Evaluation of accessible regions of Escherichia coli fimH mRNA through computational prediction and experimental investigation
-
Elnaz Harifi Mood
,
Alireza Japoni-Nejad
,
Mohammadreza Asadi Karam
,
Mohammad Pooya
,
Saeid Bouzari
,
Nader Shahrokhi
Abstract
Background and Objectives: This study aimed to investigate the accessible regions of the fimH mRNA using computational prediction and dot-blot hybridization to increase the effectiveness of antisense anti-virulence therapeutics against Uropathogenic Escherichia coli.
Materials and Methods: We predicted the secondary structure of the E. coli fimH mRNA using the Sfold and Mfold Web servers and RNA structure 5.5 program. Considering the predicted secondary structure, accessible regions in mRNA of fimH were determined and oligonucleotides complementary to these regions were synthesized and hybridization activity of those oligonucleotides to the fimH Digoxigenin (DIG) labeled mRNA was assessed with dot-blot hybridization.
Results: When searching the fimH gene in the GenBank database, two lengths for this gene was discovered in different strains of E. coli. The difference was related to the nine bases in the first part of the gene utilizing either of two translation initiation sites. Based on the bioinformatics analyses, five regions lacking obvious stable secondary structures were selected in mRNA of fimH. The result of dot-blot hybridization exhibited strongest hybridization signal between the antisense oligonucleotide number one and fimH labeled mRNA, whereas hybridization signals were not seen for the negative control.
Conclusion: The results obtained here demonstrate that the region contains start codon of fimH mRNA could act as the potential mRNA target site for anti-fimH antisense therapeutics. It is recommended in the future both of utilizing translation initiation sites be targeted with antisense oligomers compounds.
1. Barber AE, Norton JP, Wiles TJ, Mulvey MA. Strengths and limitations of model systems for the study of urinary tract infections and related pathologies. Microbiol Mol Biol Rev 2016;80:351-367.
2. Tabasi M, Asadi Karam MR, Habibi M, Yekaninejad MS, Bouzari S. Phenotypic assays to determine virulence factors of uropathogenic Escherichia coli (UPEC) isolates and their correlation with antibiotic resistance pattern. Osong Public Health Res Perspect 2015;6:261-268.
3. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 2015;13:269-284.
4. Wilson ML, Gaido L. Laboratory diagnosis of urinary tract infections in adult patients. Clin Infect Dis 2004;38:1150-1158.
5. Loubet P, Ranfaing J, Dinh A, Dunyach-Remy C, Bernard L, Bruyère F, et al. Alternative therapeutic options to antibiotics for the treatment of urinary tract infections. Front Microbiol 2020;11:1509.
6. Saint S, Kowalski CP, Kaufman SR, Hofer TP, Kauffman CA, Olmsted RN, et al. Preventing hospital-acquired urinary tract infection in the United States: a national study. Clin Infect Dis 2008;46:243-250.
7. Asadi Karam MR, Habibi M, Bouzari S. Urinary tract infection: pathogenicity, antibiotic resistance and development of effective vaccines against Uropathogenic Escherichia coli. Mol Immunol 2019;108:56-67.
8. Schwartz DJ, Kalas V, Pinkner JS, Chen SL, Spaulding CN, Dodson KW, et al. Positively selected FimH residues enhance virulence during urinary tract infection by altering FimH conformation. Proc Natl Acad Sci U S A 2013;110:15530-15537.
9. Habibi M, Asadi Karam MR, Bouzari S. Transurethral instillation with fusion protein MrpH. FimH induces protective innate immune responses against uropathogenic Escherichia coli and Proteus mirabilis. APMIS 2016;124:444-452.
10. Bien J, Sokolova O, Bozko P. Role of uropathogenic Escherichia coli virulence factors in development of urinary tract infection and kidney damage. Int J Nephrol 2012;2012:681473.
11. Cozens D, Read RC. Anti-adhesion methods as novel therapeutics for bacterial infections. Expert Rev Anti Infect Ther 2012;10:1457-1468.
12. Hung CS, Dodson KW, Hultgren SJ. A murine model of urinary tract infection. Nat Protoc 2009;4:1230-1243.
13. Vogel J. An RNA biology perspective on species‐specific programmable RNA antibiotics. Mol Microbiol 2020;113:550-559.
