Engineered polypeptide injection system and application thereof in targeted delivery of antibacterial peptide

Abstract

The invention discloses an engineered polypeptide injection system and application thereof in targeted delivery of antibacterial peptides, and belongs to the technical field of biology. The engineered polypeptide injection system provided by the invention can accurately deliver the target polypeptide to a targeted infection site, reduce whole body exposure, reduce toxicity and give full play to the effect of the target polypeptide. The invention also provides application of the engineered polypeptide injection system in preparation of a product for eliminating mycobacteria in target cells and preparation of a product for preventing and / or treating lung diseases and / or symptoms caused by the mycobacteria, and target polypeptides Granulysin, HNP1, and LysA and / or LysB derived from mycobacteriophage D29 or MS6 are delivered to macrophages, so that the lung diseases and / or symptoms caused by the mycobacteria are prevented and treated, and the lung diseases and / or symptoms caused by the mycobacteria are prevented and treated. The mycobacteria can be effectively removed.

Description

Technical Field

[0001] This invention relates to engineered peptide injection systems and their use in targeted delivery of antimicrobial peptides, specifically involving targeted delivery of antimicrobial peptides to macrophages via engineered eCIS to eliminate intracellular mycobacteria, belonging to the field of biotechnology. Background Technology

[0002] Mycobacterium tuberculosis (Mtb) is an intracellular bacterium and a major pathogen causing tuberculosis (TB). Currently, antibiotics are commonly used to treat TB, but the treatment cycle is long, patient compliance is poor, and with the increasing prevalence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), the treatment of TB faces greater challenges, urgently requiring the development of novel anti-tuberculosis drugs. In recent years, antimicrobial peptides (AMPs), as a novel antibacterial agent, have gradually become a hot topic in TB treatment research due to their broad-spectrum antibacterial activity, low drug resistance potential, and immunomodulatory functions. [1, 2] However, the application of AMPs is fraught with challenges. On the one hand, effective AMPs still need further exploration, and AMPs themselves have low stability and are easily hydrolyzed by proteases. The administration method is also a major issue, with low bioavailability for systemic, intravenous, and oral administration. Production costs, immunogenicity, cytotoxicity, and other potential adverse reactions also hinder their clinical application. To improve the stability and bioavailability of AMPs, the targeted delivery of antimicrobial peptides using nanocarriers such as polymers and liposomes has received increasing research and attention.

[0003] The Photorhabdus virulence cassette (PVC) of the non-symbiotic photorhabdus asymbiotica is a typical example of extracellular contractile injection systems (eCISs). [3] PVC exerts its killing effect by injecting bacterial virulence factors into target cells. This "syringe"-like system has now been modified into a highly efficient protein delivery tool, achieving effective delivery to human cells and the mouse brain. [4]Currently, there are modifications and applications that enhance the targeting specificity of PVC, but no reports have been found on its targeting specificity against macrophages, especially alveolar macrophages, and there are currently no studies evaluating the feasibility of using PVC as a novel delivery method for AMPs. Summary of the Invention

[0004] The problem the invention aims to solve

[0005] Antimicrobial peptides hold great potential in the treatment of tuberculosis, but their clinical application faces two major challenges: first, poor stability, making them prone to protein hydrolysis and degradation; and second, limited targeting efficiency, resulting in low bioavailability and potential systemic toxicity. [2, 10, 11] .

[0006] Currently, there is still much to be explored regarding AMPs that can eliminate intracellular mycobacteria, their effectiveness needs to be verified, and there is a lack of effective methods for delivering antimicrobial peptides into the cell. Furthermore, there are no studies on applying PVC to deliver AMPs, and no studies have investigated its effect on intracellular mycobacteria.

[0007] Therefore, this invention first identified AMPs that can effectively eliminate intracellular mycobacteria and developed an engineered PVC-AMP delivery system targeting macrophages. This system can deliver AMPs into macrophages to exert their effect of eliminating intracellular mycobacteria. Solving the aforementioned problems, PVC can load AMPs into its inner tube, allowing for injection into target cells after recognition. This not only protects the loaded AMPs from protease degradation and improves stability but also enhances the targeting of its action. Using the PVC complex to load peptides such as D29 LysA, D29 LysB, MS6 LysA, MS6 LysB, Granulysin, and HNP1 into its inner tube, it exhibits a killing effect on mycobacteria while showing no significant cytotoxicity to macrophages.

[0008] For in vivo applications, previous studies have shown that direct nebulization of endolysin leads to significant instability and impaired bioactivity.

[12] However, compared to intravenous and oral administration, nebulized inhalation can deliver the drug directly to the site of lung infection, thereby achieving higher drug concentrations in the lungs and fewer systemic side effects. [13, 14] Therefore, this invention employs pulmonary nebulization to simulate the natural infection process of mycobacteria. Direct nebulization is also used as the administration route for the PVC-AMP delivery system. Unexpectedly, this administration method preserved the anti-mycobacterial effects of the peptides, particularly D29 LysA and D29 LysB, in vivo, demonstrating that the PVC complex maintains its structural integrity and the stability of the load during nebulization, which is crucial for its clinical application.

[0009] Solution for solving the problem

[0010] The present invention provides the use of the polypeptide injection system in the following (i) and / or (ii):

[0011] (i) Prepare a product that eliminates intracellular mycobacteria in target cells;

[0012] (ii) To prepare products for the prevention and / or treatment of lung diseases and / or conditions caused by mycobacteria;

[0013] The polypeptide injection system comprises a PVC complex of non-symbiotic photobacteria and a polypeptide loaded in the PVC complex.

[0014] The polypeptide includes antimicrobial peptides, which include cell-derived Granulysin, HNP1, LysA derived from Mycobacterium phage D29, LysB derived from Mycobacterium phage D29, LysA derived from Mycobacterium phage MS6, and / or LysB derived from Mycobacterium phage MS6.

[0015] [2]. According to the use described in [1], wherein the PVC complex comprises structural proteins and regulatory factors;

[0016] The structural proteins include Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15 and Pvc16.

[0017] The regulatory factors include those derived from the LysR protein family.

[0018] [3]. According to the use described in [2], wherein the receptor recognition site of the Pvc13 is modified to other specific recognition sequences;

[0019] The other specific recognition sequences include the CD11b binding domain sequence and / or the RGD-PK7 dual-ligand domain (R7) sequence of the recombinant adenovirus serotype 5 (Ad5) vector;

[0020] Optionally, the CD11b binding domain sequence comprises the sequence shown in SEQ ID NO:16;

[0021] Optionally, the RGD-PK7 dual ligand domain (R7) sequence comprises the sequence shown in SEQ ID NO:15.

[0022] [4]. According to the use described in [3], wherein the receptor recognition site comprises amino acid residues 403 to 476 of the Pvc13 protein.

[0023] [5]. According to any one of [1] to [4], wherein the Granulysin, HNP1, LysA derived from mycobacterial phage D29, LysB derived from mycobacterial phage D29, LysA derived from mycobacterial phage MS6, and LysB derived from mycobacterial phage MS6 respectively contain the sequences shown in SEQ ID NO:13~14, 9~12.

[0024] [6]. The use according to any one of [1] to [5], wherein the polypeptide is loaded into the PVC complex by fusing it with a signal peptide at its N-terminus, the signal peptide comprising the N-terminal signal peptide of effector Pnf or Pdp1.

[0025] [7]. According to the use described in [6], wherein the N-terminal signal peptide of the Pnf comprises the sequence shown in SEQ ID NO:17, and / or,

[0026] The N-terminal signal peptide of Pdp1 contains the sequence shown in SEQ ID NO:18.

