WO2023101639A1

WO2023101639A1 - Antimicrobial agent for lysis of s. aureus

Info

Publication number: WO2023101639A1
Application number: PCT/TR2022/051108
Authority: WO
Inventors: Tulin OZBEK; Serap DERMAN; Murat TOPUZOGULLARI; Hande HANCER; Senanur DOKUZ; Berna ERDOGDU
Original assignee: Yildiz Teknik Universitesi; Yildiz Teknoloji Gelistirme Bolgesi Teknopark As
Priority date: 2021-11-30
Filing date: 2022-10-10
Publication date: 2023-06-08

Abstract

The present application proposes an antimicrobial formulation that includes one or more of the peptide sequences that are submitted as SEQ ID NO 6, SEQ ID NO 7 ve SEQ ID NO 8 as antimicrobial agent.

Description

ANTIMICROBIAL AGENT FOR LYSIS OF S. AUREUS

Technical field

The present invention relates to an antimicrobial biomaterial including recombinant protein and/or peptide units. In particular, the present invention relates to an antimicrobial biomaterial for lysis of Staphylococcus aureus.

Background of the Invention

Treatment of infections with antibiotics started by Alexander Fleming in 1928 by using penicillin, and a golden era took place until 1940's. Yet, antibiotics resistance against penicillin and methicillin is followed by multidrug resistant, extensively drug resistant and pan drug resistant bacterial strains, indicating the emergence of a "post antibiotic era" in which the infections will not be treatable (Hauser, A.R., Mecsas, J., and Moir, D.T. 2016. "Beyond antibiotics: New therapeutic approaches for bacterial infections", Clinical Infectious Diseases. 63 (1); doi: 10.1093/cid/ciw200). S. aureus infections that cause acute diseases such as bacteraemia and skin diseases, and biofilm-related numerous chronic diseases (Raafat D, Otto M, Reppschlager K, Iqbal J, Holtfreter S. Fighting Staphylococcus aureus Biofilms with Monoclonal Antibodies. Trends Microbiol. 2019 Apr;27(4):303-322. doi: 10.1016/j.tim.2018.12.009), are considered amongst most widespread causes of ever-increasing hospital- acquired infections. 2020 UHESA (National Hospital Infections Surveillance Network) report of Turkish Public Health Institution states that, in the distribution of antimicrobial resistant pathogens in bacteria that cause nosocomial infections, the second rank is occupied by methicillin resistant coagulase negative staphylococcuses (66.51%) and the seventh rank is occupied by methicillin resistant S. aureus (MRSA) (39.23%) (Hekimoglu CH, Batir E, Gbzel EY, Altun D., "Ulusal Saglik Hizmet ili§kili Enfeksiyonlar Surveyans Agi (USHiESA) Ozet Raporu 2020", (2020)). In the light of these information, it is clear that the staphylococcus-related nosocomial infections are extremely important and that related treatment actions shall be successfully conducted.

Nowadays, it is observed that infections are intensively treated by using antibiotics and antibiotic cocktails. Apart from that, considering several factors such as the disease type, prognosis, patient's age and present chronic diseases and treatment costs, the following approaches are also commenced to find commercial implementation: use of antibodies, use of probiotics with antibiotics and/or alternatives thereof, use of bacteriophage lysins with antibiotics and/or alternatives thereof, direct use of wild type bacteriophage cocktails, application of antibiotic treatment along with immune stimulation means such as phenyl butyrate, vitamin D and oral bacterial extracts, production of vaccines directed to novel targets, and application of increased efficiency nano-antibiotics (Czaplewski L, Bax R, Clokie M, Dawson M, Fairhead H, Fischetti VA, Foster S, Gilmore BF, Hancock RE, Harper D, Henderson I R, Hilpert K, Jones BV, Kadioglu A, Knowles D, Olafsdottir S, Payne D, Projan S, Shaunak S, Silverman J, Thomas CM, Trust TJ, Warn P, Rex JH. Alternatives to antibiotics-a pipeline portfolio review. Lancet Infect Dis. 2016 Feb;16(2):239-51. doi: 10.1016/51473-3099(15)00466-1. Epub 2016 Jan 13. PMID: 26795692; doi: 10.1016/51473-3099(15)00466-1) (Hauser, A.R., Mecsas, J., and Moir, D.T. 2016. "Beyond antibiotics: New therapeutic approaches for bacterial infections", Clinical Infectious Diseases. 63 (1); doi: 10.1093/cid/ciw200) (Hussain, 5., Joo, J., Kang, J. et al. Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy. Nat Biomed Eng 2, 95-103 (2018); doi: 10.1038/s41551-017-0187-) (Altamirano F. L. G., and Barr J., 2019, "Phage Therapy in the Postantibiotic Era", ASM Journals, Clinical Microbiology Reviews, Vol. 32, No. 2; doi: 10.1128/CMR.00066-18).

Brief description of the invention

Object of the present invention is to overcome the problems discussed in the current state of the art discussed above. In particular, the present invention aims to propose antimicrobial agents that effect lysis of resistant S. aureus strains that cause nosocomial infections, and to propose disinfectants as well as drugs that contain said agents.

The present invention proposes antimicrobial agents obtained by starting from Bacteriophage K, for use in prevention or treatment of infections related to resistant S. aureus. Within this context, trans membrane domain (TMD) (SEQ ID NO 2) on Holin protein (SEQ ID NO 1) of Bacteriophage K is produced using recombinant protein technology, and proposed as an antimicrobial agent for opening pores on the membrane of S. aureus. Further within the present context, as an antimicrobial agent, the present invention proposes three different peptide sequences (SEQ I D NO 3, SEQ ID NO 4 and SEQ ID NO 5) and their combinations that include, along with the TMD of said Holin protein, peptide sequences extending towards the C-terminus. Further within the present context, as an antimicrobial agent, the present invention proposes three different peptide sequences (SEQ I D NO 6, SEQ ID NO 7 and SEQ I D NO 8) and their combinations that include, along with the TMD of said Holin protein, peptide sequences extending towards the C-terminus. Further within the present context, as an antimicrobial agent, the present invention proposes further sequences (SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11) that are obtainable using recombinant protein technology with the above-mentioned TM D by peptide synthesis, and their conjugation-related combinations.

Detailed description of the invention

Holin proteins are known as bacteriophage-coded (in particular, DNA carrying) bacterial cytoplasmic membrane perforating small hydrophobic trans membrane proteins, that play an important role in determination of timing of host cell lysis towards the end of phage infection cycle. Holins that are synthesized at a late stage of infection, harmlessly accumulate in the inner membrane in the form of homodimers; and upon reaching a critical concentration, pore formation is triggered, thereby effecting a decline in proton movement force in cell membrane, thereby controlling the access of endolysins to the cell wall; thus, they are considered as the simplest biological clock.

Although Holins have a low genomic sequence similarity; they have a partly similar secondary structure property due to the distribution of polar, loaded and hydrophobic residues. Holins contain a small N-terminus, followed by 1 to 3 of hydrophobic trans membrane domains (TM D) that are connected to each other via a loop region, and a hydrophilic, positive loaded C-terminus. The Holin gene that is focussed on in the present application can also be named as Bacteriophage K Holin protein or abbreviated as HolFajK, is the Holin gene of Bacteriophage K that is a lytic virulent phage that infects S. aureus bacteria, which is a pathogen that is the cause of numerous diseases in humans and animals, and that develops multiple resistance against antibiotic treatments. Sequence listing of HolFajK is presented with SEQ ID NO 1.

Amongst holins that are classified in accordance with the number of TM Ds, HolFajK is in the group of Class II holins, and unlike Class I holins, it causes lysis of bacteria even in the absence of the enzyme endolysin. This is because HolFajK, as in the case of HoIGpll protein of D29 (Class II) phage that infects Mycobacterium tuberculosis and Mycobacterium smegmatis, causes cellular lysis of E. coll and also of M. smegmatis, even when it is expressed to a low extent, in the absence of endolysins. Hence TM D (SEQ ID NO 2) domain of HolFajK is expected to be effective as an antimicrobial agent. Furthermore, it is envisaged that amino acid sequences of rHolFajK-1, rHolFajK-2 and rHolFajK-3 (SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5, respectively) have synergistic effect, that are obtainable by dividing the polypeptide domain that extends towards the carboxy terminus outside the TMD of HolFajK, and then attaching these consecutively to the TMD.

