Polypeptide for regulating antibacterial function of sepsis and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to a polypeptide for regulating the antibacterial function of sepsis and application of the polypeptide. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1, and the polypeptide can hinder combination of NLRC3 and PKA-Calpha, increase interaction between PKA phosphorylation and Beclin-1 containing a Run structural domain and increase the expression level of protein containing a cysteine-rich structural domain, further promote LAP formation and an intracellular sterilization function, and does not intervene in NLRC3 expression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a polypeptide for regulating the antibacterial function of sepsis and its application. Background Technology

[0002] Secondary infections are a significant cause of death in patients with sepsis, influenza, and obesity. Secondary infections in sepsis patients can significantly increase their mortality rate, while obesity and inflammatory states such as influenza are also associated with a significant increase in susceptibility to infection.

[0003] NLRC3, a member of the NOD-like receptor, is an immune checkpoint protein primarily expressed in the cytoplasm of immune cells such as monocytes and macrophages. Its core function is to negatively regulate the immune response by inhibiting key immune signaling pathways. Overexpression of NLRC3 during tuberculosis, lymphocytic choriomeningitis virus infection, or the immunosuppressive phase of sepsis can reduce the host's ability to clear pathogens, thereby increasing the risk of secondary infections and death.

[0004] Although NLRC3 can serve as a target for treating NLRC3-mediated secondary infections such as sepsis or obese sepsis, traditional methods of intervening in NLRC3 expression may undermine its potential protective effects by reducing NLRC3 expression levels. This is because, in systemic lupus erythematosus, multiple sclerosis, and septic storms, NLRC3 can alleviate tissue and organ damage caused by excessive activation of immune cells by inhibiting pathways such as NF-κB and PI3K-mTOR. Decreased NLRC3 expression levels may promote inflammatory responses, leading to immune damage or organ injury.

[0005] Therefore, how to prevent or treat secondary infections caused by NLRC3 overexpression without interfering with NLRC3 expression is of great significance for restoring immune balance, enhancing anti-infection defense, and reducing mortality, and is an urgent problem to be solved. Summary of the Invention In order to prevent or treat secondary infections caused by NLRC3 overexpression without interfering with NLRC3 expression, this invention provides a polypeptide for regulating antibacterial function in sepsis and its application.

[0006] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides a polypeptide for regulating the antibacterial function of sepsis, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Secondly, the present invention provides a coding gene that encodes the aforementioned polypeptide.

[0008] Thirdly, the present invention provides an expression vector containing the above-mentioned coding gene, wherein the expression vector can express the above-mentioned polypeptide in a host cell.

[0009] Fourthly, the present invention provides an engineered cell comprising the above-mentioned coding gene or the above-mentioned expression vector, wherein the engineered cell is capable of expressing the above-mentioned polypeptide.

[0010] Fifthly, the present invention provides a method for producing polypeptides, comprising the following steps: Culture the above-mentioned engineered cells; Engineered cells were lysed, and proteins were collected and purified to obtain peptides.

[0011] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a polypeptide and a pharmaceutically acceptable carrier, said pharmaceutical composition for treating sepsis.

[0012] In a seventh aspect, the present invention provides the use of the above-mentioned polypeptide in the preparation of a drug for treating sepsis.

[0013] In conjunction with the seventh aspect of the invention, in some embodiments, the sepsis is characterized by a significant increase in the expression level of NLRC3 protein in the patient's immune cells. Further, the immune cells are bone marrow-derived macrophages.

[0014] In conjunction with the seventh aspect of the invention, in some embodiments, the drug for treating sepsis is used to modulate the antibacterial function of sepsis.

[0015] In conjunction with the seventh aspect of the present invention, in some embodiments, the dosage form of the drug is one or more of the following: oral preparation, intravenous injection, intramuscular injection, subcutaneous injection, nebulized inhaler, nasal spray, and oral spray.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The polypeptide provided by this invention can enhance the bactericidal ability of immune cells without interfering with NLRC3 expression. When used to treat sepsis, it can avoid secondary infection, reduce the risk of immune homeostasis being disrupted, and has good drug safety. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0018] Figure 1A statistical graph of NLRC3 mRNA levels in ordinary bone marrow-derived macrophages (BMDMs) after different stimulations in Example 1.

[0019] Figure 2 This is a comparative statistical chart showing the bactericidal ability of ordinary BMDMs after different stimuli in Example 1; where A: representative images of bacterial colony-forming units (CFU) in cell lysates of each group; B: statistical chart of bacterial colony-forming units (CFU) in cell lysates of each group.

[0020] Figure 3 This is a comparative statistical chart showing the bactericidal abilities of ordinary BMDMs and BMDMs with the NLRC3 gene knocked out in Example 1; where A: representative images of bacterial colony-forming units (CFU) in cell lysates of each group; B: statistical chart of bacterial colony-forming units (CFU) in cell lysates of each group.

[0021] Figure 4 The image shows the staining of Rubicon and LAP formation in LPS and palmitic acid-treated macrophages after NLRC3 knockout in Example 1. A: The effects of various stimuli on Rubicon distribution and LAP formation in wild-type cells; B: The effect of NLRC3 knockout on Rubicon distribution and LAP formation is shown by comparing wild-type and knockout macrophages.

[0022] Figure 5 This is a comparison of Rubicon protein expression levels in macrophages treated with LPS and palmitic acid after NLRC3 knockout in Example 1; where A: Western blotting analysis under different treatment conditions; B: Western blotting analysis of mice with different genotypes.

[0023] Figure 6 This is a schematic diagram of the key interaction interface formed between amino acids 96-98 of NLRC3 and amino acids 241-250 of PKA-Cα in the complex in Example 2.

[0024] Figure 7 This is a comparison of the effects of NLRC3 and PKA-Cα interaction and NLRC3 overexpression on intracellular Rubicon protein expression levels in Example 2. A shows the verification results of the interaction between NLRC3 and PKA-Cα in cells; B shows that NLRC3 overexpression reduces intracellular Rubicon protein levels; Vector is a control empty vector without the NLRC3 sequence, representing the control group.