[0027] [8]. The use according to any one of [1] to [7], wherein the removal of intracellular mycobacteria from the target cells comprises reducing the number of mycobacteria in the target cells;

[0028] The target cells include macrophages.

[0029] [9]. The use according to any one of [1] to [8], wherein the lung diseases and / or conditions caused by mycobacteria include lung pathological damage and inflammation.

[0030]

[10] . According to any one of [1] to [9], wherein the mycobacteria include Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis attenuated strain H37Ra, Mycobacterium tuberculosis INH resistant strain H37Ra, and Mycobacterium tuberculosis standard strain H37Rv.

[0031]

[11] . Use according to any one of [1] to

[10] , wherein the product comprises a drug or a drug composition;

[0032] The drug or drug composition may also include one or more pharmaceutically acceptable carriers.

[0033]

[12] . According to the use described in

[11] , the administration of the drug or drug composition includes oral, transdermal, nebulized inhalation, intramuscular, subcutaneous and intravenous injection, preferably nebulized inhalation.

[0034]

[13] . A pharmaceutical composition comprising a polypeptide injection system as described in any one of [1] to

[12] , and,

[0035] Optional, pharmaceutically acceptable carrier.

[0036] The effects of the invention

[0037] This invention provides a novel drug delivery strategy for tuberculosis treatment. By modifying the receptor recognition site of the PVC complex, it can precisely target the site of infection, which is beneficial to achieve therapeutic effects while reducing systemic exposure and toxicity.

[0038] Meanwhile, this invention also utilizes delivery strategies to deliver peptides such as D29 LysA, D29 LysB, MS6 LysA, MS6 LysB, Granulysin, and HNP1 to the target site, all of which can maintain the stable performance of the peptides and play an effective antibacterial role. Attached Figure Description

[0039] Figure 1 To verify the effectiveness of PVC modification and delivery.

[0040] Figure 2 For AMP screening and validation.

[0041] Figure 3 To verify the delivery performance of the modified PVC-CD11b.

[0042] Figure 4 Validation of the anti-intracellular mycobacterial efficacy of D29 LysA and D29 LysB delivered to PVC-CD11b.

[0043] Figures 5-6 In vivo efficacy validation of D29 LysA and D29 LysB delivery to PVC-CD11b. Detailed Implementation

[0044] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0045] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0046] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0047] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0048] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0049] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0050] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0051] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0052] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0053] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0054] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, Joseph Frank et al. “Molecular Cloning: A Laboratory Manual.” (2001). (Hereinafter referred to as “Sambrook”). Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0055] The term "contractile injection system (CIS)" refers to a diverse but evolutionarily related family of macromolecular devices that utilize retractable sheaths to deliver nucleic acids and proteins. Contractile injection systems help microorganisms transport a variety of effector molecules extracellularly to gain a survival advantage (NMI Taylor, MJvan Raaij, and PG Leiman, Contractile injection systems of bacteriophages and related systems. Mol Microbiol, 2018. 108 (1): p. 6-15.). Typical CISs include the retractable tails of bacteriophages T4, P2, and Mu. Besides retractable phages, retractable injection systems similar to phage tails are also prevalent in bacteria and archaea. For example, the Type VI secretion system (T6SS) mediates intercellular communication and plays a role in cell defense (NMITaylor, MJ van Raaij, and PG Leiman, Contractile injection systems of bacteria and related systems. Mol Microbiol, 2018. 108 (1): p. 6-15.). Contractile injection systems also include extracellular contractile injection systems (eCIS).

[0056] The term "extracellular retractable injection system" or "eCIS" is a retractable injection system that can be released extracellularly and attack target cells from outside space. Extracellular contractile injection systems include bacterial tailocin / pyocin, and the *Photorhabdus virulence cassette* (PVC) found in the genus *Photorhabdus* (G. Yang et al., *Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth. J Bacteriol, 2006. 188(6): p.2254-61.), and the antifeeding prophage (Afp) (A. Desfosses, H. Venugopal, T. Joshi, J. Felix, M. Jessop, H. Jeong, J. Hyun, JB Heymann, MRH Hurst, I. Gutsche, and AK Mitra, *Atomic structures of an entire contractile injection system in both the extended and contracted states. Nature Microbiology, 2019. 4(11): p.). 1885-1894. ) and Metamorphosis-associatedcontractile structure (MAC) (NJ Shikuma et al., Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science, 2014. 343(6170): p. 529-33. ) et al.

[0057] Photorhabdus asymbiotica is a non-symbiotic bacterium belonging to the genus Photorhabdus. While bacteria in the genus Photorhabdus are typically considered insect pathogens, non-symbiotic photorhabdus can infect humans (P. Wilkinson et al., Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens. BMC Genomics, 2009. 10: p. 302).

[0058] The term "PVC" generally refers to a retractable injection system produced by *Photorhabdus asymbiotica*. In non-symbiotic *Photorhabdus asymbiotica*, such as *Photorhabdus asymbiotica* ATCC43949, the PVC is a protein complex exceeding 10 MDa, structurally similar to a simplified T4 phage tail. It comprises a hexagonal base-plate complex with six filaments and a 117 nm long sheath trunk with a cap structure. The sheath contains an inner tube in which effector proteins are loaded. The PVC can be released extracellularly by the bacteria to exert its effects. PVC is generally considered to be toxic to eukaryotic cells because it can transfer effector proteins into insect hemocytes and promote actin aggregation (G. Yang et al., Photorhabdus virulence cassettesconfer injectable insecticidal activity against the wax moth. J Bacteriol, 2006. 188 (6): p. 2254-61.). In the context of this invention, PVC refers to a non-symbiotic photobacterial bacterium (e.g., Photorhabdus asymbiotica ATCC43949) or a retractable injection system for host cells described herein that can penetrate human cell membranes to deliver polypeptides or proteins within the sheath of the PVC into the cytoplasm of human cells.

[0059] In the context of this invention, a "PVC gene cluster" refers to adjacent genes, including structural genes and downstream effector genes, that encode a complete PVC system within the genome of a non-symbiotic photobacterial bacterium (e.g., Photobacterium asymbiotica ATCC43949). The genome of the non-symbiotic photobacterial bacterium ATCC43949 contains five PVC gene clusters: PVC-I, PVC-II, PVC-III, PVC-IV, and PVC-V. Each PVC gene cluster contains one or more downstream genes that potentially encode effector factors.

[0060] A "signal peptide" generally refers to a peptide chain that guides the transfer of a synthesized polypeptide or protein toward a target. In the context of this invention, a "signal peptide" is a peptide chain capable of guiding the polypeptide to be delivered into the lumen of the sheath of a PVC complex.

[0061] In this invention, "effect factors" are bacterial secretory proteins produced by bacteria and transported into plant or animal cells via the secretion system, playing a role in recognition or pathogenicity. Structurally, effect factors can be divided into signaling regions and functional regions.

[0062] The term "effect factor downstream of the photobacterial virulence box gene cluster" refers to secreted proteins downstream of the photobacterial virulence box (PVC) gene cluster in the genome of *Photobacterium* that can be transported to target cells.