14. Skvortsova YV, Salina EG, Burakova EA, Bychenko OS, Stetsenko DA, Azhikina TL. A new antisense phosphoryl guanidine oligo-2′-O-methylribonucleotide penetrates into intracellular mycobacteria and suppresses target gene expression. Front Pharmacol 2019;10:1049.
15. Sully EK, Geller BL. Antisense antimicrobial therapeutics. Curr Opin Microbiol 2016;33:47-55.
16. Oh E, Zhang Q, Jeon B. Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni. J Antimicrob Chemother 2014;69:375-380.
17. Sturge CR, Felder-Scott CF, Pifer R, Pybus C, Jain R, Geller BL, et al. AcrAB–TolC inhibition by peptide-conjugated phosphorodiamidate Morpholino oligomers restores antibiotic activity in vitro and in vivo. ACS Infect Dis 2019;5:1446-1455.
18. Alajlouni RA, Seleem MN. Targeting Listeria monocytogenes rpoA and rpoD genes using peptide nucleic acids. Nucleic Acid Ther 2013;23:363-367.
19. Xia Y, Xiong Y, Li X, Su X. Inhibition of biofilm formation by the antisense peptide nucleic acids targeted at the motA gene in Pseudomonas aeruginosa PAO1 strain. World J Microbiol Biotechnol 2011;27:1981-1987.
20. Jackson A, Jani S, Sala CD, Soler-Bistué AJ, Zorreguieta A, Tolmasky ME. Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes. Biol Methods Protoc 2016;1(1):bpw001.
21. Nejad AJ, Shahrokhi N, Nielsen PE. Targeting of the essential acpP, ftsZ, and rne genes in carbapenem-resistant Acinetobacter baumannii by antisense PNA precision antibacterials. Biomedicines 2021;9:429.
22.Good L, Stach JE. Synthetic RNA silencing in bacteria–antimicrobial discovery and resistance breaking. Front Microbiol 2011;2:185.
23. Liang S, He Y, Xia Y, Wang H, Wang L, Gao R, et al. Inhibiting the growth of methicillin-resistant Staphylococcus aureus in vitro with antisense peptide nucleic acid conjugates targeting the ftsZ gene. Int J Infect Dis 2015;30:1-6.
24. Wang H, He Y, Xia Y, Wang L, Liang S. Inhibition of gene expression and growth of multidrug-resistant Acinetobacter baumannii by antisense peptide nucleic acids. Mol Biol Rep 2014;41:7535-7541.
25. Da F, Yao L, Su Z, Hou Z, Li Z, Xue X, et al. Antisense locked nucleic acids targeting agrA inhibit quorum sensing and pathogenesis of community‐associated methicillin‐resistant Staphylococcus aureus. J Appl Microbiol 2017;122:257-267.
26. Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res 2002;12:656-664.
27. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31:3406-3415.
28. Mathews DH, Turner DH. Prediction of RNA secondary structure by free energy minimization. Curr Opin Struct Biol 2006;16:270-278.
29. Ding Y, Chan CY, Lawrence CE. Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res 2004;32(Web Server issue):W135-W141.
30. Reuter JS, Mathews DH. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 2010;11:129.
31. Eisel D, Seth O, Grünewald-Janho S, Kruchen B, Rüger B (2008). DIG application manual for filter hybridization. Roche Diagnostics GmbH. Mannheim. Germany.
32. Hart SM, Basu C. Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana. J Biomol Tech 2009;20:96-100.
33. Cusumano CK, Pinkner JS, Han Z, Greene SE, Ford BA, Crowley JR, et al. Treatment and prevention of urinary tract infection with orally active FimH inhibitors. Sci Transl Med 2011;3:109ra115.
34. Jarvis C, Han Z, Kalas V, Klein R, Pinkner JS, Ford B, et al. Antivirulence isoquinolone mannosides: optimization of the biaryl aglycone for FimH lectin binding affinity and efficacy in the treatment of chronic UTI. ChemMedChem 2016;11:367-373.
35. Schembri MA, Kjaergaard K, Sokurenko EV, Klemm P. Molecular characterization of the Escherichia coli FimH adhesin. J Infect Dis 2001;183 Suppl 1:S28-S31.
36. Bai H, You Y, Yan H, Meng J, Xue X, Hou Z, et al. Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene. Biomaterials 2012;33:659-667.
37. Wojciechowska M, Równicki M, Mieczkowski A, Miszkiewicz J, Trylska J. Antibacterial peptide nucleic acids—facts and perspectives. Molecules 2020;25:559.