The present application proposes HolFajK and features/variations thereof, as antimicrobial agent. Within this context, said HolFajK is proposed as antimicrobial agent that is produced in parts (SEQ ID NO 2, SEQ I D NO 3, SEQ ID NO 4 and SEQ ID NO 5), or as a whole (SEQ ID NO 5). Further within the same context, peptide sequences (SEQ ID NO 6, SEQ ID NO 7 ve SEQ ID NO 8) that are synthesized by being selected from the chain that extends towards the C-terminus of HolFajK, are proposed as antimicrobial agents. Further within the same context, sequences (SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11) that are obtainable by conjugation of the peptide sequences (pl, p2, p3) with the HolFajK TMD that is produced via recombinant protein technology, are proposed as antimicrobial agents.

The present invention proposes antimicrobial agents and production methods thereof, based on the lytic effect of Holin protein in the Staphylococcus virus K (which can be also named as bacteriophage K or phage K) on various S. aureus strains. Sequence listing of Holin protein is submitted as the sequence I D number 1.

Within the present context, sequence listing of the trans membrane domain (shortly, TMD) of said Holin protein is submitted as the sequence ID number 2. Said TM D that corresponds to a part (24-73 aa.) of the Holin protein can be produced using recombinant technology, and can be also named as rHolFajK-TMD.

Further within the present context, a first combined sequence that includes a first peptide sequence that is positioned on the C-terminus of the Holin protein along with the TMD, can be produced using recombinant technology. Said first combined sequence (a first antimicrobial agent) is abbreviated as rHolFajK-pl, and is submitted as the sequence ID number 3.

Further within the present context, a second combined sequence that includes along with the TMD, the first peptide sequence and a second peptide sequence that are on the C-terminus of the Holin protein, can be obtained using recombinant technology. Said second combined sequence (a second antimicrobial agent) is shortly referred to as rHolFajK-pl-p2, and submitted as the sequence ID number 4.

Further within the present context, a third combined sequence that includes along with the TMD, the first peptide sequence, the second peptide sequence and a third peptide sequence that are on the C-terminus of the Holin protein, can be obtained using recombinant technology. Said third combined sequence (a third antimicrobial agent) is shortly referred to as rHolFajK-pl-p2-p3, and submitted as the sequence I D number 5.

Within the scope of the present application, the above-mentioned first peptide sequence (shortly referred to as pl, submitted as the sequence ID number 6) is synthesized and proposed as a fourth antimicrobial agent for antimicrobial use.

Likewise, within the scope of the present application, the above-mentioned second peptide sequence (shortly referred to as p2, submitted as the sequence ID number 7) is synthesized and proposed as a fifth antimicrobial agent for antimicrobial use.

Likewise, within the scope of the present application, said third peptide sequence (shortly referred to as p3, submitted as the sequence ID number 8) is synthesized and proposed as a sixth antimicrobial agent for antimicrobial use.

Further within the scope of the present application, for being proposed as a seventh antimicrobial agent, the recombinant TMD that is submitted with the sequence I D number 2 is conjugated with the synthesized first peptide sequence, and thus a first conjugated sequence is obtained. Said first conjugated sequence is shortly referred to as rHolFajK-pl, and a version thereof that does not include cysteine residue is submitted as the sequence I D number 9. Depending to the selected production technique, for instance in the case where maleimide-thiol (cysteine) addition reaction is used, said first conjugated sequence can also include a cysteine residue.

Likewise, further within the scope of the present application, for being proposed as an eighth antimicrobial agent, the recombinant TMD that is submitted with the sequence ID number 2 is conjugated with the synthesized second peptide sequence that is submitted as the sequence I D number 7, and thus a second conjugated sequence is obtained. Said second conjugated sequence is shortly referred to as rHolFajK-p2, and a version thereof that does not include cysteine residue is submitted as the sequence I D number 10. Depending to the selected production technique, for instance in the case where maleimide-thiol (cysteine) addition reaction is used, said second conjugated sequence can also include a cysteine residue.

Likewise, further within the scope of the present application, for being proposed as a nineth antimicrobial agent, the recombinant TMD that is submitted with the sequence ID number 2 is conjugated with the synthesized third peptide sequence that is submitted as the sequence ID number 8, and thus a third conjugated sequence is obtained. Said third conjugated sequence is shortly referred to as rHolFajK-p3, and a version thereof that does not include cysteine residue is submitted as the sequence ID number 11. Depending to the selected production technique, for instance in the case where maleimide-thiol (cysteine) addition reaction is used, said third conjugated sequence can also include a cysteine residue.

Further within the scope of the present application, one or more further antimicrobial agents are proposed that are envisaged to show synergistic effect and that are obtained by combining one or more of the above-mentioned first to nineth antimicrobial agents. The inherent ability of bacteriophages to specify and infect bacterial hosts renders them ideal antimicrobial candidates for pathogen-specific treatment in food (decontamination of foods), agriculture (bio preservative), biotechnology (designing carrier systems, vaccines and drugs) and medicine (infection treatment, biological diagnosis, drug targeting, disinfectants).

Holin proteins that are synthesized at a late stage of infection caused in the bacteria by the phage, harmlessly accumulate at the inner membrane in the form of homodimers; and upon reaching a critical concentration a triggering takes place to pore formation, and causes cellular lysis by achieving a decrease in the force of proton movement in the cell membrane.

Resistant S. aureus strains are prominent in distribution of nosocomial infection resistant pathogens distribution in Turkiye. The present invention proposes an efficient and unique treatment agent based on phage-related lysis with antimicrobial peptide approach, that can be effective in treatment of diseases caused by said strains. The antimicrobial agent that is proposed within the scope of the present invention can be in the form of protein and/or peptide.

Holin proteins are classified based on their trans membrane domain (TMD) counts, and holins that are in the Class II holins group cause direct bacterial lysis even in the absence of the endolysin enzyme. Accordingly, the present invention proposes the SEQ ID NO 5 that is obtainable the recombinant path, and that corresponds to Holin protein (SEQ ID NO 1, shortly referred to as HolFajK) of Staphylococcus virus K which is a lytic virulent that infects S. aureus which is a pathogen that causes various diseases in human and animals and that develops multiple resistance against antibiotics treatment. Hence, within the context of the present application, an antimicrobial agent or a treatment agent is proposed that contains the Holin protein (SEQ ID NO 5 that is identical or equivalent to SEQ ID NO 1) that has the aforementioned potential effect. The context of the present application also encompasses the production of the Holin protein and its domain that shows an effective antimicrobial property (a part thereof that includes one or more of peptide sequences pl, p2 and p3, and that is positioned in the C-terminus of the TMD), with recombinant DNA technology and chemical synthesis; and it is possible to identify most advantageous versions or combinations that show for instance, synergistic effect, by evaluating the activities of the Holin proteins that are obtainable in various structures and sizes.

TMD domain of the Holin protein of Bacteriophage K can be produced with recombinant method, and the amino acid sequences that are in the form of three different parts (units) positioned in the C-terminus that constitutes the basis of the antimicrobial activity, can be obtained in the form of peptide sequences via peptide synthesis. Said three parts of peptide sequences in the C-terminus are hereby named as pl, p2 and p3, respectively. Hence, a group of antimicrobial substances can be obtained for use in lysis of S. aureus.

The peptide sequences (pl, p2 and p3) that are to be synthesized can be evaluated either protein dependent by being individually conjugated with rTMD (as rTMD-pl; rTM D-p2; rTMD-p3), or protein independent [as peptide sequences (pl-p2-p3)], in view of their potentials as antimicrobial agents. Hence, the most effective formulation and peptide sequence can be specified in terms of antimicrobial activity. By respectively attaching the genomic sequences of said peptide sequences (pl, p2, p3) to the gene sequence of rTMD, it is possible to produce 3 different recombinant proteins. Recombinant protein formulations of respective polypeptides can be thus obtained, and that can be respectively referred to as rHolFajK-pl, rHolFajK-pl-p2 and rHolFajK-pl-p2-p3. As a result, possible synergistic effects of said peptides with each other can be determined by consecutively bonding of peptides using recombinant protein production technology, instead of synthetic method.

As an example: i) TMD domain (SEQ ID NO 2) of Holin protein of Bacteriophage K can be obtained with recombinant technique; II) the amino acid sequence outside the Bacteriophage K TM D, that extends towards the carboxy terminal (24-73 aa.) can be divided into three, and structures can be obtained that include one or more of pl, p2 and p3 bonded to the TMD, using recombinant technique; thereby obtaining one or more of the SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5 (these sequences can be respectively referred to as rHolFajK-pl, rHolFajK-pl-p2 and rHolFajK-pl-p2-p3); ill) one or more of the peptide sequences that are submitted as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 and that are also respectively referred to as pl, p2 and p3 can be synthesized; one or more amongst these can be conjugated to SEQ ID NO 2, thereby obtaining one or more of the sequences that are respectively submitted as SEQ ID NO 9, SEQ I D NO 10 ve SEQ ID NO 11; iv) antimicrobial formulations directed to S. aureus lysis can be thus enabled, that include one or more of the products obtained at the steps i-iii above.

