ES2827850B2 - VIRAL PROTEINS WITH ANTIBACTERIAL ACTIVITY AGAINST Escherichia coli - Google Patents

Description

VIRAL PROTEINS WITH ANTIBACTERIAL ACTIVITY AGAINST Escherichia coli

field of invention

The present invention falls within the general field of genetic engineering and, in particular, refers to viral proteins that have been modified by adding a polycationic amino acid tail to their N-terminal end, in such a way that they have specific antibacterial activity. against Escherichia coli ( E. coli) without the need for previous treatments to permeabilize the envelope.

state of the art

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 at the end of the reproductive cycle of the virus, thus causing cell lysis and the consequent release of new bacteriophages produced during its intracellular stage. In order to access their target in the cell wall, these enzymes need the participation of other proteins, called holins (Gutiérrez D, Fernández L, Rodríguez A, and García P (2018). Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus? MBio.

9(1)), which form pores in the cytoplasmic membrane.

Endolysins (along with holins) are one of the most diverse families of proteins known. Structurally, they can be formed by a single globular domain, or composed of one or two cell wall binding domains and a catalytic domain (García P, Rodríguez L, Rodríguez A, and Martínez B (2010). Food biopreservation: promising strategies using bacteriocins, bacteriophages and endolysins. Trends Food Sci Technol. 21(8): 373-382; Oliveira H, Melo LDR, Santos SB, Nóbrega FL, Ferreira EC, Cerca N, Azeredo J, and Kluskens LD (2013). Molecular aspects and comparative genomics of bacteriophage endolysins. J Virol. 87(8): 4558-70).

Due to their ability to lyse bacteria, endolysins have been proposed as alternative antibacterial agents to the use of antibiotics, since the use of the latter has led to the appearance of resistance, considerably reducing their effectiveness (Love MJ, Bhandari D, Dobson RCJ, and Billington C (2018).Potential for Bacteriophage Endolysins to Supplement or Replace Antibiotics in Food Production and Clinical Care. Antibiotic (Basel, Switzerland). 7(1)).

Additionally, the broad spectrum of action of many antibiotics can lead to side effects during the treatment of infections, such as the alteration of the patient's natural microbiota by affecting different species in addition to the causative agent (Love MJ, Bhandari D, Dobson RCJ , and Billington C (2018). Potential for Bacteriophage Endolysins to Supplement or Replace Antibiotics in Food Production and Clinical Care. Antibiot (Basel, Switzerland). 7(1); O'Flaherty S, Ross RP, and Coffey A (2009) Bacteriophage and their lysins for elimination of infectious bacteria: Review article. FEMS Microbiol Rev. 33(4): 801-819).

However, the use of endolysins as an antibacterial agent in the specific group of Gram-negative (G-) bacteria has several limitations. In the first place, due to problems of accessibility to its target in the cell wall due to the presence of an outer lipid membrane (EM). To alleviate this problem, a permeabilizing treatment of the EM could be used or polycationic tails (of amino acids with a net positive charge) added to the endolysin that help to overcome the electrostatic repulsions due to the network of negative charges on the cell surface (Walmagh M, Briers Y, Santos SB dos, Azeredo J, and Lavigne R (2012). Characterization of Modular Bacteriophage Endolysins from Myoviridae Phages OBP, 201^2-1 and PVP-SE1. PLoS One. 7(5): e36991). Second, there is very little variability in the cell wall composition of the different G- species, compromising specificity (Love MJ, Bhandari D, Dobson RCJ, and Billington C (2018). Potential for Bacteriophage Endolysins to Supplement or Replace Antibiotics in Food Production and Clinical Care Antibiot (Basel, Switzerland) 7(1) Zampara A, Sorensen MCH, Grimon D, Antenucci F, Briers Y, and Brondsted L (2018) Innolysins: A novel approach to engineer endolysins to kill Gram-negative bacteria. bioRxiv. 408948).

For the reasons stated above, derived from the problems that generally affect the use of endolysins in G-, there is therefore a need to provide proteins with adequate lytic activity that do not require permeabilizing treatments, and that are specific to a specific group of bacteria.

Brief description of the invention

The present invention solves the problems described in the state of the art, since it provides proteins with specific antibacterial activity against E. coli without the need for previous treatments to permeabilize their envelope.

Thus, in a first aspect, the present invention relates to a polypeptide with endolysin activity (hereinafter, the peptide of the present invention) consisting of the amino acid sequence according to SEQ ID NO: 4. The polypeptide of the The present invention comprises a polycationic amino acid tail at the N-terminus (SEQ ID NO: 4). In particular, it comprises a polycationic histidine tag.