[0025] Figure 8 The image shows the co-precipitation of NLRC3 with PKA through its NBD domain (61~616aa) in Example 2.

[0026] Figure 9 This is an immunoblotting analysis diagram of the interaction between the NBD domain (61~616aa) of NLRC3 and the full-length PKA-Cα protein or the protein with amino acid deletion at positions 241~250 in Example 2.

[0027] Figure 10 This is a schematic diagram of the three-dimensional structure of the NBD domain of NLRC3 in Example 3.

[0028] Figure 11 This is a schematic diagram of the molecular docking model between Q5K (red) and the NBD domain (light tan) of NLRC3 in Example 3.

[0029] Figure 12 This is the Q5K mass spectrometry verification image from Example 3.

[0030] Figure 13 The graph shows the binding constant analysis of the NBD domain of Q5K and NLRC3 in Example 4; where A: time-response curve of Q5K binding to NLRC3; B: concentration-response curve of Q5K binding to NLRC3.

[0031] Figure 14 This is a comparison of the interaction levels between NLRC3 and PKA-Cα in cells treated with PBS, TAT, or Q5K in Example 5; where TAT is the penetrating peptide, and Flag and HA are protein tags, respectively.

[0032] Figure 15 This is a comparison of Rubicon protein expression levels in cells where PRKACA was knocked down in Example 5; where TAT is the penetrating peptide, representing the control group, "-" indicates no sh-PRKACA plasmid was added, and "+" indicates the addition of the sh-PRKACA plasmid.

[0033] Figure 16 The image shows the staining pattern of Rubicon colocalization with bacteria in the cells of the TAT control group and Q5K group in Example 5; TAT is the penetrating peptide, representing the control group.

[0034] Figure 17 This is a statistical chart showing the effective concentration range of Q5K for antibacterial effect in Example 6, including colony counts.

[0035] Figure 18 This is a comparison chart of the effective concentration range of Q5K for antibacterial effect in Example 6, obtained by immunoblotting.

[0036] Figure 19 CC of Q5K to BMDMs in Example 7 50 Value statistics chart.

[0037] Figure 20 This is a comparison chart of the effects of Q5K on the survival rate of secondary infections in septic or obese septic mice in Example 8; where A: represents the effect of Q5K intervention on survival rate in a normal-weight septic mouse model; B: represents the effect of Q5K intervention on survival rate in an obese septic mouse model.

[0038] In the above figures, BSA represents culture medium containing bovine serum albumin, PBS represents culture medium containing phosphate buffer, Palmitic acid represents palmitic acid stimulation, LPS represents lipopolysaccharide stimulation, P. aeruginosa represents Pseudomonas aeruginosa infection, and NLRC3 represents... + / + This indicates that NLRC3 was not knocked out. - / - The diagram represents NLRC3 knockout, DAPI represents a 4',6-diamidinyl-2-phenylindole fluorescent staining of the cell nucleus, Merge represents an overlay fusion diagram, Rubicon represents the intracellular localization of Rubicon, TAT represents a penetrating peptide, Tubulin represents microtubules, PRKACA represents the gene encoding PKA-Cα protein, IP represents immunoprecipitation, sh-PRKACA represents PRKACA silenced, Lyaste represents lysis buffer, Flag represents Flag tag, and HA represents HA tag. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] In the terminology of this invention, PKA is an abbreviation for protein kinase A, PKA-Cα is an abbreviation for the catalytic subunit Cα of protein kinase A, and PRKACA is the gene name of PKA-Cα.

[0041] As described in the background section, the core function of NLRC3 is to negatively regulate the immune response by inhibiting key immune signaling pathways. Its reduced expression may disrupt immune homeostasis. Therefore, there is an urgent need to find a solution to prevent or treat secondary infections caused by NLRC3 overexpression without interfering with NLRC3 expression.

[0042] Studies have shown that LC3-associated phagocytosis (LAP) can rapidly degrade intracellular pathogens in the mechanism of pathogen clearance. The LAP process is initiated by a protein containing a Run domain and interacting with Beclin-1, and containing a cysteine-rich domain (hereinafter referred to as Rubicon). Rubicon recruits microtubule-associated protein 1 light chain 3 (LC3) to form LAP bodies, accelerating phagosome maturation and fusion with lysosomes, thereby significantly improving the rate and efficiency of pathogen clearance. Rubicon is the rate-limiting factor for LAP, and its stability directly determines LAP activity; however, it is susceptible to ubiquitination degradation, which reduces LAP function.

[0043] This invention experimentally demonstrates that NLRC3 can bind to PKA-Cα. This binding blocks the phosphorylation of downstream signaling pathways by PKA, ultimately leading to a decrease in the ubiquitination level of the LAP initiation molecule Rubicon, reducing LAP execution efficiency, and thus weakening the ability of immune cells to clear pathogens. The amino acid sequence of PKA-Cα is shown in SEQ ID NO:2: MGNAAAAKKGSEQESVKEFLAKAKEDFLKKWESPAQNTAHLDQFERIKTLGTGSFGRVMLVKHKETGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVPGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSLDLIYRDLKPENLLIDQQ GYIQVTDFGFAKRVKGRTWTLCGTPEYLAPEIILSKGYNKAVDWWALGVLIYEMAAGYPPFFADQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDIKNHKWFATTDWIAIYQRKVEAPFIPKFKGPGDTSNFDDYEEEEIRVSINEKCGKEFSEF (human source).

