CN112375707B

CN112375707B - Escherichia coli highly subjected to O-acetylation modification by capsular polysialic acid and application thereof

Info

Publication number: CN112375707B
Application number: CN202011276058.0A
Authority: CN
Inventors: 杨静华; 周慧; 金城
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2022-03-01
Anticipated expiration: 2040-11-13
Also published as: CN112375707A

Abstract

The invention discloses Escherichia coli modified by capsular polysialic acid through high O-acetylation and application thereof. The Escherichia coli is Escherichia coli (Escherichia coli), the strain number of the Escherichia coli is U9-41 Variant, and the registration number of the Escherichia coli in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 19571. The strain can be used for producing natural high-level O-acetylation modified alpha 2-8-polysialic acid, and is used for research and development of polysaccharide vaccines and preparation of corresponding antibodies; on the other hand, the strain can also be used for scientific research to reveal the biological function and pathogenic mechanism of capsular polysialic acid O-acetylation modification.

Description

Escherichia coli highly subjected to O-acetylation modification by capsular polysialic acid and application thereof

Technical Field

The invention relates to the field of microorganisms, and particularly relates to escherichia coli containing capsular polysialic acid and modified by high O-acetylation and application thereof.

Background

Escherichia coli K1 strain (Escherichia coli K1, E.coli K1) is the main pathogenic bacterium causing neonatal sepsis, sepsis and purulent meningitis, and is also the common pathogenic bacterium causing urinary tract infection in women. In addition, the strain also causes respiratory tract infection and septicemia of poultry and causes death of large-area poultry, thereby bringing loss to poultry breeding enterprises. The strain is fixedly planted on the mucosal surface of the gastrointestinal tract, penetrates through the mucosa to invade blood, resists the killing of antibodies, complements and macrophages in serum, proliferates in the blood in large quantity, reaches a certain threshold value, and causes septicemia; sometimes the inflammatory reaction is activated to cause immune dysfunction to cause sepsis; if bacteria invade the central nervous system across the blood-brain barrier, they infect the pia mater, the spinal cord membrane and the cerebrospinal fluid, eventually causing meningitis. Currently, despite antibacterial drug therapy, the mortality rate caused by the escherichia coli K1 strain is still as high as 15% -40%, and survivors often have permanent nervous system sequelae including hearing impairment, mental retardation, and focal neurological deficit.

The surface of Escherichia coli is coated with a layer of Capsular polysaccharide (also called Capsular antigen or K antigen). The K antigens are divided into 80 types according to the difference of polysaccharide structures. Among them, the K1 capsular polysaccharide (K1 antigen) has the same sugar chain structure as the adhesion molecule (NACM) on the surface of human nerve cells, and can enable bacteria to escape from the recognition and elimination of the host immune system through a Molecular simulation mechanism, so that the K1 capsular polysaccharide is a key pathogenic factor of bacteria and is also a target site for developing antibodies and saccharide vaccines for resisting and preventing meningitis infection. In addition, polysialic acid has a large amount of negative charges and has hydrophilicity, so that the distance between cell membranes is increased and the ability to adhere to each other is lost, thereby affecting the interaction between cells. In some tumor tissues, polysialic acid is highly expressed, so that the separation and the brain metastasis of cancer cells are promoted, and polysialized NACM in serum is expected to become a clinical marker of neurocytoma.

The K1 capsular polysaccharide is composed of Polysialic acid (PSA) connected by alpha 2-8, namely a linear polysaccharide long chain formed by polymerization of sialic acid monomer (Neu5Ac) connected by alpha 2-8 glycosidic bonds, and the molecular formula is (Neu5Ac alpha 2-8Neu5Ac) n. Thus, the K1 capsular polysaccharide is also known as K1 capsular polysialic acid. Sialic acid is the only acidic monosaccharide containing 9 carbon atoms in nature and is a natural derivative of N-acetylneuraminic acid. Sialic acid molecules are frequently modified by O-acetylation (FIG. 1), by deprotonation of the hydroxyl group of the carbon atom by the action of an acetyltransferase, and substitution by the acetyl group of acetyl-CoA. The O-acetylation modification typically occurs at the 7 th, 8 th or 9 th carbon atom of the sialic acid molecule. Furthermore, the O-acetyl group can move between these carbon atoms as the basicity of the environmental acid changes. In the long chain of sialic acid, the different number of sialic acid monomers modified by O-acetylation results in different levels of O-acetylation of the capsular polysialic acid.

