Observing the Interactions between Antimicrobial Proteins and Bacterial Cell Membranes

According to World Health Organization (WHO), antimicrobial resistance is the ability of a microorganism to resist an antimicrobial drug that was used earlier for treating microbial infections. Establishing the interactions between microbes and drugs is part of the solution to overcome this antimicrobial problem. Here, polarized FTIR spectroscopy and Specac’s IR polarizers play an integral role.

This resistance is a fundamental property of the microbe, not the person who has been infected by a microbe. There is an ongoing global crisis, where antibiotics used for fighting relatively simple infections could become ineffective which may lead to a serious deadly outcome. According to the WHO, every year, 480,000 people are infected with multi-drug resistant TB worldwide.

However, the lack of non-toxic and effective antibiotics to fight microbial infections warrants the need for routine surgeries, which can be fundamentally risky. In other words, all but the most important procedures turn out to be complicated risk-benefit decisions.

In 2014, the Review on Antimicrobial Resistance (AMR) was undertaken in the UK which shows that if antimicrobial resistance is not controlled, approximately 300,000,000 people across the globe may die over the next 35 years and that global economic output will reduce by $100 trillion.

The Rise of Antibiotic Resistance

Most of the antibiotics are naturally occurring compounds to which microorganisms have been exposed to for millions of years. Therefore, bacteria have in-built mechanisms that allow them to develop resistance against antimicrobial drugs. Conversely, in today’s era of human medicine, there has been rampant development of strain-resistant bacteria as a result of misused and over-prescribed antibiotics.

Across the Western world, human use of antibiotics only represents 30% of the total, while the remainder is utilized by the meat industry to enable intensive factory farming. In the agricultural field, antibiotics are used for infection prophylaxis and as growth promoters in food production which has become a serious problem. As a result, both the EU and FDA have demanded food producers to adopt probiotic methodologies.

Discovering New Antibiotics

Despite ongoing discovery programs, difficulties in antibacterial discovery have limited the output of new antibacterial drug classes to extremely low levels in the past 25 years. During this period, the antibacterial product pipeline has been controlled by better analogs of antimicrobial drug classes discovered in the past. While this resulted in the development of novel antibiotics, it has hindered new drug discovery and also there has been a discovery void since the early 2000s.

In eukaryotic cells, a key part of host defense is the formation of antimicrobial proteins. The phagocytic cells, epithelial layers, and body fluids of multicellular animals literally contain hundreds of antimicrobial peptides. Larger antimicrobial proteins (usually containing over 100 amino acids) can either be nutrient-binding proteins or lytic enzymes or contain sites that are known to target microbial macromolecules. Similarly, smaller antimicrobial peptides are also significant and are likely to form either a novel class of antibiotics or act by affecting the function or structure of microbial cell membranes.

Antimicrobial proteins, besides their antiviral, antibacterial and antifungal activities, also play a role in inflammatory processes and immunomodulatory activities and some of them are also active against tumor cells.

The following are three main theories about how the target microbes are destroyed by the disruption of membrane integrity (by a compound, for example, an antimicrobial protein):

• The equilibration of extracellular and intracellular ion concentrations via the disrupted membrane drain the energy effects
• Metabolic function is disrupted by antimicrobial peptides that enter the target cell and bind to intracellular molecules
• Cell lysis is caused by peptides producing pores that allow water to pass, but prevent the passage of osmotically active substances

Antimicrobial peptides are traditionally defined as polypeptide antimicrobial substances that contain less than 100 amino acid residues (synthesized by ribosomes). This differentiates these peptides from most peptide antibiotics of fungi and bacteria, which usually include exotic amino acids and are produced by specific metabolic pathways. New classes of antibiotics can be developed and tested by studying the interaction between the lipid membranes and antimicrobial proteins of microbes.

Using Polarized FTIR to Investigate Drug-Microbe Interactions

Raman spectroscopy and Polarized Fourier transform infrared (FTIR) are suitable techniques for observing structural changes and even the most subtle interactions in the range of picoseconds. Polarized ATR FTIR is designed to deliver superior data on the orientation of lipid backbones (CH₂ chains), but during interaction studies, it also provides information on specific bonds within them.

The main determinants of the backbone structure of proteins and peptides are dihedral angles. FTIR combined with polarized Raman spectroscopy gives adequate data to acquire the dihedral angles of tripeptides without having to establish more data from computational studies. Therefore, infrared spectroscopy is not dependent on an extrinsic label and provides a clear understanding of local secondary structure. It is particularly suited for studies related to thin-film Langmuir types.