[0044] Based on the above experimental results, this invention provides a polypeptide for regulating the antibacterial function of sepsis. The polypeptide contains a binding domain that specifically binds to amino acid residues 96-98 of NLRC3. These amino acid residues are located at the interaction interface between NLRC3 and PKA. Therefore, after the polypeptide specifically and competitively binds to these amino acid residues, it can block the binding of NLRC3 to PKA, relieving the inhibition of PKA phosphorylation activity by NLRC3, thereby enhancing the phosphorylation of downstream signaling pathways by PKA, ensuring the expression level of the LAP initiator molecule Rubicon, and thus maintaining normal LAP execution efficiency. This ensures that the ability of immune cells to clear pathogens is not affected by the increased expression level of NLRC3. In some embodiments of this invention, the binding domain is composed of the amino acid sequence 241-250 of PKA-Cα. Figure 6 As shown, amino acids 96-98 of NLRC3 and amino acids 241-250 of PKA-Cα form a key interaction interface in the complex. After amino acids 96-98 of NLRC3 bind to the polypeptide of the present invention, they can no longer bind to PKA. Some embodiments of the present invention provide polypeptides that are polypeptide analogs of PKA-Cα, which are part of PKA and contain amino acids 241-250 of PKA-Cα, and can compete with normal PKA for binding to amino acids 96-98 of NLRC3. Further, the amino acid sequence of this polypeptide is shown in SEQ ID NO:1, SEQ ID NO:1: YGRKKRRQRRRGSGQIYEK, named Q5K, or a variant with ≥80% sequence identity to SEQ ID NO:1, and said variant retains the specific binding ability to amino acid residues 96-98 of NLRC3. Figure 11 As shown, Q5K can specifically bind to the NBD domain of NLRC3. As shown in Example 4, the binding constant of Q5K to the NBD domain of NLRC3 is 5.82 × 10⁻⁶. -5 M and the two have a very strong specific interaction.

[0045] This invention also provides a coding gene encoding the aforementioned polypeptide, and an expression vector containing the coding gene. The coding gene is cloned into a high-efficiency expression system to construct the expression vector. The expression vector can express the aforementioned polypeptide in host cells, or the expression vector can be transformed into engineered competent cells, and the polypeptide can be industrially produced through high-density fermentation and large-scale purification processes. As shown in Example 3, this invention inserts the gene encoding Q5K into the fusion protein expression vector pET-GST for prokaryotes to construct a polypeptide expression plasmid. The polypeptide expression plasmid is then transformed into BL21 competent cells for induced expression to obtain Q5K.

[0046] The pharmaceutical composition provided by this invention comprises the aforementioned polypeptide and a pharmaceutically acceptable carrier, and is used to treat sepsis. This pharmaceutical composition can reverse the ability of NLRC3 to cause secondary infections without interfering with NLRC3 expression, thereby preventing or treating secondary bacterial infections in sepsis.

[0047] In some embodiments of the present invention, the carrier is one or more of liposomes, lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, viral carriers, cell carriers, and exosome carriers.

[0048] This invention provides the use of the above-mentioned polypeptide in the preparation of drugs for treating sepsis.

[0049] In some embodiments of the present invention, the sepsis is characterized by a significant increase in the expression level of NLRC3 protein in the patient's immune cells. Further, the immune cells are bone marrow-derived macrophages.

[0050] In some embodiments of the present invention, the dosage form of the drug is one or more of the following: oral preparation, intravenous injection, intramuscular injection, subcutaneous injection, nebulized inhaler, nasal spray, and oral spray.

[0051] This invention verifies the pathogenesis of NLRC3-mediated secondary infections, as well as the therapeutic effect and specific mechanism of action of Q5K, through the following examples. In the following examples, the experimental samples used were wild-type C57 mice, THP cell lines, and mouse bone marrow-derived macrophages (BMDMs). Isopropyl-β-D-thiogalactoside (IPTG), used to induce monoclonal colonies in Example 3, is a commonly used inducer, mainly used to activate the lactose operon in prokaryotes, thereby promoting the expression of exogenous genes. NLRC3 gene knockout BMDMs are NLRC3... - / - Bone marrow-derived macrophages extracted from mice.

[0052] Example 1: Effects of NLRC3 knockout on the bactericidal ability of monocytes and macrophages and LAP body formation 1. This invention first investigates the effects of different stimuli on the expression level of NLRC3 in ordinary BMDMs. The cells in each group were treated as follows: Normal BMDMs were cultured in DMEM high glucose medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (P / S, Gibco) and incubated in a constant temperature incubator at 37°C and 5% CO2.

[0053] BSA+PBS group: The cells were cultured in the above-mentioned medium containing BSA (5% fatty acid-free) for 48 hours. Specifically, after culturing the cells in the above-mentioned medium containing BSA (5% fatty acid-free) for 24 hours, an equal volume of PBS was added to the LPS solvent and the cells were cultured for another 24 hours.

[0054] Pal+PBS group: The obesity environment was simulated by stimulation with unsaturated fatty acids. Specifically, cells were stimulated with palmitic acid (Pal, dissolved in 5% fatty acid-free BSA) for 24 hours, and then an equal volume of PBS was added to continue culturing for another 24 hours.

[0055] BSA+LPS group: Endotoxin tolerance was simulated by lipopolysaccharide (LPS). Specifically, cells were cultured in the above-mentioned medium with BSA (no fatty acids, 5%) for 24 hours, and then LPS was added to stimulate the cells for another 24 hours.

[0056] Pal+LPS group: BMDMs were cultured in a medium containing Pal for 24 hours, and then LPS was added for 24 hours of stimulation.

[0057] The results showed that, compared with the BSA+PBS group, the NLRC3 mRNA levels in the Pal+PBS group and the BSA+LPS group were significantly increased, and the NLRC3 mRNA level in the Pal+LPS group was further increased. Figure 1 The results suggest that in obese environments and under conditions of endotoxin tolerance, the expression level of NLRC3 in BMDMs is significantly upregulated, which may be involved in regulating the antibacterial function of cells.

[0058] 2. This invention further clarifies the effect of NLRC3 on the bactericidal ability of macrophages by evaluating the killing efficiency of BMDMs against Pseudomonas aeruginosa (ATCC 27853) under different stimuli: 2.1 Pseudomonas aeruginosa was cultured in LB liquid medium until mid-log phase, then the concentration was adjusted to 10. 4 CFU / mL, for later use.