The O-acetylation modification changes the physicochemical characteristics of K1 capsular polysialic acid. The O-acetyl group is an uncharged polar group, increases the hydrophobicity of capsular polysaccharide, changes the steric structure of polysaccharide, and enhances the resistance of bacteria to lysozyme and sialic acid hydrolase. Furthermore, the O-acetylated modified sugar structure may become a new antigenic determinant, stimulating the body to produce new antibodies. Thus, the O-acetylation modification alters the antigenicity and immunogenicity of the K1 capsular polysaccharide. In addition, clinical studies have shown that O-acetylation of capsular polysialic acid is associated with sepsis, and most of the E.coli K1 strains isolated from the patient's blood are O-acetylation modified capsular polysialic acid strains. Thus, the O-acetylation modification of K1 capsular polysialic acid enhances the virulence of the bacterium. However, under natural conditions, the majority of E.coli K1 strains have low levels of O-acetylation of capsular polysialic acid. Only under special conditions, the O-acetylation level of capsular polysaccharide changes to adapt to the new environmental changes, so that the bacteria can survive.

Treatment of bacterial meningitis relies primarily on antibiotics, but with the emergence of resistant strains of antibiotics, the first-line efficacy of drugs is almost lost. Therefore, the development of saccharide vaccines of capsular polysialic acid is very important for preventing and controlling neonatal meningitis infection and is imminent. And the high level of O-acetylation modified K1 capsular polysaccharide can enhance the antigenicity and immunogenicity of saccharide vaccine. Therefore, the discovery of E.coli strains containing naturally synthesized highly O-acetylated modified capsular polysialic acid is important for the development of highly effective saccharide vaccines and the preparation of specific antibodies.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the O-acetylation level of capsular polysialic acid or provide an Escherichia coli strain containing capsular polysialic acid with high O-acetylation modification.

The invention provides Escherichia coli (Escherichia coli), the strain number of which is U9-41 Variant, and the registration number of which in China general microbiological culture Collection center is CGMCC No. 19571. Hereinafter, referred to as Escherichia coli U9-41 Variant.

The invention also provides a microbial inoculum for preparing the polysialic acid with high O-acetylation modification, which contains Escherichia coli U9-41 Variant.

The active ingredient of the microbial inoculum can be Escherichia coli U9-41 Variant, and can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the microbial inoculum can be determined by those skilled in the art according to the effect of the microbial inoculum.

The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, a marker (such as fluorescent protein and chromoprotein), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like can be added into the microbial inoculum.

The following uses of Escherichia coli U9-41 Variant also belong to the scope of the present invention:

the application of Escherichia coli U9-41 Variant in preparing vaccine for preventing diseases caused by Escherichia coli infection; and/or

Application of Escherichia coli U9-41 Variant in preparing O-acetylated modified polysialic acid.

The invention also provides a method for preparing polysialic acid, which comprises extracting O-acetylated modified polysialic acid from Escherichia coli U9-41 Variant.

The invention also provides O-acetylated modified polysialic acid, wherein the O-acetylated modified polysialic acid is obtained from Escherichia coli U9-41 Variant. The following applications of the polysialic acid also belong to the protection scope of the invention:

the application of O-acetylation modified polysialic acid in preparing antibody; and/or

The application of O-acetylation modified polysialic acid in preparing vaccine for preventing diseases caused by Escherichia coli infection.

The invention also provides an antibody, wherein the antibody is an antibody against the O-acetylation modified polysialic acid.

The invention also provides a vaccine for preventing diseases caused by Escherichia coli infection, wherein the vaccine comprises the O-acetylated modified polysialic acid.

