Engineered gram-negative rod-shaped bacterium and use thereof in vaccine

Abstract

Provided are a novel attenuating strategy for Klebsiella pneumoniae and a use thereof in a vaccine. Specifically, provided is an engineered Gram-negative rod-shaped bacterium. The gene of an endogenous phospholipid transfer system of the Gram-negative rod-shaped bacterium is knocked out, so that the pathogenicity of hypervirulent Klebsiella pneumoniae can be greatly reduced; and the attenuated strain can be used for preventing and / or treating Gram-negative rod-shaped bacterial infections or diseases related thereto, and inhibiting the expression of the gene or protein (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) of the phospholipid transfer system, and can also be used for preventing and / or treating Gram-negative rod-shaped bacterial infections or diseases related thereto.

Description

An engineered Gram-negative rod-shaped bacterium and its application in vaccines Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a novel attenuation strategy for Klebsiella pneumoniae and its application in vaccines. Background Technology

[0002] Klebsiella pneumoniae is a common Gram-negative rod-shaped bacterium with a capsular polysaccharide layer. It lacks a flagellar system and motility. Clinically, Klebsiella pneumoniae is prone to drug resistance and commonly causes iatrogenic nosocomial infections. In recent years, its clinical isolation rate has been increasing annually, making it one of the leading causes of hospital-acquired infections and death in critically ill patients. The specific pathogenic mechanism of Klebsiella pneumoniae is unclear; it does not contain clearly defined protein virulence factors. The main pathogenic factor is the capsular polysaccharide on the bacterial surface, and the production of capsular polysaccharide is highly correlated with pathogenicity. In 1986, a highly virulent variant of Klebsiella pneumoniae with thickened capsula first appeared in my country and East Asia. This is a typical highly virulent superbug that has appeared in multiple countries and regions in recent years and shows a trend of spread. It has been identified by the World Health Organization as a priority pathogen with a potential global pandemic risk. Highly virulent Klebsiella pneumoniae can infect healthy humans and animals with intact immune systems, causing community-acquired pneumonia and multi-organ infections. Currently, there are no effective vaccines for humans or veterinary use. Highly virulent Klebsiella pneumoniae infection, accompanied by disseminated lesions, can rapidly metastasize to the eyes, lungs, and central nervous system, leading to serious conditions such as pneumonia, liver abscess, gastroenteritis, endophthalmitis, and meningitis. Characteristics of highly virulent Klebsiella pneumoniae infection include rapid disease progression, multiple foci of infection, and poor prognosis. Statistics show that the mortality rate for patients with invasive syndrome is as high as 3% to 31%, while the mortality rate for patients with bacteremia can even exceed 35%.

[0003] Currently, there are no effective preventative vaccines against the various infectious diseases caused by Klebsiella pneumoniae, and these vaccines remain in the laboratory research stage. Due to the numerous capsular polysaccharide types and complex surface antigen variations in clinical strains of Klebsiella pneumoniae, and the lack of conserved protein candidate antigens, developing a broad-spectrum and effective vaccine is extremely challenging, resulting in long development cycles and high costs for novel vaccines. Furthermore, the increasing drug resistance of Klebsiella pneumoniae, which is generally resistant to many commonly used antibiotics, coupled with the lack of effective anti-infective drugs, poses significant difficulties for clinical treatment, causing substantial losses to patients and the animal husbandry industry.

[0004] Therefore, there is an urgent need in this field to discover key protein virulence factors on the surface of Klebsiella pneumoniae and to rapidly develop effective vaccines or anti-infective drugs based on these key pathogenic factors for the prevention and treatment of human and animal diseases caused by superbug infections. Summary of the Invention

[0005] The purpose of this invention is to provide a genetically engineered strain that can effectively reduce the virulence and pathogenicity of bacteria, is simple and convenient to operate, low in cost, and can be used for the rapid development of attenuated vaccines to prevent Klebsiella pneumoniae infection.

[0006] In a first aspect, the present invention provides an engineered Gram-negative rod-shaped bacterium in which the gene for the endogenous phospholipid transport system of the Gram-negative rod-shaped bacterium has been knocked out.

[0007] In another preferred embodiment, the Gram-negative rod-shaped bacteria include pathogens such as Klebsiella pneumoniae, pathogenic Escherichia coli, Enterobacter cloacae, and Acinetobacter baumannii.

[0008] In another preferred embodiment, the Klebsiella pneumoniae includes a highly virulent Klebsiella pneumoniae with a capsule.

[0009] In another preferred embodiment, the phospholipid transport system is selected from the group consisting of MlaA, MlaB, MlaC, MlaD, MlaE, MlaF, or combinations thereof.

[0010] In another preferred embodiment, the sequence of the gene encoding MlaA is SEQ ID NO.1.

[0011] In another preferred embodiment, the sequence of the gene encoding MlaB is SEQ ID NO.2.

[0012] In another preferred embodiment, the sequence of the gene encoding MlaC is SEQ ID NO.3.

[0013] In another preferred embodiment, the sequence of the gene encoding MlaD is SEQ ID NO.4.

[0014] In another preferred embodiment, the sequence of the gene encoding MlaE is SEQ ID NO.5.

[0015] In another preferred embodiment, the sequence of the gene encoding MlaF is SEQ ID NO.6.

[0016] A second aspect of the present invention provides a pharmaceutical composition comprising the engineered Gram-negative rod-shaped bacteria described in the first aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipients.

[0017] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0018] In another preferred embodiment, the vaccine composition is monovalent or polyvalent.

[0019] In another preferred embodiment, the pharmaceutical composition further comprises an adjuvant, preferably various aluminum adjuvants.

[0020] In another preferred embodiment, the molar or weight ratio of engineered Gram-negative rod-shaped bacteria and adjuvant (such as aluminum) in the pharmaceutical composition is between 1:100, preferably between 1:40 and 1:60.

[0021] In another preferred embodiment, the pharmaceutical composition includes a single drug, a combination drug, or a synergistic drug.

[0022] In another preferred embodiment, the dosage form of the pharmaceutical composition is liquid, solid, or gel.

[0023] In another preferred embodiment, the pharmaceutical composition is administered by means selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral / nasal spray and nebulized inhalation.

[0024] A third aspect of the present invention provides a vaccine composition comprising the engineered Gram-negative rod-shaped bacteria described in the first aspect of the present invention, and an immunologically acceptable carrier and / or excipient.

[0025] In another preferred embodiment, the vaccine composition further contains an adjuvant.

[0026] In another preferred embodiment, the adjuvant includes particulate and non-particulate adjuvants.

[0027] In another preferred embodiment, the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or combinations thereof.

[0028] In another preferred embodiment, the non-particulate adjuvant is selected from the group consisting of: muramyl dipeptide and its derivatives, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products such as mycobacteria (tuberculosis bacillus, BCG), pumilus, pertussis bacillus, propolis, or combinations thereof.

[0029] In another preferred embodiment, the adjuvant includes alumina, saponins, Quil A, muramyl dipeptide, mineral oil or vegetable oil, vesicle-based adjuvants, nonionic block copolymers or DEAE dextran, and cytokines.

[0030] In another preferred embodiment, the vaccine composition comprises an injectable formulation.

[0031] The fourth aspect of the present invention provides the use of engineered Gram-negative rod-shaped bacteria as described in the first aspect of the present invention, pharmaceutical compositions as described in the second aspect of the present invention, or vaccine compositions as described in the third aspect of the present invention, (a) for the preparation of antibodies against Gram-negative rod-shaped bacteria; and / or (b) for the preparation of medicaments for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0032] In another preferred embodiment, the diseases associated with the Gram-negative rod-shaped bacterial infection include bacterial pneumonia, liver abscess, gastroenteritis, endophthalmitis, meningitis, bacteremia, and sepsis.

[0033] In another preferred embodiment, the Gram-negative rod-shaped bacteria include Klebsiella pneumoniae, pathogenic Escherichia coli, Enterobacter cloacae, and Acinetobacter baumannii.