[0063] The LysR-type transcription regulator (LTTR) family derives its name from LysR, the regulatory activator of the lysine receptor lysA. They are highly conserved and ubiquitous in bacteria, with homologous proteins also found in archaea and eukaryotes. Through sequence similarity and DNA-binding domain conservation comparisons, at least nine functionally similar transcription regulators have been identified in bacteria such as *Escherichia coli*, *Salmonella enterica* serovar Typhimurium, *Rhizobium spp.*, and *Enterobacter cloacae*. Initially, LTTRs were described as transcription activators of single transcription genes. Subsequent studies have shown that they are a class of collective transcription regulators in bacteria, functioning as activators or repressors of transcription genes. LysR regulators play a regulatory role in the PVC gene cluster. LysR proteins are involved in cell invasion and adhesion in *P. asymbiotica*.

[0064] As used in this article, the term "peptide" refers to a molecule consisting of three or more amino acid molecules linked together by peptide bonds.

[0065] In this specification, the term "linker" can refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that connects two molecules or parts (e.g., two components of a protein complex), such as two domains of a fusion protein, like a signal peptide and an effector protein. A linker can be located between or on either side of two groups, molecules, or other parts and is connected to each of them via a covalent bond or non-covalent interaction, thereby connecting the two. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can be one or more amino acids (e.g., a peptide or a protein). In some embodiments, the length of the linker can be about 5 to 100 amino acids, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 amino acids. In some embodiments, the length of the linker can be about 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, or 450 to 500 amino acids. Longer or shorter linkers are also conceivable.

[0066] The term "antimicrobial peptide" refers to peptides with broad-spectrum antipathogenic activity that can rapidly kill pathogens. Antimicrobial peptides typically consist of more than 20 amino acid residues. Their targets include Gram-negative bacteria, Gram-positive bacteria, fungi, parasites, and tumor cells. Depending on their origin, antimicrobial peptides can include insect antimicrobial peptides, mammalian antimicrobial peptides, amphibian antimicrobial peptides, antimicrobial peptides derived from fish, mollusks, crustaceans, plants, and bacteria. These antimicrobial peptides can be defensins, Cecropin A and its analogues, Magainins, Melitiin, Cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemipteracin, bactenecin, Cecropin, etc.

[0067] As used herein, the term "treatment" refers to a clinical intervention, as described herein, aimed at reversing or alleviating a disease or condition or one or more symptoms thereof, delaying the onset of a disease or condition or one or more symptoms thereof, or inhibiting the progression of a disease or condition or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after the disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of the disease. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., given a history of symptoms and / or given genetic or other susceptibility factors). Treatment may also continue after the symptoms have subsided, for example, to prevent or delay their recurrence. As used herein, the term "prevention" means: before the onset of a disease, by exposing (e.g., administering) the subject to the polypeptide injection system, medicine, pharmaceutical composition, etc. of the present invention, thereby reducing the symptoms of the disease compared to when there is no exposure, without implying the necessity of completely suppressing the disease.

[0068] As used in this invention, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0069] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not exhibit excessive toxicity upon administration. In some embodiments, pharmaceutically acceptable carriers are those well known in the art, including at least one of solvents, solubilizers, co-solvents, emulsifiers, flavoring agents, odorants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants, and preservatives.

[0070] The technical solution of the present invention will be described in detail below:

[0071] This invention first selected LysA (i.e., D29 LysA) and LysB (i.e., D29LysB) derived from mycobacterial phage D29. Currently, one study has evaluated the activity of D29 LysB against intracellular mycobacteria in vitro. [9]However, there are no studies on its specific applications and effects, and no reports on the anti-intracellular mycobacterial activity of D29 LysA. Therefore, this invention attempts to use modified PVC complexes PVC-R7 and PVC-CD11b to load D29 LysA and D29LysB and deliver them to macrophages, evaluating their delivery antibacterial effects in in vitro cell infection experiments and in vivo mouse infection models. Similarly, this invention also selected LysA (i.e., MS6 LysA) and LysB (i.e., MS6 LysB) derived from mycobacterial bacteriophage MS6, and cell-derived Granulysin and HNP1 to verify the delivery effect of the delivery system.

[0072] Experimental results showed that D29 LysA, D29 LysB, MS6 LysA, MS6 LysB, Granulysin, and HNP1 could all be delivered to target cells by both delivery systems and effectively exerted their anti-mycobacterial effects within the target cells. Specifically, PVC-R7, in addition to its recognition of various human cells (such as A549 and 293T), also... [4] In addition, it can effectively recognize macrophages (including human THP-1-derived macrophages, mouse alveolar macrophage line MH-S, and mouse primary alveolar macrophages (AMs)), which have not been reported in existing technologies. Meanwhile, in vitro experiments showed that PVC-R7 loading and delivery of D29 LysA and D29 LysB to macrophages can exert antibacterial activity against mycobacteria (including Mycobacterium smegmatis (Ms), Mycobacterium tuberculosis attenuated strain H37Ra, and Mycobacterium bovis (BCG) strain). While PVC-CD11b cannot recognize A549 and HeLa cells, it exhibits excellent recognition and delivery to THP-1, MH-S, and AMs cells, demonstrating higher specificity compared to PVC-R7. In vitro cell experiments showed that D29 LysA and D29 LysB possess antibacterial activity against Ms, H37Ra, and even INH-resistant H37Ra in macrophages. In vivo experiments showed good antibacterial effects against the highly virulent strain of Mycobacterium tuberculosis H37Rv, reducing bacterial load in mouse lungs by approximately 18-fold and effectively alleviating lung inflammation in mice. These findings indicate that using PVC-CD11b to deliver D29 LysA and LysB is an effective therapeutic strategy against Mycobacterium tuberculosis infection, providing a novel treatment approach for precision tuberculosis management.

[0073] <Polypeptide Injection System>

[0074] Based on this, the present invention provides a polypeptide injection system comprising a PVC complex of non-symbiotic photobacteria and a polypeptide loaded in the PVC complex.

[0075] In some embodiments, the PVC complex comprises structural proteins and regulatory factors.

[0076] In some embodiments, the structural proteins include Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15, and Pvc16.

[0077] In some specific implementations, the genes encoding the structural protein are shown as PAU_03353 to PAU_03338.

[0078] In some implementations, the regulatory factors include those derived from the LysR protein family, encoded by the gene PAU_RS16560.

[0079] In this invention, LysA and LysB are endosomalins derived from mycobacterial phage D29 and / or MS6, and Granulysin and HNP1 are cell-derived short peptides. This invention has experimentally verified that they have the function of clearing mycobacteria. Therefore, in some specific embodiments of this invention, the polypeptides include antimicrobial peptides, and the antimicrobial peptides include LysA and / or LysB derived from mycobacterial phage D29.

[0080] In some embodiments, D29 LysA and D29 LysB respectively comprise sequences as shown in SEQ ID NO:9 and SEQ ID NO:10:

[0081] SEQ ID NO:9

[0082] TLIVTRDHAQWVHDMCRARAGNRYGYGGAFTLNPRDTTDCSGLVLQTAAWYGGRKDWIGNRYGSTESFRLDHKIVYDLGFRRLPPGGVAALGFTPVMLVGLQHGGGGRYSHTACTLMTMDIPG GPVKVSQRGVDWESRGEVNGVGVFLYDGARAWNDPLFHDFWYLDAKLEDGPTQSVDAAEILARATGLAYNRAVALLPAVRDGLIQADCTNPNRIAMWLAQIGHESDDFKATAEYASGDAYDTR TDLGNTPEVDGDGRLYKGRSWIMITGKDNYRDFSRWAHGRGLVPTPDYFVVHPLELSELRWAGIGAAWYWTVERPDINALSDRRDLETVTRRINGGLTNLDDRRRRYNLALAVGDQLLTLIGD DDELADPTIQRFIREIHGALFNTVVTQSPYGDPQNPDGSEPRSNLWQLHELIKNGDGMGHARYVEESARAGDLRELERVVRAAKGLGRDRSPEFIARARNVLAQIEAANPEYLQAYIARNGAL*