As mentioned above, the polysialic acid has an O-acetyl content of 38 to 50%. The O-acetyl content is the O-acetylation level, and represents the percentage of the content of O-acetylated sialic acid in the capsular polysialic acid and the content of total sialic acid (sialic acid and O-acetylated sialic acid). For example, when the O-acetyl content of capsular polysialic acid is measured by HPLC, a polysialic acid sample is first hydrolyzed with an acid into sialic acid monomers including sialic acid molecules and O-acetylated sialic acid molecules and the components are separated by HPLC; then, comparing the retention time of the standard product when the peak appears to identify sialic acid molecules in the sample and different O-acetylation modified sialic acid molecules; the peak area of the response value of the sample peak reflects the content of the sample, so that the O-acetylated sialic acid content of the capsular polysialic acid is (peak area of total O-acetylated sialic acid/peak area of total sialic acid) X100%.

As mentioned above, the disease caused by E.coli infection may be sepsis, meningitis, urinary tract infection and/or respiratory tract infection of poultry, etc.

As described above, the antibodies can be used to prepare detection reagents useful for detecting capsular polysialic acid, O-acetylated capsular polysialic acid, or diagnosing diseases caused by E.coli infection, and the like.

The Escherichia coli U9-41 Variant provided by the invention can be used for producing natural high-level O-acetylation modified alpha 2-8-polysialic acid, and is used for developing polysaccharide vaccines and preparing corresponding antibodies. On the other hand, the strain can also be used for scientific research to reveal the biological function and pathogenic mechanism of capsular polysialic acid O-acetylation modification.

Deposit description

The strain name is as follows: escherichia coli

Latin name: escherichia coli

The strain number is as follows: u9-41 Variant

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 2020, 04 and 13 months

Registration number of the preservation center: CGMCC No.19571

Drawings

Fig. 1 shows the structure of the e.coli K1 capsular polysialic acid chain.

Fig. 2 is a synthetic pathway of e.coli K1 capsular polysialic acid.

FIG. 3 shows the neuO gene structure and translational regulation, where A is the structure and B is the translational regulation.

FIG. 4 is a nucleotide sequence alignment of the neuO gene of E.coli K1 strain U9-41(WT) and its Variant (Variant) of example 1, wherein WT has 38 repeats at the 5' end of the neuO gene; variant has 39 repeated sequences at the 5' end of neuO gene.

FIG. 5 is a growth curve of E.coli of example 2.

FIG. 6 is a high performance liquid chromatogram of sialic acid and O-acetylated sialic acid of example 3, wherein the abscissa is retention time (min) and the ordinate is response value (millivolts). Neu5Ac is a sialic acid molecule, Neu5, 7Ac2 is a sialic acid molecule with the 7-carbon atom modified by O-acetylation; neu5, 9Ac2 is a sialic acid molecule with the carbon atom at position 9 modified by O-acetylation; neu5, 7(8), 9Ac3 is a sialic acid molecule with carbon atoms at

positions

7, 8 and 9 modified by O-acetylation; r represents other reagent molecules.

FIG. 7 is a graph of the assay for capsular polysialic acid and O-acetylated sialic acid of example 3.

FIG. 8 shows the results of the E.coli adherence and infestation experiments of example 4.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The synthesis pathway of K1 capsular polysialic acid is shown in FIG. 2, and comprises intracellular synthesis of sialic acid monomer (Neu5Ac) from UDP-N-acetylglucosamine (UDP-GlcNAc), acetylation of sialic acid monomer under the action of O-acetyltransferase (NeuD), activation of acetylated sialic acid under the action of bifunctional enzyme (NeuA), deacetylation of the acetylated sialic acid group to produce activated sialic acid monomer (CMP-Neu5Ac), and synthesis of alpha 2-8 linked polysialic acid ([ Neu5Ac alpha 2-8Neu5Ac) under the action of polymerase (NeuS)]_n). Only a few partially activated acetylated sialic acids (CMP-O-acetyl-Neu5Ac) were incorporated into the polysialic acid synthesis, resulting in a small amount of acetylated modified polysialic acid. Therefore, the acetylation level of capsular polysialic acid sugar chain of Escherichia coli strain K1 was low. Importantly, the genome also has an O-acetyltransferase (NeuO) which can specifically carry out O-acetylation modification on the synthesized alpha 2-8 connected polysialic acid long chain and is the main mode of acetylation of K1 capsular polysialic acid. However, the catalytic activity of NeuO is variable.