[0034] In another preferred embodiment, the drug is also used for one or more purposes selected from the group consisting of:

[0035] (a) Significantly reduced capsular polysaccharide production in Gram-negative rod-shaped bacteria;

[0036] (b) Significantly reduced the viscosity of capsular polysaccharides in Gram-negative rod-shaped bacteria;

[0037] (c) Reduced ability to infect and colonize in mammals;

[0038] (d) Reduce pathogenicity in mammals.

[0039] In another preferred embodiment, the Gram-negative rod-shaped bacterial infection includes Klebsiella pneumoniae infection, pathogenic Escherichia coli infection, Enterobacter cloacae infection, and Acinetobacter baumannii infection.

[0040] In another preferred embodiment, the mammal includes humans or non-human mammals.

[0041] In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, and rabbits) and primates (such as monkeys).

[0042] In another preferred embodiment, the antibody includes an antibody against Klebsiella pneumoniae.

[0043] The fifth aspect of the present invention provides a method for generating an immune response against Gram-negative rod-shaped bacteria, comprising the steps of administering to a desired subject the engineered Gram-negative rod-shaped bacteria of the first aspect of the present invention, the pharmaceutical composition of the second aspect of the present invention, or the vaccine composition of the third aspect of the present invention.

[0044] In another preferred embodiment, the object includes a human or a non-human mammal.

[0045] In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, and rabbits) and primates (such as monkeys).

[0046] In another preferred embodiment, the method induces the production of neutralizing antibodies against Gram-negative rod-shaped bacteria in the subject.

[0047] The sixth aspect of the present invention provides a treatment method for administering engineered Gram-negative rod-shaped bacteria as described in the first aspect of the present invention, a pharmaceutical composition as described in the second aspect of the present invention, or a vaccine composition as described in the third aspect of the present invention to a subject in need.

[0048] In another preferred embodiment, the treatment method includes gene therapy.

[0049] The seventh aspect of the present invention provides the use of an inhibitor of a phospholipid transport system gene or its encoded protein for the preparation of a medicament for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0050] In another preferred embodiment, the Gram-negative rod-shaped bacterial infection includes Klebsiella pneumoniae infection, pathogenic Escherichia coli infection, Enterobacter cloacae infection, and Acinetobacter baumannii infection.

[0051] In another preferred embodiment, the diseases associated with the Gram-negative rod-shaped bacterial infection include bacterial pneumonia, liver abscess, gastroenteritis, endophthalmitis, meningitis, bacteremia, and sepsis.

[0052] In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, antisense oligonucleotides, peptide nucleic acids (PNA), morpholino oligonucleotides (PMO), nucleic acid aptamers, CRISPR reagents, or combinations thereof.

[0053] In another preferred embodiment, the inhibitor comprises a non-coding small RNA.

[0054] In another preferred embodiment, the phospholipid transport system is selected from the group consisting of MlaA, MlaB, MlaC, MlaD, MlaE, MlaF, or combinations thereof.

[0055] In another preferred embodiment, the phospholipid transport system is derived from bacteria, preferably from Gram-negative rod-shaped bacteria, and more preferably from Klebsiella pneumoniae.

[0056] In another preferred embodiment, the phospholipid transport system protein includes wild-type phospholipid transport system protein and mutant phospholipid transport system protein.

[0057] In another preferred embodiment, the mutant includes a mutant form in which the function of the encoded protein is not altered after mutation (i.e., the function is the same as or substantially the same as the wild-type encoded protein).

[0058] In another preferred embodiment, the phospholipid transport system protein includes an active fragment of a phospholipid transport system or a derivative thereof.

[0059] In another preferred embodiment, the active fragment or its derivative has at least 90% homology with the phospholipid transport system, preferably 95%, more preferably 98% or 99%.

[0060] In another preferred embodiment, the active fragment or its derivative has at least 80%, 85%, 90%, 95%, or 100% of mlaA activity.

[0061] In another preferred embodiment, the MlaA protein has the accession number WP_002913362.1 (Klebsiella pneumoniae accession number).

[0062] In another preferred embodiment, the MlaB protein has the accession number WP_004174142.1 (Klebsiella pneumoniae accession number).

[0063] In another preferred embodiment, the MlaC protein has the accession number WP_038432601.1 (Klebsiella pneumoniae accession number).

[0064] In another preferred embodiment, the MlaD protein has the accession number WP_002918387.1 (Klebsiella pneumoniae accession number).

[0065] In another preferred embodiment, the MlaE protein has the accession number WP_004150949.1 (Klebsiella pneumoniae accession number).

[0066] In another preferred embodiment, the MlaF protein has the accession number WP_004150950.1 (Klebsiella pneumoniae accession number).

[0067] In another preferred embodiment, the inhibitor inhibits the activity and / or expression level of phospholipid transport system proteins.

[0068] An eighth aspect of the present invention provides a pharmaceutical composition comprising:

[0069] (a) Inhibitors of phospholipid transport system genes or their encoded proteins; and

[0070] (b) Pharmaceutically acceptable carriers.

[0071] In another preferred embodiment, the pharmaceutical composition further includes:

[0072] (c) Other medications for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0073] In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, antisense oligonucleotides, peptide nucleic acids (PNA), morpholino oligonucleotides (PMO), nucleic acid aptamers, CRISPR reagents, or combinations thereof.

[0074] In another preferred embodiment, the inhibitor comprises a non-coding small RNA.

[0075] In another preferred embodiment, the content of component (a) in the pharmaceutical composition is 1%-99%, more preferably 10%-90%, and even more preferably 30%-70%.

[0076] In another preferred embodiment, the content of component (c) in the pharmaceutical composition is 1%-99%, more preferably 10%-90%, and even more preferably 30%-70%.

[0077] In another preferred embodiment, the component (a) and optional component (c) constitute 0.01-99.99 wt% of the total weight of the pharmaceutical composition, more preferably 0.1-90 wt%, and even more preferably 1-80 wt%.

[0078] In another preferred embodiment, the other drugs for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases include meropenem, tigecycline, levofloxacin, and polymyxin.

[0079] In another preferred embodiment, the dosage form of the pharmaceutical composition includes injectable dosage forms and oral dosage forms.

[0080] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

[0081] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0082] A ninth aspect of the present invention provides a medicine box, comprising:

[0083] (a1) A first container, and an inhibitor of a phospholipid transport system gene or its encoded protein located in the first container, or a drug containing an inhibitor of a phospholipid transport system gene or its encoded protein.

[0084] In another preferred embodiment, the pillbox further includes:

[0085] (b1) A second container, and other medications for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases located in the second container, or medications containing other medications for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0086] In another preferred embodiment, the first container and the second container may be the same or different containers.

[0087] In another preferred embodiment, the drug in the first container is a single-agent formulation containing an inhibitor of a phospholipid transport system gene or its encoded protein.

[0088] In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing other drugs for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0089] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injectable dosage form.

[0090] In another preferred embodiment, the kit also includes instructions for use.

[0091] In another preferred embodiment, the specification includes one or more descriptions selected from the group consisting of:

[0092] (a) Using inhibitors of phospholipid transport system genes or their encoded proteins for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases;

[0093] (b) Inhibitors of phospholipid transport system genes or their encoded proteins are used in combination with other drugs for the prevention and / or treatment of Gram-negative baculopathic infections or related diseases.

[0094] The tenth aspect of the present invention provides the use of the pharmaceutical composition described in the eighth aspect of the present invention or the medicament described in the ninth aspect of the present invention for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0095] In another preferred embodiment, the pharmaceutical composition or kit comprises (a) an inhibitor of a phospholipid transport system gene or the protein encoded therein; and (b) optionally other medicaments for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases; and (d) a pharmaceutically acceptable carrier.

[0096] In another preferred embodiment, the pharmaceutical composition or kit contains (b) an inhibitor of the phospholipid transport system gene or its encoded protein; and optionally other drugs for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases, comprising 0.01-99.99 wt%, more preferably 0.1-90 wt%, and more preferably 1-80 wt% of the total weight of the pharmaceutical composition or kit.

