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Introduction

Pseudomonas bacteria, which belong to the family Pseudomonadaceae (1), are gram-negative rods and are commonly found in diverse environments, including soil, water and vegetation (2). Their ability to survive in extreme conditions is accompanied by their pathogenicity in immunocompromised individuals, such as those suffering from cystic fibrosis (CF) or acquired immunodeficiency syndrome (3). Patients with CF often develop high levels of circulating Exotoxin A antibodies, which have been associated with increased mortality (4).

Pseudomonas infections are multifactorial due to the numerous virulence factors these bacteria produce, and can result in a variety of diseases, including septicemia, urinary tract infections, pneumonia, chronic lung infections, endocarditis, dermatitis and osteochondritis (5). Clinical isolates of P. aeruginosa are often multidrug-resistant, posing considerable challenges for infection management (6). These infections typically progress in three stages: Bacterial adhesion and colonization, local invasion, and disseminated systemic disease (7).

P. aeruginosa secretes various virulence factors, including pyocyanin, elastase, exoenzyme S, phospholipase C, Exotoxin A and siderophores, all of which contribute to tissue damage and bacteremia (5). Among these, Exotoxin A is the most virulent, causing direct tissue damage and necrosis (8). It functions by enzymatically transferring a nicotinamide adenine dinucleotide molecule onto elongation factor 2, thereby halting polypeptide chain elongation and leading to cell death (9). A number of exotoxin genes are carried by mobile genetic elements such as bacteriophages, and are associated with a number of human diseases (10). Transduction between phages and bacteria can result in the rapid evolution of new pathogens, which may have major consequences for public health (9).

Exotoxin A, encoded by the toxA gene, is produced as a 71-kDa precursor and secreted as a 66-kDa toxin via the type II secretion system. Its expression is regulated by environmental factors such as temperature, amino acids and aeration, with optimal production observed at 32˚C in iron-deficient media (11). The toxin exerts its effects through a three-step mechanism: i) Binding to the α2-macroglobulin receptor, a member of the low-density lipoprotein receptor family; ii) internalization via endocytosis; and iii) cleavage by a protease, followed by translocation to the cytosol, where it ADP-ribosylates elongation factor 2, thereby blocking protein synthesis and causing cell death (12).

The potential of microbial-derived proteins, particularly bacterial toxins, has garnered interest as an innovative approach to cancer therapy. Bacterial toxins can selectively target and disrupt tumor cells, offering a promising alternative to current cancer therapies (13). Exotoxin A produced by P. aeruginosa has been shown to exhibit notable specificity for cancer cells by inhibiting protein synthesis and inducing apoptosis. This specificity is attributed to the altered metabolic and signaling pathways characteristic of malignant cells, which increase their susceptibility to bacterial toxins compared with that of normal cells (11). Recent advances in genetic engineering have enhanced the therapeutic potential of bacterial toxins. Through targeted mutations, the efficacy and selectivity of Exotoxin A can be improved, reducing potential side effects and improving cancer cell specificity. Furthermore, understanding the regulatory mechanisms governing Exotoxin A expression and secretion is crucial for the development of a safe and precisely controlled therapeutic approach (13).

Despite the promise of bacterial toxins in oncology, several challenges remain, particularly those associated with biosafety, immunogenicity and delivery mechanisms (14). The introduction of Exotoxin A as a cancer therapeutic requires a thorough investigation of its pharmacokinetics, dosage optimization and potential immune responses in human subjects. Furthermore, ethical considerations regarding the use of bacterial toxins in clinical settings must be addressed to ensure their safe and effective application in cancer therapy (15).

Despite the potency of current cancer treatments, treatment failure in certain patients may occur, necessitating alternative therapeutic strategies. Bacterial toxins, such as Exotoxin A, have demonstrated promising anticancer effects by specifically targeting cancer cells, including MCF-7 breast cancer cells (16). In addition, Exotoxin A has been found to inhibit the formation of pre-cancerous lesions induced by potent carcinogens (17). The present study aimed to explore the potential of P. aeruginosa-derived Exotoxin A as an anticancer agent by inducing genetic modifications to enhance its production and cytotoxic efficacy. By presenting preliminary insights into the genetic variations that influence Exotoxin A synthesis and evaluating the cytotoxicity of Exotoxin A in a breast cancer cell line, the present study contributes to the growing body of knowledge regarding microbial-based cancer therapeutics.

