Skip to main content
SpringerLink
Log in

Towards understanding single-channel characteristics of OccK8 purified from Pseudomonas aeruginosa

  • Original Article
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

Antibiotic resistance in Gram-negative bacteria causes serious health issues worldwide. Bacteria employ several resistance mechanisms to cope with antimicrobials. One of their strategies is to reduce the permeability of antibiotics either through general diffusion porins or substrate-specific channels. In this study, one of the substrate-specific channels from Pseudomonas aeruginosa, OccK8 (also known as OprE), was investigated using single-channel electrophysiology. The study also includes the investigation of permeability properties of several amino acids with different charged groups (i.e. arginine, glycine and glutamic acid) through OccK8. We observed four different conformations of the same OccK8 channel when inserted in lipid bilayers. This is in contrast to previous studies where heterologous expressed OccK8 in E. coli showed only one conformation. We hypothesized that the difference in our study was due to the expression and purification of the native channel from P. aeruginosa. The single-channel uptake characteristics of the porin showed that negatively charged glutamic acid preferentially interacted with the channel while the positively charged arginine molecule showed infrequent interaction with OccK8. The neutral amino acid glycine did not show any interaction at the physiological conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Ballestero S, Fern’ndez-Rodriguez A, Villaverde R, Escobar H, Perez-Diaz J, Baquero F (1996) Carbapenem resistance in Pseudomonas aeruginosa from cystic fibrosis patients. J Antimicrob Chemother 38:39–45

    Article  CAS  Google Scholar 

  • Benkerrou D, Ceccarelli M (2018) Free energy calculations and molecular properties of substrate translocation through OccAB porins. Phys Chem Chem Phys 20:8533–8546

    Article  CAS  Google Scholar 

  • Boheim G, Kolb H-A (1978) Analysis of the multi-pore system of alamethicin in a lipid membrane. J Membr Biol 38:99–150

    Article  CAS  Google Scholar 

  • Carter M (2015) Guide to research techniques in neuroscience. Elsevier, United State

    Google Scholar 

  • Chevalier S et al (2017) Structure, function and regulation of Pseudomonas aeruginosa porins. FEMS Microbiol Rev 41:698–722

    Article  CAS  Google Scholar 

  • Dufrechou M, Cl P-L, Fo-X S, Vernhet A (2012) Stability of white wine proteins: combined effect of pH, ionic strength, and temperature on their aggregation. J Agric Food Chem 60:1308–1319

    Article  CAS  Google Scholar 

  • Eren E, van den Berg B (2012) Crystal Structure of Pseudomonas aeruginosa OccK8 (OprE). https://www.rcsb.org/structure/4FRX. Accessed 7 Sept 2020

  • Eren E et al (2012) Substrate specificity within a family of outer membrane carboxylate channels. PLoS Biol 10:e1001242

    Article  CAS  Google Scholar 

  • Eren E, Parkin J, Adelanwa A, Cheneke B, Movileanu L, Khalid S, Van den Berg B (2013) Toward understanding the outer membrane uptake of small molecules by Pseudomonas aeruginosa. J Biol Chem 288:12042–12053

    Article  CAS  Google Scholar 

  • Fazeli H, Akbari R, Moghim S, Narimani T, Arabestani MR, Ghoddousi AR (2012) Pseudomonas aeruginosa infections in patients, hospital means, and personnel’s specimens. J Res Med Sci 17:332

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fernández L, Hancock RE (2012) Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance. Clin Microbiol Rev 25:661–681

    Article  Google Scholar 

  • Ghai I, Ghai S (2018) Understanding antibiotic resistance via outer membrane permeability. Infect Drug Resist 11:523

    Article  CAS  Google Scholar 

  • Gill M, Simjee S, Al-Hattawi K, Robertson B, Easmon C, Ison C (1998) Gonococcal resistance to β-lactams and tetracycline involves mutation in loop 3 of the porin encoded at thepenb locus. Antimicrob Agents Chemother 42:2799–2803

