الفهرس 
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Abstract
Exotoxin A (PE toxin) is the most venomous virulence determinant expressed by P. aeruginosa strains due to its ADP-ribosyl transferase cytotoxicity. The ADP-ribosyl transferase activity of PE toxin acts upon eukaryotic elongation factor 2 (eEF-2) at its dipthamide apex blocking the alternating movement of acylated tRNA and deacylated tRNA. This in turn halts the peptide chain elongation and terminates the protein synthesis within the affected cells resulting in cellular apoptosis. Such effect made it the toxin of choice during the construction of immunotoxins for targeted cancer therapy.
This work explored ADP-ribosyl transferase activity of PE toxin isolated from clinical Pseudomonas aeruginosa isolates using NAD+ and guanyl hydrazone derivative, 4-nitrobenzylidine aminoguandine (NBAG) and the impact of gamma radiation on it. The NBAG was the acceptor of the ADP-ribose moiety from NAD+ instead of wheat germ extract elongation factor 2 and radiolabeled NAD+. Afterwards, the nucleotide sequence that encodes catalytic domain Ib/III along with the furin cleavage site of exotoxin A retrieved from clinical Pseudomonas aeruginosa isolates (PE24 moiety) was cloned into pET22b(+) for production by E. coli BL21 (DE3). The ADP-ribosylation capability of the recombinant PE24 extract was explored on NBAG and on in vitro cultured carcinoma cell lines. Moreover, the impact of conjugating radiotherapy with the expressed PE24 toxin moiety on the growth and proliferation of HEPG2 carcinoma cell lines was investigated.
A total of 40 clinical P. aeruginosa isolates were retrieved from pus samples, cultivated on Cetrimide agar and four isolates were identified by 16s rRNA. The isolates were then screened for the presence of toxA gene by conventional PCR. A total of 32 isolates harbored toxA gene and the identity of the amplified amplicon was confirmed by Sanger sequencing analysis. The NBAG was synthesized from aminoguanidine bicarbonate and 4-nitrobenzaldehyde. The synthesis reaction was successful as confirmed by the different spectroscopic techniques (UV, FTIR, C13-NMR, APCI and ESI spectroscopy). The NBAG was then used along with NAD+ to test ADP-ribosyl transferase activity of PE toxin retrieved from P. aeruginosa isolates.
The PE toxin was then extracted from some selected P. aeruginosa isolates and purified by dialysis prior to ADP-ribosyl transferase assay. Before the addition of NBAG/NAD+ mixture to purified exotoxin A extract, the extract was reduced by DTE to activate catalytic domain III of PE-toxin since it’s secreted in its proenzyme form and DTE facilitate proteolytic cleavage that initiate its enzymatic activity. After reduction with DTE and addition of NBAG/NAD+ mixture, the reaction tubes were examined by UV spectroscopy and HPLC against reference reaction tubes that lacked exotoxin A extract.
A prominent shift in λmax of NBAG to a higher wavelength (380 nm) was observed after incubation with PE extract. This shift suggested the ADP-ribosylation of NBAG by PE toxin and the deprotonation of its guanidine group. Further, analysis of reaction tube by HPLC denoted the rise of new peaks after the main peak of NBAG which is assigned to the ADP-ribosylation of NBAG by PE toxin. The peaks around retention time (" ~ "2-4) suggested the hydrolysis of NAD+ and the breakage of glycosidic bond C-N releasing ADP-ribose sugar by PE toxin. The precipitate formed following the addition of PE extract to NBAG/NAD+ mixture by FTIR spectroscopy denoted the reduction of NH amine stretch which support the acceptance of ADP-ribosyl moiety by the NBAG and that the ADP-ribosyl transferase reaction took place. Both UV spectroscopy and HPLC analysis of NBAG/NAD+ post exposure to PE toxin confirmed its ADP-ribosyl transferase activity.
The ADP-ribosyl transferase cytotoxicity of PE toxin was explored on Hep-2 cells using SRB quick cytotoxicity screening assay. The quick cytotoxicity screening assay revealed that 100 µg/ml induced significant cytotoxic effect on cultured cells indicating that the toxin exerted its activity intracellular and ADP ribosylated eEF-2 blocking the protein synthesis and inducing cellular apoptosis.
The impact of low dose gamma radiation on ADP-ribosyl transferase activity of PE toxin was also investigated and it was noted that ADP-ribosyl transferase activity of PE toxin on NBAG/NAD+ mixture in both UV spectroscopy and HPLC reaction conditions was reduced. Moreover, the IC50 values of PA 22 exotoxin A extract was increased after exposure to low dose gamma radiation. This effect might be attributed to oxidative modification incurred by ionizing radiation in cellular proteins that disrupts their functions. Oxidative changes are usually associated with conformational changes that alter the enzymatic activity. It was also noted that exposure of P. aeruginosa to low dose gamma radiation was associated with adenine base deletion in toxA gene at the three tested radiation doses, 5, 10 and 24 Gy that might be attributed to induction of abasic sites by gamma radiation and consequently altered amino acid sequence in the protein extract which in turn affects the enzymatic activity of Exotoxin A protein extract.