Materials and methods

Isolation and bacterial identification

A total of 20 clinical P. aeruginosa isolates were collected from Qassim University Hospital and King Fahad Specialist Hospital (both Al-Qassim, Saudi Arabia) between October 2023 and January 2024. These isolates were donated by the hospitals for research purposes. Samples were obtained under aseptic conditions prior to antibiotic treatment or 3 days after the cessation of antibiotic therapy. The isolates were cultured on MacConkey agar (Oxoid, Ltd.; Thermo Fisher Scientific, Inc.) and incubated at 37˚C for 24 h. Colonies were subsequently subcultured on cetrimide agar (Oxoid, Ltd.; Thermo Fisher Scientific, Inc.) to assess pyocyanin pigment production (18). Bacterial identification was performed based on morphological characteristics (Gram staining) and biochemical tests, including the triple sugar iron (TSI) (19), glucose fermentation, citrate utilization and urease tests (20). The identities of the isolates were confirmed using the Sensititre™ Complete Automated Antimicrobial Susceptibility Testing System (Thermo Fisher Scientific, Inc.), which includes 32 biochemical tests for gram-negative bacteria.

Molecular characterization. SDS-PAGE analysis

Total cellular proteins were extracted from all isolates and analyzed via SDS-PAGE (Bio-Rad Laboratories, Inc.). Bacterial pellets were suspended in protein extraction buffer consisting of 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.01% bromophenol blue (Sigma-Aldrich; Merck KGaA), then boiled at 95˚C for 5 min to lyse the cells and denature proteins. Protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. Equal amounts of protein (50 µg) were loaded onto a 12% polyacrylamide gel and then subjected to electrophoresis at 175 V for 1 h. The gels were stained with 0.2% Coomassie Blue R-250 (Uptima; Interchim) 1 h at room temperature. Densitometric analysis of the gel bands was performed using Image Lab software 6.1 (Bio-Rad Laboratories, Inc.).

Detection of Exotoxin A gene by PCR. PCR was conducted to amplify the Exotoxin A gene using specific primers (Table I). DNA was extracted using a DNeasy Kit (Qiagen, Inc.) following the manufacturer's instructions. Each 25-µl reaction mixture contained 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTP mix, 1 µM each primer, 1-unit Taq DNA polymerase (Thermo Fisher Scientific, Inc.), and 50 ng genomic DNA. The thermocycling conditions consisted of an initial denaturation step at 94˚C for 5 min, followed by 35 cycles of denaturation at 94˚C for 1 min, annealing at 60˚C for 45 sec, and extension at 72˚C for 3 min, with a final extension at 72˚C for 5 min. Amplified PCR products were resolved by 1.5% agarose gel electrophoresis (21), stained with ethidium bromide, and visualized under UV transillumination.

Table I Primer sequences used in PCR analysis.

Table I

Primer sequences used in PCR analysis.

Primer	Sequence (5'-3')	Target size (Ref.)	GenBank accession no.
S1-F	GAC AAC GCC CTC AGC ACC AGC	367 bp (21)	NC_002516.2
S1-R	CGC TGG CCC ATT CGC TCC AGC GCT

[i] F, forward; R, reverse.

Mutation induction

Six wild-type Pseudomonas isolates (numbers 1, 4, 8, 14, 16 and 20) were selected based on dendrogram analysis and suspended in 5 ml M9 minimal medium (Sigma-Aldrich; Merck KGaA). To induce genetic variability, UV irradiation at 260 nm was applied, as this wavelength is able to introduce point mutations and small deletions by promoting the formation of pyrimidine dimers, which can lead to errors in DNA repair (22,23). This method is widely used for bacterial mutagenesis as it generates a broad spectrum of mutations while maintaining cell viability under optimized exposure conditions (14). The isolates were exposed to UV light at 260 nm for 5 sec from a height of 15 cm at room temperature (22-25˚C) in the absence of daylight or ambient light. After irradiation, the cells were incubated in the dark at room temperature for 1 h to allow DNA repair processes to occur. Following this, 15 ml nutrient agar (Oxoid, Ltd.; Thermo Fisher Scientific, Inc.) was poured onto the cells on glass plates, which were then incubated at 37˚C for 21 h. The surviving colonies (putative mutants) were screened for the exotoxin A gene by PCR. To evaluate the impact of UV-induced mutations on exotoxin A production, 20 mutants from each isolate (120 mutants in total) were randomly selected for evaluation.