The product according to the present invention can be employed in pharmaceutics as a raw material in preparing drug compositions for use in oral application, or as a final product in drug compositions for use in spray application. The antimicrobial substances that are disclosed within the context of the present application, or their combinations are suitable for use in high-capacity production of industrial product in factories that serve in medical and pharmaceutical fields.

The mode of administration for the antimicrobial biomaterials according to the present invention can be considered in accordance with antibiotics (e.g.; vancomycin, oxacillin): IV - intravenous, IM - intramuscular, oral administration, and topical administration as e.g., spray or ointment. For example, on Staphylococcus aureus related scars that occur on the body in the case of diabetes or traumatization, bacterial cell membrane can be depolymerized by administration of spray or ointment, or by injection for deep penetration; thereby bacteria can be lysed and thus the infection can be treated by rapid eradication of the bacteria. In other words, antimicrobial disinfectant formulations in the form of spray, antimicrobial drug formulations for intravenous administration, antimicrobial drug formulations for intramuscular administration, antimicrobial drug formulations for oral administration and antimicrobial drug formulations for topical administration that include one or more of the proposed antimicrobial substances are also considered within the scope of the present application.

Accordingly, the present application proposes an antimicrobial formulation comprising as antimicrobial agent one or more selected from those submitted as the sequence listing number 6 (SEQ ID NO 6), sequence listing number 7 (SEQ ID NO 7) and sequence listing number 8 (SEQ ID NO 8), respectively. These peptide sequences are hereby referred to as pl, p2 and p3, respectively.

In the antimicrobial formulation according to the present invention, pl and p2 can be included in combination, and thereby a synergistic effect can be obtained. Hence, a preferred embodiment of the formulation according to the present invention includes the first peptide sequence and the second peptide sequence in combination.

In the antimicrobial formulation according to the present invention, pl and p3 can be included in combination, and thereby a synergistic effect can be obtained. Hence, a preferred embodiment of the formulation according to the present invention includes the first peptide sequence and the third peptide sequence in combination.

In the antimicrobial formulation according to the present invention, p2 and p3 can be included in combination, and thereby a synergistic effect can be obtained. Hence, a preferred embodiment of the formulation according to the present invention includes the second peptide sequence and the third peptide sequence in combination.

In the antimicrobial formulation according to the present invention, pl, p2 and p3 can be included in combination, and thereby a synergistic effect can be obtained. Hence, a preferred embodiment of the formulation according to the present invention includes the first peptide sequence, the second peptide sequence and the third peptide sequence in combination.

The present application further proposes an antimicrobial formulation comprising one or more amino acid sequences selected from sequence listing number 3 (SEQ ID NO 3), sequence listing number 4 (SEQ ID NO 4), sequence listing number 5 (SEQ ID NO 5), sequence listing number 9 (SEQ ID NO 9), sequence listing number 10 (SEQ ID NO 10) and sequence listing number 11 (SEQ ID NO 11), respectively. These amino acid sequences are hereby referred to as rHolFajK-pl, rHolFajK-pl-p2, rHolFajK-pl-p2-p3, rTMD-pl, rTMD-p2 and rTM D-p3, respectively; and it is observable that each of these include at least one of the sequences that are referred to pl, p2 and p3.

Each of the above-mentioned rHolFajK-pl, rHolFajK-pl-p2 and rHolFajK-pl-p2-p3 can be produced using recombinant technology starting from the Staphylococcus virus K Holin protein that is submitted with the sequence listing number 1 (SEQ ID NO 1). On the other hand, each of the rTM D-pl, rTMD-p2 and rTMD-p3 can be produced by respectively conjugating pl, p2 and p3 with the (recombinant-) TMD that is submitted with the sequence listing number 2 (SEQ ID NO 2) and that can be produced using recombinant technology starting from the Staphylococcus virus K Holin protein that is submitted with the sequence listing number 1 (SEQ ID NO 1). Each of the pl, p2 and p3 that are respectively submitted as sequence listing number 6 (SEQ ID NO 6), sequence listing number 7 (SEQ ID NO 7) and sequence listing number 8 (SEQ ID NO 8) are synthetically obtainable (e.g., using solid phase peptide synthesis).

Synergistic combinations:

The antimicrobial formulation according to the present invention can include rTMD-pl and/or rHolFajK-pl in combination with rTMD-p2, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 3 and/or SEQ ID NO 9 in combination with SEQ ID NO 10.

The antimicrobial formulation according to the present invention can include rTMD-pl and/or rHolFajK-pl in combination with rTMD-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 3 and/or SEQ ID NO 9 in combination with SEQ ID NO 11.

The antimicrobial formulation according to the present invention can include rTMD-p2 in combination with rTM D- p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 10 in combination with SEQ I D NO 11.

The antimicrobial formulation according to the present invention can include rTMD-pl and/or rHolFajK-pl in combination with rTMD-p2 and rTM D-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 3 and/or SEQ ID NO 9 in combination with SEQ I D NO 10 and SEQ ID NO 11.

The antimicrobial formulation according to the present invention can include rTMD-pl and/or rHolFajK-pl in combination with TM D-pl-p2, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 3 and/or SEQ ID NO 9 in combination with SEQ ID NO 4.

The antimicrobial formulation according to the present invention can include rTMD-pl and/or rHolFajK-pl in combination with rHolFajK-pl-p2-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 3 and/or SEQ ID NO 9 in combination with SEQ I D NO 5.

The antimicrobial formulation according to the present invention can include rTMD-pl and/or rHolFajK-pl in combination with rHolFajK-pl-p2 and rHolFajK-pl-p2-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 3 and/or SEQ I D NO 9 in combination with SEQ ID NO 4 and SEQ ID NO 5.

The antimicrobial formulation according to the present invention can include rTMD-p2 in combination with rHolFajK-pl-p2, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 10 in combination with SEQ ID NO 4.

The antimicrobial formulation according to the present invention can include rTMD-p2 in combination with rHolFajK-pl-p2-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 10 in combination with SEQ ID NO 5.

The antimicrobial formulation according to the present invention can include the combination of rTMD-p2, rHolFajK-pl-p2 and rHolFajK-pl-p2-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes the combination of SEQ ID NO 10, SEQ ID NO 4 and SEQ I D NO 5. The antimicrobial formulation according to the present invention can include rTMD-p3 in combination with rHolFajK-pl-p2, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 11 in combination with SEQ ID NO 4.

The antimicrobial formulation according to the present invention can include rTMD-p3 in combination with rHolFajK-pl-p2-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes SEQ ID NO 11 in combination with SEQ ID NO 5.

The antimicrobial formulation according to the present invention can include the combination of rTMD-p3, rHolFajK-pl-p2 and rHolFajK-pl-p2-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes the combination of SEQ ID NO 11, SEQ ID NO 4 and SEQ ID NO 5.

The antimicrobial formulation according to the present invention can include rHolFajK-pl-p2 in combination with rHolFajK-pl-p2-p3, and thereby show a synergistic effect. Accordingly, a preferred embodiment of the formulation according to the present invention includes the combination of SEQ ID NO 4 and SEQ I D NO 5.

EXAMPLES

The examples presented herein merely aim exemplification of various embodiments within the scope of the present invention and their industrial applicability; without setting a basis for limiting the intended scope of protection by the present application. Example 1 is directed to application in terms of terminological and implementational aspects; whereas the subsequent examples are presented for better understanding of various embodiments according to the present invention, their combinations and uses.

Example 1: Industrial applicability

Example 1 includes presentation related to production and industrial applicability within the context of the present invention. The brand names and other names that are hereby referred to are non-binding and merely presented for exemplification of the commercial availability of the related products and services.

