Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa

Document Type: Original Article

Authors

1 Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran

2 Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran

3 Department of Biotechnology, Laboratory of Microbiology and Immunology of Infectious Diseases, Para Medicine Faculty, Guilan University of Medical Sciences, Rasht, Iran

4 Biotechnology Research Center, Drug Design and Bioinformatics Group, Pasteur Institute of Iran, Tehran, Iran

5 Departments of Immunology, Pasteur Institute of Iran, Tehran, Iran

Abstract

Objective(s): Infection with Pseudomonas aeruginosa has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel Pseudomonas aeruginosa type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides.
Materials and Methods: We surveyed the expression of the PilQ380-705-PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each pilA domains. The  transformants were  expressed in Escherichia coli BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays.
Results: The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results.
Conclusion: Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ380-705-PilA fusion protein could be a reliable candidate vaccine against P. aeruginosa infection.

Keywords


1. Talwalkar JS, Murray TS. The Approach to Pseudomonas aeruginosa in Cystic Fibrosis. Clin Chest Med 2016; 37:69-81.

2. Peluso L, de Luca C, Bozza S, Leonardi A, Giovannini G, Lavorgna A, et al. Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization. BMC Microbiol 2010; 10:1471-2180.

3. Jolly AL, Takawira D, Oke OO, Whiteside SA, Chang SW, Wen ER, et al. Pseudomonas aeruginosa-induced bleb-niche formation in epithelial cells is independent of actinomyosin contraction and enhanced by loss of cystic fibrosis transmembrane-conductance regulator osmoregulatory function. MBio 2015; 6:02533-02514.

4. Bucior I, Pielage JF, Engel JN. Pseudomonas aeruginosa pili and flagella mediate distinct binding and signaling events at the apical and basolateral surface of airway epithelium. PLoS Pathog 2012; 8:5.

5. Wehbi H, Portillo E, Harvey H, Shimkoff AE, Scheurwater EM, Howell PL, et al. The peptidoglycan-binding protein FimV promotes assembly of the Pseudomonas aeruginosa type IV pilus secretin. J Bacteriol 2011; 193:540-550.

6. Farinha MA, Conway BD, Glasier LM, Ellert NW, Irvin RT, Sherburne R, et al. Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice. Infect Immun 1994; 62:4118-4123.

7. Ayers M, Sampaleanu LM, Tammam S, Koo J, Harvey H, Howell PL, et al. PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin. J Mol Biol 2009; 394:128-142.

8. Burrows LL. Weapons of mass retraction. Mol Microbiol 2005; 57:878-888.

9. Tang H, Kays M, Prince A. Role of Pseudomonas aeruginosa pili in acute pulmonary infection. Infect Immun 1995; 63:1278-1285.

10. Tammam S, Sampaleanu LM, Koo J, Sundaram P, Ayers M, Chong PA, et al. Characterization of the PilN, PilO and PilP type IVa pilus subcomplex. Mol Microbiol 2011; 82:1496-1514.

11. Bohn YS, Brandes G, Rakhimova E, Horatzek S, Salunkhe P, Munder A, et al. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection. Mol Microbiol 2009; 71:730-747.

12. Hackbarth C, Hodges RS. Synthetic peptide vaccine development: designing dual epitopes into a single pilin peptide immunogen generates antibody cross-reactivity between two strains of Pseudomonas aeruginosa. Chem Biol Drug Des 2010; 76:293-304.

13. Kus JV, Tullis E, Cvitkovitch DG, Burrows LL. Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology 2004; 150:1315-1326.

14. Tammam S, Sampaleanu LM, Koo J, Manoharan K, Daubaras M, Burrows LL, et al. PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA. J Bacteriol 2013; 195:2126-2135.

15. Hoang HH, Nickerson NN, Lee VT, Kazimirova A, Chami M, Pugsley AP, et al. Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries. MBio 2011; 2:00246-00211.

16. Pelicic V. Type IV pili: e pluribus unum? Mol Microbiol 2008; 68:827-837.

17. Nudleman E, Kaiser D. Pulling together with type IV pili. J Mol Microbiol Biotechnol 2004; 7:52-62.

18. Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expr Purif 2013; 87:129-135.

19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-685.

20. Faezi S, Sattari M, Mahdavi M, Roudkenar MH. Passive immunisation against Pseudomonas aeruginosa recombinant flagellin in an experimental model of burn wound sepsis. Burns 2011; 37:865-872.

21. Castric P, Cassels FJ, Carlson RW. Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan. J Biol Chem 2001; 276:26479-26485.

22. Kao DJ, Hodges RS. Advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for Pseudomonas aeruginosa. Chem Biol Drug Des 2009; 74:33-42.

23. Hahn HP. The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review. Gene 1997; 192:99-108.

24. Qutyan M, Henkel M, Horzempa J, Quinn M, Castric P. Glycosylation of pilin and nonpilin protein constructs by Pseudomonas aeruginosa 1244. J Bacteriol 2010; 192:5972-5981.

25. Ohama M, Hiramatsu K, Miyajima Y, Kishi K, Nasu M, Kadota J. Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice. FEMS Immunol Med Microbiol 2006; 47:107-115.

26. Turnbull L, Whitchurch CB. Motility assay: twitching motility. Methods Mol Biol 2014; 1149:73-86.

27. Kao DJ, Churchill ME, Irvin RT, Hodges RS. Animal protection and structural studies of a consensus sequence vaccine targeting the receptor binding domain of the type IV pilus of Pseudomonas aeruginosa. J Mol Biol 2007; 374:426-442.

28. Sheth HB, Glasier LM, Ellert NW, Cachia P, Kohn W, Lee KK, et al. Development of an anti-adhesive vaccine for Pseudomonas aeruginosa targeting the C-terminal region of the pilin structural protein. Biomed Pept Proteins Nucleic Acids 1995; 1:141-148.

29. Balasingham SV, Collins RF, Assalkhou R, Homberset H, Frye SA, Derrick JP, et al. Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis. J Bacteriol 2007; 189:5716-5727.

30. Varner CT, Rosen T, Martin JT, Kane RS. Recent advances in engineering polyvalent biological interactions. Biomacromolecules 2015; 16:43-55.