[0059] 2.2 Ordinary BMDMs and BMDMs with the NLRC3 gene knocked out were respectively divided into groups of 1×10⁻⁶. 4 / wells were inoculated into 96-well plates and incubated for 12 hours; then, they were divided into groups for treatment: BSA+PBS group: The cells were cultured in the above-mentioned medium containing BSA (5% fatty acid-free) for 48 hours. Specifically, after culturing the cells in the above-mentioned medium containing BSA (5% fatty acid-free) for 24 hours, an equal volume of PBS was added to the LPS solvent and the cells were cultured for another 24 hours.

[0060] Pal+PBS group: The obesity environment was simulated by stimulation with unsaturated fatty acids. Specifically, cells were stimulated with palmitic acid (5% fatty acid-free BSA solution) for 24 hours, and then an equal volume of PBS was added to continue culturing for another 24 hours.

[0061] BSA+LPS group: Endotoxin tolerance was simulated by LPS. Specifically, cells were cultured in the above-mentioned medium with BSA (no fatty acids, 5%) for 24 hours, and then LPS was added to stimulate the cells for another 24 hours.

[0062] Pal+LPS group: After culturing in medium containing Pal for 24 hours, LPS was added for another 24 hours of stimulation.

[0063] After the culture was completed, Pseudomonas aeruginosa from step 2.1 was added at a concentration of MOI=1, and the culture was continued for 2 hours. After washing, extracellular bacteria were removed by adding gentamicin-containing medium, and the culture was continued for 6 hours. Cells were lysed and plated to count intracellular surviving bacteria.

[0064] The results showed that, compared with the BSA+PBS group, the cell bactericidal ability of the Pal+PBS group and the BSA+LPS group was significantly reduced, and the cell bactericidal ability of the Pal+LPS group was further reduced. Figure 2 A and Figure 2 B); In the BSA+LPS group or the Pal+LPS group, the bactericidal ability of NLRC3 knockout cells was significantly restored compared with that of NLRC3 gene non-knockout cells (B). Figure 3 A and Figure 3 B).

[0065] 3. This invention uses confocal microscopy to observe the formation of LAP bodies in order to further clarify the role of LAP bodies in the influence of NLRC3 on the bactericidal ability of macrophages: 3.1 Pseudomonas aeruginosa expressing GFP was cultured in LB liquid medium and the concentration was adjusted to 10 after reaching mid-log phase. 4 CFU / mL, for later use.

[0066] 3.2 Ordinary BMDMs and BMDMs with the NLRC3 gene knocked out were respectively divided into groups of 1×10⁻⁶. 4 / wells were seeded in confocal dishes and incubated for 12 h; then they were treated in separate groups: BSA+PBS group: The cells were cultured in the above-mentioned medium containing BSA (5% fatty acid-free) for 48 hours. Specifically, after culturing the cells in the above-mentioned medium containing BSA (5% fatty acid-free) for 24 hours, an equal volume of PBS was added to the LPS solvent and the cells were cultured for another 24 hours.

[0067] Pal+PBS group: The obesity environment was simulated by stimulation with unsaturated fatty acids. Specifically, cells were stimulated with palmitic acid (5% fatty acid-free BSA solution) for 24 hours, and then an equal volume of PBS was added to continue culturing for another 24 hours.

[0068] BSA+LPS group: Endotoxin tolerance was simulated by LPS. Specifically, cells were cultured in the above-mentioned medium with BSA (no fatty acids, 5%) for 24 hours, and then LPS was added to stimulate the cells for another 24 hours.

[0069] Pal+LPS group: After culturing in medium containing Pal for 24 hours, LPS was added for another 24 hours of stimulation.

[0070] After the culture is completed, add the bacterial solution obtained in step 3.1 at a concentration of MOI=1 and continue to culture for 1 hour.

[0071] 3.3 Cells in each group were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton-X, and blocked with 2% BSA. They were then incubated sequentially with Rubicon primary antibody and Alexa Fluor 594-labeled secondary antibody, and the nuclei were counterstained with DAPI. Images were acquired using a Leica (STELLARIS 8) confocal microscope.

[0072] The results showed that in the cells of the Pal+PBS group and the BSA+LPS group, Rubicon was unable to effectively localize around the bacteria to form LAP bodies. Figure 4 A), while in BMDMs with the NLRC3 gene knocked out, this phenomenon was mitigated ( Figure 4 B). The above results suggest that NLRC3 weakens the antibacterial function of BMDMs by interfering with the formation of LAP bodies.

[0073] 4. To further clarify the role of Rubicon in LAP body formation, this invention uses sucrose density gradient centrifugation to separate intracellular phagosomes of BMDMs that have been stimulated with palmitic acid and lipopolysaccharide and infected with Pseudomonas aeruginosa (MOI=1) for 1 hour.

[0074] BSA+PBS group: The cells were cultured in the above-mentioned medium containing BSA (5% fatty acid-free) for 48 hours. Specifically, the cells were cultured in the above-mentioned medium containing BSA (5% fatty acid-free) for 24 hours, and then PBS was added to continue the culture for another 24 hours.

[0075] Pal+PBS group: The obesity environment was simulated by stimulation with unsaturated fatty acids. Specifically, cells were stimulated with palmitic acid (5% fatty acid-free BSA solution) for 24 hours, and then an equal volume of PBS was added to continue culturing for another 24 hours.

[0076] BSA+LPS group: Endotoxin tolerance was simulated by LPS. Specifically, cells were cultured in the above-mentioned medium with BSA (no fatty acids, 5%) for 24 hours, and then LPS was added to stimulate the cells for another 24 hours.

[0077] Pal+LPS group: After culturing in medium containing Pal for 24 hours, LPS was added for another 24 hours of stimulation.