[0097] The eleventh aspect of the present invention provides a method for preventing and / or treating Gram-negative rod-shaped bacterial infections or related diseases, comprising:

[0098] Administer an inhibitor of a phospholipid transport system gene or its encoded protein to the desired subject; or the pharmaceutical composition described in the eighth aspect of the present invention or the medicament described in the ninth aspect of the present invention.

[0099] In another preferred embodiment, the object includes a human or non-human mammal suffering from a Gram-negative rod-shaped bacterial infection or a related disease.

[0100] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0101] In another preferred embodiment, the inhibitor of the phospholipid transport system gene or its encoded protein is administered simultaneously or sequentially with optional other drugs for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0102] The twelfth aspect of the present invention provides a method for screening candidate substances for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases, the method comprising the steps of:

[0103] (a) In the test group, the test compound was added to the cell culture system, and the expression level (E1) and / or activity (A1) of phospholipid transport system protein in the cells of the test group were observed; in the control group, the test compound was not added to the same cell culture system, and the expression level (E0) and / or activity (A0) of phospholipid transport system protein in the cells of the control group were observed.

[0104] If the expression level (E1) and / or activity (A1) of phospholipid transport system proteins in the test group are significantly lower than those in the control group, it indicates that the test compound is a candidate substance for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases that inhibits the expression and / or activity of phospholipid transport systems.

[0105] In another preferred embodiment, the expression level of the phospholipid transport system protein is determined by quantitative real-time PCR or immunohistochemistry.

[0106] In another preferred embodiment, the method further includes the step of:

[0107] (b) For the candidate substances obtained in step (a), further test their therapeutic effects on Gram-negative rod-shaped bacterial infections or related diseases; and / or further test their downregulation of phospholipid transport system proteins.

[0108] In another preferred embodiment, the method includes step (c): applying the candidate substance identified in step (a) to a mammalian model and determining its effect on infection with or related to Gram-negative rod-shaped bacteria in mammals.

[0109] In another preferred embodiment, the mammal is a mammal suffering from Gram-negative rod-shaped bacterial infection or a related disease.

[0110] In another preferred embodiment, “significantly lower than” means E1 / E0 ≤ 1 / 2, more preferably ≤ 1 / 3, and even more preferably ≤ 1 / 4.

[0111] In another preferred embodiment, “significantly lower than” means A1 / A0 ≤ 1 / 2, more preferably ≤ 1 / 3, and even more preferably ≤ 1 / 4.

[0112] In another preferred embodiment, the cells comprise prokaryotic cells.

[0113] In another preferred embodiment, the cells comprise bacterial cells.

[0114] In another preferred embodiment, the cells comprise Gram-negative rod-shaped bacterial cells.

[0115] In another preferred embodiment, the cells are cells cultured in vitro.

[0116] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0117] In another preferred embodiment, the candidate substance is selected from the group consisting of antibodies, small molecule compounds, antisense oligonucleotides, peptide nucleic acids (PNA), morpholino oligonucleotides (PMO), nucleic acid aptamers, CRISPR reagents, or combinations thereof.

[0118] In another preferred embodiment, the candidate substance includes the non-coding small RNA ArcZ.

[0119] The thirteenth aspect of this invention provides a method for screening candidate substances for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases, the method comprising the steps of:

[0120] (i) Contacting the candidate material with phospholipid transport system proteins; and

[0121] (ii) Determine the functional effect of the candidate substance on a phospholipid transport system protein, wherein the functional effect indicates that the compound prevents and / or treats Gram-negative rod-shaped bacterial infections or related diseases.

[0122] In another preferred embodiment, the phospholipid transport system protein comprises a recombinant phospholipid transport system protein.

[0123] In another preferred embodiment, the functional effect is measured in vitro.

[0124] In another preferred embodiment, the functional effect is determined by measuring the expression of the phospholipid transport system.

[0125] In another preferred embodiment, the expression level of the phospholipid transport system is determined by quantitative real-time PCR or immunohistochemistry.

[0126] In another preferred embodiment, the functional effect is measured in prokaryotic cells.

[0127] In another preferred embodiment, the prokaryotic cell comprises a bacterial cell.

[0128] In another preferred embodiment, the prokaryotic cells comprise Gram-negative rod-shaped bacterial cells.

[0129] In another preferred embodiment, the functional effect is the inhibition of the expression or activity of the phospholipid transport system.

[0130] In another preferred embodiment, the candidate substances include: antibodies, small molecule compounds, peptide nucleic acids (PNA), morpholino oligonucleotides (PMO), oligonucleotides, nucleic acid aptamers, and CRISPR reagents.

[0131] In another preferred embodiment, the candidate substance includes the non-coding small RNA ArcZ.

[0132] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0133] Figure 1 shows the construction of a genetically engineered strain that lacks the mlaA gene.

[0134] Figure 2 shows the protein imprinting assay used to detect the expression of MlaA protein.

[0135] Figure 3 shows the determination of bacterial capsule polysaccharide content by uronic acid quantification experiment.

[0136] Figure 4 shows the viscosity of Klebsiella pneumoniae capsular polysaccharide as quantitatively analyzed by sedimentation experiments;

[0137] **: p-value < 0.01, Student's t-test.

[0138] Figure 5 shows the results of in vitro and in vivo competition experiments between wild-type and genetically engineered strains lacking mlaA; **: p-value < 0.01, Mann-Whitney U test.

[0139] Figure 6 shows the bacterial load of Klebsiella pneumoniae in major organs of mice after infection, determined by colony counting; (left) Klebsiella pneumoniae count in the lungs; (middle) Klebsiella pneumoniae count in the spleen; (right) Klebsiella pneumoniae count in the liver. **: p-value < 0.01, Mann-Whitney U test.

[0140] Figure 7 shows the survival curves of mice infected with Klebsiella pneumoniae.

[0141] Figure 8 shows that the non-coding small RNA ArcZ can significantly reduce the number of bacteria and the degree of infection in mouse lungs by targeting and inhibiting the expression of MlaA.

[0142] Figure 9 shows that the attenuated strain with mlaA knocked out has good safety and vaccine protection. Detailed Implementation

[0143] Through extensive and in-depth research, the inventors unexpectedly discovered that knocking out genes (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) of the endogenous phospholipid transport system in Gram-negative baculoform bacteria can yield attenuated strains. These strains can be used (a) to prepare antibodies against Gram-negative baculoform bacteria; and / or (b) to prepare drugs for the prevention and / or treatment of Gram-negative baculoform bacterial infections or related diseases. Furthermore, the inventors also discovered that inhibiting the expression of genes or proteins of the phospholipid transport system (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) can also be used to prevent and / or treat Gram-negative baculoform bacterial infections or related diseases. Based on these findings, the inventors completed this invention.

[0144] Gram-negative rod-shaped bacteria

[0145] Gram-negative rod-shaped bacteria are a type of prokaryotic microorganism without a nucleus and possessing a double cell membrane. They are rod-shaped, typically 1-3 micrometers in length, and are classified as Gram-negative bacteria according to the Gram staining method. After Gram staining, the bacteria appear red. Common Gram-negative bacteria include *Escherichia coli*, *Klebsiella pneumoniae*, *Shigella*, *Salmonella typhi*, *Yersinia*, *Pseudomonas aeruginosa*, and *Neisseria*. The pathogenicity of these bacteria is usually related to their cell wall composition, such as capsular polysaccharides and lipopolysaccharides.

[0146] Phospholipid transport system

[0147] The phospholipid transport system (Mla) is a unique membrane protein system of Gram-negative bacteria, consisting of a protein complex formed by six proteins: MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF. Its main function is to maintain the asymmetry of lipids in the outer membrane of Gram-negative bacteria and participate in the reverse transport pathway of phospholipids in the outer membrane. Specifically, the outer membrane lipoprotein MlaA is responsible for removing mislocated phospholipids from the outer membrane and further completing the reverse transport of phospholipids through the periplasmic protein MlaC and the MlaFEDB complex in the inner membrane. There is also evidence that the Mla system has a anterograde phospholipid movement function. By precisely managing the distribution of cell membrane lipids, the phospholipid transport system ensures that Gram-negative bacteria maintain the asymmetry of their outer membrane lipids, preserving the intact outer membrane protective barrier and helping bacteria resist external threats such as antibiotics and bile salts.