Work Package 1. Recombinant production of HolFajK and various embodiments thereof

Objective 1.1. Production of 5. aureus and growing the same in culture medium

S. aureus that includes Bacteriophage K for being used in the exemplary work is within the prior art knowledge and is available, e.g., in culture collections for the related profession groups. Double Agar Overlay technique can be used in growing of said phage in culture medium, and in storing of the same (Obuchowski, M. and Stopa, M. 1993. "Quick and efficient method for recovering of bacteriophages from soft agar after their propagation by the plate lysate technique.", Acta Biochimica Polonica. 40 (1); PMID: 8372579) (Kropinski, A.M., Mazzocco, A., Waddell, T.E., Lingohr, E., and Johnson, R.P. 2009. "Enumeration of bacteriophages by double agar overlay plaque assay.", Methods in Molecular Biology (Clifton, N.J.). 501; doi: 10.1007/978-l-60327-164-6_7) (Lungren, M.P., Christensen, D., Kankotia, R., Falk, L, Paxton, B.E., and Kim, C.Y. 2013. "Bacteriophage K for reduction of Staphylococcus aureus biofilm on central venous catheter material", Bacteriophage. 3 (4); doi: 10.4161/bact.26825). Briefly, bottom and top agar can be prepared by addition of 3 mM MgCh ve 4 mM CaCh, respectively. A serial dilution can be prepared by adding phage lysate (10⁶ — IO¹¹ PFU titer) into a tube that includes Brain Heart Infusion fluid growth medium in which S. aureus is grown. Top agar at 46-48 °C can be introduced into these dilution tubes, and bottom agar can be rapidly spread onto the plate, and incubated overnight at 37 °C. At the end of the incubation, the plates that show lysis can be selected and SM buffer (5 M NaCI, 1 M MgSCU, 1 M Tris-HCI [pH 7.5) 2% (v/v) chloroform can be added thereonto, and then incubated at 37 °C for 4 hours. High titer phage solution can be then taken from the plate for centrifuging. Upon passing a filter of 0.22 pm, 50% (v/v) can be added and then stored at -80 °C.

Objective 2. Production of gene domain of Bacteriophage K Holin protein

1.2a. Bacteriophage K DNA isolation

Direct kit procedure can be applied using Norgen phage DNA isolation kit (CAT No: 46800), (User's manual: Norgen 2010. "Phage DNA Isolation Kit", (905), 3-4). Briefly, phage lysate (> 1 x 108 pfu/m) can be incubater at 75 °C for 5 min., and upon ceasing the DNase activity, Lysis Buffer B can be added and vortex can be applied for e.g., 10 seconds. Lysate can be incubated at e.g., 65 °C for e.g., 15 minutes; and then isopropanol can be added and vortex can be applied, followed by spin column step. After washing steps, elution solution can be added and then centrifuge can be applied, and the DNA samples can be then stored at -20 °C.

1.2b. Amplification of Holin gene

In silico analyses related to Bacteriophage K Holin protein gene sequence specify the ORF domain of the gene; >YP_009041295.1 holin [Staphylococcus virus K], as 504 bp (NCBI). For the amplification product of the related Holin gene; 20 pl FajK DNA (200 pg/mL), 10 x PCR buffer, 1,5 mM MgCh, 1.0 mM dNTPs, 200 nm forward and reverse primers, 10% glycerol and U% Taq polymerase enzyme; 100 pL total reaction volume can be used. PCR conditions can be implemented by using High-Fidelity DNA polymerase enzyme and buffer, and by determination of annealing temperature for each of the primers (obtained from Thermo Scientific company).

The amplified product can be run on agarose gel electrophoresis and observed, and then recovered in gel using DNA extraction kit ("Zymo Zymoclean_Gel_DNA_Recovery_Kit_protocol" valid as of the year 2021). The amount and purity of DNA can be controlled using Nanodrop spectrophotometer (Thermo- at ratio of A260/280 and within the range between 1.8-2.00), then can be taken into stock at -20 °C.

Objective 1.3. Transfer of recombinant Holin genes of different sizes to BL21 (DE3) pLysS E. co// host, and expression thereof

1.3a. Transfer of recombinant Holin genes of different sizes to expression vectors

In the related work, commercially available pLATE 51 expression vector can be used for cloning and expression of Holin genes (Cloning and expression kit is commercially available as of November 2021, e.g., from Thermo Scientific company under the product name aLICator LIC cloning and expression kit and under the product code #K1251). At transferring in accordance with the kit that is used, the ratio of PCR product to vector can be 0.5:1 or 2:1. 0.5 or 2 pL of fresh PCR product, 1 pL of saline solution, 1 pL of vector, 5 pL of sterile water, thus 6 pL can be incubated at room temperature, for instance for 5 minutes. Afterwards, this mixture can be added onto competent BL21 (DE3) pLysS E. coli cells that are kept in ice. Said cells that are kept in ice for 10 to 30 min can be subjected to instant heat shock at 42 °C for 30 sec, and right afterwards placed into ice. 250 pL of SOC medium can be added into the respective tubes and then incubation can be applied for 1 hour at 37 °C.

Then the output can be spread onto LB agar media that include ampicillin. Respective Petri dishes can be incubated overnight at 37 °C, and then 5-6 of the colonies on Petri dish surfaces can be taken for being subjected to plasmid purification. After transformation, the colonies that are grown in ampicillin selective growth medium, can be verified with colony PCR and sequence analyses.

1.3b. Expression of recombinant Holin genes of different sizes on BL21(DE3) pLysS E. coli host;

Expression can be performed from colonies that are verified in the previous step. After incubation for two hours in ampicillin LB medium at 37 °C, and when the OD reaches to the level of 0.5-0.8 in the spectrophotometer, the culture can be divided to two different tubes. Expression can be induced by addition of 0.2 mM of isopropyl thiogalactoside (IPTG) into one of the tubes.

500 pl of aliquot can be taken from each culture and transferred into centrifuge tube, then can be centrifuged at high speed at 30 sec. Both of the supernatants and pellets can be frozen at -20 °C. These are samples obtained at the zero point. The tubes can be then incubated in a shaker stove at 37 °C for 24 hours. 500 pl aliquots can be taken from cultures at 15-30-60^th minutes and at 2^nd-4^th-8^th-16^th-24^th hours, transferred into centrifuge tubes and subjected to high-speed centrifuge, thereby separately obtaining supernatants and pellets, that can be then stored at -20 °C. The collected supernatants and pellets can be loaded to SDS-PAGE, and it can be determined whether the specified protein sizes are reached, by mixing with a master mix that includes proteins with specified sizes.

The accuracy of recombinant Holin proteins that are produced, can be then checked using western blot method.

Objective 1.4. Purification of recombinant Holin proteins of different sizes;

1.4a.

Ni-NTA Purification System can be used in purification of recombinant Holin proteins of different sizes. The recombinant protein that includes "His-tag", solution for separation from other proteins that belong to the bacterium, can be at first mixed with Ni-NTA agarose resin. Afterwards, by washing the resin with a suitable amount of washing solution, a great majority of bacterial proteins can be removed from the solution. Recombinant proteins that are bonded to the resin can be removed from the resin using elution buffer with 400 mM of imidazole (pH=8). Then, the elution buffer can be controlled in terms of size, by loading to SDS-PAGE; and also controlled with primary antibodies that bond to "his-tag"s, using western blot method.

For increasing the extent of purity and for removing "His-tag", TEV enzyme can be added into the solution. This enzyme bonds to the 6 amino acid domain that is disposed in-between the "His-tag" and the recombinant protein, and can remove the "His-tag" from the protein by cutting the carboxyl group of the glutamine amino acid at an end thereof. Then, imidazole can be removed from the solution by dialysis. The solution can be mixed again with Ni-NTA resin (that is commercially obtainable, e.g., as of November 2021, from Thermo Scientific company, under the name HisPur™ Ni-NTA Resin and with the reference number 88222), and the His-Tag and TEV enzyme remain bonded to the resin, and the recombinant protein remains at the solution side; thereby the solution is separated from His-tag and TEV enzyme.

1.4b. HPLC purification;

The determination of impurity of the protein that is to be produced with recombinant method, can be performed with HPLC device using C8 column, in accordance with the principle of reverse phase chromatography. In the case where the impurity ratio of the protein that is subjected to characterisation, is below 80%; a pre-purification is to be applied using a C18 column, and then the purification can be applied with a semi-preparative HPLC device in which a C8 column is used (Zhao, Q., Sun, L., Liang, Y., Wu, Q., Yuan, H., Liang, Z., et al. 2012. "Prefractionation and separation by C8 stationary phase: Effective strategies for integral membrane proteins analysis", Taianta. 88; doi: 10.1016/j. taianta.2011.11.035). Water that contains formic acid, and acetonitrile can be used as mobile phase.

1.4c. Endotoxin assay of pure proteins;

For determination of presence of toxins in the purified proteins, LAL (lumilus amebocyte lysate) test can be used, which is also known as bacterial endotoxin assay. This assay is an in vitro analysis that is used for determination of the presence and concentration of bacterial endotoxins in drugs and biological products. The process is conducted in accordance with LAL assay protocol, and at a last stage thereof, absorbance measurements can be made at 545 nm, to calculate the endotoxin values of test samples with regard to a calibration curve that is prepared based on endotoxin standard which is subjected to serial dilution (Karl D Brune, Darren B Leneghan, Iona J Brian, Andrew S Ishizuka, Martin F Bachmann, Simon J Draper , Sumi Biswas , Mark Howarth, 2016. "Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization", Sci Rep.l9;6:19234. doi: 10.1038/srepl9234).

