Abstract
This work explores the ADP-ribosyltransferase activity of Pseudomonas (P.) aeruginosa exotoxin A using the guanyl hydrazone derivative, nitrobenzylidine aminoguanidine (NBAG) and the impact of gamma radiation on its efficacy. Unlike the conventional detection methods, NBAG was used as the acceptor of ADP ribose moiety instead of wheat germ extract elongation factor 2. Exotoxin A was extracted from P. aeruginosa clinical isolates and screened for toxA gene using standard PCR. NBAG was synthesized using aminoguanidine bicarbonate and 4-nitrobenzaldehyde and its identity has been confirmed by UV, FTIR, Mass and 13C-NMR spectroscopy. The ADP-ribosyl transferase activity of exotoxin A on NBAG in the presence of Nicotinamide adenine dinucleotide (NAD+) was recorded using UV spectroscopy and HPLC. In vitro ADP-ribosyl transferase activity of exotoxin A protein extract was also explored by monitoring its cytotoxicity on Hep-2 cells using sulforhodamine B cytotoxicity assay. Bacterial broths were irradiated at 5, 10, 15, 24 Gy and exotoxin A protein extract activity were assessed post exposure. Exotoxin A extract exerted an ADP-ribosyltransferase ability which was depicted by the appearance of a new ʎmax after the addition of exotoxin A to NBAG/NAD+ mixture, fragmentation of NAD+ and development of new peaks in HPLC chromatograms. Intracellular enzyme activity was confirmed by the prominent cytotoxic effects of exotoxin A extract on cultured cells. In conclusion, the activity of Exotoxin A can be monitored via its ADP-ribosyltransferase activity and low doses of gamma radiation reduced its activity. Therefore, coupling radiotherapy with exotoxin A in cancer therapy should be carefully monitored.
Highlights
Pathogenic Pseudomonas (P.) aeruginosa possess virulence factors that aggravate the severity of their infections and contribute to series outcomes
We aimed to explore the ADP-ribosyltransferase activity for exotoxin A isolated from clinical P.s aeruginosa isolates using N AD+ and the guanyl hydrazone derivative, 4-nitrobenzylidine aminoguandine (NBAG) and the impact of gamma radiation on it
Exotoxin A gene was prevalent in 80% of the recovered clinical isolates
Summary
Pathogenic Pseudomonas (P.) aeruginosa possess virulence factors that aggravate the severity of their infections and contribute to series outcomes. Exotoxin A is the most prevalent and toxic virulence factor among pathogenic P. aeruginosa species that acquires ADPribosyltransferase activity It is the reason behind the increased mortality rates among experimentally infected animals where a single injection of 80 ng was sufficient to induce severe liver necrosis and swelling, hemorrhage in the lungs and kidneys within 48 h of exposure (Wretlind and Pavlovskis 1981). The ADP-ribosylation pathway of PE toxin follows an SN1 nucleophilic substitution mechanism where it first attaches itself to NAD+ via active site loop L4 (aa 483–490 of domain III) (Yates and Merrill 2004). EF-2 becomes ADP-ribosylated and inactive, halting the protein synthesis within the affected cell via their inability to elongate polypeptide chains This induces irreversible cellular death (Armstrong et al 2002; Jørgensen et al 2005; Michalska and Wolf 2015). The guanyl hydrazones derivatives have been deployed in the detection of the ADP-ribosyltransferase activity of Cholera toxin and ribosyl-transferases retrieved from animal tissues (Soman et al 1983)
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