CN109182367B

CN109182367B - Application of MSMEG-6171 in regulation of sensitivity of mycobacterium smegmatis to antibiotics

Info

Publication number: CN109182367B
Application number: CN201810907735.0A
Authority: CN
Inventors: 魏文静; 巫株华; 周琳; 郭卉欣; 陈亮; 陈瑜晖; 陈燕梅
Original assignee: CENTER FOR TUBERCULOSIS CONTROL OF GUANGDONG PROVINCE
Current assignee: CENTER FOR TUBERCULOSIS CONTROL OF GUANGDONG PROVINCE
Priority date: 2018-08-10
Filing date: 2018-08-10
Publication date: 2022-03-04
Anticipated expiration: 2038-08-10
Also published as: CN109182367A

Abstract

The invention discloses a method for regulating the sensitivity of mycobacterium smegmatis to antibiotics, which is characterized by comprising the following steps: the expression level of MSMEG _6171 is regulated. The inhibition of MSMEG _6171 expression can enhance the sensitivity of the mycobacterium smegmatis to antibiotics, and the promotion of MSMEG _6171 overexpression can weaken the sensitivity of the mycobacterium smegmatis to antibiotics. Provides a new method for regulating the sensitivity of mycobacterium smegmatis to antibiotics.

Description

Application of MSMEG-6171 in regulation of sensitivity of mycobacterium smegmatis to antibiotics

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of MSMEG-6171 in regulation of sensitivity of Mycobacterium smegmatis to antibiotics.

Background

The mycobacterium smegmatis (M.smegmatis) belongs to gram-positive saprophytic bacteria, has the characteristics of rapid growth, no pathogenicity, high homology with mycobacterium tuberculosis (M.tuberculosis) genes, similar cell structure and the like, is more applied to research of mycobacterium infection and related immunology, and is a relatively ideal experimental model. Meanwhile, the recombinant human mycobacterium tuberculosis vaccine also has a expansibility application in non-mycobacterial infection and other related immunity researches.

The mycobacterium smegmatis has a lipid-rich cell wall, is complex in composition and diverse in function, and is an important barrier for the mycobacterium to resist the pressure of the external environment and escape from host immune attack. The mycobacterium smegmatis cell wall is composed of two parts: an inner layer and an outer layer. The outer layer is the soluble component of the cell wall consisting of lipids and proteins, into which long and short chain fatty acids are embedded. The inner layer is composed of Peptidoglycan (PG), Arabinogalactan (AG), and Mycolic Acid (MA). It has been found that more and more proteins are involved in the synthesis of cell walls, and these proteins affect the production of cell walls by participating in the synthesis of fatty acid acyl chains, glycopeptide lipids (GPLs), peptide modification, and the assembly of various synthetic enzymes on cell membranes, etc., thereby affecting the resistance of bacteria to drugs. Many anti-mycobacterial drugs are specifically used against this good protective barrier.

MSMEG _6171 is a protein with unknown function in Mycobacterium smegmatis, the amino acid sequence of MSMEG _6171 is shown as SEQ ID No.1, the coding gene is shown as SEQ ID No.2, MSMEG _6171 protein function research has not been reported, and the homologous protein in Mycobacterium tuberculosis is Rv3660 c. A study of England. K in 2011 shows that when the Rv3660c is over-expressed, the generation of a diaphragm can be inhibited to cause bacterial fibrillation, the study discovers that the over-expression of the protein can cause the mRNA expression quantity of some cell regulatory factors to be remarkably changed through transcriptomics analysis, the regulatory factors comprise dormancy regulators and some sigma factors which are considered to play a role in adaptive metabolism, and simultaneously discovers that the Rv3660c is deleted to offset the expression quantity change of the regulatory factors and cause the bacterial shortening. There are currently few reports of the regulation of antibiotic susceptibility in bacteria by msreg _6171 and its homologous protein Rv3660 c.

Disclosure of Invention

The invention aims to provide application of MSMEG-6171 in regulation of sensitivity of Mycobacterium smegmatis to antibiotics.