Work Package 2. Synthesis and characterization of HolFajK based peptides

Peptide sequences pl (DYLKPRDSKDPNEFVQWQANANNTST), p2 (FEI DSYENNAEPDTDDSDEVPAIEDEI) and p3 (DGGSAPSQDEEDTEEHGKVFAEEEVK) that are obtained by dividing the polypeptide sequence that extends towards the C-terminus outside the TMD sequence into three, can be synthesized with microwave assisted solid phase peptide synthesis method using CEM Liberty device (Arayici, P.P., Acar, T., Maharramov, A.M., Karahan, M., and Mustafaeva, Z.A. 2017. "SYNTHESIS AND CHARACTERIZATION OF BIOCONJUGATES OF ANTIGENIC RABIES VIRUS PEPTIDE EPITOPE WITH POLYACRYLIC ACI D", Fresenius Environmental Bulletin. 26 (4)) (Acar, T., Pelit Arayici, P., Ucar, B., Karahan, M., and Mustafaeva, Z. 2019. "Synthesis, Characterization and Lipophilicity Study of Brucella abortus' Immunogenic Peptide Sequence That Can Be Used in the Future Vaccination Studies", International Journal of Peptide Research and Therapeutics. 25 (3); doi: 10.1007/sl0989-018-9739-0) (Arayici, P.P., Acar, T., Ucar, B., Karahan, M., Arslan, B.A., and Mustafaeva, Z. 2019. "Rabies Virus 31D Peptide-[P(VP-co-AA)] Conjugates: Synthesis, Characterization and Cytotoxicity Evaluation", ChemistrySelect. 4 (32); doi: 10.1002/slct.201901375) (Ucar, B., Acar, T., Pelit Arayici, P., Mustafaeva, Z., and Demirkol, M.O. 2018. "[68Ga]Ga-antiCADl: Radiosynthesis and first imaging study on rats", Kafkas Universitesi Veteriner Fakultesi Dergisi. 24 (4); doi: 10.9775/kvfd.2018.19487) (Derman, S., Kizilbey, K., Mansuroglu, B., and Mustafaeva, Z. 2014. "Synthesis and characterization of Canine parvovirus (CPV) VP2 W-7L-20 synthetic peptide for synthetic vaccine", Fresenius Environmental Bulletin) (Ucar, B., Acar, T., Pelit-Arayici, P., Demirkol, M. O., Akdeste, Z.M. 2017. "A NEW RADIO- THERANOSTIC AGENT CANDIDATE: SYNTHESIS AND ANALYSIS OF (ADH-1) c-EDTA CONJUGATE", Fresenius Environmental Bulletin. 26 (2), 4751-4758); by applying maleimide modification (maleimide-peptide-COOH) at the N terminal ends thereof (Marchan, V., Ortega, S., Pulido, D., Pedroso, E., and Grandas, A. 2006. "Diels-Alder cycloadditions in water for the straightforward preparation of peptide-Oligonucleotide conjugates", Nucleic Acids Research. 34 (3); doi: 10.1093/nar/gnj020) (Santoso, B., Lam, S., Murray, B.W., and Chen, G. 2013. "A simple and efficient maleimide-based approach for peptide extension with a cysteine-containing peptide phage library", Bioorganic and Medicinal Chemistry Letters. 23 (20); doi: 10.1016/j.bmcl.2013.08.032) . The steps to be followed in peptide synthesis (Acar, T., Pelit Arayici, P., Ucar, B., Karahan, M., and Mustafaeva, Z. 2019. "Synthesis, Characterization and Lipophilicity Study of Brucella abortus' Immunogenic Peptide Sequence That Can Be Used in the Future Vaccination Studies", International Journal of Peptide Research and Therapeutics. 25 (3); doi: 10.1007/S10989-018-9739-0) (Ucar, B., Acar, T., Pelit-Arayici, P., Demirkol, M. O., Akdeste, Z.M. 2017. "A NEW RADIO-THERANOSTIC AGENT CANDIDATE: SYNTHESIS AND ANALYSIS OF (ADH-1) c-EDTA CONJUGATE", Fresenius Environmental Bulletin. 26 (2), 4751-4758) (Ozdemir, Z.O., Karahan, M., Karabulut, E., and Mustafaeva, Z. 2010. "Characterization of foot-and-month disease virus's viral peptides with LC-ESI-MS", Journal of the Chemical Society of Pakistan. 32 (4)) are disclosed in detail within the scope of the Work Package 2.

2.2a. Examination of the peptide sequences to be synthesized, and preparation of the device program accordingly:

Amino acid, solution and other substances' amounts that are required for synthesis of pl, p2 and p3 peptide sequences can be calculated using the device by entering the desired amounts and scales.

2.2b. Preparation of the resin to be used:

In synthesizing of each of the peptides (pl, p2, p3), a respective pre-loaded resin can be used, in which the initial amino acid that is positioned on the carboxyl terminus of the respective peptide is pre-loaded to the resin. The resin that bears the suitable amino acid (that is, respective resins that are loaded with Threonin (Thr, T) a. a. for pl, Isoleucin (He, I) a. a for p2, and Lysin (Lys, K) aa.) are kept for at least 3 hours in DMF/DCM for swelling in a shaker, thereby enabling the exposure of active ends on the resin that is a polymeric particle.

2.2c. Preparation of activator, deprotection solution, etc.:

HOBt.H2O + HCTU can be used as activator, DIEA + NMP can be used as activator base, piperidine + DM F can be used as deprotection solution (for removing the protection), 6-maleimidohexanoic acid can be used for N terminal end modification.

2.2d. Initiation of the synthesis, tracking of the operations:

The synthesis can be initiated after checking the device. The highest temperature values that are reached during where the device performs the peptide synthesis operation, can be tracked particularly upon the bonding of the first amino acid. The temperature profile can be checked when the synthesis of the respective sequence is complete.

2.2e. Separation of the synthesized peptide sequence from the respective resin:

The synthesized peptide sequence can be separated from the respective resin using cleavage cocktail [TFA/EDT/Thioanisol/H2O (90/2,5/2,5/5) v/v]. Side chain protection groups of commercially obtained amino acids can also be removed during this operation. In the cleavage cocktail, TFA is the active substance, and the rest of the substances are used for suppressing (scavenger) the ions that are formed under the effect of TFA. For the cleaving operation, peptide that is bonded to resin is slightly shaken in the cocktail for three hours, and at the end of this period it can be ensured that the peptide separates from the resin and transfers into the TFA cocktail. The cocktail is then filtered, and the filtrate can be evaporated in an evaporator. The solution that is in a gelled form can be transferred into a centrifuge tube. Cold (-20 °C) diethyl ether can be added into the solution, settled as white powder by centrifugation, and then washed using cold ether. The raw peptide can be dried under vacuum and stored at -40 °C (Guides that are used: Millipore 2014. "Novabiochem® Fmoc resin cleavage protocols", Nova Biochem Darmstadt, Almanya.; Norgen 2010. "Phage DNA Isolation Kit", (905), 3-4).

2.2f. Determination of molecular weight of the raw peptide using LC-ESI-MS:

Molecular weight of the peptide can be determined using LC-MS system that includes electrospray ionisation (ESI) probe and that performs separation using reverse phase chromatography (RP-HPLC). Electrospray Ionisation Mass Spectrometry (ESI-MS) can be used for mass specification. In accordance with the solubility of the raw peptide, a peptide solution of 1 mg/mL can be prepared in a solvent such as water, acetonitrile, ethanol and methanol, in which the peptide has the most favourable solubility. The peptide can be characterised using molecular weight determination of the peptide, in reverse-phase column, with the specified mobile phase (usually, water- acetonitrile gradient).

In this work, for the synthetic peptide characterisation; an LC-MS system (Shimadzu LC-MS 2010 EV) with electrospray ionisation (ESI) can be used that includes Teknokroma Tracer Exel 120 ODS-A 5 pm HPLC column with a length of 20 cm and with an inlet diameterl of 2.1 mm. The prepared peptide samples can be injected into the chromatographic system. The mobile phase A (water, 0.1% (h/h) formic acid) and mobile phase B (acetonitrile, 0.1% (h/h) formic acid) can be applied as gradient with a flow rate of 0.2 mL/min. The following elution program can be applied (and if necessary, the gradient conditions can be altered): 0-5 min, 20% B; 5-15 min, 20-40% B; 15- 30 min, 40-80% B; 30-35 min, 80-20% B. The ESI can be run in positive ion mode within the range of 200-2000 m/z. The capillary temperature can be kept at 250°C. The flowrate of the nebulisation gas (N2) can be 1.5 L/min.