[0148] In this invention, the terms "invention protein," "phospholipid transport system protein," and "MlaA, MlaB, MlaC, MlaD, MlaE, MlaF protein" are used interchangeably and all refer to proteins or polypeptides having the amino acid sequences MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF. These include MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF proteins with or without a starting methionine. Furthermore, the term also includes the full-length MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF proteins and their fragments. The MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF proteins referred to in this invention include their complete amino acid sequences, their secreted proteins, their mutants, and their functionally active fragments.

[0149] The full-length proteins of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF of Klebsiella pneumoniae are 253 aa, 96 aa, 211 aa, 183 aa, 260 aa, 270 aa, 260 aa, and 269 aa, respectively, and their accession numbers are WP_002913362.1, WP_004174142.1, WP_038432601.1, WP_002918387.1, WP_004150949.1, and WP_004150950.1, respectively.

[0150] The full-length proteins of pathogenic Escherichia coli MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF are 251 aa, 97 aa, 211 aa, 183 aa, and 270 aa, respectively. Their amino acid sequences are shown in SEQ ID NO. 7-12.

[0151] The full-length proteins of Enterobacter cloacae MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF are 250 aa, 97 aa, 211 aa, 182 aa, and 260 aa, respectively. Their amino acid sequences are shown in SEQ ID NO. 13-18.

[0152] The full-length proteins of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF from Acinetobacter baumannii are 299 aa, 95 aa, 213 aa, 226 aa, 259 aa, and 272 aa, respectively. Their amino acid sequences are shown in SEQ ID NO. 19-24.

[0153] In this invention, the terms "MlaA, MlaB, MlaC, MlaD, MlaE, MlaF genes" and "MlaA, MlaB, MlaC, MlaD, MlaE, MlaF polynucleotides" are used interchangeably and both refer to nucleic acid sequences having the nucleotide sequences of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF.

[0154] The full-length nucleotide sequences of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF of Klebsiella pneumoniae are 762bp, 291bp, 636bp, 552bp, 783bp, and 813bp, respectively, and their gene sequences are SEQ ID NO.1-6.

[0155] Having obtained the amino acid fragments of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF, the nucleic acid sequences encoding them can be constructed, and specific probes can be designed based on the nucleotide sequences. Full-length nucleotide sequences or fragments thereof can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF disclosed in this invention, especially the open reading frame sequences, and bacterial genomic DNA can be used as a template to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0156] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0157] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0158] Currently, the DNA sequence encoding the protein of this invention (or its fragments, derivatives) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0159] Using conventional recombinant DNA technology, the polynucleotide sequences of this invention can be used to express or produce recombinant MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF polypeptides. Generally, the following steps are involved:

[0160] (1). Transform or transduce suitable host cells using the polynucleotides (or variants) encoding the polypeptides MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF of the present invention, or using a recombinant expression vector containing the polynucleotides;

[0161] (2) Host cells cultured in a suitable culture medium;

[0162] (3) Isolate and purify proteins from culture media or cells.

[0163] In this invention, the polynucleotide sequences MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF can be inserted into recombinant expression vectors for recombinant protein expression. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0164] Methods well known to those skilled in the art can be used to construct expression vectors containing coding DNA sequences for MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF, and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequences can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0165] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as tetracycline or ampicillin resistance for Escherichia coli, or dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture.

[0166] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0167] The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a human cell.

[0168] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0169] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0170] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0171] When used to refer to, for example, cells, nucleic acids, proteins, or vectors, the term "recombinant" means a cell, nucleic acid, protein, or vector that has been modified by introducing a heterologous nucleic acid or protein or by altering a native nucleic acid or protein, or a cell derived from a cell that has undergone said modification. Thus, for example, recombinant cells express genes that are not present in native (non-recombinant) cell forms, or express native genes that are abnormally expressed, poorly expressed, or not expressed at all.

[0172] Preparation of vaccine composition

[0173] The present invention also provides a method for preparing a vaccine composition, specifically comprising the following steps:

[0174] The engineered Gram-negative rod-shaped bacteria prepared according to this invention are mixed with a pharmaceutically acceptable vaccine adjuvant to form a vaccine composition.

[0175] In another preferred embodiment, the adjuvant is an aluminum adjuvant or a GLA adjuvant, with aluminum adjuvant being more preferred.

[0176] Composition and method of application

[0177] The present invention also provides a composition comprising: (i) engineered Gram-negative rod-shaped bacteria prepared by the method of the present invention, and (ii) pharmaceutically or immunologically acceptable excipients or adjuvants. In this invention, the term "comprising" means that various components may be used together or present in the composition of the present invention. Therefore, the terms "consistent with..." and "consisting with..." are included in the term "comprising".

[0178] The compositions of the present invention include pharmaceutical compositions and vaccine compositions. The compositions of the present invention can be monovalent or polyvalent.

[0179] The pharmaceutical or vaccine compositions of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc.

[0180] (i) Pharmaceutical Composition

[0181] The pharmaceutical composition of the present invention comprises an effective amount of engineered Gram-negative rod-shaped bacteria prepared by the method of the present invention.

[0182] As used herein, the term "effective dose" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. This effect can be detected, for example, by antigen levels. Therapeutic effects also include a reduction in physiological symptoms. The precise effective dose for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is not useful to predetermine an accurate effective dose. However, for a given condition, the effective dose can be determined using routine laboratory methods.

[0183] For the purposes of this invention, an effective dosage is approximately 0.2 μg / kg to 2 μg / kg administered to an individual.

[0184] Pharmaceutical compositions may also contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent (e.g., recombinant protein or other therapeutic agent). This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0185] Pharmaceutically acceptable carriers in a composition may include liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc. Typically, the composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be formulated as a solid form suitable for reconstitution into solutions or suspensions, or as a liquid excipient, prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0186] (ii) Vaccine composition

[0187] The vaccine compositions of the present invention can be prophylactic (i.e., for infection prevention) or therapeutic. The vaccine compositions comprise immune antigens (including the engineered Gram-negative rod-shaped bacteria of the present invention) and are generally combined with pharmaceutically acceptable carriers, including any carrier that does not itself induce antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those skilled in the art. Additionally, these carriers can act as immunostimulants (“adjuvants”). Furthermore, the antigens can also be conjugated with bacterial toxoids (such as toxoids of pathogens such as diphtheria, tetanus, cholera, and Helicobacter pylori).

[0188] Preferred adjuvants for enhancing the efficacy of immune compositions include, but are not limited to: (1) aluminum salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO 90 / 14837), (b) SAF, and (c) Ribi. TMThe adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvants; (4) Freund complete adjuvant (CFA) and Freund incomplete adjuvant (IFA); (5) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; (6) detoxified variants of bacterial ADP-ribosylated toxins (e.g., cholera toxin CT, pertussis toxin PT, or Escherichia coli heat unstable toxin LT), see, for example, WO93 / 13302 and WO92 / 19265; and (7) other substances that enhance the effect of the composition as immunostimulants.

[0189] Vaccine compositions, including immunogenic compositions (e.g., may include antigens, pharmaceutically acceptable carriers, and adjuvants), typically contain diluents such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., may be present in these carriers.

[0190] More specifically, vaccines, including immunogenic compositions, contain an immunologically effective amount of an immunogenic peptide, as well as the other required components mentioned above. An "immunologically effective amount" refers to the amount administered to an individual as a single or partial dose that is effective for treatment or prevention. This dosage can be determined based on the individual's health and physiological condition, the individual's class (e.g., human), the individual's immune system's ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. This dosage is expected to be within a relatively wide range and can be determined through routine laboratory testing.

[0191] Typically, vaccine compositions or immunogenic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or liquid excipients prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects.