Purification of Exotoxin A

The isolates were cultivated in trypticase soy broth (TSB; BD Difco™; Becton, Dickinson and Company). Exotoxin A was purified by precipitation using 70% saturated ammonium sulfate. Following dialysis, the proteins were purified by Sephadex G-150 column chromatography (GE Healthcare Technologies, Inc.). A pre-packed column (HiPrep™ 16/60 Sephadex G-150; GE Healthcare Life Sciences) with a bed volume of 120 ml was equilibrated with sodium citrate buffer (0.05 M, pH 6.5), and the purification was carried out at room temperature (22-25˚C). Proteins were eluted with the same buffer at a flow rate of 1 ml/min. A total of fifteen fractions (2 ml/fraction) were then collected at 15-min intervals and the optical density (OD) at 280 nm was measured to monitor protein elution. Fractions containing Exotoxin A were pooled, concentrated, and dialyzed against 0.05 M sodium citrate buffer, pH 6.5. The concentration of purified protein was determined using BCA assay and the protein was analyzed by SDS-PAGE to confirm a molecular weight of 66 kDa (24).

MCF-7 cell culture

The MCF-7 breast cancer cell line was obtained from the American Tissue Culture Collection (ATCC). Cells were maintained in Dulbecco's Modified Eagle's Medium with high glucose and 1% L-glutamine HEPES buffer (ATCC) supplemented with heat-inactivated 10% fetal bovine serum (v/v) (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Cells were incubated at 37˚C in a humidified atmosphere containing 5% CO2. Cells were regularly sub-cultured and passaged every 3 days to maintain exponential growth.

Cytotoxicity assay

Cell toxicity was monitored by determining the effect of the test samples on cell morphology and viability. The MCF-7 breast cancer cell line was seeded at a density of 1x104 cells per well in 96-well plates and incubated at 37˚C in a humidified incubator with 5% CO2. After 24 h, various concentrations of crude and purified Exotoxin A (1.56-50 µg/ml) were added to the wells. Following an additional 24-h incubation, MTT reagent (Elabscience Bionovation Inc.) was added, and the plates were incubated for 2 h. After removing the medium, DMSO (Thermo Fisher Scientific, Inc.) was added to dissolve the formazan crystals, and the absorbance was measured at 540 nm using a microplate reader (BioTek; Agilent Technologies, Inc.). Half-maximal inhibitory concentration (IC50) values were determined using nonlinear regression analysis, and Exotoxin A concentrations are presented on a logarithmic scale (µg/ml) (25,26).

Statistical analysis

Protein concentrations and IC50 values were calculated using GraphPad Prism 10 software (Dotmatics). Data are presented as the mean ± SEM. The IC50 values for Exotoxin A in the MTT cytotoxicity assay were calculated using non-linear regression (log Exotoxin A concentration vs. normalized cell viability). The inhibitory responses of different concentrations of crude and pure isolated Exotoxin A proteins were compared using two-way ANOVA followed by Bonferroni's multiple comparisons test.

Results

Identification of P. aeruginosa isolates

The 20 P. aeruginosa isolates were confirmed based on their morphological, biochemical and molecular characteristics. Gram staining showed that all isolates were gram-negative rods (Fig. 1A). Biochemical characterization confirmed that these isolates were P. aeruginosa, based on standard tests, including the urease, citrate and TSI tests (Fig. 1B and C). In addition, the isolates produced the characteristic pyocyanin pigment on cetrimide agar (Fig. 1D), and PCR amplification verified the presence of the Exotoxin A gene in all isolates.

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.

Protein profiling and classification of isolates

SDS-PAGE analysis of total cellular proteins revealed distinct protein banding patterns among the isolates, with protein sizes ranging from 10 to 260 kDa. Variations in band intensity and distribution indicate genetic diversity among the isolates (Fig. 2A), which was further analyzed using numerical clustering.

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.

The 20 isolates were classified into six distinct groups based on their protein banding profiles. A dendrogram was constructed, in which the isolates were grouped based on band similarities (Fig. 2B). Isolates within the same cluster exhibited nearly identical protein expression patterns, suggesting shared genetic characteristics. The clusters were defined as follows: 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; and group 6, isolates 18-20. Isolate 4 was identified a strong candidate for further mutation analysis, based on its strong protein expression of Exotoxin A revealed by the SDS-PAGE analysis.

Detection of Exotoxin A gene by PCR

PCR was performed to detect the Exotoxin A gene in six selected wild-type isolates (1, 4, 8, 14, 16 and 20; Fig. 3A), one from each group. A 367-bp PCR amplicon, characteristic of the Exotoxin A gene, was identified in all tested wild-type isolates. Following UV mutagenesis, PCR analysis revealed that mutant 4* exhibited additional DNA bands, which are likely attributable to modifications in the primer binding sites (Fig. 3B). Based on the findings of the SDS-PAGE and PCR analyses, mutant 4* and wild-type isolate 4 were selected for exotoxin A purification and cytotoxicity assay.

