Role of an hpd gene in the oxidative stress response of a human pathogenic bacterium Pseudomonas aeruginosaTrishna Manandhar
( M.Sc. )
Pseudomonas aeruginosa is a gram negative bacterial pathogen associated with high morbidity and mortality. This ubiquitous bacterium act as the opportunistic pathogen to human particularly causing nosocomial infections in hospitalized patients, immunocompromised hosts and patients with burn wound infections as well as cystic fibrosis. Among many virulence factors, the ability to survive the oxidative stress is one of the important system by which this versatile bacterium can evade host defenses. Homogentisate production, augmented by 4-Hydroxyphenylpyruvate dioxygenase (HPPD), is considered significant front line defense mechanism by which the bacteria can detoxify free radicals and protection against organic hydroperperoxide. Here we identify hpd (PA0242) gene that is involved in conversion of 4-hydroxyphenylpyruvate (4HPP) to 2, 5-dihydroxyphenylacetate (homogentisate) that provide bacteria with added ability to fight against oxidative stress response and overcome host-pathogen interactions. To investigate the functional role of P. aeruginosa hpd (PA0242) gene in protection against oxidative stress response, the study was carried out with the principle objectives to investigate the phenotypic characterstics of hpd gene, its expressional analysis under oxidative stress and role of homogentisate in detoxification of hydroperoxide radicals. In order to study the physiological role of hpd (PA0242) gene in of P. aeruginosa, mutant strain of this gene was created by Cre-lox system. Complementation system for hpd (PA0242) expression in ∆hpd strain was done by introducing the pBBR-Gm plasmid harboring full length hpd (PA0242) gene with ribosome binding site. The oxidant killing assay revealed that the mutant strain was sensitive to the oxidative stress caused by organic hydrperoxides (OHPs). The survival capability of mutant strain of hpd showed 3 and 4 log reduction upon the killing of cells by 5 mM and 10 mM t-BOOH respectively and 1-2 log reduction upon the treatment 5 mM CHP. The phenotype was shown to restore its ability to resist the oxidative stress in the complementary strain. To investigate the expressional analysis of hpd (PA0242) and its possible regulation by putative transcriptional regulator tetR gene, reverse transcription end point PCR was carried out. Expressional analysis of hpd (PA0242) show that expression of this gene is organic hydroperoxide inducible and is induced by treatment of cells with 250 µM CHP and 250 µM t-BOOH. The experimental results showed that the tetR gene that is also induced by organic hydroperoxides, which is suggestive of the redox sensing ability of this possible transcription regulator. Moreover, the generation of expected intergenic regional band upon induction of cells with organic hydriperoxides is suggestive of the speculation that hpd and tetR were transcribed in an operon. Hydroperoxide degradation ability of homogentisate was carried using modified Ferrous Oxidation Xylenol-Orange (FOX) assay. It was experimentally shown that homogentisate was able to reduce the amount of hydroperoxides generated by hydrogen peroxides (H2O2) by 60.7% and linoleic hydroperoxide (LOOH) by 60.6 %.