[0078] Specific procedure: After infection at different time points (0-2 hours), cells were collected by centrifugation at 55 g and 4°C for 7 minutes. The pellet was resuspended in homogenization buffer containing protease inhibitors and processed using Duracell technology. Cells were homogenized 10 times using a Grind stainless steel homogenizer to lyse them. The lysate was centrifuged at 1500 rpm for 5 minutes to remove nuclei and unlysed cells. The supernatant was then subjected to sucrose gradient ultracentrifugation (Beckman SW 41 rotor, 28400 rpm, 4°C, 2 hours), and phagosome fractions were collected from the 55%–65% sucrose interface. This fraction was adjusted to an 11% sucrose concentration with sucrose-free homogenization buffer and placed on a 15% Ficoll mat for a second ultracentrifugation (10000 rpm, 4°C, 40 minutes). After discarding the supernatant, the phagosomes were concentrated again by centrifugation at 10000 rpm, 4°C for subsequent Western blotting and other analyses.

[0079] The results showed that, compared with the BSA+PBS group, the level of Rubicon protein in the phagosome components of cells in the Pal+PBS group and the BSA+LPS group was significantly decreased. Figure 5 A). In the BSA+LPS group or Pal+LPS group, the Rubicon protein level was significantly increased in NLRC3 knockout cells compared to NLRC3 non-knockout cells. Figure 5 B). This indicates that Rubicon expression is reduced during LAP body formation, thereby affecting the normal formation of LAP bodies; NLRC3 interferes with LAP body formation.

[0080] This invention demonstrates the effects of palmitic acid and lipopolysaccharide on the expression level of NLRC3 in BMDMs through Example 1, clarifies the effect of NLRC3 on the bactericidal ability of BMDMs treated with palmitic acid and lipopolysaccharide, and verifies the conclusion that NLRC3 weakens the antibacterial function of BMDMs by interfering with the formation of LAP bodies.

[0081] Meanwhile, Example 1 clarifies the role of Rubicon in LAP body formation. Rubicon expression is reduced during LAP body formation, thereby affecting the normal formation of LAP bodies.

[0082] Example 2: NLRC3 and PKA-Cα interaction promotes decreased Rubicon expression 1. Rubicon is a key regulatory protein in the formation of LAP bodies, and its stability is regulated by ubiquitination degradation mechanism.

[0083] First, phosphorylated kinases that may affect the ubiquitination process were screened. Based on the functional characteristics of each domain of NLRC3 in signal transduction, the focus was on its nucleotide-binding domain (NBD). The amino acid sequence of the NLRC3-PKA-Cα complex was predicted using the AlphaFold3 online platform (https: / / www.alphafoldserver.com), and the results showed stable interactions between the two at multiple amino acid sites (van der Waals overlap ≥0.4 Å).

[0084] Further visualization analysis of the complex's spatial conformation using ChimeraX 1.8 software revealed that amino acids 96-98 of NLRC3 and 241-250 of PKA-Cα form a key interaction interface in the complex. Figure 6 ).

[0085] 2. To verify the above predictions, Flag-PKA-Cα and GFP-NLRC3 expression plasmids were constructed and co-transfected into 293T cells. Forty-eight hours later, immunoprecipitation was used to detect the binding of the two plasmids.

[0086] Specifically, the method for constructing Flag-PRKACA and GFP-NLRC3 expression plasmids includes the following steps: mRNA is extracted from HeLa cells, and total RNA is reverse transcribed into first-strand cDNA (i.e., a whole cDNA library) using universal Oligo / random primers and reverse transcriptase. Using this cDNA as a template, high-fidelity PCR amplification is performed using PRKACA and NLRC3-specific primers. The products are separated by electrophoresis (130V, 20 min), purified, and then digested with SalI and NotI restriction endonucleases for 4 h. T4 DNA ligase is then used to ligate the Flag and GFP tag vectors (Miaoling, P33800; P51456) to construct transient expression plasmids. The Flag-PRKACA and GFP-NLRC3 expression plasmids are co-transfected into 293T cells, and cell lysates are collected after 48 h for immunoprecipitation.

[0087] The results showed that NLRC3 and PKA-Cα interacted significantly within the cell. Figure 7 A); at the same time, compared with the control group, NLRC3 overexpression significantly reduced the level of intracellular Rubicon protein (A). Figure 7(B) This suggests that NLRC3 may promote the degradation of Rubicon protein by binding to PKA-Cα.

[0088] 3. Further clarify the interacting structural domains through truncated volume experiments.

[0089] According to existing reports, NLRC3 is divided into three structural domains: 1~60aa, 61~616aa, and 617~1065aa.

[0090] Using the FLAG-NLRC3 overexpression plasmid (Miaoling P36112) as a template, high-fidelity PCR amplification was performed with primers specific to each domain. The products were separated by electrophoresis (130V, 20 min), purified, and then digested with SalI and NotI restriction endonucleases for 4 h. They were then ligated with the pLV3-CMV-FLAG vector (Miaoling, P33800) using T4 DNA ligase to construct transient expression plasmids for three different domains of NLRC3 (1~60aa, 61~616aa, 617~1065aa). These three domain expression plasmids were co-transfected into 293T cells with the Flag-PRKACA expression plasmid. Cell lysates were collected after 48 h and subjected to immunoprecipitation.

[0091] like Figure 8 As shown, NLRC3 interacts with PKA-Cα through its NBD structural domain (61~616aa). 4. To verify this direct interaction, GST-labeled NLRC3 NBD domain (61-616aa) protein and FLAG-labeled full-length PKA-Cα protein (FL) were purified and obtained.

[0092] Specifically, using the FLAG-NLRC3 overexpression plasmid (Miaoling P36112) as a template, high-fidelity PCR amplification was performed using specific primers for each domain (1~60aa, 61~616aa, 617~1065aa) (1~60aa: upstream primer (SEQ ID NO:3): ATGAGGAAGCAAGGGGTGCGGACGG, downstream primer (SEQ ID NO:4): CCCGCTGGGGCCCTGCAGCAATGAC; 61-616aa: upstream primer (SEQ ID NO:5): TCAAGGATACAGAGGCACCGCAAGG, downstream primer (SEQ ID NO:6): CCTGCAGGTGTCCGACGCCTGTGCC; 617-1065aa: upstream primer (SEQ ID NO:7): GAGGCCAACCTGTCCCTGAGCCTCA, downstream primer (SEQ ID NO:8): TGCTCCCACGTGCACTGTTGAAATG). The product was separated by electrophoresis (130V, 20min), recovered and purified, then digested with SalI and NotI restriction endonucleases for 4h, and ligated with T4 DNA ligase to pET-GST vector (addgene, 42049) to construct NLRC3 truncated somatic protein expression plasmid.

