Polypeptide for regulating sepsis antibacterial function and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a polypeptide for regulating antibacterial function of sepsis and application thereof. An amino acid sequence of the polypeptide is shown as SEQ ID NO:1, the polypeptide can hinder NLRC3 from being combined with PKA-Ca, increase the expression level of PKA phosphorylation and Beclin-1 interaction containing a Run domain and a protein containing a cysteine-rich domain, and then promote the formation of LAP and intracellular bactericidal function, and does not interfere with NLRC3 expression.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a polypeptide for regulating antibacterial function of sepsis and application thereof. BACKGROUND

[0002] Secondary infection is an important cause of death in patients with diseases such as sepsis, influenza and obesity. Once secondary infection occurs in patients with sepsis, the mortality of the patients can be significantly improved, and obesity and inflammation such as influenza are also accompanied by a significant increase in susceptibility to infection.

[0003] NOD-like receptor member NLRC3 is an immune checkpoint protein mainly expressed in the cytoplasm of immune cells such as monocytes and macrophages, and the core function is to negatively regulate immune response by inhibiting key immune signaling pathways. In the immunosuppression period of tuberculosis, lymphocytic choriomeningitis virus infection or sepsis, NLRC3 overexpression will reduce the ability of the host to clear pathogens, thereby increasing the risk of secondary infection and death.

[0004] Although NLRC3 can be used as a target for treating secondary infection mediated by NLRC3, such as sepsis or obesity sepsis, the traditional method of interfering with the expression of NLRC3 may damage its potential protective effect due to the reduction of NLRC3 expression level. The reason is that in systemic lupus erythematosus, multiple sclerosis and sepsis inflammatory storm, NLRC3 can reduce tissue and organ damage caused by excessive activation of immune cells by inhibiting NF-κB, PI3K-mTOR and other pathways, and once the expression level is reduced, it may promote the inflammatory response, leading to immune damage or organ damage.

[0005] Therefore, how to prevent or treat secondary infection caused by NLRC3 overexpression without interfering with the expression of NLRC3 is of great significance for restoring immune balance, enhancing anti-infection defense and reducing mortality, and is a problem to be solved at present. SUMMARY

[0006] In order to prevent or treat secondary infection caused by NLRC3 overexpression without interfering with the expression of NLRC3, the application provides a polypeptide for regulating antibacterial function of sepsis and application thereof.

[0007] The technical scheme provided by the application is as follows:

[0008] In a first aspect, the application provides a polypeptide for regulating antibacterial function of sepsis, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1.

[0009] In a second aspect, the application provides a coding gene for coding the above-mentioned polypeptide.

[0010] In a third aspect, the present application provides an expression vector comprising the above-mentioned coding gene, wherein the expression vector can express the above-mentioned polypeptide in a host cell.

[0011] In a fourth aspect, the present application provides an engineered cell comprising the above-mentioned coding gene or the above-mentioned expression vector, wherein the engineered cell can express the above-mentioned polypeptide.

[0012] In a fifth aspect, the present application provides a method for producing a polypeptide, comprising the following steps:

[0013] culturing the above-mentioned engineered cell;

[0014] lyzing the engineered cell, collecting and purifying the protein to obtain the polypeptide.

[0015] In a sixth aspect, the present application provides a pharmaceutical composition comprising the polypeptide and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is used for treating sepsis.

[0016] In a seventh aspect, the present application provides use of the above-mentioned polypeptide in the preparation of a medicament for treating sepsis.

[0017] In combination with the seventh aspect of the present application, in some embodiments, the sepsis is manifested by a significant increase in the expression level of NLRC3 protein in immune cells of a patient. Further, the immune cells are bone marrow-derived macrophages.

[0018] In combination with the seventh aspect of the present application, in some embodiments, the medicament for treating sepsis is used for regulating the antibacterial function of sepsis.

[0019] In combination with the seventh aspect of the present application, in some embodiments, the dosage form of the medicament is one or more of an oral dosage, an intravenous injection, a muscle injection, a subcutaneous injection, an aerosol inhalation, a nasal spray, and a buccal spray.

[0020] Compared with the prior art, the present application has at least the following beneficial effects:

[0021] The polypeptide provided by the present application can enhance the bactericidal capacity of immune cells without interfering with the expression of NLRC3, and the use of the polypeptide for treating sepsis can avoid secondary infection and reduce the risk of breaking immune homeostasis, thereby having good drug safety. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only exemplary, and other drawings can be derived from the provided drawings without creative labor for those skilled in the art.

[0023] Figure 1 Statistical chart of NLRC3 mRNA level after different stimulations for the ordinary bone marrow-derived macrophages (BMDMs) in Example 1.

[0024] Figure 2 Statistical chart of bactericidal ability after different stimulations for the ordinary BMDMs in Example 1; wherein, A: representative pictures of bacterial colony-forming units (CFU) of cell lysates of each group; B: statistical chart of bacterial colony-forming units (CFU) of cell lysates of each group.

[0025] Figure 3 Statistical chart of bactericidal ability of ordinary BMDMs and NLRC3 gene-knocked-out BMDMs in Example 1; wherein, A: representative pictures of bacterial colony-forming units (CFU) of cell lysates of each group; B: statistical chart of bacterial colony-forming units (CFU) of cell lysates of each group.