[0083] SEQ ID NO:10

[0084] SKPWLFTVHGTGQPDPLGPGLPADTARDVLDIYRWQPIGNYPAAAFPMWPSVEKGVAELILQIELKLDADPYADFAMAGYSQGAIVVGQVLKHHILPPTGRLHRFLHRLKKVIFWGNPMRQKGFAHS DEWIHPVAAPDTLGILEDRLENLEQYGFEVRDYAHDGDMYASIKEDDLHEYEVAIGRIVMKASGFIGGRDSVVAQLIELGQRPITEGIALAGAIIDALTFFARSRMGDKWPHLYNRYPAVEFLRQI*

[0085] In other embodiments, the antimicrobial peptide further comprises LysA and / or LysB from mycobacterial phage MS6, wherein MS6 LysA and MS6 LysB comprise sequences as shown in SEQ ID NO:11 and SEQ ID NO:12, respectively:

[0086] SEQ ID NO:11

[0087] TTKDQVAQITIAEAKARGYTRSECLAIMSTFYQESGWNDTIWDPTHTTYGIAQQDGSYPHRFDGAAAQIKGFFDKLDVWRAKPGASTDIWLNICWMQQAPNWPSADYWYANGRRAYLTEIKSRIATVTPYLDKYWPADGGTAVPDEPRPDFNEFPIWSNNNSARSGKPTMFLIHTQEGGGGDAAAENLAKWF Question

[0088] SEQ ID NO:12

[0089] RIDGQYVGLGPGDRSDEIRKIKAFMRRKFSYAATLADTEFYDEAMTAVVAEMQSRYNTAGQLRDGLYIPGIINAETKYVMGYLSRPVIDTRPVLFTVCGTGVPWWVGPDADTARAVEDQYLWQPIGYPAAPFPMGRSITAGITEAHNQANRWRERIETHGTALAGY SQGAVVLSELWMNHIAPEDGSLRWMKPHVRKAVTWGNPNRELGHVWADHGGSPMAPSNTQGVSSNGMRNTPDWWRDYAHQGDLYACTEPGDTQEVRNAIWQIVRDLDLFTGPDSLLAQVIELAQAPLPETIAITRAILDAGMFFAKRTGPHVDYNPQPAIDYLRT*

[0090] In other embodiments, the antimicrobial peptide further comprises granulin and human neutrophil defensin 1 (HNP1). In some embodiments, the granulin and HNP1 comprise sequences as shown in SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0091] SEQ ID NO:13

[0092] GRDYRTCLTIVQKLKKMVDKPTQRSVSNAATRVCRTGRSRWRDVCRNFMRRYQSRVTQGLVAGETAQQICEDLR*

[0093] SEQ ID NO:14

[0094] DCYCRIPACIAGERRYGTCIYQGRLWAFCC*

[0095] PVC can recognize cells through its tail fibrin Pvc13, but its recognition range is limited and it cannot recognize all types of cells. By modifying the receptor-binding domain of PVC, its specific recognition of different target cells can be achieved. In some embodiments of the present invention, the receptor recognition site of Pvc13 is modified to other specific recognition sequences to specifically recognize different target cells. Specifically, the receptor recognition site is positions 403-476 of the Pvc13 protein.

[0096] In some specific embodiments, other specific recognition sequences include the RGD-PK7 dual-ligand domain (R7) sequence of the recombinant adenovirus serotype 5 (Ad5) vector. In some embodiments, the RGD-PK7 dual-ligand domain sequence comprises the sequence shown in SEQ ID NO:15:

[0097] SEQ ID NO:15

[0098] NDKLTLWTTPAPSPNCRLNAEKDAKLTLVLTKCGSQILATVSVLAVKGSLAPISGTVQSAHLIIRFDENGVLLNNSFLDPEYWNFRNGDLTEGTAYTNAVGFMPNLS AYPKSHGKTAKSNIVSQVYLNGDKTKPVTLTITLNGTQETGDTTACDCRGDCFCGPSAYSMSFSWDWSGHNYINEIFATSSYTFSYIAQEGSGSGSGSGSKKKKKKK

[0099] In other specific embodiments, additional specific recognition sequences include CD11b binding domain sequences, which comprise the sequence shown in SEQ ID NO:16:

[0100] SEQ ID NO:16

[0101] DLGKKLLEAARAGQDDEVRILMANGADVNAIDFFGSTPLHLAAELGHLEIVEVLLKNGADVNADDILGDTPLHLAADFGHLEIVEVLLKNGADVNASDAFGLTPLHLAANAGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQ

[0102] In some alternative embodiments, the additional specific recognition sequence is connected to the receptor recognition site at both ends via adapters. In some alternative embodiments, the adapters include GGSGGGGSGG (SEQ ID NO:23).

[0103] In some embodiments, the polypeptide is loaded into the PVC complex using a signal peptide, the signal peptide comprising an N-terminal signal peptide of an effector factor Pnf or Pdp1, for example, 50 amino acids at the N-terminus.

[0104] In some alternative embodiments, the N-terminal signal peptide is fused to the N-terminus of the polypeptide.

[0105] In some exemplary embodiments, the N-terminal signal peptide (i.e., C50) of the Pnf comprises the sequence shown in SEQ ID NO:17.

[0106] SEQ ID NO:17

[0107] MLKYANPQTVATQRTKNTAKKPPSSTSFDGHLELSNGENQPYEGHKIRKI

[0108] In some exemplary embodiments, the N-terminal signal peptide (i.e., S50) of the Pdp1 comprises the sequence shown in SEQ ID NO:18.

[0109] SEQ ID NO:18

[0110] MPRYANYQINPKQNIKNSHGKSSSSDFSSGYLSFSNNSLDDPFIRQQVKR

[0111] <Composition, Use>

[0112] The present invention also provides for the use of the polypeptide injection system described above in the following (i) and / or (ii):

[0113] (i) Prepare a product that eliminates intracellular mycobacteria in target cells.

[0114] (ii) Prepare products for the prevention and / or treatment of lung diseases and / or conditions caused by mycobacteria.

[0115] In some implementations, the removal of intracellular mycobacteria from target cells includes reducing the number of mycobacteria within the target cells.

[0116] In some preferred embodiments, the target cells include macrophages. In some optional embodiments, the target cells include human THP-1-derived macrophages, mouse alveolar macrophage cell lines, and mouse primary alveolar macrophages.

[0117] In some implementations, the lung diseases and / or conditions caused by mycobacteria include pathological damage and inflammation of the lungs.

[0118] In this specification, pathological damage refers to changes such as cell or tissue atrophy, degeneration, and necrosis, as well as functional decline or loss, caused by various pathogenic factors exceeding the adaptive tolerance of cells or tissues.

[0119] In some exemplary embodiments, the lung inflammation includes increased TNF-α expression due to mycobacterial infection.

[0120] In some embodiments, the mycobacteria include Mycobacterium smegmatis, attenuated strain H37Ra of Mycobacterium tuberculosis, INH-resistant strain H37Ra of Mycobacterium tuberculosis, Mycobacterium bovis, and standard strain H37Rv of Mycobacterium tuberculosis.

[0121] In some embodiments, the product includes a drug or a drug composition.

[0122] In some alternative embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable" means that when the molecular bulk and composition are appropriately administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions. Further, the pharmaceutically acceptable carrier includes one or more of solvents, solubilizers, co-solvents, emulsifiers, flavoring agents, odor-enhancing agents, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants, and preservatives.