[0093] The pET-GST vector (purchased from Addgene, 42049) and the plasmid encoding the GST-NLRC3 truncated variants (1~60aa, 61~616aa, 617~1065aa) (pET-GST vector addgene, 42049) were transformed into BL21 competent cells. The cells were induced at 18°C ​​for 16 hours with isopropyl-β-D-thiogalactoside (1 mM) to express the target protein. After cell lysis, the fusion protein was captured using GST affinity magnetic beads, eluted with 10 mM glutathione-Tris-HCl buffer, and dialyzed to obtain purified protein. Simultaneously, FLAG-PRKACA was expressed by transfecting HEK293T cells. Total protein was extracted using NP-40 lysis buffer, immunoprecipitated with anti-FLAG agarose beads, and highly purified FLAG-RKA-Cα protein was obtained through competitive elution with 3×FLAG peptides. The purified GST-NLRC3 protein and the full-length FLAG-PKA-Cα protein with amino acid deletions at positions 241-250 were co-incubated overnight at 4°C. GST agarose beads were then added and incubated in PBS containing a protease inhibitor for 2 hours, followed by pull-down assays. The mixture was thoroughly washed with PBS and analyzed using Western blotting.

[0094] The results showed that the NBD domain (61-616aa) of NLRC3 directly binds to the full-length PKA-Cα protein or the protein with amino acid deletions at positions 241-250. Figure 9 ).

[0095] Example 3: Building Q5K Figure 10 A schematic diagram of the three-dimensional structure of the NBD domain of NLRC3; Figure 11 This is a molecular docking model of Q5K (red) and the NBD domain (light tan) of NLRC3.

[0096] Based on the amino acid sequence of Q5K YGRKKRRQRRRGSGQIYEK (SEQ ID NO:1), its encoding DNA sequence was chemically synthesized and cloned into the pET-GST vector (addgene, 42049) by enzyme digestion and ligation to construct the Q5K polypeptide expression plasmid.

[0097] Specifically, 2 μL of the Q5K peptide expression plasmid was transformed into BL21(DE3) competent cells and plated on LB agar plates containing the appropriate antibiotics. The cells were then incubated upside down at 37°C for 17 hours. Single colonies were picked and inoculated into 4 mL of LB medium containing antibiotics. The cells were incubated at 37°C with shaking at 220 rpm until the OD600 reached 0.6–0.8. The cells were then divided into two groups: the induction group was treated with isopropyl-β-D-thiogalactoside for 6 hours to induce expression, while the control group received no treatment. The cells were collected by centrifugation, resuspended in buffer, and then sonicated on ice. The supernatant and precipitate were mixed with reducing loading buffer, boiled, centrifuged, and then analyzed by SDS-PAGE.

[0098] Western blotting was performed using a wet transfer method to transfer the protein onto a PVDF membrane. The membrane was then incubated sequentially with a His-tagged polyclonal antibody (AP0032) and an HRP-labeled secondary antibody (BS13278), followed by imaging and detection. For scale-up culture, seed culture was inoculated at a ratio of 1:100 into 500 mL of LB medium. After induction, the cells were collected by centrifugation. The cells were sonicated at a ratio of 1:20 (w / v). The lysate was then subjected to Ni column chromatography with gradient elution using equilibration buffer, 50 mM, and 350 mM imidazole buffer. The final eluate was dialyzed at a ratio of 1:1000, and then SUMO enzyme (1 mg sample + 10 μL enzyme solution) was added and digested at 4°C for 16 h. The digestion product was purified again using a Ni column, and the crosslinking fluid was collected to obtain the target protein. Further purification was achieved using reversed-phase high-performance liquid chromatography (RP-HPLC). The obtained protein was identified by mass spectrometry and then lyophilized for storage.

[0099] Protein spectroscopy analysis results ( Figure 12 The results showed that the amino acid sequence of the purified protein was consistent with the Q5K design sequence, indicating that the polypeptide had been successfully synthesized.

[0100] This invention further provides verification examples to illustrate the effectiveness of Q5K: Example 4: Q5K can specifically bind to the NBD domain of NLRC3. The Q5K provided by this invention can specifically bind to the NBD domain of NLRC3.

[0101] Specifically, the surface plasmon resonance (SPR) constant was determined using a Biacore 8 k instrument (Cytiva, USA) equipped with a CM5 sensor chip. The buffer solution was 0.5% Tween-20 phosphate buffer (PBST) at pH 7.4.

[0102] The instrument operation procedure was performed according to the instruction manual. First, 100 μL of the 100 mM GST-NLRC3 truncated variant (61-616 aa.) purified in Example 2 was immobilized on the sensor chip of the Biacore 8k instrument. Q5K was bound to the sensor chip at a rate of 20 μL / min for 120 s, and then dissociated in PBST analyte buffer for 180 s. The binding of Q5K at 0.78 to 200 μM (0.78125, 3.1256, 12.5, 50, 200 μM) was tested (see...). Figure 13 A), to determine the binding constant.

[0103] The results show that the binding constant (K) between Q5K and the NBD domain of NLRC3 is... D Approximately 5.82 × 10 -5 M (see) Figure 13 B). This indicates that Q5K has a very strong specific interaction with the NBD domain of NLRC3.

[0104] Example 5: Verification Method for the Mechanism of Action of Q5K To verify the effect of Q5K on the binding of NLRC3 to PKA and its influence on Rubicon-mediated LC3-associated phagocytosis (LAP) formation, an NLRC3 overexpression vector with the FLAG tag and a PKA-Cα overexpression vector with the HA tag (PKA-Cα overexpression plasmid) were first constructed using blank vectors (purchased from Miaoling, FLAG: P33800; HA: P1750).