[0192] (iii) Route of administration and dosage

[0193] The composition can be administered directly to a subject. The subject can be a human or a non-human mammal, preferably a human. When used as a vaccine, the virus-like particles of the present invention can be administered directly to an individual using known methods. These vaccines are typically administered via the same route of administration as conventional vaccines and / or by mimicking the pathogen infection pathway.

[0194] The routes of administration for the pharmaceutical or vaccine compositions of the present invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, vaginal, oral, or other parenteral routes. Routes of administration may be combined if necessary, or adjusted according to the disease condition. The vaccine compositions may be administered in single or multiple doses, and may include booster doses to induce and / or maintain immunity.

[0195] Virus-like particle vaccines should be administered in an "effective amount," meaning that the amount of virus-like particles is sufficient to elicit an immune response and effectively protect the host against infection by Gram-negative rod-shaped bacteria via the chosen route of administration.

[0196] The amount of virus-like particles selected in each vaccine dose is determined based on the amount that can elicit a protective immune response without significant side effects. Typically, after infecting host cells, each dose of vaccine is sufficient to contain approximately 1 μg-1000 μg, preferably 1 μg-100 μg, and more preferably 10 μg-50 μg of protein or VLP. The optimal dosage of a specific vaccine can be determined using standard research methods, including antibody titers and other responses in the subjects. Whether a booster dose is needed can be determined by monitoring the level of immunity provided by the vaccine. A booster dose may be required after assessing antibody titers in the serum. Administration of adjuvants and / or immunostimulants can enhance the immune response to the proteins of the present invention. A preferred method is to administer the immunogenic composition by injection via a parenteral (subcutaneous or intramuscular) route.

[0197] The term "functional effect" includes the measurement of parameters, such as chemical or phenotypic effects, that are indirectly or directly affected by the biomarkers of this invention. Therefore, functional effects include immediate signaling events, such as transcriptional activation or phosphorylation, or more indirect effects, such as gene expression or apoptosis.

[0198] "Functional effects" include in vitro, in vivo, and ex vivo activities.

[0199] "Determining functional effects" means determining compounds that increase or decrease parameters of genes (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) that are indirectly or directly affected by the phospholipid transport system of this invention, for example, determining physical, chemical, or phenotypic effects. Such functional effects can be determined by any method known to those skilled in the art, such as by changes in spectrophotometric properties (e.g., fluorescence, absorption, refractive index); hydrodynamics (e.g., shape), chromatography; or protein solubility properties; determining inducible markers or transcriptional activation of markers; determining changes in enzyme (e.g., kinase) activity; increasing or decreasing the ability of cells to proliferate, apoptosis, or cell cycle arrest; determining changes in cell surface markers. Functional effects can be assessed by many methods known to those skilled in the art, such as microscopy for quantitative or qualitative determination of morphological changes, determination of changes in RNA or protein levels of other genes expressed in cells, determination of RNA stability, identification of downstream or reporter molecule gene expression (CAT, luciferase, β-gal, GFP, etc.), for example by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, etc.

[0200] The term "inhibitor" in biomarkers refers to inhibitory molecules identified using in vitro and in vivo assays of genes or proteins of the phospholipid transport system (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF). An "inhibitor" is, for example, a compound that binds to, partially blocks, or completely blocks the activity of, reduces, prevents, delays, inactivates, desensitizes, or downregulates the activity or expression of genes or proteins of the phospholipid transport system (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF).

[0201] Inhibitors also include genetically modified genes of phospholipid transport systems (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) or their proteins, for example, in forms with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, RNAi and siRNA molecules, small organic molecules, etc. Such assays for inhibitors include, for example, expressing genes of phospholipid transport systems (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) or their proteins in vitro, in cells, or in cell extracts; administering a putative modulatory compound; and then measuring the functional effect on activity, as described above.

[0202] The extent of activity alteration is detected by comparing a sample or assay of a gene or protein containing a phospholipid transport system (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) treated with a potential inhibitor with a control sample without the inhibitor. The control sample (untreated or treated with a known modulator) is assigned as 100% relative protein activity. Inhibition of a gene or protein containing a phospholipid transport system (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) is achieved when the activity relative to the control is approximately 80%, preferably 50%, more preferably 25-0%.

[0203] Inhibitors also include agents designed to reduce the mRNA levels of genes (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) or their proteins in the phospholipid transport system (e.g., antisense molecules, ribozymes, DNases, small repressive RNAs, etc.) or to reduce the translation levels from mRNA (e.g., translation blockers, such as antisense molecules complementary to other sequences on the translation initiation site or mRNA molecule). In some embodiments, known methods are used to identify the nucleic acid sequences of genes (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) or their proteins that inhibit the phospholipid transport system. Such inhibitors may include, but are not limited to, siRNA oligonucleotides and antisense oligonucleotides. Inhibitor siRNAs or antisense oligonucleotides can be used to specifically target the expression of genes (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) or their proteins in the phospholipid transport system.

[0204] In some implementations, RNA interference is used to generate small double-stranded RNA, small interfering RNA (siRNA), or short hairpin RNA (shRNA) inhibitors to affect the expression of genes or proteins of the phospholipid transport system (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF), typically by cleaving and disrupting their homologous RNA. Small interfering RNA (siRNA or shRNA, used interchangeably) is usually a 19-22 nt double-stranded RNA. siRNA can be obtained by chemical synthesis or via DNA vector-based RNAi technology. Using DNA vector-based siRNA technology, a small DNA insert (approximately 70 bp, encoding a short hairpin RNA targeting the target gene) is cloned into a commercially available vector. The vector containing the insert can be transfected into cells, expressing the short hairpin RNA. The hairpin RNA is rapidly processed by cellular mechanisms into a 19-22 nt double-stranded RNA (siRNA). siRNA is generally inserted into a suitable RNAi vector because synthetically prepared siRNA tends to have lower stability and is less effective in transfection.

[0205] siRNA can be prepared using methods and algorithms described in, for example, the following references: Wang et al., (2004) A Web-based Design Center for Vector-based siRNA and siRNA cassette Bioinformatics. (In press); Khvorova et al., (2003) Cell. 115(2): 209-16; Harborth et al., (2003) Antisense Nucleic Acid Drug Dev. 13(2): 83-105; Reynolds et al., (2004) Nat Biotechnol. 22: 326-30; and Ui-Tei et al., (2004) Nucleic Acids Res. 32: 936-48. Other tools for constructing siRNA sequences are available online, such as siRNA TargetFinder and Construct Builder (available from GenScript), Oligo Design and Analysis Tools (available from Integrated DNA Technologies), or siDESIGN™ Center (available from Dharmacon, Inc.). It is recommended to use the ORF (Open Reading Frame) as the target selection region, preferably 50-100 nt downstream of the start codon, for siRNA construction.

[0206] Compound drug composition and medicine box

[0207] This invention provides compound pharmaceutical compositions containing (a) an inhibitor of a phospholipid transport system gene or its encoded protein; and (b) a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated as injections, for example, using physiological saline or an aqueous solution containing glucose and other excipients prepared by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under sterile conditions. The pharmaceutical compositions of this invention can also be formulated as powders for nebulized inhalation. A preferred dosage form is an injectable formulation. Furthermore, the pharmaceutical compositions of this invention can be used with other therapeutic agents.

[0208] The present invention also provides a medicine box for the prevention and / or treatment of diseases related to abnormal skin structures, the medicine box comprising:

[0209] (a1) A first container, and an inhibitor of a phospholipid transport system gene or its encoded protein located in the first container, or a drug containing an inhibitor of a phospholipid transport system gene or its encoded protein.

[0210] The pharmaceutical compositions and kits of the present invention are suitable for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases.

[0211] The formulation of this invention can be taken three times a day to once every ten days, or in a sustained-release manner once every ten days. The preferred method is once daily, as this facilitates patient adherence and significantly improves patient compliance.

[0212] When taking this medication, in most cases the total daily dose should be lower than (or in a few cases equal to or slightly greater than) the usual daily dose of each individual drug. Of course, the effective dose of the active ingredient may vary depending on the administration method and the severity of the disease being treated.

