scholarly journals Isolation and Characterizations of A Novel Recombinant scFv Antibody Against Exotoxin-A of Pseudomonas Aeruginosa

2020 ◽  
Author(s):  
Zahra Shadman ◽  
Safar Farajnia ◽  
Mohammad Pazhang ◽  
Mohammadreza Tohidkia ◽  
Leila Rahbarnia ◽  
...  

Abstract Background: Pseudomonas aeruginosa is known as the leading cause of nosocomial infections especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections.Methods: In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was conducted to identify a novel human scFv antibody against domain I of P. aeruginosa exotoxin A from a human scFv phage library. For this, the recombinant domain I of exotoxin A was expressed in E. coli and purified by Ni-NTA column. A novel screening procedure was conducted to prevent the elimination of rare specific clones. Based on polyclonal phage ELISA results, the fifth round of biopanning was selected to identify specific phage clones.Results: Two positive clones were found by monoclonal phage. The phage clone with high reactivity was evaluated by ELISA and western blot. The purified scFv also showed high reactivity with full length exotoxin.Conclusions: In conclusion, the purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified can be the groundwork for development of a novel therapeutic agent for control of P. aeruginosa infections.

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zahra Shadman ◽  
Safar Farajnia ◽  
Mohammad Pazhang ◽  
Mohammadreza Tohidkia ◽  
Leila Rahbarnia ◽  
...  

Abstract Background Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. Methods The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. Results Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. Conclusions The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.


2013 ◽  
Vol 11 (3) ◽  
pp. 193-98 ◽  
Author(s):  
Asghar Tanomand ◽  
Shahin Najar Peerayeh ◽  
Safar Farajnia ◽  
Jafar Majidi

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sirijan Santajit ◽  
Watee Seesuay ◽  
Kodchakorn Mahasongkram ◽  
Nitat Sookrung ◽  
Sumate Ampawong ◽  
...  

Abstract Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.


2017 ◽  
Vol 13 (3) ◽  
pp. 206-221
Author(s):  
Hadi Rahman Rasheed Al-Taai ◽  
◽  
Zainab Mohammed Hameed ◽  
Izdehar Mohammed Jasim

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Syed A. K. Shifat Ahmed ◽  
Michelle Rudden ◽  
Sabrina M. Elias ◽  
Thomas J. Smyth ◽  
Roger Marchant ◽  
...  

AbstractPseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI’s) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.


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