As shown in FIG. 3, the neuO gene has a unique repetitive sequence region (poly Ψ domain) at the 5' end, which is formed by repeating an AAGACTC heptabase sequence. Only when the number of repetitions (VNTR) is a multiple of 3, the neuO gene is normally transcribed and translated and is able to synthesize the complete active O-acetyltransferase, i.e., neuO is in the "on" state (neuO-on); if the number of repeats is not a multiple of 3, the neuO gene will undergo a frame shift mutation during transcription, resulting in a stop codon that cannot be translated into O-acetyltransferase, i.e., neuO is in the "off" state (neuO-off). Moreover, studies have demonstrated that the catalytic activity of neuO enzymes increases linearly with the number of repeats of the repeat sequence when neuO is "on". This way of regulation of the strain Escherichia coli K1 leads to a more frequent change in the O-acetylation level, i.e.to a different level of modification or non-modification or modification of the capsular polysialic acid by O-acetylation.

Generally, the acetylation level of the E.coli K1 capsular polysialic acid is only 2-4%, which is synthesized by NeuD and belongs to the inherent expression of bacteria. Bacteria are affected by factors that alter the copy number of the repeat sequence in the poly Ψ region of the neuO gene, which causes the gene to "open" and produce an active enzyme. The more the repetition times, the stronger the enzymatic activity and the higher the O-acetylation level of polysialic acid.

EXAMPLE 1 obtaining of E.coli Strain U9-41 Variant

1. Escherichia coli K1 strain U9-41 (serotype O2: K1: H4) was purchased from the national collections of medical cultures (No. 44277), and hereinafter referred to as the wild type strain.

2. The bacterium was inoculated into 3mL of LB medium and cultured at 37 ℃ and 200rpm for 16 hours. The cells were collected by centrifugation.

3. The genomic DNA of the bacterium was extracted.

(1) 3mL of overnight-cultured bacterial liquid was put into a centrifuge tube and centrifuged at 5000rpm for 3 minutes to collect the cells. The bacteria were resuspended in 250. mu.L of 50mM Tris (pH8.0) buffer and centrifuged at 5000rpm for 3 minutes to collect the cells.

(2) The bacteria were resuspended in 250. mu.L of 50mM Tris (pH8.0) buffer, 10. mu.L of 0.4M EDTA was added, 20 minutes at 37 ℃ and 10. mu.L of 20mg/mL lysozyme was added, and the action was continued for 30 minutes to lyse the bacteria.

(3) Protein was degraded by addition of 1.5uL of 20mg/mL proteinase K.

(4) Add 15. mu.L of 10% SDS and water bath at 50 ℃ for 2 hours.

(5) Add 3. mu.L of 10mg/mL RNase and wash in water at 65 ℃ for 30 minutes to remove RNA.

(6) The solution was transferred to a centrifuge tube using a tip-off pipette tip, 250. mu.L phenol, chloroform, isoamyl alcohol (25: 24: 1) was added, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was transferred to the centrifuge tube and the extraction was repeated once to remove the protein.

(7) Then 250. mu.L of chloroform/isoamyl alcohol (24: 1) was added thereto, and centrifuged at 12000rpm for 10 minutes to remove phenol. The supernatant was transferred to a clean centrifuge tube.

(8) Add 600. mu.L of pre-cooled absolute ethanol and centrifuge at 12000rpm for 10 minutes to pellet the DNA. And 70% ethanol washing DNA to remove salt ion.

(9) The genomic DNA was dissolved in 100. mu.L of TE.

4. Obtaining the sequence of the neuO gene of the bacterium

(1) First, the sequences of neuO genes in NCBI nucleic acid database (GenBank) are searched and compared, and most of neuO genes are found to be positioned between sialK1 gene and intS gene, so that upstream primer NeuO-1 (5'-CGGGAGCATCATTGTTGATGAG-3') and downstream primer NeuO-2 (5'-TTATTGCGTGAGCTTCGCATG-3') are designed according to the two genes, and the neuO genes are amplified by PCR by taking genome as a template.