[0026] Figure 4 Staining chart of Rubicon and LAP formation of LPS and palmitic acid-treated macrophages after NLRC3 knockout in Example 1; wherein, A: effects of various stimulations on Rubicon distribution and LAP formation in wild-type cells; B: effects of NLRC3 knockout on Rubicon distribution and LAP formation by comparison between wild-type and knockout macrophages.

[0027] Figure 5 Comparison chart of phagosome Rubicon protein expression level of LPS and palmitic acid-treated macrophages after NLRC3 knockout in Example 1; wherein, A: Western Blot analysis under different treatment conditions; B: Western Blot analysis of mice of different genotypes.

[0028] Figure 6 Schematic diagram of NLRC3 amino acids 96-98 and PKA-Cα amino acids 241-250 forming a key interaction interface in a complex in Example 2.

[0029] Figure 7 Comparison chart of NLRC3 and PKA-Cα interaction and effect of NLRC3 overexpression on intracellular Rubicon protein expression level in Example 2; wherein, A: verification results showing that NLRC3 and PKA-Cα interact in cells; B: NLRC3 overexpression reduces the level of intracellular Rubicon protein; Vector is a control empty vector without NLRC3 sequence, representing a control group.

[0030] Figure 8Figure for immunoprecipitation of NLRC3 through its NBD domain (61-616 aa) with PKA in Example 2.

[0031] Figure 9 Figure for immunoblotting analysis of the interaction of NBD domain of NLRC3 (61-616 aa) with full-length protein of PKA-Ca or protein with deletion of amino acids 241-250 in Example 2.

[0032] Figure 10 Figure for the three-dimensional structure of NBD domain of NLRC3 in Example 3.

[0033] Figure 11 Figure for the molecular docking model of Q5K (red) and NBD domain of NLRC3 (light yellow) in Example 3.

[0034] Figure 12 Figure for mass spectrometry verification of Q5K in Example 3.

[0035] Figure 13 Figure for analysis of the binding constant of Q5K and NBD domain of NLRC3 in Example 4; wherein, A: time-response curve of Q5K binding to NLRC3; B: concentration-response curve of Q5K binding to NLRC3.

[0036] Figure 14 Figure for comparison of the interaction level of NLRC3 and PKA-Ca in cells treated with PBS or TAT, Q5K in Example 5; wherein, TAT is a penetrating peptide, and Flag and HA are protein tags.

[0037] Figure 15 Figure for comparison of the expression level of Rubicon protein in cells in which PRKACA is knocked down in Example 5; wherein, TAT is a penetrating peptide, and "-" indicates that no sh-PRKACA plasmid is added, and "+" indicates that sh-PRKACA plasmid is added.

[0038] Figure 16 Figure for staining of the co-localization of Rubicon and bacteria in cells in the TAT control group and the Q5K group in Example 5; wherein, TAT is a penetrating peptide, and represents the control group.

[0039] Figure 17 Figure for colony counting of the effective concentration range of the antibacterial effect of Q5K in Example 6.

[0040] Figure 18 Figure for comparison of immunoblotting detection of the effective concentration range of the antibacterial effect of Q5K in Example 6.

[0041] Figure 19 Figure for the effect of Q5K on the CC50 Value statistics chart.

[0042] Figure 20 Figure for comparison of the effect of Q5K on the survival rate of secondary infection in sepsis or obese sepsis mice in Example 8; wherein, A: represents the effect of Q5K intervention on the survival rate in the normal weight sepsis mouse model; B: represents the effect of Q5K intervention on the survival rate in the obese sepsis mouse model.

[0043] In the above figures, BSA represents bovine serum albumin in the culture medium, PBS represents phosphate buffer solution in the culture medium, Pal, Palmitic acid represents palmitic acid stimulation, LPS represents lipopolysaccharide stimulation, P. aeruginosa represents Pseudomonas aeruginosa infection, NLRC3 + / + represents NLRC3 not knocked out, NLRC3 - / - represents NLRC3 is knocked out, DAPI represents 4', 6-diamidino-2-phenylindole fluorescent staining to mark the nucleus, Merge represents superimposed fusion chart, Rubicon represents the localization of Rubicon in the cell, TAT represents a penetrating peptide, Tubulin represents tubulin, PRKACA represents a gene encoding PKA-Cα protein, IP represents immunoprecipitation, sh-PRKACA represents PRKACA is silenced, Lyaste represents lysate, Flag represents Flag tag, and HA represents HA tag. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0045] In the terms of the present application, PKA is the abbreviation of protein kinase A, and PKA-Cα is the abbreviation of the catalytic subunit Cα of protein kinase A; PRKACA is the gene name of PKA-Cα.

[0046] As described in the background, the core function of NLRC3 is to negatively regulate immune response by inhibiting key immune signaling pathways, and the decrease of its expression may break the immune homeostasis, therefore, it is urgent to find a solution to prevent or treat secondary infection caused by overexpression of NLRC3 without interfering with the expression of NLRC3.

[0047] Studies have shown that in the mechanism of eliminating intracellular pathogens, LC3-related phagocytosis (LAP) can quickly achieve degradation of intracellular pathogens. The LAP process is initiated by Beclin-1 containing Run domain interacting with Rubicon containing cysteine-rich domain (hereinafter referred to as Rubicon), Rubicon recruits microtubule-associated protein 1 light chain 3 (LC3) to form LAP bodies, accelerates phagosome maturation, and fuses with lysosomes, thereby significantly improving the rate and efficiency of pathogen clearance. Rubicon is a LAP rate-limiting factor, and its stability directly determines the LAP activity, but it is easily degraded by ubiquitination and reduces the function of LAP.