[0105] Specifically, using PKA-Cα overexpression plasmid (Miaoling P92701) and NLRC3 overexpression plasmid (Miaoling P36112) as templates, high-fidelity PCR amplification was performed using specific primers (PKA-Cα: upstream primer (SEQ ID NO:9): GTCGACATGGGCAACGCCGCCGCCGCCAAGA; downstream primer (SEQ ID NO:10): GCGGCCGCCTACTCGAGAAACTCAGAAAACT. NLRC3: upstream primer (SEQ ID NO:11): ATGAGGAAGCAAGGGGTGCGGACGG; downstream primer (SEQ ID NO:12): TGCTCCCACGTGCACTGTTGAAATG.) The products were separated by electrophoresis (130V, 20min), recovered and purified, and then digested with SalI and NotI restriction endonucleases for 4h. T4 DNA ligase and blank vectors (purchased from Miaoling, FLAG: P33800; HA: P1750) were used to construct FLAG-NLRC3 overexpression plasmid and HA-PKA-Cα overexpression plasmid.

[0106] FLAG-NLRC3 overexpression plasmid and HA-PKA-Cα overexpression plasmid were simultaneously transiently transfected into 293T cells; 48 hours later, the cells were divided into groups and treated accordingly, and cell lysates were collected for co-immunoprecipitation (Co-IP); the specific grouping and treatment procedures are as follows: Control group: treated with PBS for 12 hours.

[0107] TAT group: Add membrane-penetrating peptide (TAT, 10 μM) for 12 hours.

[0108] Q5K group: Add Q5K (10μM) for 12 hours.

[0109] like Figure 14 The results showed that, compared with the control group or the TAT group, the interaction level between NLRC3 and PKA-Cα in the Q5K group cells was significantly reduced, suggesting that Q5K interferes with the binding of NLRC3 and PKA.

[0110] The THP-1 (ATCC, TIB-202) cell line was induced into macrophages; and the macrophages were induced into M0 macrophages for 24 hours using phorbol ester (Sigma, 37558-16-0) at 100 ng / ml. To further verify the role of PKA-Cα in the Q5K-mediated NLRC3-mediated Rubicon degradation process, a PRKACA knockdown vector (shPRKACA, SEQ ID NO:13: 5'-AGCGTGAAAGAATTCTTAGCC-3') was constructed and transfected using pLKO.1 (purchased from Miaoling, P0258) to specifically inhibit PKA activity. Control cells were transfected with the empty vector (pLKO.1, purchased from Miaoling, P0258). Cells were cultured in DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (P / S, Gibco) at 37°C in a 5% CO2 incubator. After 48 hours, the medium was replaced with PAL and cultured for 24 hours, followed by LPS stimulation for another 24 hours. Finally, the following stimulation was performed: TAT group: Add membrane-penetrating peptide (TAT, 10 μM) for 12 hours.

[0111] Q5K group: Add Q5K (10μM) for 12 hours.

[0112] After processing, total cellular protein was extracted, and the expression level of Rubicon was detected by Western blotting.

[0113] The results showed that, compared with the control group, the expression level of Rubicon protein was significantly reduced in cells with PRKACA knockdown. This indicates that NLRC3 mediates the ubiquitination and degradation of Rubicon by interacting with PKA-Cα and affecting its signaling pathway, while Q5K stabilizes Rubicon protein levels by blocking this interaction. Figure 15 ).

[0114] To further evaluate the effect of Q5K on Rubicon-associated LAP formation, confocal microscopy was used to observe the colocalization of Rubicon and bacteria after macrophages phagocytosed Pseudomonas aeruginosa. The specific steps were as follows: 1×10⁻⁶ Q5K cells were injected with 10⁻⁶ Q5K cells into a 10⁻⁶ LAP cell. 4 One BMDM was seeded in a confocal microscope culture dish and cultured at 37°C in a 5% CO2 incubator until fully adherent. Treatment with 200 μM palmitic acid for 24 hours simulated a high-fat environment, followed by treatment with 100 ng / mL LPS for 24 hours to establish an immune tolerance model. Each group was treated with 10 μM Q5K or an equal volume of PBS for 2 hours, followed by treatment with 1×10⁻⁶ LPS. 7 Infect cells with fluorescent Pseudomonas aeruginosa (PA) at CFU / mL for 1 hour. Fix cells and perform immunofluorescence staining to label the Rubicon protein.

[0115] like Figure 16The results showed that, compared with the control group, Rubicon and bacteria were significantly co-localized in the cells of the Q5K group, suggesting that Q5K can enhance Rubicon stability and promote the formation of LC3-related phagocytosis, thereby improving the host cell's clearance efficiency of intracellular pathogens.

[0116] In summary, Q5K can block the binding of NLRC3 and PKA, inhibit the degradation effect of its downstream signals on Rubicon, thereby maintaining Rubicon stability and promoting LAP formation.

[0117] Example 6: Validation of the bactericidal ability of Q5K as a drug in in vitro experiments. To assess the effective concentration range of Q5K's antibacterial activity, bone marrow-derived macrophages were prepared at a concentration of 1×10⁻⁶. 4 Cells were seeded in 96-well plates and cultured for 12 hours. Then, Q5K peptide solutions at concentrations of 0, 0.625, 1.25, 2.5, 5, 10, and 20 μM were added, and incubation continued for 2 hours. Subsequently, *Pseudomonas aeruginosa* expressing GFP were infected with an MOI of 1 and co-cultured at 37°C for 2 hours. After washing three times with pre-warmed PBS, the medium was replaced with gentamicin-containing medium to remove extracellular bacteria, and the cells were cultured for another 12 hours. Cells were lysed using 0.5% Triton-X, and intracellular bacteria were collected. Half of the lysate was used for LB agar plating and colony counting analysis. Figure 17 The remainder is used for immunoblotting detection. Figure 18 ).

[0118] Experimental results showed that Q5K exhibited significant antibacterial activity at concentrations greater than 2.5 μM, effectively enhancing the ability of macrophages to clear intracellular Pseudomonas aeruginosa.

