Combatting antimicrobial resistance using novel small fusion proteins

Antibiotic resistance is a public health burden worldwide. Alternatives to antibiotics that can counter the detrimental effects of resistant ‘superbugs’ are urgently needed. Dr Xristo Zarate at the Autonomous University of Nuevo Leon in Mexico and his collaborators have worked extensively on developing novel small carrier proteins, like SmbP and CusF3H+, for recombinant protein and peptide expression and purification in bacteria. The team encourages the scientific community to use these proteins to improve the production of recombinant peptides with biological activity, especially antimicrobial properties.

Clinically relevant ‘superbugs’ like Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species have developed genetic and epigenetic mechanisms to counteract powerful broad-spectrum antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin. Frequent use of prescription antibiotics in clinical practices is one of the key factors driving antibiotic resistance. Hence, there is an urgent need for potent alternatives to counter the detrimental effects of these superbugs.

Antimicrobial peptides and the immune system

Antimicrobial peptides (AMPs) are a class of small proteins crucial to the innate immune systems of several organisms, with a wide range of protective effects against multiple classes of microorganisms such as bacteria, fungi, and viruses. While our immune system produces and utilises AMPs naturally, there is an urgent need to design more potent AMPs to protect us from superbugs. Novel AMPs are mostly engineered using genetic materials from different organisms, equipping them with the best defence features from the organisms involved.

On entering the host system, the AMPs stimulate the host’s innate immune response to provide the first line of defence against pathogens. There are two mechanisms to curb the organism’s pathogenic properties: firstly, AMPs can disrupt its cell wall and plasma membrane by forming pores along these membranes wherein the positively charged (cationic) AMPs bind to the negatively charged (anionic) pathogenic cell walls. Secondly, AMPs can genetically modify the pathogen by inhibiting DNA replication and protein synthesis.

Examples of such AMPs include defensins, LL-37, gramicidin D, histatins, and renalexin. These AMPs are biochemically cationic, amphipathic (with water-soluble/polar and water-insoluble/non-polar components on their borders to help them bind better to a variety of microbial cell walls) and composed of 10–100 amino acid residues. Apart from being naturally synthesised in human or multicellular hosts, AMPs are also synthesised by single-celled prokaryotes, to prevent competition from or to outgrow other microbes in a competitive ecosystem for survival.

Dr Xristo Zarate of the Autonomous University of Nuevo Leon in Mexico and his team of researchers genetically engineered novel fusion proteins. Their proteins can aid in the development of more powerful and novel AMPs for human therapeutic benefit to meet their growing demand in the antimicrobial peptide market.

How do fusion proteins fit the memo?

Fusion proteins are a class of recombinant proteins which help to improve the downstream processing of novel AMPs by increasing their solubility, stabilising them, reducing the formation of metabolic wastes (inclusion bodies), and helping in tagging and purification processes. Fusion proteins with affinity for metal ions can help purify recombinant AMPs using metal tags in chromatography – a process known as immobilised metal affinity chromatography (IMAC). The most common fusion protein candidates include maltose binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide isomerase (PDI), glutathione S-transferase (GST), and thioredoxin (TRX).

The most common organism that scientists use to generate recombinant AMPs are Escherichia coli (E. coli) cells. This is due to their low costs, high yield of proteins, operational ease, and ease of purification of the final product. However, most protein purification processes come across challenges like high molecular weight, impaired solubility, challenges associated with affinity chromatography, and the feasibility of the protocol needed to separate it from the protein of interest. It is crucial to design fusion proteins keeping in mind the specific characteristics of the target AMP. There is no ‘one size fits all’ theory applicable in this regard. Hence, fusion proteins ticking all the boxes for specific conditions would be ideal for purifying AMPs with maximum potency and purity.

Curbing antimicrobial resistance

Zarate and his team of researchers have isolated two novel fusion proteins – CusF3H+ and small metal-binding protein (SmbP) – from E. coli and Nitrosomonas europaea, respectively, to deliver pure forms of AMP with clinical significance. These two fusion proteins were chosen because of their small molecular weight and ability to reduce the formation of inclusion bodies, bind to metal ions, and produce purer proteins.

SmbP acts as a metal ‘scavenger’ which can bind to several metal ions, including copper (Cu(II)), nickel (Ni(II)), zinc (Zn(II)), and even iron (Fe(III)) and free the cells of such metallic impurities. CusF3H+ is an enhanced version of the fusion protein CusF. CusF is a periplasmic protein, part of the CusCBFA efflux complex which binds the toxic ions silver (Ag(I)) and copper (Cu(I)) to expel them from the cell; it also binds copper (Cu(II)) and nickel (Ni(II)) which allows the purification of recombinant proteins tagged with CusF3H+ to be purified with the help of IMAC.

The cytoplasmic space on the inside of a cell is home to a more diverse family of cellular, enzymatic, and metabolic proteins, DNA, and endotoxins compared to the periplasm (the space between the cytoplasm and the outer membrane). Hence, sometimes, using the periplasmic over cytoplasmic space ensures that the AMPs that need to be expressed and purified face fewer obstacles in the form of metabolic enzymes and toxins, making the downstream processes of purification and isolation easier.

Zarate and collaborators then used the fusion proteins CusF3H+ and SmbP to design, produce, and purify novel recombinant AMPs. They engineered a multifunctional hybrid antimicrobial peptide LL-37_Renalexin with the help of a flexible GS peptide linker. In another study, the team designed a soluble recombinant protein with the mature, bioactive Bin1b (beta-defensin, originally found in rats) peptide synthesised using the E. coli strain SHuffle. Both recombinant proteins were purified using IMAC and subsequently cleaved with the enzyme enterokinase to separate the fusion protein from LL-37_Renalexin and Bin1b.

The purified peptides displayed antimicrobial activity against major pathogens. The purified, mature bioactive Bin1b was able to reduce gram-negative organisms (including E. coli) by ~80% while it removed ~50% of gram-positive organisms (such as S. aureus). The LL-37_Renalexin was also shown to be a potent AMP wherein it reduced 85% of all major bacterial strains like E. coli, S. aureus, Klebsiella pneumoniae, and methicillin-resistant S. aureus (MRSA) – all of which are known to be the most notorious, resistant pathogens. Moreover, this reduction came at a significantly lower concentration of AMP as compared to the previously isolated (non-hybrid) peptides, thus showing the potential of recombinant AMPs purified with metal-binding novel fusion proteins.

Moving ahead

Considering that conventional antibiotics have become futile in treating some of the deadliest microbial pathogens, it is time to adapt to new technologies which offer the option of selecting bioactive molecules with potent antibiotic activity to help reduce the global disease burden of antibiotic resistance.

Zarate’s work gives us an insight into one such novel method – the use of small metal-binding carrier proteins CusF3H+ and SmbP, to purify AMPs for clinical use.