[0048] Experiments prove that NLRC3 can bind PKA-Cα, which will block the phosphorylation of PKA to the downstream signal pathway, ultimately leading to the decrease of the ubiquitination level of Rubicon, the LAP execution efficiency, and the ability of immune cells to clear pathogens. The amino acid sequence of PKA-Cα is shown as SEQ ID NO: 2:

[0049] MGNAAAAKKGSEQESVKEFLAKAKEDFLKKWESPAQNTAHLDQFERIKTLGTGSFGRVMLVKHKETGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVPGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSLDLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGTPEYLAPEIILSKGYNKAVDWWALGVLIYEMAAGYPPFFADQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDIKNHKWFATTDWIAIYQRKVEAPFIPKFKGPGDTSNFDDYEEEEIRVSINEKCGKEFSEF (human).

[0050] Based on the above experimental results, the present application provides a polypeptide for regulating the antibacterial function of sepsis, which comprises a binding domain specifically binding to the 96th-98th amino acid residues in NLRC3, the 96th-98th amino acid residues in NLRC3 are located at the interaction interface of NLRC3 and PKA, therefore, after the polypeptide specifically competes with the 96th-98th amino acid residues in NLRC3, the binding of NLRC3 and PKA is blocked, the inhibition of NLRC3 on the phosphorylation activity of PKA is released, the phosphorylation of PKA on the downstream signaling pathway is enhanced, the expression level of LAP starting molecule Rubicon is ensured, and then the normal LAP execution efficiency is maintained, and the ability of immune cells to remove pathogens is not affected by the increase of the expression level of NLRC3. In some embodiments of the present application, the binding domain is composed of the 241st-250th amino acid sequence of PKA-Cα. As shown in Figure 6 the 96th-98th amino acids in NLRC3 and the 241st-250th amino acids of PKA-Cα form a key interaction interface in the complex, and after the 96th-98th amino acids in NLRC3 bind to the polypeptide of the present application, they can no longer bind to PKA. The polypeptide provided in some embodiments of the present application is a polypeptide analogue of PKA-Cα, which is a part of PKA and comprises the 241st-250th amino acids of PKA-Cα, and can compete with normal PKA to bind to the 96th-98th amino acids in NLRC3. Further, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, SEQ ID NO: 1: YGRKKRRQRRRGSGQIYEK, named Q5K, or a variant having ≥80% sequence identity with SEQ ID NO: 1, and the variant retains the specific binding ability to the 96th-98th amino acid residues in NLRC3. As shown in Figure 11 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 -5 M, both of which have very strong specific interaction.

[0051] The present application also provides a coding gene encoding the above-mentioned polypeptide, and an expression vector comprising the above-mentioned coding gene. The coding gene is cloned into a high-efficiency expression system to construct an expression vector, and the expression vector can express the above-mentioned polypeptide in a host cell, or the expression vector is transformed into an engineered competent cell, and through high-density fermentation and large-scale purification process, the industrial production of the polypeptide can be realized. As shown in Example 3, the gene encoding Q5K is inserted into the fusion protein expression vector pET-GST for prokaryotes to construct a polypeptide expression plasmid, the polypeptide expression plasmid is transformed into BL21 competent cells, and induction expression is carried out, and then Q5K can be obtained.

[0052] The pharmaceutical composition provided by the present application comprises the polypeptide and a pharmaceutically acceptable carrier, and is used for treating sepsis. The pharmaceutical composition can reverse the ability of NLRC3 to cause secondary infection, thereby preventing or treating secondary bacterial infection of sepsis without interfering with the expression of NLRC3.

[0053] In some embodiments of the present application, the carrier is one or more of a liposome, a lipid nanoparticle, a polymer nanoparticle, an inorganic nanoparticle, a viral vector, a cell carrier, and an exosome carrier.

[0054] The present application provides the use of the polypeptide in the preparation of a medicament for treating sepsis.

[0055] In some embodiments of the present application, the sepsis is manifested by a significant increase in the expression level of NLRC3 protein in immune cells of the patient. Further, the immune cells are bone marrow-derived macrophages.

[0056] In some embodiments of the present application, the dosage form of the medicament is one or more of an oral dosage, an intravenous injection, a muscle injection, a subcutaneous injection, an aerosol inhalation, a nasal spray, and a buccal spray.

[0057] The present application verifies the pathogenesis of NLRC3-mediated secondary infection, 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 are wild-type C57 mice, THP cell lines, and bone marrow-derived macrophages (BMDMs) of mice. The isopropyl-β-D-thiogalactopyranoside (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 foreign genes. BMDMs with NLRC3 gene knocked out are NLRC3 - / - Bone marrow-derived macrophages removed from mice.

[0058] Example 1: Effect of NLRC3 knockout on the bactericidal ability of mononuclear macrophages and the formation of LAP bodies

[0059] 1. The present application first discusses the effect of different stimuli on the expression level of NLRC3 in ordinary BMDMs, and each group of cells is treated as follows:

[0060] Normal BMDMs are cultured in DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (P / S, Gibco) in a constant temperature incubator at 37°C and 5% CO2.