The technical scheme adopted by the invention is as follows:

a method for regulating the sensitivity of Mycobacterium smegmatis to antibiotics is realized by regulating the expression level of MSMEG-6171.

Further, inhibition of MSMEG _6171 expression increases the sensitivity of Mycobacterium smegmatis to antibiotics, and promotion of MSMEG _6171 overexpression decreases the sensitivity of Mycobacterium smegmatis to antibiotics.

Further, the expression level of MSMEG _6171 is the expression level of MSMEG _6171 protein.

Further, the antibiotic is an antibiotic acting on cell wall synthesis of Mycobacterium smegmatis.

Further, the antibiotic is any one or more of vancomycin, cefotaxime, cefoxitin and cephapridine.

The application of the reagent for regulating the expression level of MSMEG-6171 in the preparation of a kit for regulating the sensitivity of Mycobacterium smegmatis to antibiotics.

Further, a kit for enhancing the sensitivity of Mycobacterium smegmatis to antibiotics is prepared using an agent that inhibits MSMEG _6171 expression, and a kit for attenuating the sensitivity of Mycobacterium smegmatis to antibiotics is prepared using an agent that promotes MSMEG _6171 overexpression.

The invention has the beneficial effects that:

the invention discloses a relation between MSMEG _6171 and antibiotic sensitivity of mycobacterium smegmatis, and particularly inhibits MSMEG _6171 expression to enhance antibiotic sensitivity of mycobacterium smegmatis and promotes MSMEG _6171 overexpression to weaken antibiotic sensitivity of mycobacterium smegmatis. The invention provides a novel method for regulating the sensitivity of mycobacterium smegmatis to antibiotics.

Drawings

FIG. 1 is an agarose electrophoresis picture of the PCR product of the MSMEG _6171 gene;

FIG. 2 shows PCR verification of MSMEG _6171 over-expressed Mycobacterium smegmatis bacterial liquid,

lanes

1, 2 and 3 are all positive strains;

FIG. 3 shows the result of Western Blot analysis of MSMEG _6171-3X Flag fusion protein expression, lane 1: protein marker; lane 2: no-load comparison; lane 3: overexpressing a recombinant strain;

FIG. 4 is an agarose electrophoresis of the MSMEG _6171 deleted Mycobacterium smegmatis PCR product, lane 1: a deletion strain; lane 2: no-load comparison; m: d2000 DNA marker;

FIG. 5 shows the results of Pull Down experiments;

FIG. 6 is a fluorescent staining chart, wherein A is wild type M.smegmatis, B is M.smegmatis with MSMEG _6171 over-expression, C is M.smegmatis with MSMEG _6171 deletion, and D is anaplerotic strain.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Example 1

The MSMEG-6171 protein is expressed by using a mycobacterium-escherichia coli shuttle plasmid pMV261-3X flag (New use of BCG for recombinants vaccines, Nature,1991.351(6326): 456-60; publicly available from tuberculosis control center in Guangdong province), and the mycobacterium smegmatis over-expressing the MSMEG-6171 is obtained.

One, construction of MSMEG _6171 expression plasmid

Mycobacterium smegmatis mc²155 as a template by a forward primer 5' -agaattcatgccgtcgagtcgtaagg-3 '(SEQ ID No.3) and reverse primer 5' -taaaagcttccgccgcctcccgcacag-3' (SEQ ID No.4) to obtain MSMEG _6171 gene coding fragment, the result of gel electrophoresis detection is shown in figure 1, the MSMEG _6171 gene coding fragment is connected to pMV261-3X Flag vector by EcoRI, HindIII and T4 ligase of NEB company, and MSMEG _6171-3X Flag fusion protein expression plasmid pMV261-HtpG-3X Flag is obtained. The recombinant plasmid was sequenced and confirmed to be correct.

Secondly, preparing pMV261-MSMEG _6171-3X Flag Mycobacterium smegmatis

(1) Mycobacterium smegmatis mc²155 strains in containing 0.5% glycerol and 0.05% Tween 80 7H9 liquid medium at 37 degrees C shaking culture for 3 days, 1% transfer fresh medium, 37 degrees C shaking culture to logarithmic phase (OD)₆₀₀About 0.6), the cells were collected and washed with 10% sterile glycerol (deionized water) for 3 times to prepare electroporation competence.