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.

Purification and molecular weight conformation of Exotoxin A

Wild-type isolate 4 and mutant 4* were subjected to cultivation, precipitation and dialysis, followed by the purification of Exotoxin A by column chromatography. Protein concentration was estimated by UV absorbance at 280 nm and verified using the BCA protein assay (Thermo Fisher Scientific, Inc.). The highest yield was observed between fractions 4 and 12 based on OD280 measurements, corresponding to the Exotoxin A peak (Table II). As shown in Fig. 4, SDS-PAGE analysis revealed a distinct 66 kDa protein band in the purified fractions, confirming successful isolation. Notably, mutant isolate 4* exhibited a higher protein yield than wild-type isolate 4 as determined by optical density at 280 nm and confirmed by SDS-PAGE densitometric analysis.

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.

Table II Protein concentration of crude and purified Exotoxin A from isolates 4 and 4*.

Table II

Protein concentration of crude and purified Exotoxin A from isolates 4 and 4*.

	Crude Exotoxin A		Purified Exotoxin A
Isolate no.	OD 280 nm, mean ± SEM	Concentration, µg/ml	OD 280 nm, mean ± SEM	Concentration, µg/ml
4	0.48±0.01	15.40	0.47±0.01	15.10
4*	0.49±0.01	15.70	0.48±0.01	15.30

[i] OD, optical density. For purified samples, the values represent the pooled and concentrated fractions (4-12) eluted from the Sephadex G-150 column. Protein levels were estimated by UV absorbance at 280 nm and confirmed using the BCA protein assay (Thermo Fisher Scientific, Inc.). Values are presented as mean ± SEM.

Cytotoxicity of Exotoxin A on MCF-7 cells

The cytotoxic effect of Exotoxin A on MCF-7 cells was assessed using the MTT assay (Fig. 5). Both crude and purified Exotoxin A demonstrated marked dose-dependent cytotoxicity against the MCF-7 cells. The IC50 values for the crude toxin were for 13.1 and 12.0 for isolate 4 and mutant 4*, respectively. After purification, the cytotoxicity increased substantially, with IC50 values of 4.9 and 3.6 µg/ml for isolate 4 and mutant 4*, respectively (Fig. 5B). Further comparison of the MTT assay results at various toxin concentrations confirmed that purified Exotoxin A was significantly more cytotoxic than its crude counterpart (Fig. 6). A clear dose-dependent cytotoxic effect was observed, with concentrations ≥6.56 µg/ml leading to a marked reduction in viability, particularly for the purified isolates. The differences between crude and purified Exotoxin A were statistically significant at multiple concentrations (P<0.05). Notably, mutant 4* (Fig. 6B) exhibited a stronger cytotoxic effect than isolate 4, demonstrated by a more pronounced decline in cell viability with increased concentration (Fig. 6A). These findings suggest that purified Exotoxin A derived from mutant 4 is a particularly potent inhibitor of MCF-7 cell growth. These findings highlight the potential of purified Exotoxin A, particularly that derived from mutant 4*, as a promising antitumor agent.

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.

Discussion

Malignancy is a complicated disease characterized by interconnected dysregulated metabolic pathways that promote tumor development and the evasion of immune surveillance. Genetic mutations contribute to the formation of tumors, which are rapidly proliferating cells that bypass normal regulatory mechanisms (13). The search for effective anticancer drugs remains challenging due to the ability of cancer cells to resist apoptosis and escape immune detection. Secondary metabolites derived from microorganisms have played a critical role in the discovery of new chemotherapeutics, with bacterial toxins showing promise due for the selective targeting of cancer cells (27).

Exotoxin A, a well-characterized virulence factor of P. aeruginosa, has shown marked potential for anticancer applications due to its ability to inhibit protein synthesis and induce apoptosis in cancer cells (28). The present study confirmed the presence of Exotoxin A in all tested P. aeruginosa isolates, supporting the findings of Aljebory (29), which demonstrated the widespread presence of the exotoxin A gene in clinical isolates. However, lower prevalences of Exotoxin A have been noted, for example, by Ismail et al (30), who reported a prevalence of 72%. These variations in prevalence are likely due to differences in sampling locations and infection control measures.

In the present study, molecular characterization of the isolates using SDS-PAGE revealed clear differences in protein banding patterns, which were used to classify the isolates into six distinct groups. The purification of Exotoxin A was achieved by ammonium sulfate precipitation followed by Sephadex G-150 chromatography, yielding a purified protein with a molecular mass of ~66 kDa, in agreement with previous studies (15,31).