2.2g. Production of the intended sequence in a pure state using preparative HPLC:

Each peak fraction can be separately collected using the preparative HPLC device with the gradient that provides the most favourable separation. The pure peptide can be obtained in the form of powder by evaporating an amount of the organic phase in the solution using a rotary evaporator and then by drying using a lyophilisator. As an external alternative, MALDI-TOF-MS analysis can also be applied for determination of net molecular weight of the peptides that are obtained in a pure state.

Purification of the synthesized peptide can be performed using preparative reverse phase high performance liquid chromatography (RP-HPLC, SPD-M20A, FRC-10A, LC- 8A, CBM-20A, Shimadzu, Tokyo, Japan). In the device that is equipped with Shim-pack PRC-ODS HPLC column (20 mm x 25 cm) and UV-PDA detector, gradient elution can be performed with mobile phase A (water, 0.1% (h/h) formic acid) and mobile phase B (acetonitril, 0.1% (h/h) formic acid). The gradient elution can be programmed as follows: 0-5 min, 20% B; 5-15 min, 20-40% B; 15-30 min, 40- 60% B; 30-35 min, 60-20% B with flowrate of 14 mL/min. In the case where the separation of peptide peaks is at an insufficient extent, the peptide peaks can be obtained by applying a different gradient.

Work Package 3. Conjugation of rTMD and synthesized peptide sequences and characterisation

For the conjugation, a protocol can be followed that is within the literature (Hutchins, B.M., Kazane, S.A., Staflin, K., Forsyth, J.S., Felding-Habermann, B., Smider, V. V., et al. 2011. "Selective formation of covalent protein heterodimers with an unnatural amino acid", Chemistry and Biology. 18 (3); doi: 10.1016/j.chembiol.2011.01.006). Briefly, possible disulphide bonds that can occur in the rTM D protein can be reduced using tris(2-carboxyethyl) phosphine (immobilised on Agarose CL-4B) (TCEP resin). Thus, thiol groups of cysteine amino acid at the C- terminus can be exposed for maleimide conjugation. To this end, a 100 pM solution of rTMD can be prepared in saline phosphate buffer (PBS pH 7.4).

Afterwards, a solution that includes TCEP at a ratio that corresponds to 2 folds of the protein can be added and smoothly stirred at 4 °C for 2 hours. Then, this solution can be added with a solution that includes 300 pM of pl peptide in PBS, and the conjugation reaction can be continued for 3 hours, thereby the rTMD-pl conjugate can be formed (this operation can also be performed for p2 and p3 peptides, to obtain rTMD-p2 and rTM D-p3, respectively). After the conjugation, the non-reacted free polypeptides can be separated using a membrane centrifuge tube (MWCO 10 kDa), thereby the conjugate can be purified.

The resulting conjugates can be characterised using SDS-PAGE electrophoresis and MALDI-TOF mass spectrophotometer. In SDS-PAGE electrophoresis, rTMD (~9 kDa), pl (~3 kDa), p2 (~3 kDa), p3 (~3 kDa) and conjugates (~12 kDa) can be run together, and the relative amount of the conjugate at desired molecular weight zone (~12 kDa) can be determined using ImageJ software. The non-bonded peptides (pl, p2 or p3) can be separated from the solution in which the conjugation is performed, and the amounts of free peptides (pl, p2 or p3) can be determined using HPLC (C8 column), thus the amounts of bonded peptides (pl, p2 or p3) can be determined. Furthermore, molecular weights of the conjugates can be determined using MALDI-TOF mass spectrophotometer.

Work Package 4. In vitro antibacterial analysis of Holin-based recombinant protein formulations and peptide sequences/formulations that are synthesized

In vitro antibacterial activities of synthesized recombinant proteins/peptides/rTMD-peptide formulations can be evaluated on S. aureus (ATCC- 25923), S. aureus (ATCC29213-oxacilin sensitive), MSSA (ATCC BAA-2419- methicillin sensitive), M RSA (ATCC43300-methicillin and oxacillin resistant), h-VISA (ATCC®-700698 vancomycin semisensitive), VISA (S. aureus, Strain HIP07256). These strains are within the prior art knowledge, and can be obtained from persons or institutions that are active in the professional field related to the present application. The methods to be used in determination of antibacterial activity are specified based on CLSI/2018 standard. The samples to be used in determination of antibacterial activity are: rTMD, rHolfajK-1, rHolfajK-2, rHolfajK-3, pl, p2, p3, rTMD-pl, rTMD-p2, and rTMD-p3. Control groups to be used in determination of antibacterial activity are: vancomycin antibiotic is positive control, PBS and bacterial study culture alone are negative controls.

Detailed explanation related to the Broth Microdilution Method to be used in the study is available in literature (Arasoglu, T., Derman, S., and Mansuroglu, B. 2016. "Comparative evaluation of antibacterial activity of caffeic acid phenethyl ester and PLGA nanoparticle formulation by different methods", Nanotechnology. T1 (2); doi: 10.1088/0957-4484/27/2/025103) (Arasoglu, T., Mansuroglu, B., Derman, S., Gumus, B., Kocyigit, B., Acar, T., et al. 2016. "Enhancement of Antifungal Activity of Juglone (5-Hydroxy-l,4-naphthoquinone) Using a Poly(d, I -lactic- co-glycolic acid) (PLGA) Nanoparticle System", Journal of Agricultural and Food Chemistry; doi: 10.1021/acs.jafc.6b03309 ) (Arasoglu, T., Derman, S., Mansuroglu, B., Uzunoglu, D., Kopyigit, B., Gumu§, B., et al. 2017. "Preparation, characterization, and enhanced antimicrobial activity: Quercetin-loaded PLGA nanoparticles against foodborne pathogens", Turkish Journal of Biology; doi: 10.3906/biy-1604-80) (Bruno, M., Modica, A., Catinella, G., Canli, C., Arasoglu, T., and elik, S. 2019. "Chemical composition of the essential oils of Centaurea tomentella Hand.-Mazz. and C. haussknechtii Boiss. (Asteraceae) collected wild in Turkey and their activity on microorganisms affecting historical art craft", Natural Product Research. 33 (8); doi:

10.1080/14786419.2018.1463531). The MIC values are to be specified in pg/ml, and the response of strains to the samples can be evaluated in accordance with EUCAST/2019 standard, as susceptible, intermediate and resistant.

Work Package 5. Evaluation of in vitro biocompatibilities of synthesized Holin-based recombinant protein formulations and peptide sequences

The test samples to be evaluated in the present work package, are the samples of which the antibacterial activities are determined, in other words, M IC values are specified in the Work Package 4; the samples that do not show activity can be disregarded. In vitro biocompatibility test can be performed using human fibroblast cell line (e.g., commercially available ATCC BJ CRL-2522™). The cells can be incubated in tissue culture vials that include TPMI 1640 supported with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 pg/mL streptomycin) under standard conditions (37°C, 5% CO2). The cell growth can be achieved at a logarithmic phase by addition of fresh growth medium once every two days during incubation. The cell viability can be evaluated prior to cytotoxicity test, using trypan blue, arranged within the range between 93-95% (Sktadanowski, M., Golinska, P., Rudnicka, K., Dahm, H., and Rai, M. 2016. "Evaluation of cytotoxicity, immune compatibility and antibacterial activity of biogenic silver nanoparticles", Medical Microbiology and Immunology. 205 (6); doi: 10.1007/s00430-016-0477-7). For evaluation of cytotoxicity of the formulations at the cell viability analysis, fibroblast cells at a density of 2 x 10⁵/mL (100 uL) can be distributed to 96-well plates and test samples with increasing concentrations (that can be specified regarding MIC values to be obtained) can be added thereonto. The cells on which no test sample is added, form positive control for viability; whereas the cells that are treated with 2% saponin can be used as negative control for cell lysis. The plates can be incubated for 24 hours under standard conditions (37°C, 5% CO2). After the incubation, the cell viability can be evaluated using MTT (3- (4,5- dimethylthioazolyl) -2,5-diphenyltetrazolium bromide) colorimetric technique. This technique relies on the principle of conversion of water soluble MTT compound into an insoluble formazan crystal. Live cells that have an active metabolism converts MTT into formazan, yet dead cells have lost this ability. Therefore, the colour formation defines only the presence of live cells. The measured absorbance (590 nm) is proportional to the count of live cells. Optical density (OD 950 nm) is determined using a spectrophotometer, and then the I C50 values can be calculated using GraphPad which is a statistics software (Sktadanowski, M., Golinska, P., Rudnicka, K., Dahm, H., and Rai, M. 2016. "Evaluation of cytotoxicity, immune compatibility and antibacterial activity of biogenic silver nanoparticles", Medical Microbiology and Immunology. 205 (6); doi: 10.1007/s00430-016-0477-7).