(2) 10 mul of plasmid (100 ng/. mu.L) pMV261-HtpG-3X Flag and pMV261-3X Flag are respectively added into 300 mul of competence, the mixture is transferred to an electric rotating cup, parameters of an electric converter are set to be 2.5kV, 25 muF and 1000 omega, 800 mul of precooled fresh culture medium is added after electric shock is finished, and the bacteria are recovered by shaking culture at 37 ℃ for 2 h.

(3) Taking the recovered bacteria 13 coating 7H10 solid culture medium containing kanamycin 30 mu g/mL, standing and culturing for 3 days at 37 ℃ to obtain the mycobacterium smegmatis recombinant bacterium pMV261-MSMEG _6171-3X Flag/mc²155 and pMV261-3X Flag/mc² 155。

(4) Selecting recombinant bacteria pMV261-MSMEG _6171-3X Flag/mc²155 and pMV261-3X Flag/mc²155 single colony, inoculating 7H9 liquid culture medium containing kanamycin 30 mu g/mL, 0.5% glycerol and 0.05% Tween 80, performing shake culture at 37 ℃ for 3 days, sucking 50 mu L of bacterial liquid, and centrifuging to precipitate thallus; 10 mu L of deionized water is used for resuspending the bacteria, 1 mu L of deionized water is used for PCR reaction, the primers are a forward primer and a reverse primer used for constructing the MSMEG _6171 expression plasmid, and whether the MSMEG _6171 gene is successfully introduced into the mycobacterium smegmatis is verified, and the result is shown in figure 2.

Thirdly, detecting whether the MSMEG-6171-3X Flag fusion protein is expressed or not by using Western Blot

20 ug of pMV261-MSMEG _6171-3X Flag/mc²155 cell lysate was subjected to Western Blot detection, and the result is shown in FIG. 3, which can obtain an obvious fluorescence signal, and prove that MSMEG _6171-3X Flag is indeed expressed.

Example 2

The MSMEG-6171 gene knockout strain is constructed by a homologous gene recombination method.

TABLE 1 recombinant plasmids relating to homologous gene recombination

Construction of suicide plasmid

(1) PCR amplification of MSMEG _6171 upstream and downstream homology arms: MSMEG-6171 gene and its upstream and downstream DNA sequences are used to design primer 6171-UF: 5' -cccaagcttatgtagtcacgcatccggtcc-3' (SEQ ID No.5) (underlined sequence indicates a HindIII site), 6171-UR: 5'-gagtgcctcgtcgagacccggttcgtcgcatccgatcac-3' (SEQ ID No.6), and the MSMEG _6171 gene upstream homology arm (Up,855bp) is obtained by using M.smegmatis genomic DNA as a template for amplification. With 6171-DF: 5'-gtgatcggatgcgacgaaccgggtctcgacgaggcactc-3' (SEQ ID No.7) and 6171-DR: 5'-ttaattaacggtgttgagcgcggcg-3' (SEQ ID No.8) (underlined sequence indicates PacI site), and the MSMEG _6171 gene downstream homology arm (Down,849bp) was amplified using M.smegmatis genomic DNA as a template. And respectively purifying and recovering the upstream and downstream homologous arm fragments, and amplifying the overlapped fragments by using the upstream homologous arm and the downstream homologous arm as templates and using 6171-UF and 6171-DR as primers by using an overlap PCR method.

(2) The well-connected upstream and downstream homology arms are inserted into a p1NIL vector by utilizing HindIII and PacI enzyme cutting sites, then a gene fragment for screening in a pGOAL19 vector is cut by PacI enzyme and recovered, and the fragment is connected into a p1NIL recombinant vector containing the homology arms.

(3) Transferring the suicide particles into E.coli Top10, carrying out PCR and sequencing verification, and upgrading the particles for later use.