Following the confirmation of ADP-ribosyl transferase activity of PE toxin extract retrieved from P. aeruginosa isolates, the nucleotide sequence encoding domain Ib/III i.e. PE24 moiety was amplified and cloned into pET22b(+) plasmid. During the amplification reaction, the restriction sites for NdeI and EcoR1 was introduced to facilitate the digestion and ligation by T4 DNA ligase reaction. Post digestion and ligation of pET22b(+) and PE24 amplicons, the constructs (pET22b(+)-PA 16 and pET22b(+)-PA 22) were transformed into E. coli BL21 (DE3) by CaCl2/ heat shock transformation technique. The transformed isolates were then grown in LB agar plates containing 100 μg/ml ampicillin that allowed the growth of pET22b(+)-PA16 and pET22b(+)-PA22 transformed E. coli BL21 (DE3) only.
The recombination technique was proved successful by colony PCR, sequencing analysis and re-digestion of (pET22b(+)-PA16 and pET22b(+)-PA22) constructs by NdeI and EcoR1 where the PE24 insert was electrophoresed on agarose gel. Expression of PE24 moiety by E. coli BL21 (DE3) was initiated by cultivation in LB broth containing 0.5 mM IPTG. Protein extracts from E. coli BL21, (pET22b(+)-PA16 and pET22b(+)-PA22) harboring E. coli BL21 and empty pET22b(+) containing E. coli BL21 were electrophoresed through sodium dodecyl sulfate–polyacrylamide gel and PE24 insert appeared among (pET22b(+)-PA16 and pET22b(+)-PA22) harboring E. coli BL21 extracts only at " ~ " 30KDa. This confirmed the expression of PE24 moiety by E. coli BL21 (DE3) post transformation by (pET22b(+)-PA16 and pET22b(+)-PA22) constructs.
The recombinant PE24 moiety extracts retrieved from pET22b(+)-PA 16/PA 22 harboring E. coli BL21 (DE3) was then subjected to several in vitro assays. First, the ADP ribosyl transferase activity of PE24 extract was tested using NBAG/NAD+ mixture as described earlier using different spectroscopic techniques (UV, FTIR, and C13-NMR) and HPLC. A similar shift to higher λmax (" ~ "370 nm) was observed post exposure of NBAG/NAD+ mixture to PE24 extract suggesting the ADP-ribosylation of NBAG. Both FTIR and C13-NMR spectroscopic analysis of reaction mixture confirmed the ADP-ribosylation of NBAG.
In similar manners, HPLC chromatograms of NBAG/NAD+ mixtures treated by the recombinant PE24 extracts elucidated that ADP-ribosylation reaction took place. The rise of new peaks before NBAG peak proposed the hydrolysis of NAD+ and breakage of glycosidic C-N bond by the recombinant PE24 moiety. Interestingly, the recombinant PE24 moiety was able to produce higher amounts of ADP-ribosylated NBAG than that the whole toxin retrieved from P. aeruginosa species. This might be attributed to the removal of the steric hindrance posed by both domains Ia and II that reduce the enzymatic activity of catalytic domain III.
The impact of low dose gamma radiation on the recombinant PE24 expression by E. coli BL21 (DE3) was also explored by SDS-PAGE, UV spectroscopy and HPLC. In SDS-PAGE, it was noted that expression of PE24 moiety was halted as noted by absence of the PE24 band. Further, ADP-ribosylating capability was reduced in a dose dependent manner as irradiated plasmids are known for their low transformation efficacies and expression profiles. Additionally, exposure of PE24 moiety to low concentrations of metal salts was also reflected by reduction in ADP-ribosylating activity of PE24 moiety retrieved from E. coli BL21 (DE3).This effect might be attributed to the conformational changes in protein induced by metal ions that lowered its enzymatic activity.
The ADP-ribosyl transferase cytotoxicity of the recombinant PE24 extract was then assessed on different in vitro cultured cancer cell lines by different cytotoxicity assays. The recombinant PE24 moiety exhibited prominent cytotoxic effects on the adherent cell lines, HEPG2, MCF-7, A375, and Kasumi-1 cell suspension with IC50 values below 10 μg/ml with R2 values ranging from 0.8105 to 0.9977 suggesting the good inhibitory effect of PE24 moiety on cancer cells. It was also noted that 10 μg/ml of PE24 extract showed " ~ "90% cell viability on normal OEC cells. This indicates that the PE24 moiety incurs less cytotoxic effects on the normal cells as previously reported.
Furthermore, combining low dose paclitaxel (5 μg/ml) to PE24 was associated with synergistic effect where a prominent reduction in IC50 value by 17.5% in the case of HEPG2 and 41.1% for MCF-7 were recorded. This synergistic effect might be attributed to enhanced cellular uptake of PE24 moiety by cancer cells post-exposure to paclitaxel.
On the contrary to previous reports, combining low dose gamma radiation with the recombinant PE24 toxin moiety was associated with antagonistic effect. The IC50 value of PE24 was increased by 27.2% and 56% among HEPG2 cells pre-exposed to gamma radiation at 5 and 24 Gy, respectively. Irradiating HEPG2 cells post-exposure to PE24 was associated with an elevation in IC50 values by 8.3% and 26.3% at 5 and 24 Gy, respectively. The antagonist effect incurred by low dose gamma radiation might be attributed to the stemness effect exhibited by gamma irradiated HEPG2 cells that enhanced their differentiation and alters their interaction with the surrounding microenvironment, affecting their quiescence, proliferation, and regeneration and rendered them more resistant to external treatment.