Gallant et al (32) demonstrated that optimizing culture conditions, such as by supplementing the medium with glycerol and monosodium glutamate, enhances Exotoxin A production. While the present study employed UV-induced mutagenesis rather than media optimization, the increased Exotoxin A production observed in mutant P. aeruginosa isolates highlights the potential of experimental modifications to enhance toxin yield.

The introduction of mutations via UV exposure led to a marked increase in Exotoxin A production by mutant 4* compared with that by the corresponding wild-type isolate, suggesting that the mutation may have upregulated the gene responsible for toxin production or altered regulatory elements involved in its expression. This is consistent with previous research indicating that bacterial virulence genes are subject to environmental and genetic regulation (33). The PCR analysis of mutant 4* revealed additional DNA bands that were not present in the corresponding wild-type isolate, which may reflect genetic rearrangements that affect gene regulation or expression efficiency.

The cytotoxicity assay results demonstrated that the potency of purified Exotoxin A against MCF-7 breast cancer cells was greater than that of its crude counterpart. The IC50 values indicated that mutant 4* exhibited a 1.4-fold increase in cytotoxic activity compared with the wild-type isolate, reinforcing the hypothesis that mutations enhanced toxin production. These findings are consistent with previous research demonstrating that bacterial toxins can be engineered for improved specificity and potency in cancer therapy (34)

Bacterial toxins such as Exotoxin A are being explored as potential alternatives to conventional chemotherapy, which is often associated with systemic toxicity and the non-specific targeting of healthy tissues (35). It is hypothesized that conjugating Exotoxin A to monoclonal antibodies or tumor-specific ligands may lead to the development of targeted cancer therapies that selectively kill tumor cells while sparing normal tissues. The use of bacterial toxins in oncology is gaining traction, with current research focusing on structural modifications to enhance their stability and reduce potential immunogenicity (30).

Despite the promising results obtained in the present study, several challenges remain to be addressed before Exotoxin A can be considered a viable cancer therapeutic. The study was conducted entirely in vitro; therefore, further validation in animal models is required to assess pharmacokinetics, systemic toxicity and long-term efficacy. In addition, while UV-induced mutation increased toxin production, the exact genetic modifications responsible remain unknown. Future studies should employ whole-genome sequencing to identify the specific mutations contributing to increased expression and activity. In addition, the potential of site-directed mutagenesis to refine toxin production and improve its clinical applicability also warrants investigation.

Structural analyses, such as X-ray crystallography, may provide insights into how genetic changes influence toxin conformation and function. Furthermore, strategies for targeted delivery, such as nanoparticle conjugation, should be investigated to improve therapeutic efficacy and minimize off-target effects. It is also important to note that the current study relied solely on SDS-PAGE to verify the specific purification of Exotoxin A from the isolates. In future studies, complementary methods, such as mass spectrometry or immunodetection assays, should be used to identify and validate the purified toxin.

In conclusion, the present study highlights the potential of Exotoxin A from P. aeruginosa as a promising anticancer agent. The results demonstrate that certain genetic mutations can enhance Exotoxin A production and cytotoxicity, making it a strong candidate for further development as an anticancer therapeutic. However, additional research is necessary to optimize its application, address biosafety concerns and explore targeted delivery approaches for clinical use. While these findings provide a strong foundation for microbial toxin-based cancer therapies, future studies must prioritize in vivo validation and translational research to bring Exotoxin A-based treatments closer to clinical application.

Acknowledgements

The author wishes to thank the Deanship of Graduate Studies and Scientific Research at Qassim University for financial support. The author also expresses gratitude to Dr Nahla Azab and Dr Medhat Rehan (Qassim University) for their invaluable support, assistance with laboratory materials and guidance.

Funding

Funding: Financial support was provided by the Deanship of Graduate Studies and Scientific Research at Qassim University (grant no. QU-APC-2025-2/1).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

ISA was responsible for conceptualization, methodology and data analysis, and for writing, reviewing and editing the manuscript. ISA confirms the authenticity of all the raw data. The author has read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The author declares that they have no competing interests.

Authors' information

Dr Ibtesam S. Almami, ORCID ID: https://orcid.org/0000-0001-7876-560X.

Use of artificial intelligence tools

During the preparation of this work, artificial intelligence tools were used to improve the readability and language of the manuscript, and subsequently, the author revised and edited the content produced by the artificial intelligence tools as necessary, taking full responsibility for the ultimate content of the present manuscript.

References