[0119] Example 7: In vitro biosafety verification method for Q5K To assess the in vitro biosafety of the antibacterial Q5K of the present invention, the present invention systematically evaluated its effect on mammalian cell activity using the CCK-8 assay to determine its safe concentration range for use.

[0120] Mouse bone marrow-derived macrophages were used at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 96-well cell culture plates and pre-cultured at 37°C in a 5% CO2 incubator until complete cell adhesion. The medium was then replaced with a solution containing PAL and cultured for 24 hours, followed by LPS stimulation for another 24 hours. Subsequently, cells were grouped for further treatment. Experimental groups (multiple groups): Culture media containing different concentrations of Q5K peptide were added to the culture system to make the final concentrations 2, 4, 8, 16, 24, 48, 96, 128 and 256 μM respectively, and the culture was continued for 12 hours.

[0121] Control group: Add an equal volume of PBS to the culture system and continue culturing for 12 hours.

[0122] After incubation, carefully discard the supernatant containing peptides from each well, wash once with fresh culture medium containing 10% fetal bovine serum, and then replace with an equal volume of complete culture medium and continue incubation for 12 hours. After the specified incubation time with CCK-8 solution, measure the absorbance at 450 nm using a multi-functional microplate reader. The OD values ​​of the experimental groups were then analyzed. 450 Value / Control Group OD 450 The value (CC) × 100% represents cell viability; the concentration of the compound required to reduce cell viability by 50% is considered as CC. 50 (50% cytotoxic concentration); such as Figure 19 As shown, Q5K's CC for BMDMs 50 The value is 217.22 μM.

[0123] This value is much higher than the minimum effective concentration required for it to exert its antibacterial function, indicating that Q5K has an extremely high safety window for host mammalian cells at effective antibacterial concentrations, providing important safety evidence for its subsequent biomedical applications.

[0124] Example 8: Verification method of Q5K in vivo effects To verify the protective effect of Q5K in sepsis and obesity-related sepsis secondary infection models, 6–8-week-old C57BL / 6 mice were selected. The obese mouse model was obtained by feeding the mice with a high-fat diet (RESEARCH DIETS, catalog number 25040406) for 8 consecutive weeks.

[0125] A cecal ligation-perforated sepsis model was established according to Example 2. Normal-weight mice and obese mice were randomly divided into a control group and a Q5K group, respectively. 42 hours after cecal ligation-perforation (CLP), the Q5K group mice received a 5 mg / kg Q5K peptide solution via tail vein injection, while the control group received an equal volume of saline. 48 hours after CLP, surviving mice in each group were infused with *Pseudomonas aeruginosa* via tracheal infusion to simulate secondary lung infection following sepsis. The 7-day survival rate of each group was observed.

[0126] The results showed that in a normal-weight mouse model of septicemia with secondary lung infection, the 7-day mortality rate was approximately 65% ​​in the control group, while it decreased to approximately 35% in the Q5K group. In an obese mouse model of septicemia with secondary lung infection, Q5K intervention also significantly improved survival. Figure 20 (p<0.05).

[0127] The above results confirm that Q5K can effectively improve the survival rate of normal-weight and obese mice with sepsis, and has the potential to be developed as a preventive or therapeutic drug against secondary bacterial infections mediated by increased NLRC3 expression.

[0128] As can be seen from the above embodiments, the Q5K provided by the present invention is designed based on the structure of PKA-Cα. It can specifically bind to the NBD domain (61-616aa) of NLRC3, thereby preventing NLRC3 from binding to PKA-Cα, increasing the expression level of proteins containing cysteine-rich domains that interact with PKA phosphorylation and Beclin-1 containing the Run domain, and thus promoting LAP formation and intracellular bactericidal function, without interfering with the expression level of NLRC3. This enables the targeted development of drugs to combat NLRC3-mediated secondary bacterial infections such as sepsis or obesity sepsis.

[0129] In vitro and in vivo experiments have shown that the peptide exhibits significant antibacterial activity at concentrations greater than 2.5 μM, effectively enhancing the ability of macrophages to clear intracellular Pseudomonas aeruginosa. It can also treat NLRC3-mediated secondary infections associated with immune dysregulation, such as sepsis or obesity sepsis, and improve the survival rate of mice, demonstrating its potential as an anti-infective and sepsis treatment drug.

[0130] Furthermore, the experiment found that Q5K's CC50 was 217.22 μM, which had no effect on cell viability within a safe dosage range, indicating that it has very good drug safety. It is a very effective reagent for increasing the cell bactericidal function mediated by NLRC3 in relevant pathological states, and can be used in the preparation of drugs for the treatment or prevention of NLRC3-mediated secondary bacterial infections such as sepsis or obese sepsis.

[0131] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its spirit and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

Claims

1. A polypeptide for regulating antibacterial function in sepsis, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A gene encoding a gene, characterized in that, Encoding the polypeptide of claim 1.

3. An expression carrier, characterized in that, The expression vector contains the coding gene of claim 2 and can express the polypeptide of claim 1 in a host cell.

4. An engineered cell, characterized in that: The engineered cells contain the coding gene of claim 1 or the expression vector of claim 3, and are capable of expressing the polypeptide of claim 1.

5. A method for producing a polypeptide, characterized in that, Includes the following steps: Cultivate the engineered cells as described in claim 4; The engineered cells were lysed, and the protein was collected and purified to obtain the polypeptide described in claim 1.

6. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the polypeptide of claim 1 and a pharmaceutically acceptable carrier, and is used to treat sepsis.

7. Use of the polypeptide of claim 1 in the preparation of a drug for treating sepsis.

8. The use according to claim 7, characterized in that: The sepsis is characterized by a significant increase in the expression level of NLRC3 protein in the patient's immune cells.

9. The use according to claim 7, characterized in that: The medication for treating sepsis is used to modulate the antibacterial function in sepsis.

10. The use according to claim 7, characterized in that: The dosage form of the drug is one or more of the following: oral, intravenous, intramuscular, subcutaneous, nebulized, nasal, and oral sprays.

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