Work Package 6. Evaluation of in vivo activity of the synthesized Holin-based antibacterial agent on incision scar model

A scar model can be formed with regard to the test sample amongst the synthesized Holin-based recombinant protein/peptide sequences/formulations, that is found most effective in in vitro antibacterial and biocompatibility tests (that has the lowest MIC value, and the highest IC50 value), and the strains on which that is effective. In the case where the MIC value of the formulation obtained when selecting the treatment doses, is higher than the respective IC50 value, the experiments can be performed based on the M IC value.

Scar contraction analysis within the scope of in vivo experimental studies can be performed using molecular analyses and histopathological evaluations.

Example 2: Production of pl, p2 and p3

Three different parts of the polypeptide chain / amino acid sequence that causes membrane depolymerisation on S. aureus and that extends towards the carboxy terminal outside the SEQ ID NO 2 which corresponds to Bacteriophage K TM D (24-73 aa.), can be, for instance, synthesized using a microwave assisted solid phase peptide synthesis device. The amino acid sequences of the three different parts synthesized herein are submitted as SEQ ID NO 6, SEQ I D NO 7 and SEQ ID NO 8; and herein referred to as pl, p2 and p3, respectively.

Each of the above-mentioned antimicrobial substances that are referred to as pl, p2 and p3, can be produced and characterised using a microwave assisted solid phase peptide synthesis device, starting from SEQ ID NO 6, SEQ ID NO 7 and SEQ I D NO 8, respectively.

Example 3: Production of rTM D

The TM D that is submitted with SEQ ID NO 2 can be obtained using recombinant DNA technology, starting from Staphylococcus virus K. The obtained product can be referred to as recombinant trans membrane domain, or shortly, as rTMD. The rTMD includes the first 73 amino acids of the Holin protein, submitted as SEQ ID NO 1; and can be obtained by, for instance, addition of cysteine amino acid to the C-terminus. Said rTMD deploys to the bacterial membrane of S. aureus as an anchor, thereby effecting pore formation.

Example 4: Partial Holin production by chemical method The amino acid sequences that are shortly referred to as rTMD-pl, rTMD-p2 ve rTMD-p3 are obtained for being used as antibacterial agents, by conjugation of the respective peptide sequences pl, p2 and p3 that are disclosed in Example 2 and that are produced using solid phase peptide synthesis, with rTMD, the production of which by recombinant technique is disclosed in Example 3. Antimicrobial activity of said antibacterial agents is achieved by microbial lysis that is exerted by the sequences corresponding to pl, p2 and p3 upon pore formation on S. aureus membrane on which the rTMD part is anchored.

Within this context, pl (SEQ ID NO 6), p2 (SEQ I D NO 7) ve p3 (SEQ I D NO 8) that extend towards C-terminus of the Holin protein and that can be synthetically produced in solid phase synthesis device, can be conjugated to the rTMD that is disclosed in Example 2, for instance, via maleimide-thiol (cysteine) conjugation. The respective sequences include cysteine residue in the case where the maleimide-thiol (cysteine) conjugation method is selected; on the other hand, the sequences would not include a cysteine residue in the case where alternative techniques are employed. Within this context:

Regarding SEQ ID NO 9: for instance, in the case where pl is synthesized using maleimide synthesis, the respective sequence can include a cysteine residue as 89^th amino acid (in-between Thr and Asp at the related domain in the present respective list), and thus the length of the sequence listing can be 115 instead of 114.

Regarding SEQ ID NO 10: for instance, in the case where synthetic p2 is conjugated to the recombinant TMD using maleimide synthesis, the respective sequence can include a cysteine residue as 89^th amino acid (in-between Thr and Phe at the related domain in the present respective list), and thus the length of the sequence listing can be 116 instead of 115.

Regarding SEQ ID NO 11: for instance, in the case where synthetic p3 is conjugated to the recombinant TMD using maleimide synthesis, the respective sequence can include a cysteine residue as 89^th amino acid (in-between Thr and Asp at the related domain in the present respective list), and thus the length of the sequence listing can be 115 instead of 114.

In the case where a method is selected that does not cause cysteine residue, the conjugations result in the formation of the antimicrobial agents that are referred to as rTMD-pl, rTMD-p2 and rTMD-p3, that are respectively submitted as SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11. Antimicrobial activities thereof or of their versions that contain cysteine residue due to being obtained using the maleimide conjugation technique, can be evaluated on S. aureus strains that are known to be resistant.

In other words:

The antimicrobial substance (SEQ ID NO 9) that is referred to as rTMD-pl can be obtained by conjugation of the rTMD (SEQ ID NO 2) that is explained in Example 3, with pl (SEQ ID NO 6) that is explained in Example 2. Likewise, the antimicrobial substance (SEQ ID NO 10) that is referred to as rTMD-p2 can be obtained by conjugation of the rTMD (SEQ I D NO 2) that is explained in Example 3, with p2 (SEQ ID NO 7) that is explained in Example 2. Further likewise, the antimicrobial substance (SEQ ID NO 11) that is referred to as rTMD-p3 can be obtained by conjugation of the rTM D (SEQ ID NO 2) that is explained in Example 3, with p3 (SEQ I D NO 8) that is explained in Example 2. In said conjugation operation, for instance, maleimide-thiol (cysteine) addition method can be employed. In such case it is expected that the resulting sequence listing includes a cysteine residue. Cysteine residue does not form in other techniques that are used in the related technical field, and the sequence listings that are submitted along with the present application are examples to this latter case. With the presumption of that the selected conjugation method does not have any effect on antimicrobial activity of the obtained products; the versions that to and do not have cysteine residue are considered equivalent to each other in terms of technical effect (function- way-result). In this direction it is possible to express that: each of SEQ ID NO 1 to SEQ ID NO 11 show the same technical effect with amino acids that show a respective similarity ratio of 90% or higher, in particular, with amino acids that show a respective similarity ratio of 95% or higher. It is thus envisaged that, for each of SEQ ID NO 1 to SEQ ID NO 11, the sequences with respective similarity ratios of 90% or higher and in particular, the sequences with respective similarity ratios of 95% or higher, are antimicrobial agents within the context of the present invention that lyse resistant S. aureus strains.

Some advantages of chemical peptide synthesis (over the recombinant protein synthesis that is disclosed in Example 5 below) can be listed as follows: it allows the use of D-amino acids that increase the biological activity and the proteolytic stability, it allows extended amino acid sequence modifications that are, in recombinant DNA technology, achieveable only by using alternative codons, provides accuracy in a-helix and |3-layer foldings that allow formation of secondary structures of proteins, unlike the incorrect foldings that are encountered in recombinant protein production.

Example 5: Partial or complete Holin production using recombinant DNA technology

Each of the antimicrobial substances that are submitted as SEQ ID NO 3, SEQ ID NO 4 ve SEQ ID NO 5 can be obtained using recombinant DNA technology starting from Staphylococcus virus K. Here, the SEQ ID NO 3 that is shortly referred to as rHolFajK-pl and the SEQ ID NO 4 that is shortly referred to as rHolFajK-pl-p2 can be considered as "partial Holin". The SEQ ID NO 5 that is shortly referred to as rHolFajK-pl-p2-p3 can be considered as complete Holin by being identical or equivalent to the SEQ ID NO 1.

With said rHolFajK-pl, rHolFajK-pl-p2 and rHolFajK-pl-p2-p3, upon anchoring of the TMD to the S. aureus membrane, the polypeptide chain that extends towards the carboxy terminus (that is, one or more of the -pl and/or -pl-p2 and/or pl/p2/p3 chains) achieves depolymerisation of the membrane.

Example 6: Evaluation of the antimicrobial agents according to the present invention

In an implementation of the present invention, regarding the lysis of antibiotics resistant S. aureus strains, the antimicrobial or lytic activities of the antimicrobial agents according to the present invention can be comparatively determined. The antibacterial activities of the antimicrobial agents according to the present invention or formulations thereof can be tested in vitro on resistant or sensitive S. aureus strains.

The antibacterial activities of the antimicrobial agents according to the present invention or formulations thereof can be tested in vivo on incision scar models. In this direction, incision scar models can be formed based on the skin lesions that depend on S. aureus, and then infected scar model can be formed by bacterial inoculation. The antibacterial activity of the formulation which is regarded as having the most effective MIC value can be administered three doses: under ICso, at ICso and over ICso; thereby performing topical formulation applications for 14 days. At the end of this period, the molecular parameters on resulting skin samples can be evaluated with qRT-PCR method. Said skin samples can be further subjected to histopathological analysis.

