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Innovation & Technologie Angebot

Antibacterial alternative to antibiotics with high specificity against Escherichia coli

Country of Origin: Spain
Reference Number: TOES20200113001
Publication Date: 13 January 2020

Summary

A Spanish university has modified several phage proteins (Poll-N and UK-C) that have high specificity against Escherichia coli (E. coli), but not against other Gram-negative bacteria. They could be used in the case of contaminated food, cosmetics or water, or even in the treatment of diseases (infections) caused by E. coli. Biotechnological or pharmaceutical companies interested in the commercial exploitation of the technology through license and/or technical cooperation agreements are sought.

Description

Endolysins are enzymes produced by bacteriophages (viruses that infect bacteria). The biological function of these endolysins is to hydrolyze bonds in the bacterial cell wall causing thus cell lysis and the consequent release of new bacteriophages. As a result, endolysins have been proposed as alternative to antibiotics, since the use of the latter has led to the emergence of resistances, considerably decreasing their effectiveness. 



In the case of Gram-positive (G+) bacteria, the use of endolysins as an antibacterial agent is widely established. However, in the specific group of Gram-negative (G-) bacteria it has several limitations related to problems of accessibility and specificity. 



In order to alleviate the first problem, a permeabilizing outer lipid membrane (OM) treatment could be used, or tails of amino acids with net positive charge could be added to the endolysin to help overcome electrostatic repulsions due to the network of negative charges on the cell surface.



In order to solve the specificity issue, modular proteins can be used, with a catalytic domain and a cell wall-binding domain. However, the modular proteins described for G- are less numerous than for G+ and, in addition, their larger size hampers their purification and manipulation.



In this sense, a Spanish research group has developed several modified phage proteins (named Poll-N and UK-C) with antibacterial activity highly specific against E. coli without the need for previous permeabilization treatments. 



The polypeptides (proteins) developed with endolysin activity, Poll-N and UK-C, comprise, respectively:

• An amino acid sequence according to SEQ ID NO: 3 or a derivative thereof (deletion, addition, insertion and/or substitution in this amino acid sequence), and a polycationic tail of amino acids (histidines) at the N-terminal end; and, 

• An amino acid sequence according to SEQ ID NO: 4, or a derivative thereof (deletion, addition, insertion and/or substitution in this amino acid sequence), and a polycationic tail of amino acids (histidines) at the C-terminal end.



Once cloned, expressed and purified, the resulting proteins (Poll-N and UK-C) carry a histidine tail at their N-terminal or C-terminal end, being therefore different from the original. This tail not only facilitates its purification, but also favours the endolysin contact to the cell surface, thus improving its lysis efficiency.

	

Due to their high specificity to E. coli, the developed polypeptides can be used both as antimicrobial agents to prevent contamination by E. coli (particularly in food, cosmetics, water contaminated with E. coli, etc.), and in the treatment of diseases (infections) produced by E. coli. Then, this technology could be applied in biosanitary, veterinary, biotechnological, or agri-food companies interested in antimicrobial treatments alternative to antibiotics to control the growth of E. coli.



The research group is mainly looking for companies (biotechnology or pharmacological sectors) interested in acquiring this technology for its commercial exploitation through license agreement. The company should be responsible for the clinical trials, the development to the industrial scale, its installation and its introduction into the market. The university will be ready to provide technical assistance in each step, if required.



However, the research group would be also interested in establishing technical cooperation agreements to further develop the technology, to find new applications or to adapt it to the company’s needs. The goal of this type of collaboration would be increasing the technology readiness level for a future commercial exploitation of the patent. The university would offer its support based on their know-how; while, the partner sought would provide its expertise to help improve this invention. The university would offer this partner a preferential option to acquire this technology in exclusivity.

Advantages and Innovations

The main innovative aspect of the modified phage proteins is that they do not require prior treatments of OM permeabilization. Furthermore, the addition of nucleotides at the ends facilitates their manipulation and subsequent cloning in the appropriate expression vector. Finally, another innovative aspect is the generation of anti-E. coli endolysins whose sequence is significantly different from others.



Whereas, the main advantages of the synthesized peptides, Poll-N and UK-C, are the following:



• The presence of a histidine tail at the N-terminal or C-terminal ends of the synthesized peptides facilitates their purification and improves their lysis efficiency.

• They have a high specificity against E. coli as, among all bacteria tested, their lytic action is limited to those pertaining to this species without affecting any other belonging to other species, including closely related ones.

• Compared to antibiotics, a lesser probability of resistance occurrence is anticipated.

• Their use is expected to result, at most, in negligible effects on resident microbiota during the treatment of infections.

• Simple formulation, since no previous treatments of permeabilization of the external bacterial membrane are necessary, which in turn facilitates its application.

Stage Of Development

Under development/lab tested

Stage Of Development Comment

The technology is developed at laboratory scale. 



The efficacies of purified Poll-N and UK-C endolysins have been tested by means of spot test experiments against different bacterial strains. Figure 1 (for Poll-N) and Figure 2 (for UK-C) show the appearance of growth inhibition zones produced by lysis. 



The results obtained show that both Poll-N and UK-C are capable of directly lysing the majority (92.5% for Poll-N and 91.2% for UK-C) of E. coli strains tested (159 in total).

Requested partner

- Type of partner sought: Industries.

- Specific area of activity of the partner: biotechnology; pharmaceutical; biosanitary; veterinary; agri-food.

- Task to be performed:

* In the license agreement: to buy a license for the technology, to further develop it to the industrial scale and to introduce it into the market.

* In the technical cooperation agreement: to provide their expertise in order to collaborate with the scientists on further development and improvements of the technology. The company should identify technical requirements and/or market and client’s needs in order to carry out further technical development so that the market readiness will be increased and the technology could be commercially exploited.