The PCR reaction system is as follows:

PCR amplification reaction conditions: denaturation at 95 ℃ for 30 seconds, annealing at 61 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, and after 40 cycles, incubation at 72 ℃ for 5 minutes.

Primer Synthesis Co: shanghai bioengineering GmbH

(2) The PCR product was purified with a purification kit (AXYPREP PCR clean kit) and then subjected to sequencing analysis, and the result was shown in SEQ ID NO. 1. Sequence analysis shows that the poly psi region of the neuO gene of the strain contains 38 repetitive sequences, so that O-acetyltransferase neuO cannot be synthesized.

5. Obtaining recombinant Escherichia coli K1 strain containing high-level O-acetylation modification through subculturing of bacteria

(1) A loop of Escherichia coli K1 strain U9-41 frozen at-80 ℃ is selected and streaked on LB solid medium, and cultured in an incubator at 37 ℃ for 24 hours.

(2) Single colonies on the plate of step (1) were picked, inoculated into LB liquid medium and cultured at 37 ℃ for 16 hours at 200 rpm.

(3) Inoculating the bacterial culture solution obtained in the step (2) into a new LB liquid culture medium at a ratio of 1: 100, and culturing at 37 ℃ and 200rpm for 16 hours to obtain a bacterial culture of the first passage.

(4) Inoculating the bacterial culture solution obtained in the step (3) into a new LB liquid culture medium at a ratio of 1: 100, and culturing at 37 ℃ and 200rpm for 16 hours to obtain a bacterial culture of the second passage.

(5) This procedure was repeated further and the bacteria were subcultured 5 times in succession.

(6) Serial 10-fold dilution of the bacterial culture after the 5 th passage was performed, and 100uL of 10 cells was taken⁷And 10⁸The diluted bacterial suspension was applied to LB solid medium and cultured in an aerobic incubator at 37 ℃ for 24 hours to grow a single colony.

(7) The single colony obtained in step (6) was picked up into 30uL of lysis buffer (2% Triton-X100, 2.5mg Sodium Azide, 1mL 0.1M This-HCl), acted at 80 ℃ for 5min to lyse the bacteria, and then cooled to room temperature.

1uL of bacterial lysate was used as template, and neuO gene was amplified using primers NeuO-1 and NeuO-2 and Takara Taq DNA polymerase. The PCR reaction system and reaction conditions were as described above. PCR products were purified with a kit and sent to the company for sequencing. In total, 36 single colonies were screened to obtain the sequences of 36 neuO genes.

(8) And (5) aligning and analyzing the sequences. And (3) comparing the DNA sequences obtained in the step (7) by using ClustalW, and as a result, only 1 poly psi region of the neuO gene contains 39 repetitive sequences, and the nucleotide sequence of the neuO gene is shown as SEQ ID NO. 2. This neuO gene (Variant in figure 4) was increased by only one copy of AAGACTC over the neuO gene of the wild type strain (WT in figure 4) by NCBI blast comparison, all the other being the same (figure 4).

(9) The single colony of the strain with the nucleotide sequence of the neuO gene shown as SEQ ID No.2 is inoculated into LB liquid culture medium, cultured at 37 ℃ and 200rpm for 10 hours, added with 80% glycerol with the same volume, mixed and frozen. The strain has the number of U9-41 Variant, the scientific name is Escherichia coli (Escherichia coli), the registration number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.19571, and the strain is hereinafter referred to as Escherichia coli strain U9-41 Variant.

Example 2 growth curves of wild-type and variant strains of E.coli

This example demonstrates that the growth characteristics of the variant strain of E.coli K1 strain U9-41 are almost identical to the growth characteristics of the wild type strain.

(1) A loop of the frozen stock of bacteria (wild strain, E.coli K1 strain U9-41; mutant strain, E.coli strain U9-41 Variant) was inoculated into 3mL of LB liquid medium, and the bacteria were cultured overnight at 37 ℃ and 200r/min for about 16 hours.