[0123] In some embodiments, the polypeptide injection system is mixed with one or more pharmaceutically acceptable carriers to form a formulation, the dosage form of which is not limited, as long as it enables the active ingredient to be effectively delivered into the body, including: injections, tablets, film-coated tablets, enteric-coated tablets, capsules, lozenges, granules, powders, ointments, pills, suspensions, pills, powders, sprays, drops, suppositories, creams, patches, etc.; injection dosage forms and inhalation dosage forms are preferred, and inhalation dosage forms are more preferred, such as: inhaled aerosols, dry powder inhalers, soft fog inhalers, nebulized inhalation solutions, nebulized suspensions, etc.

[0124] The drugs or pharmaceutical compositions described in this invention can be administered to patients via various routes, including but not limited to oral, transdermal, nebulized inhalation, intramuscular, subcutaneous, and intravenous injection. In some preferred embodiments, the route of administration is nebulized inhalation.

[0125] Example

[0126] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0127] (1) Cell and bacterial culture

[0128] Escherichia coli (E. coli) was cultured in Luria-Bertani (LB) broth. Plasmid DNA cloning was performed using the top 10 E. coli strains, and PVC purification was performed using the E. coli EPI300 strain. Mycobacterium smegmatis (M. smegmatis) mc 2 155. Mycobacterium tuberculosis H37Ra and H37Rv, Mycobacterium bovis BCG strain, INH-resistant H37Ra, and GFP-expressing Mycobacterium smegma were cultured in Middlebrook 7H9 liquid medium and Middlebrook 7H10 agar medium, respectively, with 10% OADC enrichment broth added to the medium. Primary mouse alveolar macrophages (AMs) were obtained by bronchoalveolar lavage (BAL). AMs, THP-1 (human monocytic leukemia cell line), MH-S (mouse alveolar macrophage cell line), and A549 (human alveolar type II epithelial cell line) were cultured in 1640 medium containing 10% FBS, and HeLa cell line was cultured in DMEM medium containing 10% FBS.

[0129] (2) Expression and purification of PVC protein (for specific plasmid information, see reference 3: Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System, and reference 8: Characterization of Photorhabdus Virulence Cassette as a causative agent in the emerging pathogen Photorhabdus asymbiotica, which are incorporated herein by reference).

[0130] ① Construction of recombinant plasmid pCNM3 expressing 16 structural genome segments of PVC:

[0131] Structural genes: PAU_03353 to PAU_03338 from the genome of Photorhabdus asymbiotica ATCC43949.

[0132] DNA fragments containing structural genes PAU_03353 to PAU_03338 were obtained from the genome of Photorhabdus asymbiotica ATCC43949 and ligated into the pRK404 vector to construct the recombinant plasmid pCNM3.

[0133] ② Construction of recombinant plasmid pBR60 expressing the LysR regulator necessary for PVC assembly and maturation: The LysR regulator (PAU_RS16560) fragment was cloned into pBR322 plasmid by double digestion with BamHI / SalI.

[0134] LysR regulator (PAU_RS16560, SEQ ID NO:1):

[0135] GTGTTTATTTCAAAAGAATTATCAAGCTTTATTGCTGTAGCTAAAAATAAATCAATAAATAAAGCAACAACGGAACTTTTTATTACACAGTCACCGATTTGCCGAAGCTTAAAAAAACTGGAATTTTCATTAGGTGTAAAACTCTTTG TAAGAAAAAGCCAGGGATTGGCTTTAACAGAAGAAGGAGAATGCTTATATAATAAAGTTTTGTCAATTTGTGAACGGCTTCAAGAAATAGAAGGTGACTACAAGAATGAAAAATCAACATACAATGATCTATATCAAAAAAATATATGA

[0136] ③ In order to modify the receptor recognition site of the PVC complex, the R7 sequence or the CD11b binding domain sequence is integrated into Pvc13 (PAU_03341) through linkers (GGSGGGGSGG) to replace the original recognition site (amino acids 403-476).

[0137] R7 nucleotide sequence (SEQ ID NO:2):

[0138] aacgataaactgaccctgtggaccacaccggcaccgagtccgaattgtcgtctgaatgcagaaaaagatgcaaaactgacactggttctgaccaaatgtggtagccagattctggcaaccgttagcgttctggcagttaaaggtagcctggcaccgattagcggcaccgttcagagcgcacatctgattattcgttttgatgaaaatggcgtgctgctgaataatagctttctggatccggaatattggaattttcgtaatggtgatctgaccgaaggcaccgcatataccaatgcagttggttttatgccgaatctgagcgcatatccgaaaagccatggtaaaaccgcaaaaagcaatattgttagccaggtgtatctgaacggcgataaaacaaaaccggttacactgaccattacactgaatggcacccaagaaaccggtgataccacagcatgtgattgtcgtggtgattgtttttgtggtccgagcgcctatagcatgagctttagctgggattggagcggtcataactatatcaatgaaatttttgcgaccagcagctacacctttagctatattgcacaagaaggtagcggtagcggttcaggtagtggttctaaaaagaagaaaaagaaaaag

[0139] CD11b binding domain nucleotide sequence (SEQ ID NO:3):

[0140] gatctgggtaaaaagctgctggaagcagcacgtgcaggtcaggatgatgaagttcgtattctgatggcaaatggtgcagatgttaatgccattgatttctttggtagcacaccgc tgcatctggcagcagaactgggtcatctggaaattgttgaagtgctgctgaaaaacggtgccgatgtgaatgcagatgatattctgggtgataccccgttacacctggcagccgat tttggccatttagaaatcgttgaggtcctgttaaagaatggtgcggacgtgaatgcctcagatgcatttggtctgacccctctgcatttagcagcaaatgctggccacctggaaa tcgtggaagttttactgaaacatggcgcagatgtgaacgcacaggataaatttggtaaaaccgcctttgatatcagcatcgataatggcaatgaagatctggcagaaatcctgcag

[0141] ④ For the PVC complex to be produced for loading cargo, a third plasmid, pBBRN, was used (the pBBRN plasmid is disclosed in Reference 5: N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system, which is incorporated herein by reference). The N-terminal signal peptide of Pnf or Pdp1 (i.e., C50 or S50) was inserted into the NdeI and BamHI restriction sites of the pBBRN plasmid, and the target sequence (TcsT, D29 LysA, D29 LysB, MS6 LysA, MS6 LysB, Granulysin, HNP1, etc.) was inserted between the BamHI and HindIII restriction sites. Simultaneously, a flag (gactacaaggacgatgatgacaag, SEQ ID NO:24) was added to the C-terminus.