[0061] BSA+PBS group: the above-mentioned culture medium added with BSA (without fatty acid, 5%) was used to culture for 48 hours, specifically, the above-mentioned culture medium added with BSA (without fatty acid, 5%) was used to culture cells for 24 hours, and then PBS with the same volume as LPS solvent was added to continue to culture for 24 hours.

[0062] Pal+PBS group: an obese environment was simulated by unsaturated fatty acid stimulation, specifically, palmitic acid (Pal, 5% BSA solution without fatty acid) was used to stimulate cells for 24 hours, and then PBS with the same volume as LPS solvent was added to continue to culture for 24 hours.

[0063] BSA+LPS group: endotoxin tolerance was simulated by lipopolysaccharide (LPS), specifically, the above-mentioned culture medium added with BSA (without fatty acid, 5%) was used to culture for 24 hours, and then LPS was added to stimulate cells for 24 hours.

[0064] Pal+LPS group: BMDMs were cultured in the culture medium containing Pal for 24 hours, and then LPS was added to stimulate for 24 hours.

[0065] The results show that, compared with the BSA+PBS group, the NLRC3 mRNA levels of the Pal+PBS group and the BSA+LPS group are significantly increased, and the NLRC3 mRNA level of the Pal+LPS group is further increased, Figure 1 which indicates that the expression level of NLRC3 in BMDMs is significantly up-regulated in the obese environment and endotoxin tolerance, and NLRC3 may be involved in regulating the antibacterial function of cells.

[0066] 2. The present application further determines the influence of NLRC3 on the bactericidal ability of macrophages by evaluating the killing efficiency of BMDMs on P. aeruginosa (ATCC 27853) under different stimulations:

[0067] 2.1 P. aeruginosa was cultured in LB liquid medium to the logarithmic phase, and then the concentration was adjusted to 10 4 CFU / mL for standby.

[0068] 2.2 Normal BMDMs and BMDMs with NLRC3 gene knocked out were respectively inoculated in 96-well plates at 1×10 4 / well, and cultured for 12 hours; then they were respectively treated in groups:

[0069] BSA+PBS group: the above-mentioned culture medium added with BSA (without fatty acid, 5%) was used to culture for 48 hours, specifically, the above-mentioned culture medium added with BSA (without fatty acid, 5%) was used to culture cells for 24 hours, and then PBS with the same volume as LPS solvent was added to continue to culture for 24 hours.

[0070] Pal+PBS group: simulate the obese environment by stimulating with unsaturated fatty acid, specifically, stimulate the cells with palmitic acid (5% fatty acid-free BSA solution) for 24 hours, then add PBS with the same volume as LPS solvent to continue culturing for 24 hours.

[0071] BSA+LPS group: simulate endotoxin tolerance by LPS, specifically, use the above-mentioned culture medium with BSA (fatty acid-free, 5%) to culture for 24 hours, then add LPS to stimulate the cells for 24 hours.

[0072] Pal+LPS group: culture with the medium containing Pal for 24 hours, then add LPS to stimulate for 24 hours.

[0073] After the culture is completed, add P. aeruginosa of step 2.1 at a concentration of MOI=1 to continue culturing for 2 hours; wash, add gentamicin-containing medium to remove extracellular bacteria, continue to culture for 6 hours, lyse the cells, and plate to count the intracellular surviving bacteria.

[0074] The results show that, compared with the BSA+PBS group, the bactericidal ability of the cells in the Pal+PBS group and the BSA+LPS group is significantly reduced, and the bactericidal ability of the cells in the Pal+LPS group is further reduced (P<0.05) Figure 2 A and Figure 2 B); and in the BSA+LPS group or the Pal+LPS group, the bactericidal ability of the NLRC3-knockout cells is significantly restored relative to the NLRC3 gene non-knockout cells (P<0.05) Figure 3 A and Figure 3 B).

[0075] 3. The present application uses confocal microscopy to observe the formation of LAP bodies to further clarify the role of LAP bodies in the bactericidal ability of NLRC3-affected macrophages:

[0076] 3.1 Adjust the concentration of P. aeruginosa expressing GFP in LB liquid medium to 10 4 CFU / mL after culturing to the mid-log phase, and reserve.

[0077] 3.2 Inoculate ordinary BMDMs and NLRC3 gene-knockout BMDMs into confocal dishes at 1×10 4 per well, and culture for 12 hours; then group and treat as follows:

[0078] BSA+PBS group: use the above-mentioned culture medium with BSA (fatty acid-free, 5%) to culture for 48 hours, specifically, use the above-mentioned culture medium with BSA (fatty acid-free, 5%) to culture the cells for 24 hours, then add PBS with the same volume as LPS solvent to continue culturing for 24 hours.

[0079] Pal+PBS group: simulate the obese environment by stimulating with unsaturated fatty acids, specifically, stimulate the cells with palmitic acid (5% fatty acid-free BSA dissolved) for 24 hours, then add PBS with the same volume as the LPS solvent to continue culturing for 24 hours.

[0080] BSA+LPS group: simulate endotoxin tolerance by LPS, specifically, use the above-mentioned culture medium with BSA (fatty acid-free, 5%) to culture for 24 hours, then add LPS to stimulate the cells for 24 hours.

[0081] Pal+LPS group: culture with the Pal-containing medium for 24 hours, then add LPS to stimulate for 24 hours.