II, alkali treatment of suicide plasmid

Taking a small amount of the stored plasmid, determining its concentration, taking about 20 μ g of plasmid according to its concentration, adding ddH₂O to 100. mu.L, then 100. mu.L of 0.4mol/L NaOH was added. 30min at 37 ℃. Adding 20 μ L of 3M sodium acetate, adding 140 μ L of isopropanol, precipitating at-80 deg.C for 1h or overnight, centrifuging, discarding supernatant, adding 700 μ L of 75% ethanol, centrifuging, discarding supernatant, and washing repeatedly. Standing at room temperature for 5min, adding 7 μ L ddH₂And dissolving the O.

Thirdly, screening single-exchange strain

Adding 7 mu L of the plasmid after alkali treatment into 350 mu L of competent cells (note that the volume of the plasmid corresponding to 200 mu L of competent cells is not more than 5 mu L), incubating for 10-20 min at 37 ℃, and then transferring the bacterial liquid into an electric cuvette. After confirming that the outer wall of the electric rotor was clean, electric shock was applied (voltage 2.5kV, resistance 1000. omega. and capacitance 25. mu.F), and 0.7mL of 7H9 liquid medium was immediately added. The cells were aspirated, placed in a 50mL centrifuge tube, and supplemented with 7H9 liquid medium to 4mL (typically, the volume of medium used for resuscitation was 10 times the competent volume). And (4) performing shake culture at 37 ℃ for 2-3 days for resuscitation. After recovery, centrifugation is carried out at 6000rpm for 15min, 200 mu L of culture medium is taken to suspend thallus, a 7H10 plate (containing OADC, 50 mu g/mL hygromycin, 50 mu g/mL kanamycin and 50 mu g/mL Xgal) is coated, and the thallus is cultured for 30-40 days in the dark to observe whether blue spots are generated or not, if yes, the single exchange is successful.

Fourth, screening double-exchange strain

And (4) culturing for 30-40 days in a dark place to observe whether the blue spots are generated, if so, indicating that the single exchange is successful, selecting the blue spots, and culturing in a 7H9 liquid culture medium. mu.L of the plate was coated with 7H10 (containing OADC, X-Gal, 2% sucrose) at 4H, 8H, 12H and 16H, respectively. The cells were incubated at 37 ℃ in an incubator protected from light for 2-3 days, white colonies were picked, and PCR was performed after 2 days of incubation in 2mL of 7H9 medium.

Complementation of mycobacterium smegmatis MSMEG-6171 deletion strain

(1) The anaplerotic plasmid is pMV361-MSMEG _ 6171.

(2) Preparation of competent cells of M.tuberculosis MSMEG-6171 deletion strain: reference is made to the preparation of the above described Mycobacterium smegmatis competent cells. The PCR identification result of MSMEG _ 6171-deleted Mycobacterium smegmatis is shown in FIG. 4.

(3) Electroporation of competent cells: refer to the above transformation procedure.

Example 3

MIC detection of 10 antibiotics by wild type Mycobacterium smegmatis, M.smegmatis deleted by MSMEG _6171, anaplerotic strain and M.smegmatis overexpressed by MSMEG _ 6171.

Sensitivity of MSMEG _6171 to antibiotics was tested by dilution. Antibiotic solutions (rifampin, isoniazid, streptomycin, vancomycin, ethionamide, cycloserine, meropenem, cephamidin, cefoxitin, cefotaxime) at different concentrations after dilution in double ratio were added to sterile 96-well polystyrene plates, respectively. Wells 1 to 11 were dosed with 10 μ L per well, well 12 was not dosed as a growth control, 3 replicates. Diluting the bacterial liquid to 0.5 McLeod ratio turbidity, diluting with 7H9 liquid culture medium at a ratio of 1: 1000, adding 90 μ L of diluted bacterial liquid into each hole, sealing, and incubating in a shaking table at 37 deg.C for 2-3H, wherein the lowest concentration capable of inhibiting bacterial growth is MICs.

