuals. This bacterium is extremely resistant to many antibiotics, making the treatments against this pathogen often unsuccessful. Azoreductases, a family of enzymes involved in the reduction of azo compounds and quinones, are found in many bacterial species including P. aeruginosa. Although the enzymatic activity of three of these enzymes has been extensively characterized, their physiological role remains unclear. In this study, the enzymatic activity as well as the effect on physiological processes such as swarming motility, biofilm formation and antibiotic resistance of known and putative azoreductase proteins from P. aeruginosa PAO1 have been investigated. Five putative azoreductase genes from P. aeruginosa PAO1 (pa0949, pa1204, pa2280, pa2580 and pa4975) were cloned and four of these (pa0949, pa1204, pa2280 and pa2580) were over expressed in E. coli strains. Recombinant proteins were purified and biochemically characterized showing the presence of FAD bound to PA1204 and PA2580 proteins. Enzymatic reaction conditions were established for each protein by determining the preferred cofactor and reductant (flavin and NAD(P)H) used by each protein. Higher reduction rates were obtained using FAD for PA1204 and PA2580, and FMN for PA0949 and PA2280, whereas NADPH was always the preferred reductant for all of the enzymes tested. Substrate specificity studies performed with azo compounds and quinones showed that PA1204, PA2280 and PA2580 recombinant proteins can reduce both classes of substrate, with higher reduction rates with quinones, whereas the recombinant PA0949 protein showed to reduce only quinone substrates. Investigation of the role of azoreductase genes on P. aeruginosa PAO1 motility, biofilm formation and antibiotic resistance was conducted using single gene transposon mutants for each of the genes paazor1, paazor2, paazor3, pa0949, pa1204, pa2280, pa2580 and pa4975. Motility analysis demonstrated greater swarming for all mutants tested compared with wild type, although both wild type and mutants showed the same growth rate. Similarly, all mutants showed higher biofilm production compared with the wild type in a short term period (24 hours), whereas no differences were observed after a longer biofilm production period (48 hours). The MICs and MBCs of several antibiotics were determined, showing that, in presence of fluoroquinolones, P. aeruginosa PAO1 azoreductase mutants exhibit higher growth inhibition (up to 127-fold) and reduced survival (up to 7 fold) compared with wild type. This suggests that azoreductase genes (or gene products) may be involved in the P. aeruginosa PAO1 resistance to antibiotic treatments, and in particular to fluoroquinolones. The findings prove that the proteins PA0949, PA1204, PA2280 and PA2580 have similar features and enzymatic functions to the already characterized paAzoR1-3 from P. aeruginosa PAO1. Therefore, these can be included in the family of azo- and quinone- oxidoreductase enzymes. The data presented here on the antibiotic resistance strongly suggest a role for azoreductase gene products in antimicrobial resistance in P. aeruginosa. These original findings provide a springboard for further investigation of azoreductases as novel targets for antimicrobial agents for this pathogen.
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