[0082] After the end of the culture, add the bacterial solution obtained in step 3.1 at a concentration of MOI=1, and continue to culture for 1 hour.

[0083] 3.3 After the cells in each group are sequentially fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton-X, and blocked with 2% BSA, they are sequentially incubated with Rubicon primary antibody and AlexaFluor 594-labeled secondary antibody, and the cell nucleus is counterstained with DAPI. Images are collected by a Leica (STELLARIS 8) confocal microscope.

[0084] The results show that in the cells of the Pal+PBS group and the BSA+LPS group, Rubicon cannot effectively localize around the bacteria to form LAP bodies Figure 4 A), while in the BMDMs with NLRC3 gene knocked out, this phenomenon is alleviated Figure 4 B). The above results suggest that NLRC3 weakens the antibacterial function of BMDMs by interfering with the formation of LAP bodies.

[0085] 4. To further clarify the role of Rubicon in the formation of LAP bodies, the present application uses sucrose density gradient centrifugation to separate the intracellular phagosome of BMDMs stimulated with palmitic acid and lipopolysaccharide and infected with Pseudomonas aeruginosa (MOI=1) for 1 hour.

[0086] BSA+PBS group: use the above-mentioned culture medium with BSA (fatty acid-free, 5%) to culture for 48 hours, specifically, use the above-mentioned culture medium with BSA (fatty acid-free, 5%) to culture the cells for 24 hours, then add PBS to continue culturing for 24 hours.

[0087] Pal+PBS group: simulate the obese environment by stimulating with unsaturated fatty acids, specifically, stimulate the cells with palmitic acid (5% fatty acid-free BSA dissolved) for 24 hours, then add PBS with the same volume as the LPS solvent to continue culturing for 24 hours.

[0088] BSA+LPS group: simulate endotoxin tolerance by LPS, specifically, culture with the above-mentioned medium added with BSA (without fatty acid, 5%) for 24 hours, and then add LPS to stimulate the cells for 24 hours.

[0089] Pal+LPS group: culture with the medium containing Pal for 24 hours, and then add LPS to stimulate for 24 hours.

[0090] Specific process: after the cells are infected at different time points (0-2 hours), collect them by centrifugation at 55 g and 4℃ for 7 minutes, resuspend the precipitate in a homogenate buffer containing protease inhibitors, and use Dura Grind the cells by strong homogenization for 10 times with a stainless steel homogenizer. Remove the cell nucleus and unlysed cells by centrifugation of the lysate at 1500 rpm for 5 minutes, and perform sucrose gradient ultracentrifugation (Beckman SW 41 rotor, 28400 rpm, 4℃, 2 hours) on the supernatant. Collect the phagosome component from the 55%-65% sucrose interface. After adjusting the component to 11% sucrose concentration with a sucrose-free homogenate buffer, perform the second ultracentrifugation (10000 rpm, 4℃, 40 minutes) on the component with a 15% Ficoll cushion. Discard the supernatant, and concentrate the phagosome by centrifugation at 10000 rpm and 4℃ again, for subsequent Western Blot analysis.

[0091] The results show that, compared with the BSA+PBS group, the Rubicon protein level in the phagosome component of the cells in the Pal+PBS group and the BSA+LPS group is significantly decreased (Fig. 1A). Figure 5 A). In the BSA+LPS group or the Pal+LPS group, the Rubicon protein level of the NLRC3 knockout cells is significantly increased compared with the NLRC3 gene unknocked cells (Fig. 1B). Figure 5 B). This indicates that Rubicon is expressed at a low level in the LAP body formation process, thereby affecting the normal formation of the LAP body; NLRC3 interferes with the formation of the LAP body.

[0092] The present application proves the influence of palmitic acid and lipopolysaccharide on the expression level of NLRC3 in BMDMs by Example 1, determines the influence of NLRC3 on the bactericidal capacity of BMDMs treated with palmitic acid and lipopolysaccharide, and verifies the conclusion that NLRC3 interferes with the formation of the LAP body, and weakens the antibacterial function of BMDMs.

[0093] Meanwhile, Example 1 determines the role of Rubicon in the LAP body formation, and Rubicon is expressed at a low level in the LAP body formation process, thereby affecting the normal formation of the LAP body.

[0094] Example 2: NLRC3 and PKA-Cα interaction promotes decreased Rubicon expression

[0095] 1. Rubicon is a key regulatory protein in the formation of LAP bodies, and its stability is regulated by ubiquitination degradation mechanism.

[0096] 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).

[0097] 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 ).

[0098] 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.

[0099] 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.

[0100] 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 7B), which indicated that NLRC3 might promote the degradation of Rubicon protein by binding to PKA-Ca.

[0101] 3. Further identified the interaction domain by truncation experiment.

[0102] According to the existing reports, NLRC3 is divided into three domains: 1-60 aa, 61-616 aa, and 617-1065 aa.

[0103] Using the FLAG-NLRC3 overexpression plasmid (Mylone P36112) as a template, each domain-specific primer was used for high-fidelity PCR amplification. The product was recovered and purified by electrophoresis separation (130 V, 20 min), and then digested with Sal I and Not I restriction endonuclease for 4 h, and then connected with pLV3-CMV-FLAG vector (Mylone, P33800) using T4 DNA ligase to construct the transient expression plasmid of NLRC3 (1-60 aa, 61-616 aa, 617-1065 aa) three different domains. The three domain expression plasmids were co-transfected into 293T cells with Flag-PRKACA expression plasmid, and the cell lysate was collected after 48 h for immunoprecipitation.