(2) The fresh bacterial culture obtained in step (1) was rejoined to 3mL of LB liquid medium at a ratio of 1: 100, the shake tube was inserted into a GENESYS 30 spectrophotometer (Thermo Scientific), the uninoculated LB medium was used as a control, photoelectric turbidimetry was performed at a wavelength of 600nm, and the initial OD was recorded. The bacteria were cultured at 37 ℃ on a shaker at 200 r/min. The glass shaking tube was taken out every 1 hour, and the OD value was measured. The measurement was continued for 16 hours.

(3) And drawing a growth curve of the escherichia coli by taking the time as an abscissa and the wavelength value of 600nm as an ordinate. As shown in FIG. 5, the growth characteristics of the E.coli variant strain and the wild strain were almost the same, except that the growth amount of the variant strain was slightly reduced as compared with the wild strain.

Example 3 detection of O-acetyl content of capsular polysialic acid in strains U9-41 and U9-41 Variant

This example identifies the O-acetyl content of capsular polysialic acid in wild-type strains (WT, i.e.E.coli K1 strain U9-41) and variants thereof (Variant, i.e.E.coli strain U9-41 Variant). The methods are modified with reference to the references.

(1) Separation and extraction of capsular polysialic acid: a loop of the bacterial frozen stock was picked and inoculated into 3mL of LB liquid medium, cultured overnight at 37 ℃ and 200 r/min. The overnight culture was re-inoculated into 3mL LB liquid medium at a ratio of 1: 100, cultured at 37 ℃ for 2h-3h at 200r/min, OD_600nmAbout 0.6. 1mL of the bacterial solution was centrifuged at 4 ℃ and 2000g for 15 min. The pellet was washed 4 times with pre-cooled 1x PBS (pH 7.4), 700. mu.L each, 4 ℃, 2000g, and centrifuged for 15min to wash out residual media components. The pellet was resuspended in 100. mu.L of pre-warmed 1 XPBS (pH 7.4) and placed in a 37 ℃ water bath for 1h, during which time the tube was flicked 10 times with a finger every 10min to release loosely bound capsular polysialic acid from the bacterial surface into the culture. Centrifuging at 4 deg.C and 10000g for 10min, removing impurities, collecting polysaccharide in supernatant, quick freezing with liquid nitrogen, and immediately freezing into dry powder in freeze dryer. Adding 100 μ L of 2mol/L acetic acid to dissolve the sample, placing in a water bath kettle at 80 deg.C for hydrolysis for 2h to change the long chain of polysialic acid into sialic acid monomer, cooling to room temperature, lyophilizing the product again, and adding 10 μ L of double distilled water to completely dissolve the sample. Capsular sialic acid samples of Escherichia coli K1 strain U9-41 and Escherichia coli strain U9-41 Variant were obtained by this method, respectively.

(2) Derivatization labeling of sialic acid samples: labeling was carried out using kit (Glyko Signal DMB labeling kit) from prozyme. Namely, 20. mu.L of DMB (1, 2-diamino-4, 5-methylenedioxybenzene dihydrochloride) is added into 5. mu.L of a sample, mixed, placed in a water bath kettle at 50 ℃ for 3 hours in a dark place for derivatization and labeling, and then 500. mu.L of double distilled water is added to terminate the reaction. The sample was again lyophilized and dissolved completely with 10 μ L of double distilled water. Meanwhile, a standard mixture of sialic acid and O-acetylated sialic acid (including Neu5Ac, Neu5, 7Ac2, Neu5, 9Ac2, Neu5, 7(8) and 9Ac3) provided by the kit is labeled by DMB, and the steps of the method are the same.