    Article  CAS  Google Scholar 

  • Gutsmann T, Heimburg T, Keyser U, Mahendran KR, Winterhalter M (2015) Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization. Nat Protoc 10:188–198

    Article  Google Scholar 

  • Guzel FD, Citak F (2018) Development of an on-chip antibiotic permeability assay with single molecule detection capability. IEEE Trans Nanobiosci 17:155–160

    Article  Google Scholar 

  • Hancock RE (1998) Resistance mechanisms in Pseudomonas aeruginosa and other non fermentative gram-negative bacteria. Clin Infect Dis 27:S93–S99

    Article  CAS  Google Scholar 

  • Jaffe A, Chabbert YA, Semonin O (1982) Role of porin proteins OmpF and OmpC in the permeation of beta-lactams. Antimicrob Agents Chemother 22:942–948

    Article  CAS  Google Scholar 

  • Kim SW et al (2020) The importance of Porins and β-lactamase in outer membrane vesicles on the hydrolysis of β-lactam antibiotics. Int J Mol Sci 21:2822

    Article  CAS  Google Scholar 

  • Lamichhane U, Islam T, Prasad S, Weingart H, Mahendran KR, Winterhalter M (2013) Peptide translocation through the mesoscopic channel: binding kinetics at the single molecule level. Eur Biophys J 42:363–369

    Article  CAS  Google Scholar 

  • Limansky AS, Mussi MA, Viale AM (2002) Loss of a 29-kilodalton outer membrane protein in Acinetobacter baumannii is associated with imipenem resistance. J Clin Microbiol 40:4776–4778

    Article  CAS  Google Scholar 

  • Liu J, Eren E, Vijayaraghavan J, Cheneke BR, Indic M, van den Berg B, Movileanu L (2012a) OccK channels from Pseudomonas aeruginosa exhibit diverse single-channel electrical signatures but conserved anion selectivity. Biochemistry 51:2319–2330

    Article  CAS  Google Scholar 

  • Liu J, Wolfe AJ, Eren E, Vijayaraghavan J, Indic M, van den Berg B, Movileanu L (2012b) Cation selectivity is a conserved feature in the OccD subfamily of Pseudomonas aeruginosa. Biochim Biophys Acta Biomembr 1818:2908–2916

    Article  CAS  Google Scholar 

  • Mahendran KR, Chimerel C, Mach T, Winterhalter M (2009) Antibiotic translocation through membrane channels: temperature-dependent ion current fluctuation for catching the fast events. Eur Biophys J 38:1141–1145

    Article  CAS  Google Scholar 

  • Markovic-Housley Z, Garavito RM (1986) Effect of temperature and low pH on structure and stability of matrix porin in micellar detergent solutions. Biochim Biophys Acta Protein Struct Mol Enzymol 869:158–170

    Article  CAS  Google Scholar 

  • Martina M, Taverna S (2014) Patch-Clamp.

  • Mishra M, Panda S, Barik S, Sarkar A, Singh DV, Mohapatra H (2020) Antibiotic resistance profile, outer membrane proteins, virulence factors and genome sequence analysis reveal clinical isolates of Enterobacter are potential pathogens compared to environmental isolates. Front Cell Infect Microbiol 10:54

    Article  CAS  Google Scholar 

  • Nikaido H (1992) Porins and specific channels of bacterial outer membranes. Mol Microbiol 6:435–442

    Article  CAS  Google Scholar 

  • Nikaido H (1994) Porins and specific diffusion channels in bacterial outer membranes. J Biol Chem 269:3905–3908

    Article  CAS  Google Scholar 

  • Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656

    Article  CAS  Google Scholar 

  • Nikaido H, Rosenberg EY (1981) Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli. J Gener Physiol 77:121–135