38. Dryselius R, Aswasti SK, Rajarao GK, Nielsen PE, Good L. The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli. Oligonucleotides 2003;13:427-433.
39. Daly SM, Sturge CR, Marshall-Batty KR, Felder-Scott CF, Jain R, Geller BL, et al. Antisense inhibitors retain activity in pulmonary models of Burkholderia infection. ACS Infect Dis 2018;4:806-814.
40. Rasmussen LC, Sperling-Petersen HU, Mortensen KK. Hitting bacteria at the heart of the central dogma: sequence-specific inhibition. Microb Cell Fact 2007;6:24.
2. Tabasi M, Asadi Karam MR, Habibi M, Yekaninejad MS, Bouzari S. Phenotypic assays to determine virulence factors of uropathogenic Escherichia coli (UPEC) isolates and their correlation with antibiotic resistance pattern. Osong Public Health Res Perspect 2015;6:261-268.
3. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 2015;13:269-284.
4. Wilson ML, Gaido L. Laboratory diagnosis of urinary tract infections in adult patients. Clin Infect Dis 2004;38:1150-1158.
5. Loubet P, Ranfaing J, Dinh A, Dunyach-Remy C, Bernard L, Bruyère F, et al. Alternative therapeutic options to antibiotics for the treatment of urinary tract infections. Front Microbiol 2020;11:1509.
6. Saint S, Kowalski CP, Kaufman SR, Hofer TP, Kauffman CA, Olmsted RN, et al. Preventing hospital-acquired urinary tract infection in the United States: a national study. Clin Infect Dis 2008;46:243-250.
7. Asadi Karam MR, Habibi M, Bouzari S. Urinary tract infection: pathogenicity, antibiotic resistance and development of effective vaccines against Uropathogenic Escherichia coli. Mol Immunol 2019;108:56-67.
8. Schwartz DJ, Kalas V, Pinkner JS, Chen SL, Spaulding CN, Dodson KW, et al. Positively selected FimH residues enhance virulence during urinary tract infection by altering FimH conformation. Proc Natl Acad Sci U S A 2013;110:15530-15537.
9. Habibi M, Asadi Karam MR, Bouzari S. Transurethral instillation with fusion protein MrpH. FimH induces protective innate immune responses against uropathogenic Escherichia coli and Proteus mirabilis. APMIS 2016;124:444-452.
10. Bien J, Sokolova O, Bozko P. Role of uropathogenic Escherichia coli virulence factors in development of urinary tract infection and kidney damage. Int J Nephrol 2012;2012:681473.
11. Cozens D, Read RC. Anti-adhesion methods as novel therapeutics for bacterial infections. Expert Rev Anti Infect Ther 2012;10:1457-1468.
12. Hung CS, Dodson KW, Hultgren SJ. A murine model of urinary tract infection. Nat Protoc 2009;4:1230-1243.
13. Vogel J. An RNA biology perspective on species‐specific programmable RNA antibiotics. Mol Microbiol 2020;113:550-559.
14. Skvortsova YV, Salina EG, Burakova EA, Bychenko OS, Stetsenko DA, Azhikina TL. A new antisense phosphoryl guanidine oligo-2′-O-methylribonucleotide penetrates into intracellular mycobacteria and suppresses target gene expression. Front Pharmacol 2019;10:1049.
15. Sully EK, Geller BL. Antisense antimicrobial therapeutics. Curr Opin Microbiol 2016;33:47-55.
16. Oh E, Zhang Q, Jeon B. Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni. J Antimicrob Chemother 2014;69:375-380.
17. Sturge CR, Felder-Scott CF, Pifer R, Pybus C, Jain R, Geller BL, et al. AcrAB–TolC inhibition by peptide-conjugated phosphorodiamidate Morpholino oligomers restores antibiotic activity in vitro and in vivo. ACS Infect Dis 2019;5:1446-1455.
18. Alajlouni RA, Seleem MN. Targeting Listeria monocytogenes rpoA and rpoD genes using peptide nucleic acids. Nucleic Acid Ther 2013;23:363-367.
19. Xia Y, Xiong Y, Li X, Su X. Inhibition of biofilm formation by the antisense peptide nucleic acids targeted at the motA gene in Pseudomonas aeruginosa PAO1 strain. World J Microbiol Biotechnol 2011;27:1981-1987.