[0142] The nucleotide sequence encoding C50 (SEQ ID NO:4):

[0143] ATGTTAAAATATGCTAATCCTCAGACCGTAGCCACACAACGTACTAAAAATACTGCGAAGAAACCGCCATCATCAACCTCTTTTGATGGGCACCTTGAACTTTCAAATGGTGAAAACCAGCCTTACGAAGGCCATAAGATTAGGAAAATC

[0144] The nucleotide sequence encoding S50 (SEQ ID NO:5):

[0145] ATGCCTAGATATGCTAATTATCAGATAAACCCCAAACAGAATATTAAAAATTCACATGGGAAATCTTCATCGTCAGATTTTTCTAGTGGGTACCTTTCATTCTCAAATAATTCGCTTGATGACCCTTTTATTCGGCAGCAAGTTAAGAGA

[0146] The nucleotide sequence encoding TcsT (SEQ ID NO:6):

[0147] GATGTTAGCTTTCGTCTGAGCGGGGCAACCAGCAGCAGCTATGGTGTTTTTATTTCTAATCTGCGTAAAGCACTGCCGAATGAACGTAAACTGTATGATATTCCGCTGCTGCGTAGTAGCCTGCCGGGTAGTCAACGTTATGCACTGATTCATCTGACCAATTATGCCGACGAAACCATCAGCGTGGCAATTGACGTTACCAATGTATATATTATGGGCTATCGTGCCGGTGATACAAGCTATTTTTTTAATGAAGCAAGCGCAACCGAAGCAGCAAAATATGTTTTTAAAGACGCCATGCGTAAAGTGACCCTGCCGTATAGTGGTAACTACGAACGCCTGCAAACAGCAGCAGGAAAAATTCGTGAAAATATCCCGCTGGGCCTGCCGGCACTGGATAGCGCAATCACCACCCTGTTTTATTATAACGCCAATTCTGCAGCAAGCGCCCTGATGGTTCTGATTCAGAGTACAAGCGAAGCAGCACGTTACAAATTTATTGAACAGCAGATTGGGAAACGTGTCGACAAAACCTTTCTGCCGTCACTGGCAATTATTAGTCTGGAAAATAGCTGGTCTGCACTGAGCAAACAAATCCAAATTGCAAGCACCAATAACGGGCAGTTTGAAAGCCCGGTTGTTCTGATTAACGCCCAAAATCAGCGTGTGACCATCACCAATGTGGACGCAGGTGTCGTTACCAGCAACATCGCCCTGCTGCTGAATCGCAATAATATGGCA

[0148] Nucleotide sequence encoding D29 LysA (SEQ ID NO:7):

[0149]

[0150] Nucleotide sequence encoding D29 LysB (SEQ ID NO:8):

[0151] AGCAAACCGTGGTTATTTACCGTTCACGGCACCGGTCAGCCGGACCCTCTGGGTCCTGGACTGCCAGCAGATACAGCAAGAGATGTTTTAGATATTTACCGTTGGCAGCCGATCGGGAATTATCCTGCAGCAGCATTTCCGATGTGGCCGTCAGTTGAAAAAGGTGTTGCAGAATTAATACTGCAGATTGAACTGAAACTGGATGCCGATCCGTATGCAGATTTCGCAATGGCGGGGTATAGCCAGGGTGCAATTGTTGTTGGTCAGGTGCTGAAACATCATATATTACCGCCGACAGGTAGACTGCATCGTTTTCTTCATAGACTGAAAAAAGTTATCTTCTGGGGGAATCCGATGAGACAAAAAGGTTTCGCACATAGTGATGAATGGATTCATCCCGTTGCCGCACCTGACACACTGGGTATTCTTGAGGATCGTTTAGAAAACCTGGAGCAGTATGGTTTTGAGGTTCGCGATTACGCACATGATGGTGACATGTATGCAAGCATTAAAGAAGATGATCTTCATGAATACGAGGTTGCAATTGGACGGATTGTGATGAAGGCAAGCGGTTTTATTGGTGGTCGTGATAGCGTTGTTGCACAGCTGATTGAGCTGGGTCAGCGCCCGATAACCGAAGGTATTGCACTGGCAGGTGCAATTATAGATGCACTGACGTTCTTTGCACGTAGCCGTATGGGTGATAAATGGCCGCATCTGTATAATCGTTATCCTGCAGTTGAATTTCTGCGCCAGATTTAA

[0152] Nucleotide sequence encoding MS6 LysA (SEQ ID NO:19):

[0153]

[0154] The nucleotide sequence encoding MS6 LysB (SEQ ID NO:20):

[0155] CGTATTGATGGTCAGTATGTTGGTCTTGGTCCGGGTGATCGTTCAGATGAAATTCGTAAAATTAAGGCATTCATGCGTCGTAAATTTTCTTATGCAGCAACCCTTGCAGATACCGAATTTTATGATGAAGCAATGACGGCAGTTGTTGCCGAGATGCAGAGCCGTTATAATACCGCAGGCCAGTTACGTGACGGTTTATATATTCCGGGTATAATTAACGCAGAAACCAAATATGTTATGGGTTATTTAAGCAGACCAGTTATTGATACAAGACCAGTTTTATTTACCGTTTGTGGTACCGGGGTTCCGTGGTGGGTGGGTCCTGATGCCGACACGGCACGTGCAGTGGAAGATCAGTATCTGTGGCAGCCGATTGGTTATCCGGCAGCGCCGTTTCCGATGGGCCGTTCTATTACCGCAGGTATTACTGAAGCGCATAATCAGGCAAATCGCTGGCGTGAACGTATTGAAACCCATGGTACTGCATTAGCGGGTTATTCACAGGGTGCAGTTGTTCTGAGTGAACTTTGGATGAATCATATTGCCCCGGAAGATGGCTCATTACGGTGGATGAAACCGCATGTTAGAAAAGCAGTTACCTGGGGTAATCCGAACCGTGAATTAGGTCATGTTTGGGCAGATCACGGTGGGAGCCCGATGGCACCTAGCAATACGCAGGGCGTTAGCAGCAATGGTATGCGTAATACACCGGATTGGTGGCGTGACTATGCGCATCAGGGTGATTTATATGCGTGTACCGAACCGGGAGATACCCAAGAAGTCCGGAACGCGATTTGGCAGATTGTTCGGGATCTGGATCTGTTTACAGGTCCGGATAGCCTGCTGGCACAAGTTATTGAGCTGGCACAGGCGCCACTGCCTGAAACCATCGCAATAACCCGTGCGATTCTGGATGCTGGTATGTTTTTTGCTAAACGTACCGGCCCGCATGTGGATTACAACCCGCAGCCTGCCATTGATTATTTACGTACCTAA

[0156] The nucleotide sequence encoding Granulysin (SEQ ID NO:21):

[0157] GGTCGTGATTATCGTACATGTCTGACAATTGTTCAGAAACTGAAAAAAATGGTGGATAAACCGACACAGCGTAGCGTTTCAAATGCAGCAACACGGGTTTGTAGAACAGGTCGGAGTCGTTGGCGTGATGTGTGTCGTAATTTTATGCGTAGATACCAAAGCCGGGTTACGCAGGGTTTAGTCGCAGGTGAAACCGCACAGCAAATATGCGAAGATCTGCGT

[0158] The nucleotide sequence encoding HNP1 (SEQ ID NO:22):

[0159] GATTGTTACTGTCGTATTCCGGCGTGTATTGCAGGTGAACGGCGGTATGGTACGTGTATTTATCAGGGGCGTCTGTGGGCGTTTTGTTGT

[0160] ⑤ PVC protein expression: Recombinant plasmid pCNM3, expressing 16 structural gene segments of PVC, and recombinant plasmid pBR60, expressing regulatory genes (LysR protein family) essential for PVC assembly and maturation, were sequentially transformed into E. coli EPI300 competent cells to obtain an EPI300 strain containing both pCNM3 and pBR60 plasmids. This strain can express and assemble mature, unloaded PVC. Transforming the pBBRN plasmid into EPI300 competent cells containing pCNM3 and pBR60 plasmids yielded a strain containing all three plasmids, capable of expressing and assembling mature PVC loaded with the target protein.