(3) Sialic acid and O-acetylated sialic acid were identified by High Performance Liquid Chromatography (HPLC), and the content of O-acetylated sialic acid was determined. The sample mixture was separated on an Agilent HC-C18HPLC (4.6 mm. times.250 mm) column with a sample volume of 20. mu.L and the column was eluted isocratically with acetonitrile, methanol and deionized water (volume ratio 9: 7: 84) for 40min at a flow rate of 0.9 mL/min. The eluted sialic acid was detected by a fluorescence detector at 373nm excitation and 448nm detection wavelength. DMB-labeled standards were also run through the column in the same manner. The sialic acid peak in the sample was determined using the peak of the standard as a reference. The results are shown in FIG. 6, wherein A in FIG. 6 is a HPLC chromatogram peak plot of each Standard (ST) for sialic acid and O-acetylated sialic acid, as a reference; b in FIG. 6 is a HPLC chromatogram peak of sialic acid and O-acetylated sialic acid in the capsule of wild-type strain (WT); FIG. 6C is a HPLC peak profile of sialic acid and O-acetylated sialic acid in the capsule of a Variant strain (Variant). The results showed that the peak of sialic acid (Neu5Ac) was high in the WT chromatogram and that the peaks of O-acetylated sialic acid molecules (including Neu5, 7Ac2, Neu5.9ac2 and Neu5, 7(8), 9Ac3) were low. Whereas the peak height of sialic acid in the chromatogram of Variant is significantly higher for O-acetylated sialic acid than for WT.

(4) The relative content of sialic acid and O-acetylated sialic acid was calculated by peak area. The experiment was independently repeated three times, and the results are shown as mean ± standard deviation (fig. 7); statistical analysis of the data was performed using GraphPad Prism 6 software. The results show that the sum of the peak areas of all sialic acids in the capsular polysaccharide of Escherichia coli K1 strain U9-41 and Escherichia coli strain U9-41 Variant is 1.87 multiplied by 10⁷±1.56×10⁶And 1.55X 10⁷±1.2×10⁶(ii) a The sum of the peak areas of all O-acetylated sialic acids was 5.33X 10⁵±6.5×10⁴And 6.81X 10⁶±1.4×10⁶(A in FIG. 7). The content of O-acetylated sialic acid was calculated according to the formula. The formula is as follows: the O-acetyl content of capsular polysialic acid (peak area of total O-acetylated sialic acid/peak areas of total sialic acid and O-acetylated sialic acid) X100%. The results showed that the O-acetyl content of capsular polysialic acid of Escherichia coli K1 strain U9-41 (i.e., wild strain) was 3% + -0.2%, and the O-acetyl content of capsular polysialic acid of Escherichia coli strain U9-41 Variant (i.e., mutant strain) was 44% + -6.1% (FIG. 7B) of (1). Thus, E.coli strain U9-41 Variant had a 14.7-fold level of O-acetylation of capsular polysialic acid as compared to the wild-type strain.

Example 4 Escherichia coli adhesion and infection experiments

This example demonstrates that high levels of O-acetylated modified capsular polysialic acid inhibit macrophage adhesion and phagocytosis of E.coli.

(1) The adhesion experiment is divided into two experimental groups, wherein the wild flora strain is Escherichia coli K1 strain U9-41, the Variant flora strain is Escherichia coli strain U9-41 Variant, the infection experiment is also divided into two experimental groups, the wild flora strain is Escherichia coli K1 strain U9-41, and the Variant flora strain is U9-41 Variant. Coli K1 strain U9-41 and E.coli strain U9-41 Variant were both cultured in LB medium at 37 ℃.

(2) A loop of frozen E.coli K1 strain U9-41 or E.coli strain U9-41 Variant was inoculated in 3mL of LB medium and cultured overnight at 37 ℃.

(3) After overnight incubation, the bacterial solution was diluted 100 times and counted on a bacterial counting plate.

(4) Murine macrophage RAW264.7 was cultured in DMEM complete medium, as follows.

(5) RAW264.7 cells were harvested, centrifuged at 1000r/min for 5min and resuspended in DMEM complete medium. At a density of 1X 10⁵Each cell/well was inoculated into a 24-well plate at 500. mu.L per well, and cultured in an incubator at 37 ℃ for 12 hours.

(6) The DMEM complete medium in the 24-well plate was aspirated and each well was washed 2 times with 500 μ L each time using DMEM minimal medium. After washing, 500. mu.L of DMEM minimal medium was added to each well.