[0104] As shown in Figure 8 , NLRC3 interacts with PKA-Ca through its NBD domain (61-616 aa).

[0105] 4. To verify the direct interaction, the GST-labeled NBD domain (61-616 aa) protein of NLRC3 and the FLAG-labeled full-length protein (FL) of PKA-Ca were purified, respectively.

[0106] Specifically, the FLAG-NLRC3 overexpression plasmid (Mingling P36112) was used as a template for high-fidelity PCR amplification with domain-specific primers (1-60 aa: upstream primer (SEQ ID NO: 3): ATGAGGAAGCAAGGGGTGCGGACGG, downstream primer (SEQ ID NO: 4): CCCGCTGGGGCCCTGCAGCAATGAC; 61-616 aa: upstream primer (SEQ ID NO: 5): TCAAGGATACAGAGGCACCGCAAGG, downstream primer (SEQ ID NO: 6): CCTGCAGGTGTCCGACGCCTGTGCC; 617-1065 aa: upstream primer (SEQ ID NO: 7): GAGGCCAACCTGTCCCTGAGCCTCA, downstream primer (SEQ ID NO: 8): TGCTCCCACGTGCACTGTTGAAATG). The product was electrophoretically separated (130 V, 20 min) and purified, then digested with Sal I and Not I restriction enzymes for 4 h, and ligated with pET-GST vector (addgene, 42049) using T4 DNA ligase to construct NLRC3 truncated protein expression plasmids.

[0107] The pET-GST vector (purchased from addgene, 42049) and plasmids encoding GST-NLRC3 truncates (1-60 aa, 61-616 aa, 617-1065 aa) were transformed into BL21 competent cells, and the target proteins were expressed by inducing with isopropyl-beta-D-thiogalactoside (1 mM) at 18°C for 16 hours. After lysing the bacterial cells, the fusion proteins were captured using GST affinity magnetic beads, eluted and dialyzed with 10 mM glutathione-Tris-HCl buffer to obtain purified proteins. Meanwhile, FLAG-PRKACA was expressed by transfecting HEK293T cells, total proteins were extracted using NP-40 lysis buffer, and high-purity FLAG-RKA-Cα protein was obtained by immunoprecipitation using anti-FLAG agarose beads and 3xFLAG polypeptide competitive elution. The purified GST-NLRC3 and FLAG-PKA-Cα proteins, full-length and 241-250 aa deletion proteins, were incubated at 4°C overnight, GST agarose beads were added and incubated in PBS containing protease inhibitors for 2 h, and pull-down experiments were performed. PBS was washed thoroughly and immunoblotting analysis was performed.

[0108] Results showed that NBD domain of NLRC3 (61-616 aa) directly bound to PKA-Ca full-length protein or the protein with deletion of amino acids 241-250 Figure 9 ).

[0109] Example 3: Construction of Q5K

[0110] Figure 10 is a schematic diagram of the three-dimensional structure of the NBD domain of NLRC3; Figure 11 is a molecular docking model of Q5K (red) and the NBD domain of NLRC3 (light yellow).

[0111] According to the amino acid sequence YGRKKRRQRRRGSGQIYEK (SEQ ID NO: 1) of Q5K, the encoding DNA sequence was chemically synthesized and cloned into the pET-GST vector (addgene, 42049) by enzyme digestion and ligation to construct a Q5K polypeptide expression plasmid.

[0112] Specifically, 2 μL of the Q5K polypeptide expression plasmid was transformed into BL21 (DE3) competent cells, which were plated on LB plates containing the corresponding antibiotic and incubated at 37°C for 17 hours. A single colony was picked and inoculated into 4 mL of LB medium containing the antibiotic, and incubated at 37°C and 220 rpm for 0.6-0.8 OD600. Then the cells were treated by induction group with isopropyl-β-D-thiogalactoside for 6 hours, and the control group was not treated. The cells were collected by centrifugation, resuspended in buffer, and then broken by ultrasonic in ice bath. The supernatant and precipitate samples were mixed with reduced loading buffer and boiled, and then analyzed by SDS-PAGE after centrifugation.

[0113] Western Blot detection was performed by wet transfer of the protein to a PVDF membrane, followed by incubation with His-tag polyclonal antibody (AP0032) and HRP-labeled secondary antibody (BS13278), and finally developed and detected. For large-scale culture, the seed liquid was inoculated into 500 mL of LB medium at a ratio of 1:100, and the cells were collected by centrifugation after induction. The cells were broken by ultrasonic at a ratio of 1:20 (mass volume ratio), and the lysate was subjected to Ni column chromatography, followed by gradient elution with equilibration buffer, 50 mM and 350 mM imidazole buffer. The final eluate was dialyzed at a ratio of 1:1000, and SUMO enzyme (10 μL of enzyme solution for 1 mg of sample) was added for enzyme digestion at 4°C for 16 h. The enzyme digestion product was purified again by Ni column, and the flow-through liquid was collected to obtain the target protein. The protein was further purified by reverse-phase high-performance liquid chromatography, and then stored after identification by mass spectrometry.