[0161] ⑥ Purification of PVC protein: The single colonies containing the three plasmids obtained above were inoculated into LB liquid medium and incubated overnight at 37°C and 220 rpm. The bacterial suspension incubated overnight was transferred to 200 mL of LB liquid medium and incubated at 30°C for 24 hours. The bacterial pellet was collected by centrifugation (12,000 rpm, 10 min) and stored at -80°C. The next day, the pellet was removed, and 35 mL of P buffer (25 mM Tris, 140 mM NaCl, 3 mM KCl, pH 7.4, 50 μg / mL DNase I, 200 μg / mL lysozyme, 0.5% Triton X-100, 5 mM MgCl2 and protease inhibitor (1×)) was added and the pellet was resuspended. The pellet was lysed at 37°C for 60 min, centrifuged (14,000 rpm, 10 min) to remove bacterial fragments, and the supernatant was subjected to ultracentrifugation (32 Ti, 32,000 rpm, 2 h, 4°C). Discard the supernatant, resuspend the precipitate in 4 mL of pre-chilled PBS at 4°C, centrifuge (14,000 rpm, 10 min, 4°C), collect the supernatant and centrifuge again at ultra-high speed (41 Ti, 200,000 g, 75 min, 4°C). Discard the supernatant, resuspend the precipitate in pre-chilled PBS at 4°C, centrifuge (14,000 rpm, 10 min, 4°C), discard the precipitate, and the supernatant is the obtained PVC solution. Quantify the protein using Nanodrop and store at 4°C for later use. Before using the PVC solution in cell or animal experiments, remove endotoxins using Triton X-114. The specific steps are as follows: add a final concentration of 1% Triton X-114, mix well, incubate at 4°C for 30 min, then incubate in a 37°C water bath for 10 min, and then centrifuge (14,000 rpm, 10 min, 37°C) to collect the supernatant solution. Repeat the above steps 3 times.

[0162] (3) Cell infection experiment

[0163] THP-1, MHS, and primary mouse mycobacterial cells (AMs) were used to evaluate the effects of intracellular mycobacterial infection and PVC-AMP complex treatment. THP-1 cells were induced into macrophages by PMA (100 nM) for 48 hours before treatment. Macrophages were infected with Ms, H37Ra, and BCG in logarithmic growth phase at multiplici of infection (MOI) of 10, 3, and 10, respectively. Four hours after infection, the supernatant was discarded, and the cells were treated with gentamicin (200 μg / ml) for 2 hours to remove extracellular mycobacteria. Then, different PVC-AMP complexes (0.8 mg / mL) were used alone or in combination for 24–48 hours. After PVC treatment, cells were lysed with 0.25% Triton X-100, and the lysates were serially diluted with PBS and plated onto Middlebrook 7H10 agar for colony-forming unit (CFU) counting.

[0164] (4) In vivo infection experiment

[0165] C57BL / 6 mice were anesthetized by intraperitoneal injection of 20 μL / g tribromoethanol, and mycobacteria were infected via tracheal nebulization using a nebulizer. Log-phase mycobacteria were used for infection, with an infection dose of Ms: 3 × 10⁻⁶. 7 CFU, H37Ra: 1×10 6 CFU, H37Rv: 5×10 4 CFU, infection volume 50 μl. After a period of infection, mice were treated with 1.0 mg of PVC sample (50 μl) per mouse using the same nebulization system. Specifically, Ms was treated once 24 h after infection, and subsequent experiments were performed 24 h later; H37Ra was treated once 7 days after infection, and subsequent experiments were performed 48 h later; H37Rv was treated once every two days 2 days after infection, up to 3 and 6 times respectively. After treatment, mouse lung tissue was collected and homogenized in sterile PBS. The serially diluted homogenate was spread on 7H10 agar medium and incubated at 37°C to count the number of mycobacteria. Formalin-fixed lung tissue was routinely embedded in paraffin and sectioned, and then stained with hematoxylin and eosin (HE) to assess the pathological condition of the mouse lungs.

[0166] Example 1: The modified PVC-R7 can recognize and deliver effector proteins to macrophages.

[0167] PVC can recognize cells through the cytokinin Pvc13, but its recognition range is limited and it cannot recognize all types of cells, including macrophages. Therefore, the receptor-binding domain of PVC was further modified to achieve its specific recognition of different target cells. Specifically, the RGD / PK7 (R7) domain of recombinant adenovirus serotype 5 vector was integrated into the cytokinin Pvc13 to construct PVC-R7.

[0168] The inventors first used the modified PVC-R7 to verify its recognition effect on macrophages (the modified PVC-R7 is as follows). Figure 1 As shown in A, NTD: N-terminal domain, CTD: C-terminal domain).

[0169] like Figure 1 As shown in Figure B, the toxin protein TcsT was successfully loaded onto PVC-R7 cells under the guidance of signal peptides C50 and S50. Subsequently, human THP-1 macrophages were treated with empty and TcsT-loaded PVC-R7 cells, respectively. Significant cell death was observed in the TcsT-loaded group. Figure 1 C), using mouse alveolar macrophage cell line (MH-S) and mouse primary alveolar macrophages (AMs) for validation, the same results were observed. Figure 1 D, Figure 1 These results demonstrate that PVC-R7 can successfully deliver effector proteins to macrophages and exert their effects.

[0170] Example 2: PVC-R7 delivers antimicrobial peptides into macrophages to kill mycobacteria.

[0171] To test whether AMP could be delivered into cells via the PVC complex and exert its effects, the inventors selected several AMPs from different sources, including LysA and LysB from mycobacterial phages D29 and MS6, and Granulysin and HNP1 from host cells.

[0172] The inventors used PVC-R7 to load and deliver these AMPs into Ms-infected THP-1 macrophages, and then tested their antibacterial effects. Figure 2 A- Figure 2As shown in Figure B, PVC-R7 successfully loaded different AMPs without significant cytotoxicity. After treatment of infected cells, D29 LysA, D29 LysB, MS6 LysA, Granulysin, and HNP1 all significantly reduced the number of intracellular Ms, demonstrating a significant scavenging effect. MS6 LysB also tended to reduce the number of intracellular Ms. The combined use of D29 LysA and D29 LysB showed a synergistic effect, achieving the most significant result. Figure 2 C, Figure 2 D). Therefore, D29LysA and D29LysB were selected for further in-depth research. Infection of cells with GFP-labeled Ms and observation of intracellular bacterial content revealed that PVC-R7 delivery of D29LysA and LysB significantly reduced intracellular GFP fluorescence intensity, indicating a reduction in the number of Ms in the cells (D). Figure 2 E, Figure 2 F). Simultaneously, qPCR detection of intracellular inflammatory factors revealed that it could reduce intracellular IL-6 and TNF-α, indicating that it can effectively alleviate cellular inflammation. Figure 2 G, Figure 2 H). Subsequently, its antibacterial effect was verified using MH-S and AMs cells, respectively, and the same results were observed: the number of intracellular Ms was significantly reduced (H). Figure 2 I, Figure 2 J).

[0173] Furthermore, the inventors verified the lethal effects of PVC-R7 delivery of D29 LysA and LysB on H37Ra and BCG, such as... Figure 2 K and Figure 2 L also showed significant bactericidal effects against H37Ra and BCG, significantly reducing the number of intracellular bacteria. These results indicate that D29 LysA and D29 LysB have a killing effect on Mycobacterium tuberculosis, and the targeted delivery of D29LysA and D29 LysB using modified PVC is a very promising new treatment method for Mycobacterium tuberculosis.

[0174] Example 3: The modified PVC-CD11b complex exhibits specific recognition of macrophages.