    Article  CAS  Google Scholar 

  • Pletzer D, Braun Y, Weingart H (2016) Swarming motility is modulated by expression of the putative xenosiderophore transporter SppR-SppABCD in Pseudomonas aeruginosa PA14. Antonie Van Leeuwenhoek 109:737–753

    Article  CAS  Google Scholar 

  • Pothula KR et al (2017) Single residue acts as gate in OccK channels. J Phys Chem B 121:2614–2621

    Article  CAS  Google Scholar 

  • Ripoll DR, Faerman CH, Axelsen PH, Silman I, Sussman JL (1993) An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase. Proc Natl Acad Sci 90:5128–5132

    Article  CAS  Google Scholar 

  • Samanta S et al (2017) How to get large drugs through small pores? Exploiting the porins pathway in Pseudomonas aeruginosa. Biophys J 112:416a

    Article  Google Scholar 

  • Samanta S et al (2018) Getting drugs through small pores: exploiting the Porins pathway in Pseudomonas aeruginosa. ACS Infect Dis 4:1519–1528

    Article  CAS  Google Scholar 

  • Schaich M et al. (2019) An integrated microfluidic platform for quantifying drug permeation across biomimetic vesicle membranes. J BioRxiv:523431

  • Stürenburg E, Sobottka I, Mack D, Laufs R (2002) Cloning and sequencing of Enterobacter aerogenes OmpC-type osmoporin linked to carbapenem resistance. Int J Med Microbiol 291:649–654

    Article  Google Scholar 

  • Tamagawa H, Morita S (2014) Membrane potential generated by ion adsorption. Membranes 4:257–274

    Article  Google Scholar 

  • Terrasse R, Winterhalter M (2018) Translocation of small molecules through engineered outer-membrane channels from Gram-negative bacteria. Eur Phys J E 41:111

    Article  Google Scholar 

  • Trapani JG, Korn S (2003) Effect of external pH on activation of the Kv1. 5 potassium channel. Biophys J 84:195–204

    Article  CAS  Google Scholar 

  • Yamano Y, Nishikawa T, Komatsu Y (1993) Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1. Mol Microbiol 8:993–1004

    Article  CAS  Google Scholar 

  • Yamano Y, Nishikawa T, Komatsu Y (1998) Involvement of the RpoN protein in the transcription of the oprE gene in Pseudomonas aeruginosa. FEMS Microbiol Lett 162:31–37

    Article  CAS  Google Scholar 

  • Yoneyama H, Yamano Y, Nakae T (1995) Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: construction of mutants with deletions in the multiple porin genes. Biochem Biophys Res Commun 213:88–95

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Authors received funding from IMI Project Translocation [The Innovative Medicines Joint Undertaking under Grant Agreement No. ~ 115525, with resources composed of financial contribution from the European Union’s seventh framework programme (FP7/2007–2013) and EFPIA companies)].

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences, Ankara Yildirim Beyazit University, 06010, Ankara, Turkey

    Fatma Dogan Guzel & Araz Norouz Dizaji

  2. School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany

    Fatma Dogan Guzel, Daniel Pletzer, Kareem Al-Nahas, Mawadah Bajrai & Mathias Winterhalter

  3. Department of Microbiology and Immunology, Centre for Microbial Diseases and Immunity Research, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada

    Daniel Pletzer

  4. Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge, CB3 0HE, UK

    Kareem Al-Nahas

Authors
  1. Fatma Dogan Guzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Pletzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Araz Norouz Dizaji
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kareem Al-Nahas
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mawadah Bajrai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mathias Winterhalter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fatma Dogan Guzel.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 126 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dogan Guzel, F., Pletzer, D., Norouz Dizaji, A. et al. Towards understanding single-channel characteristics of OccK8 purified from Pseudomonas aeruginosa. Eur Biophys J 50, 87–98 (2021). https://doi.org/10.1007/s00249-021-01498-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-021-01498-5

Keywords

Search

Navigation