Biocompatibility of the antimicrobial agents according to the present invention or formulations thereof can be subjected to in vitro evaluation. Useability of said antimicrobial agents on living systems can be thus evaluated.

Notes regarding the free text format explanations in the sequence listings are as follows:

Regarding SEQ ID NO 1: the present sequence listing (SEQ I D NO 1) is publicly available, for instance from the address https://www.ncbi.nlm.nih.gov/gene/19622108 under GENE ID: 19622108 (accessed as of 25.11.2021).

Regarding SEQ ID NO 2: the present sequence listing (SEQ ID NO 2) corresponds to trans membrane domain of Holin gene, and is obtainable by implementation of recombinant protein technology to the Holin gene that is hereby defined as SEQ I D NO 1. Sequence information related to trans membrane domain is available over CCTOP CONSTRAINED CONSENSUS TOPOLOGY PREDICTION SERVER <http://cctop.enzim.ttk.mta.hu/?_=/jobs/submit> and over HMMTOP TRANS MEMBRANE TOPOLOGY PREDICTION SERVER http://www.enzim.hu/hmmtop/html/submit.html. The verification can be performed using HHPRED BIOINFORMATICS TOOLKIT over

<https://toolkit.tuebingen.mpg.de/jobs/3517122> (JOB ID: 3517122) and over

<https://toolkit.tuebingen.mpg.de/jobs/8785893> (JOB ID: 8785893). Each of the web links submitted in the present file are available as of 25 November 2021.

Regarding SEQ ID NO 3: the present sequence listing (SEQ ID NO 3) can be obtained by combining the trans membrane domain (that is herein defined with SEQ ID NO 2) with the first peptide sequence (that is herein defined with SEQ ID NO SEQ ID NO 6) via recombinant protein technology. Said two sequences are adjacently positioned in a row with each other on the Holin gene (that is herein defined with SEQ ID NO 1).

Regarding SEQ ID NO 4: the present sequence listing (SEQ ID NO 4) can be obtained by combining the trans membrane domain (that is herein defined with SEQ ID NO 2) with the first peptide sequence (that is herein defined with SEQ ID NO SEQ ID NO 6) and the second peptide sequence (that is herein defined with SEQ ID NO SEQ I D NO 7) via recombinant protein technology. Said three sequences are adjacently positioned in a row with each other on the Holin gene (that is herein defined with SEQ ID NO 1).

Regarding SEQ ID NO 5: the present sequence listing (SEQ I D NO 5) corresponds to the Holin gene (GENE ID: 19622108, YP_009041295.1 holin [Staphylococcus virus K]) that is herein defined with SEQ ID NO 1, as a whole. The present sequence can be obtained by implementing recombinant protein technology on Holin gene that is herein defined with SEQ ID NO 1, thereby identically resulting in the combination of the trans membrane domain (that is herein defined with SEQ ID NO SEQ ID NO 2), the first peptide sequence (that is herein defined with SEQ ID NO SEQ I D NO 6), the second peptide sequence (that is herein defined with SEQ ID NO SEQ I D NO 7) and the third peptide sequence (that is herein defined with SEQ ID NO SEQ ID NO 8). These four sequences are adjacently positioned in a row with each other on the Holin gene (that is herein defined with SEQ I D NO 1).

Regarding SEQ ID NO 6: the present sequence listing (SEQ ID NO 6) corresponds to a first peptide sequence that can be synthetically obtained using solid phase peptide synthesis.

Regarding SEQ ID NO 7: the present sequence listing (SEQ ID NO 7) corresponds to a second peptide sequence that can be synthetically obtained using solid phase peptide synthesis.

Regarding SEQ ID NO 8: the present sequence listing (SEQ ID NO 8) corresponds to a third peptide sequence that can be synthetically obtained using solid phase peptide synthesis.

Regarding SEQ ID NO 9: the present sequence listing (SEQ ID NO 9) corresponds to the product of conjugation a synthetic first peptide sequence that is obtainable with solid phase peptide synthesis and that is herein defined with SEQ ID NO 6, with the Holin gene trans membrane domain that is obtainable with recombinant protein technology. The present sequence further corresponds to the sequence that is herein referred to as SEQ ID NO 3. If the first peptide sequence and the trans membrane domain that is obtainable using recombinant protein technology, are bonded with maleimide conjugation method, cysteine amino acid residue is added to the trans membrane domain as 89^th amino acid.

Regarding SEQ ID NO 10: the present sequence listing (SEQ ID NO 10) corresponds to the product of conjugation a synthetic second peptide sequence that is obtainable with solid phase peptide synthesis and that is herein defined with SEQ I D NO 7, with the Holin gene trans membrane domain that is obtainable with recombinant protein technology. If the second peptide sequence and the trans membrane domain that is obtainable using recombinant protein technology, are bonded with maleimide conjugation method, cysteine amino acid residue is added to the trans membrane domain as 89^th amino acid.

Regarding SEQ ID NO 11: the present sequence listing (SEQ ID NO 11) corresponds to the product of conjugation a synthetic third peptide sequence that is obtainable with solid phase peptide synthesis and that is herein defined with SEQ ID NO 8, with the Holin gene trans membrane domain that is obtainable with recombinant protein technology. If the third peptide sequence and the trans membrane domain that is obtainable using recombinant protein technology, are bonded with maleimide conjugation method, cysteine amino acid residue is added to the trans membrane domain as 89^th amino acid

Claims

- 22 - WO 2023/101639 PCT/TR2022/051108 Claims

1. An antimicrobial formulation for lysis of S. aureus, comprising one or more of a first peptide sequence, a second peptide sequence and a third peptide sequence that are respectively submitted as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, as antimicrobial agent.

2. Formulation according to claim 1, comprising both of the sequences that are submitted as SEQ ID NO 6 and SEQ ID NO 7.

3. Formulation according to claim 1, comprising both of the sequences that are submitted as SEQ ID NO 6 and SEQ ID NO 8.

4. Formulation according to claim 1, comprising both of the sequences that are submitted as SEQ ID NO 7 and SEQ ID NO 8.

5. Formulation according to claim 1, comprising all of the sequences that are submitted as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.

6. Formulation according to any of claims 1 to 5, wherein one or more of the sequences that are submitted as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 is bonded together with a trans membrane domain sequence that is submitted as SEQ ID NO 2.

7. Formulation according to any of claims 1 to 5, comprising one or more of the sequences that are submitted as SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11.

8. Formulation according to claim 7, comprising SEQ ID NO 5.

9. Formulation according to any of claims 1 to 8, comprising SEQ ID NO 3 and/or SEQ ID NO 9 together with the sequence submitted as SEQ ID NO 10.

10. Formulation according to any of claims 1 to 9, comprising SEQ ID NO 3 and/or SEQ ID NO 9 together with the sequence submitted as SEQ ID NO 11.

11. Formulation according to any of claims 1 to 10, comprising SEQ ID NO 3 and/or SEQ ID NO 9 together with the sequences submitted as SEQ ID NO 10 and SEQ ID NO 11.

12. Formulation according to any of claims 1 to 10, comprising SEQ ID NO 3 and/or SEQ ID NO 9 together with the sequence submitted as SEQ ID NO 4.

13. Formulation according to any of claims 1 to 12, comprising SEQ ID NO 3 and/or SEQ ID NO 9 together with the sequence submitted as SEQ ID NO 5.

14. Formulation according to any of claims 1 to 13, comprising the sequences submitted as SEQ ID NO 10 and SEQ ID NO 4.

15. Formulation according to any of claims 1 to 14, comprising the sequences submitted as SEQ ID NO 10 and SEQ ID NO 5.

16. Formulation according to any of claims 1 to 15, comprising the sequences submitted as SEQ ID NO 10, SEQ ID NO 4 and SEQ ID NO 5.

17. Formulation according to any of claims 1 to 16, comprising the sequences submitted as SEQ ID NO 11 and SEQ ID NO 4.

18. Formulation according to any of claims 1 to 17, comprising the sequences submitted as SEQ ID NO 11 and SEQ ID NO 5.

19. Formulation according to any of claims 1 to 18, comprising the sequences submitted as SEQ ID NO 11, SEQ ID NO 4 and SEQ ID NO 5.

20. Antimicrobial disinfectant formulation according to any of claims 1 to 19, in the form of spray.

21. Antimicrobial drug formulation according to any of claims 1 to 19, for intravenous administration.

22. Antimicrobial drug formulation according to any of claims 1 to 19, for intramuscular administration.

23. Antimicrobial drug formulation according to any of claims 1 to 19, for oral administration.

24. Antimicrobial drug formulation according to any of claims 1 to 19, for topical administration.