[0175] Because PVC-R7 has a broad recognition ability for multiple cell types, the inventors wanted to further improve the targeting specificity of PVC for macrophages. Therefore, the inventors modified the Pvc13 receptor-binding domain of the PVC complex by inserting a CD11b specific receptor-binding domain (…). Figure 3 A). Transmission electron microscopy (TEM) imaging showed that the PVC-CD11b complex retained its typical sheath structure, with essentially no change in morphological structure. Figure 3 B). Simultaneously, nanoparticle tracking analysis (NTA) was used to detect the particle size distribution of PVC, with the main particle size distribution located at 123 nm (…). Figure 3 C), similar in size to the reported wild-type PVC. [3] Subsequently, the inventors verified the protein loading and delivery function of PVC-CD11b. For example... Figure 3 D- Figure 3 As shown in G, PVC-CD11b successfully loaded and delivered TcsT to different macrophages (THP-1, MH-S, AMs) to exert cytotoxic effects. Furthermore, compared to PVC-R7, PVC-CD11b did not recognize A549 and HeLa cells. Figure 3 H- Figure 3 M) indicates that it has a more obvious recognition preference for macrophages expressing the CD11b receptor, which is more conducive to its targeted delivery application in vivo.

[0176] D29 LysA and D29 LysB can also be loaded into PVC-CD11b. Figure 4 A), TEM and NAT were used to observe the morphology and particle size of PVC particles. The results showed that the morphology and structure did not change significantly after loading D29 LysA and D29 LysB, and the dominant peak particle sizes were 123 nm and 133 nm, respectively. Figure 4 B, Figure 4 C). Through PVC-CD11b delivery, D29 LysA and D29LysB can still effectively clear Ms within macrophages (C). Figure 4 D). Consistent with this result, confocal microscopy observation of Ms-GFP-infected macrophages showed that it significantly reduced intracellular fluorescence intensity, further validating its anti-intracellular bacterial effect. Figure 4 E, Figure 4 F), the effectiveness of the PVC-CD11b delivery system was further confirmed using H37Ra. Figure 4 G). Furthermore, the inventors further verified its effectiveness against drug-resistant Mycobacterium tuberculosis using the INH-resistant H37Ra strain, finding it equally effective against the INH-resistant H37Ra strain. Figure 4 These results indicate that PVC-CD11b can serve as an effective transmembrane biological carrier for D29 LysA and LysB, effectively inhibiting various types of mycobacteria within macrophages and exerting an anti-mycobacterial infection effect.

[0177] Example 4: PVC-CD11b delivery of D29 LysA and LysB effectively inhibited mycobacteria in mouse lungs.

[0178] To verify the in vivo antibacterial effects of D29 LysA and LysB delivered by PVC-CD11b, the inventors constructed different mycobacterial infection mouse models using Ms and H37Ra, respectively, and administered the drugs via pulmonary nebulization. Figure 5 A). The results showed that, regardless of whether Ms infection or H37Ra infection occurred, PVC-CD11b delivery of D29 LysA and LysB effectively reduced the bacterial load in the lungs of mice. Figure 5 B, Figure 5 D), and can reduce the expression of TNF-α in mouse serum that is increased due to infection (D). Figure 5 C, Figure 5 E), indicating that it can also effectively exert antibacterial effects in vivo and reduce inflammatory responses in mice. Based on the preliminary in vivo test results, the inventors further used the highly virulent strain of Mycobacterium tuberculosis H37Rv to infect mice to verify its potential for treating tuberculosis. Considering the high virulence of H37Rv, in order to further improve the effect, the inventors administered the drug multiple times, and tested it after 3 and 6 administrations respectively. Figure 5 F). The results showed that the mice's body weight did not fluctuate significantly during infection and administration, indicating that the PVC-CD11b vector was relatively safe. Figure 5 G). After 6 administrations, D29 LysA and LysB delivered by PVC-CD11b significantly reduced the number of H37Rv in the lungs of mice, by approximately 18-fold. Figure 6 A). HE staining of lung tissue showed that it could effectively alleviate lung pathological damage and inflammation caused by H37Rv infection. Figure 6 B, Figure 6 C). In summary, these results indicate that PVC-CD11b delivery of D29 LysA and LysB can significantly inhibit intracellular mycobacterial activity and has certain therapeutic potential for tuberculosis. This delivery method represents a novel tuberculosis treatment strategy.

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[0194] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0195] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Uses of the peptide injection system in the following (i) and / or (ii): (i) Prepare a product that eliminates intracellular mycobacteria in target cells; (ii) To prepare products for the prevention and / or treatment of lung diseases and / or conditions caused by mycobacteria; in, The polypeptide injection system comprises a PVC complex of non-symbiotic photobacteria and a polypeptide loaded in the PVC complex; The polypeptide includes antimicrobial peptides, which include cell-derived Granulysin, HNP1, LysA derived from Mycobacterium phage D29, LysB derived from Mycobacterium phage D29, LysA derived from Mycobacterium phage MS6, and / or LysB derived from Mycobacterium phage MS6.

2. The use according to claim 1, wherein, The PVC complex contains structural proteins and regulatory factors; The structural proteins include Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15 and Pvc16. The regulatory factors include those derived from the LysR protein family.

3. The use according to claim 2, wherein, The receptor recognition site of the Pvc13 was modified to a different specific recognition sequence; The other specific recognition sequences include the CD11b binding domain sequence and / or the RGD-PK7 dual-ligand domain (R7) sequence of the recombinant adenovirus serotype 5 (Ad5) vector.

4. The use according to claim 3, wherein, The CD11b binding domain sequence comprises the sequence shown in SEQ ID NO:16; and / or, The RGD-PK7 dual ligand domain (R7) sequence includes the sequence shown in SEQ ID NO:

15.

5. The use according to claim 3 or 4, wherein, The receptor recognition site contains amino acid residues 403-476 of the Pvc13 protein.

6. The use according to claim 1 or 2, wherein, The Granulysin, HNP1, LysA derived from mycobacterial phage D29, LysB derived from mycobacterial phage D29, LysA derived from mycobacterial phage MS6, and LysB derived from mycobacterial phage MS6 respectively contain sequences as shown in SEQ ID NO: 13~14, 9~12.

7. The use according to claim 1 or 2, wherein, The polypeptide is loaded into the PVC complex by fusing it with a signal peptide at its N-terminus, the signal peptide including the N-terminal signal peptide of effector factors Pnf or Pdp1.

8. The use according to claim 7, wherein, The N-terminal signal peptide of the Pnf contains the sequence shown in SEQ ID NO:17, and / or, The N-terminal signal peptide of Pdp1 contains the sequence shown in SEQ ID NO:

18.

9. The use according to claim 1 or 2, wherein, The elimination of intracellular mycobacteria in target cells includes reducing the number of mycobacteria in the target cells; The target cells include macrophages.

10. The use according to claim 1 or 2, wherein, The lung diseases and / or conditions caused by mycobacteria include pathological damage and inflammation of the lungs.

11. The use according to claim 1 or 2, wherein, The mycobacteria include Mycobacterium smegmatis, Mycobacterium bovis, attenuated strain H37Ra of Mycobacterium tuberculosis, INH-resistant strain H37Ra of Mycobacterium tuberculosis, and standard strain H37Rv of Mycobacterium tuberculosis.

12. The use according to claim 1 or 2, wherein, The product includes a drug or a drug composition; The drug or drug composition may also include one or more pharmaceutically acceptable carriers.

13. The use according to claim 12, wherein, The drug or drug composition can be administered orally, transdermally, via nebulized inhalation, intramuscularly, subcutaneously, or intravenously.

14. A pharmaceutical composition comprising the polypeptide injection system as described in any one of claims 1 to 13.

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