In the present invention the term "polypeptide" is synonymous with the term "protein". In the present invention the term "polypeptide" refers to a sequence of amino acids, where said amino acids are linked together by peptide bonds.

In a particular embodiment of the present invention, the derivative of the peptide of the present invention comprises a deletion, addition, insertion and/or substitution in the amino acid sequence SEQ ID NO:4.

In another aspect, the present invention relates to an isolated nucleic acid, which encodes the polypeptide of the present invention (hereinafter, nucleic acid of the present invention). More particularly, the nucleic acid of the present invention, once cloned, encodes the polypeptide of amino acid sequence according to SEQ ID NO:4. More particularly, the nucleic acid of the present invention consists of the nucleotide sequence according to SEQ ID NO: 2.

In another aspect, the present invention relates to a vector comprising the nucleic acid of the present invention (hereinafter vector of the present invention).

In another aspect, the present invention relates to a host cell, which contains the nucleic acid of the present invention, and/or the vector of the present invention, and/or the protein of the present invention.

In another aspect of the present invention, it relates to a method for genetic transformation of a host cell by introducing the nucleic acid of the present invention to express the polypeptide of the present invention. Once expressed, the polypeptide of the present invention is purified. Genetic transformation, expression of the polypeptide of the present invention, and the purification, can be carried out by genetic engineering methods known to the person skilled in the art.

In another aspect, the present invention relates to the non-therapeutic use of the polypeptide of the present invention as an antimicrobial against E. coli. In a particular embodiment, the present invention refers to the non-therapeutic use of the polypeptide of the present invention as an antimicrobial in food, cosmetics, and water contaminated with E. coli.

In another aspect, the present invention relates to the polypeptide of the present invention for use in the treatment of diseases caused by E. coli. More in particular, for the treatment of infections caused by E. coli.

In another aspect, the present invention relates to a composition comprising the polypeptide of the present invention (hereinafter composition of the present invention). More particularly, the composition of the present invention comprises a chemically and/or pharmaceutically acceptable excipient.

In the present invention, "excipient" refers to any component that does not have therapeutic activity and that is non-toxic, mainly referring to vehicles and buffers such as saline solutions, aqueous solutions, emulsions, dyes, flavorings, flavorings, etc.

In a more particular embodiment, the present invention relates to the non-therapeutic use of the composition of the present invention as an antimicrobial against E. coli. In a particular embodiment, as an antimicrobial in food, cosmetics, water contaminated with E. coli.

In another aspect, the present invention relates to the composition of the present invention for use in the treatment of diseases caused by E. coli. More particularly, for the treatment of infections caused by E. coli .

Description of the figures

Figure 1. Shows the expression vector pCDF-1b, used to clone the endolysins of the present invention. The vector has a multiple cloning site that includes the cleavage sequences for the Ncol and BamHI restriction enzymes, a streptomycin (Sm) resistance cassette for the selection of transformants, as well as a T7 promoter and a lac operator to induce fusion. expression of the cloned gene.

Figure 2. Shows the result of a spot test experiment carried out with Poll-N at a concentration of 16 μg/mL on an E. coli strain. The halo of clarity around the point where the protein solution has been deposited indicates the growth inhibitory effect produced by endolysin.

Detailed description of the invention

Example 1: cloning and expression of the modified viral protein Poll-N.

The endolysin synthetic gene, with the nucleotide sequence identified in the present invention as SEQ ID NO:2, isolated from E. coli bacteriophage Pollock, was amplified by PCR with the primers described in Table 1 under the following conditions: 1 cycle 5 min at 95°C; followed by 25 cycles of 10 s at 95°C, 30 s at 52°C, and 2 min at 72°C, and finally 1 cycle of 10 min at 72°C. The amplification product was purified and later digested with the restriction enzyme BamHI for 3 h at 37 °C to finish with a Ncol digestion at 37 °C for 24 h. After each digestion, the enzymes were inactivated at 80 °C for 20 min, and the digestion products were likewise purified after each inactivation. The same restriction procedure was carried out with the plasmid pCDF-1b, the vector used for cloning (Figure 2), which was finally treated with alkaline phosphatase, and subsequently purified in an equivalent way. The vector and insert digests were mixed in a 1:3 ratio and ligated at 16 °C for 16 h with the enzyme bacteriophage T4 DNA ligase. The amounts of each of the DNA molecules were estimated fluorimetrically with a Qubit device (Invitrogen®).