[0213] Treatment

[0214] The present invention also provides a method for preventing and / or treating Gram-negative rod-shaped bacterial infections or related diseases using the above-described active ingredient or corresponding drug of the present invention, comprising administering to a mammal an effective amount of an inhibitor of the active ingredient (a) phospholipid transport system gene or the protein encoded thereon, or administering a pharmaceutical composition containing said active ingredient (a).

[0215] When the active ingredient of the present invention is used for the above-described uses, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the following forms: tablets, pills, capsules, dispersible powders, granules, or suspensions (containing, for example, about 0.05-5% suspension concentrate), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or administered non-gastrointestinally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspension concentrate in an isotonic medium). For example, these pharmaceutical formulations may contain about 0.01-99%, more preferably about 0.1%-90% (by weight) of the active ingredient mixed with a carrier.

[0216] The active ingredients or pharmaceutical compositions of the present invention can be administered via conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral, or local administration. Preferred routes of administration include oral, intramuscular, or intravenous administration.

[0217] From the perspective of ease of administration, preferred pharmaceutical compositions are liquid compositions, especially injectable formulations.

[0218] Furthermore, the active ingredients or drugs of the present invention can also be used in combination with other drugs for the prevention and / or treatment of Gram-negative rod-shaped bacterial infections or related diseases (such as meropenem, tigecycline, levofloxacin, polymyxin).

[0219] The main advantages of this invention are:

[0220] (1) This invention is the first to discover that knocking out genes (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) of the endogenous phospholipid transport system of Gram-negative baculoform bacteria can yield attenuated strains, which can be (a) used to prepare antibodies against Gram-negative baculoform bacteria; and / or (b) used to prepare drugs for the prevention and / or treatment of Gram-negative baculoform bacterial infections or related diseases. Furthermore, the inventors have also discovered that inhibiting the expression of genes or proteins (such as one or more of MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF) of the phospholipid transport system can also be used to prevent and / or treat Gram-negative baculoform bacterial infections or related diseases.

[0221] (2) This invention discovers a phospholipid transporter system in Klebsiella pneumoniae, which plays an indispensable role in the pathogenicity of this bacterium. Constructing genetically engineered strains of Klebsiella pneumoniae lacking this system can effectively reduce the virulence and pathogenicity of the bacteria. The process is simple, convenient, and inexpensive. The constructed engineered strains can be used for the rapid development of attenuated vaccines to prevent Klebsiella pneumoniae infection. Because the phospholipid transporter system is highly conserved in many Gram-negative bacteria, including Klebsiella pneumoniae, and is widely present in various clinical strains, and is exposed on the bacterial surface with high accessibility, it is an ideal drug target that can be used for the development of novel antibiotics and the treatment of diseases.

[0222] (3) This invention utilizes engineered strains lacking MlaA to rapidly develop attenuated or inactivated Klebsiella pneumoniae vaccines, which have high safety and practicality. Because the surface of engineered strains lacking MlaA still contains a certain amount of capsular polysaccharide, the immunogenicity of the capsular polysaccharide is preserved while the pathogenicity is reduced, which can effectively activate the host's protective immune response.

[0223] (4) Because the MlaA protein is located on the bacterial outer membrane and is directly exposed on the bacterial cell surface, drugs do not need to cross the cell membrane to bind to it, resulting in higher drug accessibility. MlaA can be used as a candidate antigen for the development of recombinant protein vaccines or mRNA vaccines, and can also be used for the development of novel anti-infective drugs or therapeutic antibodies. Compared with other proteins in the bacterial cytoplasm, MlaA is an ideal drug target and a superior surface antigen.

[0224] (5) This invention is the first to discover that the phospholipid transport system with MlaA as its core is a key pathogenic factor of Klebsiella pneumoniae.

[0225] (6) This invention is the first to discover that Klebsiella pneumoniae strains lacking the MlaA-based phospholipid transport system can be used to develop attenuated vaccines.

[0226] (7) This invention is the first to discover that the phospholipid transport system with MlaA as its core can serve as a drug target for treating Klebsiella pneumoniae infection.

[0227] (8) This invention describes a key pathogenic factor MlaA of Klebsiella pneumoniae, which plays an indispensable role in infectious diseases caused by Klebsiella pneumoniae. MlaA encodes a core component of the phospholipid transporter system. Knocking out MlaA or genetically engineered strains of this system significantly reduces virulence and pathogenicity, and can be used as a novel attenuated vaccine. In addition, the MlaA transporter system is an ideal drug target and can be used to develop novel anti-infective drugs to treat infectious diseases caused by Klebsiella pneumoniae.

[0228] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989); or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0229] Unless otherwise specified, all experimental materials and reagents used in the following examples are available from commercially available sources.

[0230] Experimental methods

[0231] (1) PCR amplification of the resistance gene fragment containing the mAA homologous arm.

[0232] Using plasmid pIJ773 (obtained from Tufts University) containing the apramycin resistance gene as a template, primers (YCO-1787 / 1788) were designed 60 bp upstream and downstream of the mlaA(VK055_RS24245) gene as homologous arms to amplify the resistance gene fragment containing the 60 bp homologous arm by PCR.

[0233] YCO-1787:

[0234] YCO-1788:

[0235] Taq PCR reaction system

[0236] Taq PCR reaction procedure

[0237] (2) Recovery and purification of PCR products

[0238] 1) Transfer the PCR reaction solution to a clean 1.5mL centrifuge tube, add 5 times the volume of Buffer B3 (a component of the SanPrep column PCR product purification kit, purchased from Shanghai Sangon Biotech), and mix thoroughly.

[0239] 2) Transfer the entire mixture into the adsorption column and centrifuge at 8000g for 30 seconds. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0240] 3) Add 500 μL of Wash Solution (a component of the SanPrep column PCR product purification kit, purchased from Shanghai Sangon Biotech) to the adsorption column and centrifuge at 9000g for 30 seconds. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0241] 4) Repeat step 3 once.

[0242] 5) Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 9000g for 1 minute.

[0243] 6) Add 15-40 μL of ddH2O to the center of the adsorption membrane, let it stand at room temperature for 1-2 minutes, and then centrifuge at 9000g for 1 minute.

[0244] (3) Preparation and electrotransfer competence

[0245] 1) Wild-type Klebsiella pneumoniae (K. pneumoniae ATCC 43816, purchased from ATCC) containing pACBSR plasmid (obtained from Tufts University) was streaked on LB agar plates containing 100 μg / ml hygromycin and incubated overnight at 28°C. The next day, a single colony was picked from the plate and inoculated into 2 mL of fresh LB medium containing hygromycin (purchased from Shanghai Sangon Biotech) and incubated overnight at 28°C and 220 rpm on a shaker.

[0246] 2) Transfer 100 μL of the bacterial culture to 10 ml of LB liquid medium containing hygromycin, and add 100 μL of 20% L-arabinose. Continue incubation at 28°C for 2.5 hours until OD (dose retardation). 600 =0.5.

[0247] 3) Centrifuge at 4000 rpm for 8 min at 4℃, discard the supernatant, and resuspend the bacterial cells in 10 ml of 10% glycerol.

[0248] 4) After washing twice, resuspend in 200 μL of 10% glycerol.

[0249] 5) Take 100 μL of bacterial suspension and put it into a pre-cooled 0.2 cm electroporation cuvette. Add 1 mg of the purified PCR product and perform transformation under standard conditions: voltage = 2.5 kV, electroporation time approximately 5.0-6.0 ms.

[0250] 6) Add 250 μL of LB medium and incubate at 37°C on a shaker for 2 hours. Spread the mixture on a 50 μg / ml apramycin plate and incubate at 37°C overnight.

[0251] (4) Validation and sequencing of positive clones of MlaA gene knockout

[0252] Positive clones grown on amphotericin B plates were validated by colony PCR. Primers YCO-1836 / 1837 were designed and synthesized based on the upstream and downstream sequences of the MlaA gene. Single colonies were selected as templates for PCR amplification. After the reaction, the results were detected by 1% agarose gel electrophoresis. The PCR products were then sequenced, and sequence alignment confirmed successful gene knockout.