(7) Adding the bacteria counted in the step 3) into the 24-well plate treated in the step 6), wherein the bacteria count is 1 multiplied by 10⁶Bacteria/well (MOI 10: 1), 5% CO at 37 ℃%₂Culturing in cell culture box for 60 min.

(8) In the adhesion experiment, the old medium was aspirated, and each well was washed 3 times with 1mL of DMEM minimal medium.

(9) In the dip-dyeing experiment, 500. mu.L of DMEM complete medium containing 200. mu.g/mL gentamicin was added and cultured at 37 ℃ for 1 hour to kill extracellular bacteria. Old medium was aspirated and each well was washed 2 times with 1mL of PBS.

(10) Adding 500 μ L of 0.5% Triton-X100 (precooling at 4 ℃) into the 24-well plate treated in the step 8) or the step 9) for 5min, blowing and beating until all cells are broken, and sucking the cells into a 1.5mL centrifuge tube.

(11) The blood plates were diluted and plated and incubated overnight at 37 ℃ in an incubator with inversion. The next day, bacterial colonies were counted and experimental results were counted.

(12) The adhesion and infection experiments of bacteria to macrophages were independently repeated three times. Statistical analysis was performed using GraphPad Prism 6 software and data are shown as mean ± standard deviation, # P < 0.05, calculated using Student's t-test. The results are shown in FIG. 8, in which A in FIG. 8 represents the adhesion and infection of the wild strain and the mutant strain to RAW264.7 cells; in FIG. 8B is the ratio of bacterial adhesion RAW 264.7; in FIG. 8, C is the ratio of bacteria invading RAW 264.7.

Analysis of the results showed that the amount of wild-type adhesion to RAW264.7 was 8300 + -1651 CFU/well; the number of adhesion of the inoculum to RAW264.7 was 4317. + -. 1179 CFU/well; the adhesion rate was calculated as CFU (number of adhered bacteria)/CFU (number of bacteria initially added per well) × 100%, so the adhesion rate of wild bacteria was 8.0%; the adhesion rate of the transformed bacteria was 4.3%. The number of wild bacteria invading RAW264.7 is 5940 +/-911 CFU/hole; the number of the transformed bacteria invading into RAW264.7 was 2760 ± 789 CFU/well, and the invasion rate was calculated as CFU (number of invading bacteria)/CFU (number of bacteria initially added per well) × 100%, so that the invasion rate of the wild bacteria was 5.9%; the invasion rate of the transformed bacteria was 2.8%. These results clearly show that high levels of O-acetylation modification of capsular polysialic acid significantly inhibit macrophage adhesion and phagocytosis of e.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

<120> Escherichia coli highly subjected to O-acetylation modification by capsular polysialic acid and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 917

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 1

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tgccatctat gtgtgcagca gccggtaatc cagcaaaaat aataaaaaga aatataatat 840

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cgaagctcac gcaataa 917

<210> 2

<211> 924

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 2

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aagactcaag actcaagact caagactcaa gactcaagac tcaagactca agactcaaga 240

ctcaagactc aagactcaag actcaagact caagactcgt tttccgttga tgataatggg 300

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attaattggg caaaagatat cattatatct agttacgtat gggtagggag aaatgtctct 720

ataatgaaag gagtatctgt tggaagcgga tctgtcattg ggtatgggag tattgtaact 780

aaagatgtgc catctatgtg tgcagcagcc ggtaatccag caaaaataat aaaaagaaat 840

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Claims

1. Escherichia coli, characterized in that: the Escherichia coli is Escherichia coli (Escherichia coli), the strain number of the Escherichia coli is U9-41 Variant, and the registration number of the Escherichia coli in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 19571.

2. A microbial inoculum for preparing O-acetylated modified polysialic acid, which is characterized in that: the microbial inoculum comprises the Escherichia coli according to claim 1.

3. Use of the escherichia coli of claim 1 for the preparation of a vaccine for the prevention of a disease caused by an escherichia coli infection.

4. Use of E.coli as claimed in claim 1 for the preparation of O-acetylated modified polysialic acid.

5. A method for preparing O-acetylated modified polysialic acid, characterized in that: the method comprises extracting O-acetylated modified polysialic acid from the Escherichia coli strain of claim 1.