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Genetic mutations enhance the production of Exotoxin A by Pseudomonas aeruginosa for use as a potential anticancer therapeutic agent

  • Authors:
    • Ibtesam S. Almami
  • View Affiliations / Copyright

    Affiliations: Department of Biology, College of Science, Qassim University, Buraydah, Al-Qassim 52571, Saudi Arabia
    Copyright: © Almami et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 161
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    Published online on: June 23, 2025
       https://doi.org/10.3892/etm.2025.12911
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Abstract

Microbial proteins have emerged as promising anticancer agents for various cancer types. Pseudomonas aeruginosa derived‑Exotoxin A is a potent virulence factor that specifically binds to the α2‑macroglobulin cell receptor. It exhibits strong cytotoxicity and potential advantages over conventional cancer treatments due to its ability to penetrate cancer cell membranes and inhibit protein synthesis. In the present study, 20 P. aeruginosa isolates collected from microbiological laboratories between October 2023 and January 2024 were characterized. The isolates were identified using classical biochemical tests, confirmed using an automated identification system, and classified into six groups based on SDS‑PAGE protein banding patterns. The gene encoding Exotoxin A was amplified in one isolate from each group using PCR, yielding a 367‑bp amplicon. To enhance the production of Exotoxin A, random mutations were introduced to the selected isolates using UV irradiation. Exotoxin A was then purified using column chromatography followed by dialysis, resulting in a product with a molecular mass of ~66 kDa as determined by SDS‑PAGE. The cytotoxic effects of crude and purified Exotoxin A were assessed against the MCF‑7 breast cancer cell line. MTT assay results revealed that the half‑maximal inhibitory concentration (IC50) of purified Exotoxin A from a wild‑type isolate was 4.9 µg/ml, whereas the corresponding mutant exhibited an IC50 of 3.6 µg/ml, indicating a 1.4‑fold increase in cytotoxic activity. The findings of the present study highlight the potential of microbial‑derived proteins in cancer therapy. However, further evaluation of these proteins is necessary to explore their therapeutic applicability.
View Figures

Figure 1

Identification of P. aeruginosa
isolates. (A) Gram-negative P. aeruginosa observed as
pink-red rods under a microscope after Gram staining
(magnification, x1,000; oil immersion). (B) Reference biochemical
identification tests for P. aeruginosa (from left to right):
Urease, citrate and TSI control agar slants. (C) Representative
experimental results for P. aeruginosa isolates: Urease test
(+, pink), citrate test (+, blue), and TSI (-) with no gas
production. (D) Characteristic blue-green pigment (pyocyanin)
production on cetrimide agar slant, indicating a positive result
for P. aeruginosa. P. aeruginosa, Pseudomonas
aeruginosa; TSI, triple sugar iron.

Figure 2

Protein profiling and dendrogram of
the P. aeruginosa isolates. (A) SDS-PAGE protein profiles of
selected P. aeruginosa isolates. (B) Dendrogram representing
the classification of 20 P. aeruginosa isolates into six
groups based on SDS-PAGE band patterns: Group 1, isolates 1-3;
group 2, isolates 4-7; group 3, isolates 8-11, group 4, isolates
12-15; group 5, isolates 16 and 17; group 6, isolates 18-20. The
SDS-PAGE analysis revealed protein sizes ranging from 10-260 kDa,
with variations in band intensity and distribution. Among these,
isolate 4 was selected for further mutation studies due to its
strong protein expression of Exotoxin A. P. aeruginosa,
Pseudomonas aeruginosa; M, protein molecular weight marker.

Figure 3

Detection of Exotoxin A gene by PCR.
PCR products obtained using primer S1 for (A) six wild-type
isolates (1, 4, 8, 14, 16, and 20) and (B) six corresponding
mutated isolates (1*, 4*, 8*, 14*, 16*, 20*). PCR analysis
confirmed the presence of a 367-bp amplicon in all tested isolates,
characteristic of the Exotoxin A gene. Following mutagenesis,
additional DNA bands were observed in mutant 4*, suggesting
modifications in primer binding sites due to genetic variations. M,
lambda DNA marker.

Figure 4

SDS-PAGE profiling of crude proteins
and purified exotoxin A from isolate 4 and mutant 4*. SDS-PAGE was
used for the comparison of protein profiles between exotoxin A in
its crude form and following purification using column
chromatography. M, protein molecular weight marker.

Figure 5

Cytotoxic effects of crude and
purified Exotoxin A against MCF-7 cells, assessed using MTT assay.
(A) Cytotoxicity of Exotoxin A from isolate 4. The IC50
values for crude and purified Exotoxin A were 13.1 and 4.9 µg/ml,
respectively, corresponding to logIC50 values of 1.125
and 0.6938, respectively. (B) Cytotoxicity of Exotoxin A from
mutant 4*, The IC50 values for crude and purified
Exotoxin A were 12.0 and 3.6 µg/ml, respectively, corresponding to
logIC50 values of 1.082 and 0.5519, respectively. Data
represent the mean ± SEM from three independent experiments
performed in duplicate. IC50 values were calculated
using non-linear regression analysis (log Exotoxin A concentration
vs. normalized cell viability). IC50, half-maximal
inhibitory concentration.

Figure 6

Cytotoxic effects of crude and
purified Exotoxin A on MCF-7 cell viability, assessed using MTT
assay. Exotoxin A from (A) isolate 4 and (B) mutant 4* was
evaluated at a range of different concentrations. Purified Exotoxin
A exhibited significantly higher cytotoxicity compared with its
crude counterpart, with mutant 4* exhibiting the greatest increase
in cytotoxicity upon purification. Statistical significance was
determined using two-way ANOVA followed by Bonferroni's multiple
comparisons tests. Data are presented as the mean ± SEM from three
independent experiments performed in duplicate. Exotoxin A
concentrations are presented on a logarithmic scale (µg/ml).
*P<0.05 and ****P<0.0001 as
indicated.
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Copy and paste a formatted citation
Spandidos Publications style
Almami IS: Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent. Exp Ther Med 30: 161, 2025.
APA
Almami, I.S. (2025). Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent. Experimental and Therapeutic Medicine, 30, 161. https://doi.org/10.3892/etm.2025.12911
MLA
Almami, I. S."Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent". Experimental and Therapeutic Medicine 30.2 (2025): 161.
Chicago
Almami, I. S."Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent". Experimental and Therapeutic Medicine 30, no. 2 (2025): 161. https://doi.org/10.3892/etm.2025.12911
Copy and paste a formatted citation
x
Spandidos Publications style
Almami IS: Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent. Exp Ther Med 30: 161, 2025.
APA
Almami, I.S. (2025). Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent. Experimental and Therapeutic Medicine, 30, 161. https://doi.org/10.3892/etm.2025.12911
MLA
Almami, I. S."Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent". Experimental and Therapeutic Medicine 30.2 (2025): 161.
Chicago
Almami, I. S."Genetic mutations enhance the production of Exotoxin A by <em>Pseudomonas aeruginosa</em> for use as a potential anticancer therapeutic agent". Experimental and Therapeutic Medicine 30, no. 2 (2025): 161. https://doi.org/10.3892/etm.2025.12911
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