[0253] YCO-1836: GATGGCAACTCGCGAAAGCC

[0254] YCO-1837:CTGATAAGCAGCGCGCCAC

[0255] Colony PCR reaction system for positive clone verification

[0256] PCR reaction procedure for positive clone verification

[0257] Example 1: Construction of a genetically engineered strain lacking MlaA

[0258] Using PCR, the upstream and downstream regions (60bp-500bp) of the mlaA gene (WP_002913362.1) were amplified as homologous arms. 1 mg of purified DNA was electroporated into Klebsiella pneumoniae (2500V, 5mS), and DNA homologous recombination was performed using the red recombinase method to construct an engineered strain with the mlaA gene knocked out (Figure 1).

[0259] Example 2: Verification of genetically engineered strains lacking mlaA

[0260] Following successful homologous recombination, the deletion of the mlaA gene was further verified in the genetically engineered strain. Western blotting and a specific antibody (anti-MlaA polyclonal antibody, catalog number PA5-144553, purchased from Thermo Fisher Scientific) were used to examine the expression of the MlaA protein in the engineered strain. The results showed that the wild-type control strain (Klebsiella pneumoniae ATCC 43816, purchased from ATCC) showed detectable MlaA protein expression, while the genetically engineered strain (ΔmlaA) showed no detectable MlaA protein expression (Figure 2), confirming the successful construction of the genetically engineered strain lacking mlaA.

[0261] Example 3: Detection of capsular polysaccharide content in genetically engineered strains

[0262] Klebsiella pneumoniae is coated with a highly viscous capsular polysaccharide, which is the main pathogenic factor of the bacterium. The thickness and yield of the capsular polysaccharide are related to the pathogenicity of Klebsiella pneumoniae. This invention found that the MlaA phospholipid transporter system can affect the yield of capsular polysaccharide on the bacterial surface; the absence of mlaA reduces the yield of capsular polysaccharide. To quantitatively analyze the yield of capsular polysaccharide, the content of uronic acid components was experimentally detected. 2 ml of overnight Klebsiella pneumoniae culture was mixed with capsular polysaccharide extraction solution (1% ZWITTERGENT 3-10 in citric acid) and incubated for 30 minutes. After high-speed centrifugation, the supernatant was collected and isopropanol was added to extract the capsular polysaccharide components. Subsequently, sulfuric acid solution containing sodium tetraborate was added, and the content of uronic acid in the sample was determined by the m-hydroxybiphenyl method. The absorbance was measured at a wavelength of 520 nm, and the uronic acid content was quantitatively determined using a standard curve of glucuronic acid. The results of uronic acid quantification experiments showed that, compared with wild-type Klebsiella pneumoniae, the capsular polysaccharide production in the genetically engineered strain lacking mlaA was significantly reduced (Figure 3).

[0263] Example 4: Detection of capsular polysaccharide viscosity in genetically engineered strains

[0264] The capsular polysaccharide on the surface of Klebsiella pneumoniae cells exhibits high viscosity, and the degree of viscosity is strongly positively correlated with the pathogenicity of the bacterium. This invention reveals that the MlaA phospholipid transporter system enhances capsular viscosity, while the absence of MlaA decreases bacterial viscosity. 1 ml of overnight bacterial culture (OD200) was used to... 600 =1.0), centrifuged at 1000g for 5 minutes, and sedimentation analysis was performed. Non-viscous bacterial cells aggregated at the bottom of the tube, while highly viscous bacterial cells remained in the supernatant. The optical density of the supernatant was measured spectrophotometrically at 600nm wavelength, allowing for quantitative analysis of the capsular viscosity of Klebsiella pneumoniae. Quantitative analysis of capsular viscosity showed that, compared to the wild-type strain, the capsular polysaccharide viscosity of the engineered strain lacking the mlaA gene was significantly reduced (Figure 4).

[0265] Example 5: Detection of the growth adaptability of genetically engineered strains in vitro and in vivo.

[0266] The phospholipid transporter system encoded by MlaA is responsible for maintaining the phospholipid asymmetry of the bacterial outer membrane. It directly binds to and removes phospholipids from the outer membrane layer, and then retrogradely transports phospholipids to the inner membrane via the MlaC and MlaBDEF protein complexes. Interference with the phospholipid transport system leads to decreased bacterial outer membrane integrity and increased permeability, making bacteria more sensitive to some antimicrobial agents, but does not affect normal bacterial growth and competitive adaptation in nutrient-rich media. Wild-type strains and genetically engineered strains lacking mlaA were mixed at a 1:1 ratio and inoculated into LB medium for overnight culture. Colony counting was used to analyze the competitive adaptation of the two strains. The results showed that in LB medium (purchased from Shanghai Sangon Biotech), the ratio of genetically engineered strains lacking mlaA to wild-type strains remained at 1:1, and both strains had the same growth capacity and competitive adaptation, with no obvious growth or survival defects (Figure 5).

[0267] Wild-type strains and genetically engineered strains lacking the mlaA gene were mixed in a 1:1 ratio and the lungs of mice (BalB / C mice, purchased from Shanghai Shengchang Biotechnology Co., Ltd.) were infected via nasal drops. Lung tissue was harvested for colony counting 48 hours after infection. Analysis revealed that the mlaA-deficient genetically engineered strain exhibited a significant competitive deficiency in mice, with a colony count only 0.1% of that of the wild-type strain. Therefore, this genetically engineered strain possesses the desirable characteristic of normal growth in vitro and reduced adaptability in vivo.

[0268] Example 6: Detection of the colonization ability of genetically engineered strains in mice.

[0269] To further examine the infectivity and colonization ability of the mlaA-deficient genetically engineered strain in mice (BalB / C mice, purchased from Shanghai Shengchang Biotechnology Co., Ltd.), mice were infected by intranasal administration of either the wild-type or ΔmlaA strain. After 48 hours, lungs, spleen, and liver were dissected, homogenized, and plated to analyze the viral load of the two strains in the mice. The results showed that compared to the wild-type strain, the number of colonies of the ΔmlaA genetically engineered strain in each organ was reduced by 3-4 orders of magnitude, with statistically significant differences (Figure 6). The experiment confirmed that the virulence of the ΔmlaA genetically engineered strain was weakened, and its infectivity and colonization ability in multiple organs of mice was significantly reduced.

[0270] Example 7: Detection of the pathogenicity of genetically engineered strains in mice

[0271] To examine whether the pathogenicity of the genetically engineered strain lacking mlaA is reduced in animals, healthy BalB / C mice (purchased from Shanghai Shengchang Biotechnology Co., Ltd.) were infected with either the wild-type strain or the ΔmlaA strain. Each mouse was inoculated with 1000 bacterial cells or an equal volume of PBS as a control via intranasal drip. Five mice were in each group. The morbidity and mortality patterns were observed, and survival curves were constructed. The results showed that infection with the wild-type Klebsiella pneumoniae strain led to the death of all mice within 3 days, while mice infected with the ΔmlaA genetically engineered strain did not show any deaths or significant disease progression, consistent with the results of the PBS negative control group (Figure 7). These results strongly suggest that the pathogenicity of the ΔmlaA genetically engineered strain is significantly reduced in animals, making it a safe, effective, and practical attenuated strain.

[0272] Example 8: Using non-coding RNA to target and inhibit mlaA has anti-infective therapeutic effects.

[0273] Studies have found that the non-coding small RNA ArcZ (RNA sequence: 5'-gagcaauccacuucuucgguugcgccacguaacaacaucacucaaacaacacuggcucaaccaccaguucccugguguuggcgcaguauucgcgcaccccggucuguccggggucauuuuuu-3') specifically targets mlaA mRNA. Therefore, ArcZ can be used as an antisense RNA drug to regulate the expression level of MlaA (Figure 8A-B). In Klebsiella pneumoniae, ArcZ binds directly to mlaA mRNA through base complementarity. Overexpression of ArcZ small RNA in wild-type strains using a strong promoter (16S rRNA) can effectively inhibit or knock down the expression level of MlaA protein (Figure 8B), while significantly reducing the ability of Klebsiella pneumoniae to infect mice and greatly reducing the viral load of Klebsiella pneumoniae in mouse lungs (Figure 8C). The results showed that overexpression of the non-coding small RNA ArcZ had a good therapeutic effect, and also demonstrated that MlaA is an effective target for anti-infective drugs.