Table 1 . Primers used in the cloning and expression of the modified Poll-N viral protein

The ligation product was transformed into chemocompetent BL21(AI) cells, prepared according to the method described by (Green and Rogers Green R, and Rogers EJ (2013). Transformation of chemically competent E. coli. Methods Enzymol. 529: 329 -36). Transformants were selected by growth on solid LB medium containing 100 µg/mL streptomycin. Subsequently, they were amplified by PCR using the corresponding primers for confirmation (Table 2), under the following conditions: 1 initial cycle of 5 min at 95 °C; followed by 25 cycles of 30 s at 94 °C, 30 s at 53 °C, 80 s at 72 °C; and 1 final cycle of 7 min at 72 °C.

The PCR products of clones carrying inserts were sequenced to confirm their identity, and in this case their expression and purification of the protein were carried out following the following procedure. One of the transformant colonies confirmed in this way was transferred to a flask with 100 mL of LB broth, incubating the culture at 37 °C and 200 rpm. When the OD600 reached a value of 0.3, protein expression was induced by the addition of IPTG and arabinose, at final concentrations of 1 mM and 0.3%, respectively. After 4 h of incubation of the culture under the same conditions, it was centrifuged at 4000 g for 30 min at 4 °C. After discarding the supernatant, cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.6; 1 mM Pefabloc; 0.1 mg/mL lysozyme; 10 μg/mL DNAse I) and sonicated on ice, administering 30 pulses of 30 s at 30 s intervals. The resulting lysate was centrifuged at 4000 g for 30 min at 4 °C, and the supernatant was filtered through a sterile 0.45 µm filter followed by a second filtration through a 0.22 µm filter. Protein was purified from the latter filtrate with a Hispur™ Ni-NTA Chromatography column (Thermo Fisher Scientific™) at 4°C. The total amount of purified protein is determined by the Bradford method (Kruger NJ (2009). The Bradford Method For Protein Quantitation. Humana Press, Totowa, NJ; pp 17-24). The product obtained was frozen in liquid nitrogen, after which it was stored at -80 °C in aliquots.

The efficacy of the purified Poll-N endolysin was tested by "spot test" experiments against different bacterial strains. Cultures of these strains grown in liquid LB medium at 37 °C up to an OD600 of 0.3 were mixed with 5 mL of LB melted at 50 °C and were then poured onto plates with solid LB medium. Once all the medium had solidified, 10 μL aliquots of suspensions of the Poll-N enzyme at different concentrations were deposited on its surface. After incubating at 37 ° C for 12 h, the appearance of growth inhibition zones produced by lysis was observed (Figure 3).

The results obtained show that Poll-N is capable of directly lysing the majority (92.5%) of E. coli strains tested (159 in total), without the need for any prior cell surface permeabilization treatment. In addition, its lytic action is limited to this species without affecting any of the tested bacteria belonging to other species, even those closely related (Table 2). Based on these results, Poll-N endolysin is confirmed as an antimicrobial agent with high specificity against E. coli .

Table 2 . Results of the "spot test" experiments with the endolysin Poll-N against different bacterial strains.

1, Appearance (+) or not (-) of clarity in growth in the spot test in the area where a concentration of endolysin of 16 μg/mL is added.

2, Ochman H, and Selander R (1984). Standard reference strains of E. coli from natural populations. J Bacteriol. 157(2): 690-693.

3, Enterotoxigenic E. coli Strains, courtesy of Dr. Anders Nilsson (Stockholm University, Sweden).

4, Enterotoxigenic E. coli strains isolated by the University of Alicante during field trips to pig farms, courtesy of DHESA (Fortuna, Murcia, Spain).

5, E. coli avian strains, courtesy of Dr. Pablo Catalá (CECAV, Castellón, Spain).

6, Avian Salmonella Strains, courtesy of Dr. Pablo Catalá (CECAV, Castellón, Spain).

7, Salmonella clinical strains, courtesy of the Microbiology service of the Hospital General Universitario de Elche (Alicante, Spain).

Claims (11)

1. Polypeptide consisting of an amino acid sequence according to the sequence SEQ ID NO:4. An isolated nucleic acid encoding a polypeptide according to claim 1. 3. Nucleic acid according to claim 2, consisting of SEQ ID NO:2. 4. Vector comprising a nucleic acid according to any of claims 2-3. 5. Host cell comprising a nucleic acid according to any of claims 2-3 or a vector according to claim 4. 6. Non-therapeutic use of the polypeptide according to claim 1 as an antimicrobial against E.coli. 7. Polypeptide according to claim 1 for use in the treatment of diseases caused by E. coli . 8. Composition comprising a polypeptide according to claim 1. Composition according to claim 8 comprising a chemically and/or pharmaceutically acceptable excipient. 10. Non-therapeutic use of the composition according to any of claims 8-9, as an antimicrobial against E. coli . 11. Composition according to any of claims 8-9, for use in the treatment of diseases caused by E. coli .