[0114] Protein mass spectrometry results Figure 12) shows that the amino acid sequence of the purified protein is consistent with the Q5K design sequence, indicating that the polypeptide has been successfully synthesized.

[0115] The present application further provides verification examples to illustrate the effect of Q5K:

[0116] Example 4: Q5K can specifically bind to the NBD domain of NLRC3

[0117] The Q5K provided by the present application can specifically bind to the NBD domain of NLRC3.

[0118] Specifically, surface plasmon resonance (SPR) constant determination interaction was completed by using a Biacore 8k instrument (Cytiva, USA) equipped with a CM5 sensor chip, and the buffer was phosphate buffered saline (PBST) with 0.5% Tween-20, pH 7.4.

[0119] The instrument operation steps were performed according to the instructions. First, 100 μL of 100 mM GST-NLRC3 truncated body (61-616 aa.) purified in Example 2 was immobilized on the sensor chip in the Biacore 8k instrument. Q5K was combined with the sensor chip at a rate of 20 μL / min for 120 s, and then dissociated in PBST analyte buffer for 180 s. Q5K was tested for binding at 0.78 to 200 μM (0.78125, 3.1256, 12.5, 50, 200 μM). See Figure 13 A), and the binding constant was determined.

[0120] The results show that the binding constant (K D ) of Q5K to the NBD domain of NLRC3 is about 5.82×10 -5 M (see Figure 13 B). This indicates that Q5K has very strong specific interaction with the NBD domain of NLRC3.

[0121] Example 5: Verification method of Q5K mechanism

[0122] To verify the effect of Q5K on the binding of NLRC3 to PKA and its effect on Rubicon-mediated LC3-related phagocytosis (LAP) formation, first, the NLRC3 overexpression vector with a FLAG tag and the overexpression PKA-Cα protein vector (PKA-Cα overexpression plasmid) with an HA tag were constructed using a blank vector (purchased from Miro, FLAG: P33800; HA: P1750), respectively.

[0123] Specifically, the PKA-Ca overexpression plasmid (Mylone P92701) and the NLRC3 overexpression plasmid (Mylone P36112) were used as templates, and specific primers (PKA-Ca: 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.) were used for high-fidelity PCR amplification. The product was recovered and purified by electrophoresis (130V, 20min), and then digested with SalI and NotI restriction endonucleases for 4h, and T4 DNA ligase was used to construct the FLAG-NLRC3 overexpression plasmid and the HA-PKA-Ca overexpression plasmid with the blank vector (purchased from Mylone, FLAG: P33800; HA: P1750).

[0124] The FLAG-NLRC3 overexpression plasmid and the HA-PKA-Ca overexpression plasmid were simultaneously transiently transfected into 293T cells; 48 hours later, the cells were treated in groups and the cell lysates were collected for immunoprecipitation (Co-IP); the group treatment operation is as follows:

[0125] Control group: treated with PBS for 12 hours.

[0126] TAT group: treated with transmembrane peptide (TAT, 10μM) for 12 hours.

[0127] Q5K group: treated with Q5K (10μM) for 12 hours.

[0128] As Figure 14 It is shown that the interaction level of NLRC3 and PKA-Ca in the Q5K group is significantly reduced compared with the control group or the TAT group, indicating that Q5K interferes with the binding of NLRC3 and PKA.

[0129] THP-1 (ATCC, TIB-202) cell line, induced into macrophages; using phorbol ester (sigma, 37558-16-0) 100ng / ml, induced into M0 macrophages for 24 hours;

[0130] To further verify the role of PKA-Ca in the process of Q5K mediating NLRC3 to degrade Rubicon, the vector pLKO.1 (purchased from Miro, P0258) was used to construct and transfect the PRKACA knockdown vector (shPRKACA, SEQ ID NO: 13: 5'- AGCGTGAAAGAATTCTTAGCC-3') to specifically inhibit PKA activity. The control group cells were transfected with empty vector (vector pLKO.1, purchased from Miro, P0258). The cells were cultured in DMEM high glucose medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (P / S, Gibco) in a constant temperature incubator at 37°C and 5% CO2, and then replaced with Pal-containing medium for 24 hours, and then added with LPS for 24 hours. Finally, the following stimulation was performed:

[0131] TAT group: add TAT (10 mM) for 12 hours.

[0132] Q5K group: add Q5K (10 mM) for 12 hours.

[0133] After the treatment, the total protein of the cells was extracted, and the expression level of Rubicon was detected by immunoblotting.

[0134] The results showed that the expression level of Rubicon protein in the cells with PRKACA knocked down was significantly reduced compared with the control group, which indicated that NLRC3 affected the ubiquitination degradation of Rubicon by interacting with PKA-Ca, and Q5K stabilized the expression level of Rubicon protein by blocking the interaction. Figure 15 ).

[0135] To further evaluate the effect of Q5K on the formation of Rubicon-related LAP, confocal microscopy was used to observe the colocalization of Rubicon and bacteria after macrophages phagocytosed P. aeruginosa. The specific steps were as follows: 1x10 4 BMDMs were inoculated in confocal microscopy special culture dishes and cultured in a 37°C, 5% CO2 incubator until they were completely adherent. The cells were treated with 200 mM palmitic acid for 24 hours to simulate a high-fat environment, and then added with 100 ng / mL LPS for 24 hours to establish an immune tolerance model. Each group was added with 10 mM Q5K or the same volume of PBS for 2 hours, and then added with 1x10 7 CFU / mL fluorescent P. aeruginosa (PA) for 1 hour. The cells were fixed and immunofluorescence staining was performed to label Rubicon protein.