20. Jackson A, Jani S, Sala CD, Soler-Bistué AJ, Zorreguieta A, Tolmasky ME. Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes. Biol Methods Protoc 2016;1(1):bpw001.
21. Nejad AJ, Shahrokhi N, Nielsen PE. Targeting of the essential acpP, ftsZ, and rne genes in carbapenem-resistant Acinetobacter baumannii by antisense PNA precision antibacterials. Biomedicines 2021;9:429.
22.Good L, Stach JE. Synthetic RNA silencing in bacteria–antimicrobial discovery and resistance breaking. Front Microbiol 2011;2:185.
23. Liang S, He Y, Xia Y, Wang H, Wang L, Gao R, et al. Inhibiting the growth of methicillin-resistant Staphylococcus aureus in vitro with antisense peptide nucleic acid conjugates targeting the ftsZ gene. Int J Infect Dis 2015;30:1-6.
24. Wang H, He Y, Xia Y, Wang L, Liang S. Inhibition of gene expression and growth of multidrug-resistant Acinetobacter baumannii by antisense peptide nucleic acids. Mol Biol Rep 2014;41:7535-7541.
25. Da F, Yao L, Su Z, Hou Z, Li Z, Xue X, et al. Antisense locked nucleic acids targeting agrA inhibit quorum sensing and pathogenesis of community‐associated methicillin‐resistant Staphylococcus aureus. J Appl Microbiol 2017;122:257-267.
26. Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res 2002;12:656-664.
27. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31:3406-3415.
28. Mathews DH, Turner DH. Prediction of RNA secondary structure by free energy minimization. Curr Opin Struct Biol 2006;16:270-278.
29. Ding Y, Chan CY, Lawrence CE. Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res 2004;32(Web Server issue):W135-W141.
30. Reuter JS, Mathews DH. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 2010;11:129.
31. Eisel D, Seth O, Grünewald-Janho S, Kruchen B, Rüger B (2008). DIG application manual for filter hybridization. Roche Diagnostics GmbH. Mannheim. Germany.
32. Hart SM, Basu C. Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana. J Biomol Tech 2009;20:96-100.
33. Cusumano CK, Pinkner JS, Han Z, Greene SE, Ford BA, Crowley JR, et al. Treatment and prevention of urinary tract infection with orally active FimH inhibitors. Sci Transl Med 2011;3:109ra115.
34. Jarvis C, Han Z, Kalas V, Klein R, Pinkner JS, Ford B, et al. Antivirulence isoquinolone mannosides: optimization of the biaryl aglycone for FimH lectin binding affinity and efficacy in the treatment of chronic UTI. ChemMedChem 2016;11:367-373.
35. Schembri MA, Kjaergaard K, Sokurenko EV, Klemm P. Molecular characterization of the Escherichia coli FimH adhesin. J Infect Dis 2001;183 Suppl 1:S28-S31.
36. Bai H, You Y, Yan H, Meng J, Xue X, Hou Z, et al. Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene. Biomaterials 2012;33:659-667.
37. Wojciechowska M, Równicki M, Mieczkowski A, Miszkiewicz J, Trylska J. Antibacterial peptide nucleic acids—facts and perspectives. Molecules 2020;25:559.
38. Dryselius R, Aswasti SK, Rajarao GK, Nielsen PE, Good L. The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli. Oligonucleotides 2003;13:427-433.
39. Daly SM, Sturge CR, Marshall-Batty KR, Felder-Scott CF, Jain R, Geller BL, et al. Antisense inhibitors retain activity in pulmonary models of Burkholderia infection. ACS Infect Dis 2018;4:806-814.
40. Rasmussen LC, Sperling-Petersen HU, Mortensen KK. Hitting bacteria at the heart of the central dogma: sequence-specific inhibition. Microb Cell Fact 2007;6:24.
| Files | ||
| Issue | Vol 13 No 5 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i5.7430 | |
| Keywords | ||
| Uropathogenic Escherichia coli; FimH protein; Target prediction; Nucleic acid hybridizations | ||
| Rights and permissions | |
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Harifi Mood E, Japoni-Nejad A, Asadi Karam M, Pooya M, Bouzari S, Shahrokhi N. Evaluation of accessible regions of Escherichia coli fimH mRNA through computational prediction and experimental investigation. Iran J Microbiol. 2021;13(5):653-663.