Thanks to its potential for monitoring conformation and aggregation of proteins and peptides, FTIR has become an important tool for investigating the interactions between protein membranes. Particularly, polarized ATR-IR has been useful for studying:

• Electric field-induced reorientation of lipid headgroups
• Insulin fibril formation
• The role of lipid type on the conformation of transmembrane receptors
• Protein orientation at solid surfaces
• Protein association with lipid bilayers

Infrared Polarizing Filters from Specac

Polarizers supplied by Specac are valuable spectroscopy sampling tools that enable the analysis of samples with key molecular orientation, for instance, the conformational change of peptide chains during interaction with lipid layers. Also, Specac polarizers are useful for investigating thin films on reflective surfaces and such situations are usually used for the analysis of proteins and membranes in a controlled environment.

A wide range of free-standing wire grid polarizers is available from Specac for FT-IR applications. These polarizers have operating wavelengths ranging from 20 μm to 10 mm, and contain a range of parallel 5 μm to 10 μm tungsten wires on a mounting frame with 12.5 μm or 25 μm wire spacing. Polarizers also come in custom shapes and sizes and can even include beryllium coated copper wire or gold-plated tungsten, if needed.

Conclusion

Antimicrobial proteins could pave the way for a whole new class of powerful and effective antibiotics. A more direct insight of the ‘way they work’ and the way they interact with the microorganisms to kill is crucial for their development.

Polarizing FT-IR is an important method for understanding the structure of peptides and how this structure changes during its interplay with microbial cell membranes.

References and Further Reading

1. WHO (April 2014). "Antimicrobial resistance: global report on surveillance 2014" (http://www.who.int/mediacentre/factsheets/fs194/en/)
2. Michael T. Osterholm and Mark Olshaker, Deadliest Enemy: Our War Against Killer Germs, 2017, Little, Brown and Company, New York.
3. The Review on Antimicrobial resistance, https://amr-review.org/ UK Prime minister 2014
4. https://www.england.nhs.uk/2016/03/antibiotic-overusage/  
5. Tomas Ganz, The Role of Antimicrobial Peptides in Innate Immunity, Integrative and Comparative Biology, Volume 43, Issue 2, 1 April 2003, Pages 300–304
6. The Pew Charitable Trusts, A Scientific Roadmap for Antibiotic Discovery, May 11, 2016 Antibiotic Resistance Project. http://www.pewtrusts.org/en/research-and-analysis/reports/2016/05/a-scientific-roadmap-for-antibiotic-discovery  
7. Lynn L. Silver, Challenges of Antibacterial Discovery, Clinical Microbiology Reviews, Jan. 2011, p. 71–109 Vol. 24, No. 1
8. Kóta Z, Páli T, Marsh D. Orientation and Lipid-Peptide Interactions of Gramicidin A in Lipid Membranes: Polarized Attenuated Total Reflection Infrared Spectroscopy and Spin-Label Electron Spin Resonance. Biophysical Journal. 2004;86(3):1521-1531.
9. Claudia Dannehl, Gerald Brezesinski, and Helmuth Möhwald, Interactions of Two Fragments of the Human Antimicrobial Peptide LL-37 with Zwitterionic and Anionic Lipid Monolayers Z. Phys. Chem. 2015; 229(7–8): 1141–1159
10. Jessica J. Li, Christopher M. Yip. Super-resolved FT-IR spectroscopy: Strategies, challenges, and opportunities for membrane biophysics, Biochimica et Biophysica Acta (BBA) – Biomembranes, Volume 1828, Issue 10, October 2013, Pages 2272–2282

About Specac Ltd

Specac manufactures an extensive range of FTIR Accessory, IR Polarizer, and Pellet Press Products for Atomic and Molecular Spectroscopy.

These products include ATR Accessories, Specular Reflectance Accessories, Diffuse Reflectance Accessories, Liquid Transmission and Gas Transmission Cells, as well as Infrared and Terahertz Wire Grid Polarizers, Bench-Top Hydraulic Presses, KBr Pellet Presses, XRF Pellet Presses, Thin Film Making Kits, and Evacuable Pellet Dies.

For online optical spectroscopy or FTIR analysis, Specac offers a comprehensive range of NIR Process Cells suitable for liquid and gas/vapour analysis.