[0136] For example, Figure 16The results show that, compared with the control group, Rubicon in the Q5K group is significantly co-localized with bacteria, indicating that Q5K can enhance the stability of Rubicon and promote the formation of LC3-related phagocytosis, thereby improving the clearance efficiency of intracellular pathogens by host cells.

[0137] In summary, Q5K can block the binding of NLRC3 and PKA, inhibit the degradation of Rubicon by downstream signals, maintain the stability of Rubicon, and promote the formation of LAP.

[0138] Example 6: Verification of the bactericidal ability of Q5K as a drug in vitro experiments

[0139] To evaluate the effective concentration range of the antibacterial effect of Q5K, bone marrow-derived macrophages were seeded at a density of 1×10 4 per well in a 96-well plate and cultured for 12 hours. Then, 0, 0.625, 1.25, 2.5, 5, 10, and 20 μM of Q5K polypeptide solution was added, respectively, and incubation was continued for 2 hours. Then, P. aeruginosa expressing GFP was infected at an MOI of 1, and co-cultured at 37°C for 2 hours. After washing three times with preheated PBS, the medium containing gentamicin was replaced to remove extracellular bacteria, and incubation was continued for 12 hours. The cells were lysed with 0.5% Triton-X, and intracellular bacteria were collected. Half of the lysate was used for LB plate coating and colony counting analysis ( Figure 17 ), and the other half was used for immunoblotting detection ( Figure 18 ).

[0140] The experimental results show that Q5K exhibits significant antibacterial activity at more than 2.5 μM, and can effectively enhance the clearance ability of macrophages against intracellular P. aeruginosa.

[0141] Example 7: Method for verifying the in vitro biological safety of Q5K

[0142] To evaluate the in vitro biological safety of the antibacterial Q5K of the present application, the present application systematically evaluated the effect of Q5K on the activity of mammalian cells using the CCK-8 method to determine the safe concentration range for use.

[0143] Mouse bone marrow-derived macrophages were seeded at a density of 1×10 4 per well in a 96-well cell culture plate and pre-cultured at 37°C in a 5% CO2 incubator until the cells were completely adherent. Then, the medium containing Pal was replaced and cultured for 24 hours, followed by LPS stimulation for 24 hours, and then the cells were grouped and treated as follows:

[0144] The experimental group (multiple groups): different concentrations of Q5K polypeptide were added to the culture system to make the final concentrations 2, 4, 8, 16, 24, 48, 96, 128, and 256 μM, respectively, and incubation was continued for 12 hours.

[0145] Control group: Add an equal volume of PBS to the culture system and continue culturing for 12 hours.

[0146] 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-mode 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.

[0147] 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.

[0148] Example 8: Verification method of Q5K in vivo effects

[0149] 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.

[0150] 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.

[0151] 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).

[0152] The above results demonstrate that Q5K can effectively improve the survival rate of sepsis normal weight mice and obese mice, and has the development potential as a preventive or therapeutic drug against secondary bacterial infection mediated by NLRC3 expression increase.

[0153] From the above examples, it is known that the Q5K provided by the present application is designed according to the structure of PKA-Cα, can be combined with the NBD domain (61-616 aa) of NLRC3 through specificity, hinder the combination of NLRC3 and PKA-Cα, increase the expression level of PKA phosphorylation and Run domain-containing Beclin-1 interaction and cysteine-rich domain-containing protein, and then promote the formation of LAP and intracellular bactericidal function, and does not interfere with the expression level of NLRC3, realizes the targeted research and development of NLRC3-mediated secondary bacterial infection drugs such as sepsis or obese sepsis.

[0154] In vivo and in vitro experiments, verification shows that the polypeptide exhibits significant antibacterial activity when greater than 2.5 μM, can effectively enhance the clearance ability of macrophages to intracellular P. aeruginosa, and can treat NLRC3-mediated secondary infection related to immune disorders such as sepsis or obese sepsis, improve the survival rate of mice, and has the development potential as an anti-infection and sepsis treatment drug.

[0155] And it is found in the experiment that the CC50 of Q5K is 217.22 μM, which has no effect on cell activity within a safe dose, indicating that it has very good drug safety, is a very effective agent for increasing the bactericidal function of cells in the pathological state mediated by NLRC3, and can be used for the preparation of drugs for the treatment or prevention of NLRC3-mediated secondary bacterial infection such as sepsis or obese sepsis.

[0156] The above examples are only exemplary embodiments of the present application and are not intended to limit the present application, and the protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements to the present application within the spirit and protection scope of the present application, and such modifications or equivalent replacements shall also be considered to fall within the protection scope of the present application.

Claims

1. A polypeptide for modulating sepsis antibacterial function, 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 vector, characterized by, 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 by: The engineered cells contain the coding gene of claim 2 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 by, 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 by: 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. 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. Use according to claim 7, characterized in that: The medication for treating sepsis is used to modulate the antibacterial function in sepsis.

10. Use according to claim 7, characterized in that: The dosage form of the drug is one or more of the following: intravenous injection, intramuscular injection, and subcutaneous injection.

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