[0274] Example 9: Genetically engineered strains lacking mlaA exhibit good vaccine protection.

[0275] The genetically engineered strain with the mlaA deletion exhibits a strong attenuation effect, significantly reducing pathogenicity in animals while retaining all bacterial surface antigen components, including capsular polysaccharides. Therefore, the ΔmlaA strain can be used as an attenuated live vaccine. To verify its vaccine efficacy, 10,000 ΔmlaA bacteria were administered intranasally to each of five healthy BalB / C mice (purchased from Shanghai Shengchang Biotechnology Co., Ltd.) for primary immunization. A booster immunization was performed 14 days later with another 10,000 bacteria administered intranasally. No mice showed disease symptoms or died within 28 days, indicating good safety. On day 30, a challenge infection experiment was conducted using wild-type Klebsiella pneumoniae strain with an intranasal dose of 1,000 bacteria. After 14 days of observation, no mice showed symptoms or died after either immunization, demonstrating that the genetically engineered strain with the mlaA deletion has 100% vaccine protection, significantly different from the unimmunized negative control group (NC) (Figures 9A-B).

[0276] Furthermore, 1,000,000 ΔmlaA bacteria were administered intranasally to each mouse (5 healthy BalB / C mice per group, purchased from Shanghai Shengchang Biotechnology Co., Ltd.). No mice showed symptoms or died within 28 days, indicating that high-dose immunization still has good safety. On day 30, a challenge infection experiment was conducted using wild-type Klebsiella pneumoniae strain with an intranasal dose of 1,000 bacteria. After 14 days of observation, no mice showed symptoms or died, indicating that using a larger dose of the strain for initial immunization is sufficient to protect mice from infection and death with a single immunization, significantly reducing the immunization schedule while ensuring vaccine efficacy (Figures 9C-D). These results fully demonstrate that the ΔmlaA genetically engineered strain is a highly safe, effective, and practical live attenuated vaccine.

[0277] Since the six proteins MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF assemble to form a protein complex and work together to perform the function of phospholipid transport, the effect of the engineered strain with MlaA deletion shown in the specific embodiments of the present invention can be achieved by deleting any one of the genes MlaB, MlaC, MlaD, MlaE, or MlaF, or by deleting any combination of the above genes.

[0278] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0279] Sequence information:

[0280] >SEQ ID NO.1 Klebsiella pneumoniae mlaA gene sequence 762bp

[0281] >SEQ ID NO.2 Klebsiella pneumoniae mlaB gene sequence 291bp

[0282] >SEQ ID NO.3 Klebsiella pneumoniae mlaC gene sequence 636bp

[0283] >SEQ ID NO.4 Klebsiella pneumoniae mlaD gene sequence 552bp

[0284] >SEQ ID NO.5 Klebsiella pneumoniae mlaE gene sequence 783bp

[0285] >SEQ ID NO.6 Klebsiella pneumoniae mlaF gene sequence 813bp

[0286] >Escherichia coli MlaA protein sequence 251aa (SEQ ID NO.7)

[0287] The MlaA protein sequence of Enterobacter cloacae is 250aa (SEQ ID NO.13).

[0288] >Acinetobacter baumannii MlaA protein sequence 299aa (SEQ ID NO.19)

[0289] >Escherichia coli MlaB protein sequence 97aa (SEQ ID NO.8)

[0290] The MlaB protein sequence of Enterobacter cloacae is 97aa (SEQ ID NO.14).

[0291] >Acinetobacter baumannii MlaB protein sequence 95aa (SEQ ID NO.20)

[0292] >Escherichia coli MlaC protein sequence 211aa (SEQ ID NO.9)

[0293] The MlaC protein sequence 211aa of Enterobacter cloacae (SEQ ID NO.15)

[0294] >Acinetobacter baumannii MlaC protein sequence 213aa (SEQ ID NO.21)

[0295] >Escherichia coli MlaD protein sequence 183aa (SEQ ID NO.10)

[0296] The MlaD protein sequence of Enterobacter cloacae is 182aa (SEQ ID NO.16).

[0297] >Acinetobacter baumannii MlaD protein sequence 226aa (SEQ ID NO.22)

[0298] >Escherichia coli MlaE protein sequence 260aa (SEQ ID NO.11)

[0299] The protein sequence 260aa of Enterobacter cloacae MlaE (SEQ ID NO.17)

[0300] >Acinetobacter baumannii MlaE protein sequence 259aa (SEQ ID NO.23)

[0301] >Escherichia coli MlaF protein sequence 269aa (SEQ ID NO.12)

[0302] The protein sequence 270aa of Enterobacter cloacae MlaF (SEQ ID NO.18)

[0303] >Acinetobacter baumannii MlaF protein sequence 272aa (SEQ ID NO.24)

Claims

1. An engineered Gram-negative bacillus, characterized in that, The gene of the endogenous phospholipid transport system of the gram-negative bacillus is knocked out.

2. A pharmaceutical composition, characterized by, The composition contains the engineered gram-negative bacillus of claim 1, and a pharmaceutically acceptable carrier and / or adjuvant.

3. A vaccine composition, characterized in that, The composition contains the engineered gram-negative bacillus of claim 1, and an immunologically acceptable carrier and / or adjuvant.

4. Use of the engineered Gram-negative bacillus bacterium of claim 1, the pharmaceutical composition of claim 2, or the vaccine composition of claim 3, wherein, (a) for the preparation of an antibody against the gram-negative bacillus; and / or (b) for the preparation of a medicament for preventing and / or treating a gram-negative bacillus infection or a disease related thereto.

5. Use of an inhibitor of a phospholipid transport system gene or a protein encoded thereby, characterized in that, for the preparation of a medicament for preventing and / or treating a gram-negative bacillus infection or a disease related thereto.

6. A pharmaceutical composition, characterized by, Comprising: (a) an inhibitor of the phospholipid transport system gene or the protein encoded thereby; and (b) a pharmaceutically acceptable carrier. Comprising:

7. A kit characterized in that, (a1) a first container, and an inhibitor of the phospholipid transport system gene or the protein encoded thereby, or a medicament containing the inhibitor of the phospholipid transport system gene or the protein encoded thereby, in the first container. for preventing and / or treating a gram-negative bacillus infection or a disease related thereto.

8. Use of a pharmaceutical composition according to claim 6 or a kit according to claim 7, characterized in that, The method comprises the steps of:

9. A method of screening for a candidate substance for preventing and / or treating a Gram-negative bacillus infection or a disease associated therewith, characterized by, (a) in a test group, adding a test compound to a culture system of cells, and observing the expression amount (E1) and / or activity (A1) of the phospholipid transport system protein in the cells of the test group; in a control group, not adding the test compound to the culture system of the same cells, and observing the expression amount (E0) and / or activity (A0) of the phospholipid transport system protein in the cells of the control group; wherein if the expression amount (E1) and / or activity (A1) of the phospholipid transport system protein in the cells of the test group is significantly lower than that of the control group, it indicates that the test compound is a candidate substance for preventing and / or treating a gram-negative bacillus infection or a disease related thereto by inhibiting the expression and / or activity of the phospholipid transport system. The method comprises the steps of:

10. A method of screening for a candidate substance for preventing and / or treating a Gram-negative bacillus infection or a disease associated therewith, characterized by, (i) contacting a candidate substance with a phospholipid transport system protein; and (ii) determining the functional effect of the candidate substance on the phospholipid transport system protein, wherein the functional effect indicates that the compound prevents and / or treats a gram-negative bacillus infection or a disease related thereto. ​

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