The results of the Minimum Inhibitory Concentrations (MIC) measurements of 10 different antibiotics revealed that isoniazid, rifampicin, streptomycin, cycloserine were at the same level for all bacteria. After the MSMEG-6171 gene is deleted, the sensitivity of the mycobacterium smegmatis to vancomycin, cefotaxime, cefoxitin and cefpyridine is enhanced, and the corresponding MIC value is reduced; meanwhile, the sensitivity of MSMEG _6171 over-expressed Mycobacterium smegmatis to vancomycin, cefotaxime, cefoxitin and cephapridine is weakened, and the corresponding MIC value is increased.

Example 4

The result of the protein interacting with MSMEG-6171 in Mycobacterium smegmatis is shown in figure 5, the protein interacting with MSMEG-6171 in the compound is identified by mass spectrometry after the differential bands are subjected to enzymolysis, and the interaction between MSMEG-6171 and enzymes related to cell wall synthesis such as fatty acid synthetase, glycerol-3-phosphate dehydrogenase and the like is found through the analysis of the MSMEG-6171 interacting protein, so that the MSMEG-6171 is used as one member of a protein compound and participates in the synthesis of bacterial cell walls.

Example 5

Fluorescence staining experiments confirmed whether MSMEG _6171 affected the formation of nuclei and septa of M.smegmatis.

The results of staining with BODIPY-labeled vancomycin and DAPI and observing with a Zeiss LSM880 fluorescence confocal microscope are shown in fig. 6. Fluorescent staining of wild Mycobacterium smegmatis, Mycobacterium smegmatis overexpressed by MSMEG _6171, Mycobacterium smegmatis deleted by MSMEG _6171 and anaplerosis shows that the generation of the septum and the nucleus of the four bacteria under in vitro culture has no obvious difference, which indicates that MSMEG _6171 has no influence on the generation of the septum and the nucleus of the Mycobacterium smegmatis.

SEQUENCE LISTING

<110> tuberculosis control center of Guangdong province

<120> use of MSMEG _6171 for modulating sensitivity of M.smegmatis to antibiotics

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 316

<212> PRT

<213> MSMEG_6171

<400> 1

Met Pro Ser Ser Arg Lys Ala Trp Leu Ser Ala Ala Ala Val Leu Leu

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Asp Ala Ser Ala Ala Arg Arg Cys Ala Asp Arg Gly Leu Pro Arg Arg

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Asp Arg Val Leu Val Ile Gly Cys Asp Glu Pro Gly Pro Ala Asp Trp

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Gln Ala Ala Ile Ala Val Gly Ala Gln His Val Val Thr Leu Pro Arg

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Gln Asp Thr Asp Leu Val Ala Ala Leu Ser Val Val Asp Glu Gly Gly

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Gly His Arg Gly Pro Val Val Ala Val Val Ala Ala Lys Gly Gly Ala

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Gly Ala Ser Val Phe Ala Ala Ala Leu Ala Leu Ser Ala Pro Gly Ala

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Leu Leu Val Asp Ala Asp Pro Trp Ser Gly Gly Ile Asp Leu Val Leu

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Gly Ser Glu Asp Gln Pro Gly Leu Arg Trp Ala Asp Leu Ala Leu Gln

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Gly Gly Arg Leu Gly Tyr Gly Ala Leu Arg Asp Ala Leu Pro Arg Arg

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Gly Glu Ile Ser Val Leu Ser Gly Gly Arg Ala Gly Val Asp Ile Thr

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Ala Ala Ala Leu His Ala Val Ile Asp Ala Gly Cys Arg Gly Ala Thr

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Leu Val Val Cys Asp Val Pro Arg Arg Ser Thr Asp Ala Ala Glu Ala

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Ala Leu Glu Ala Ala Asp Leu Val Val Val Val Ala Arg Ala Asp Val

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Met Arg Pro Gln Pro Gly Leu Asp Glu Ala Leu Glu Arg Gly Gly Leu

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Gly Val Leu Ala Gln His Pro Val Arg Glu Ala Ala

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<213> MSMEG_6171

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atgccgtcga gtcgtaaggc ctggctgtcc gcggcggccg tgctgctcga cgcgtcggcc 60

gcgcggcggt gcgccgaccg gggactgccg cgccgcgacc gggtgctcgt gatcggatgc 120

gacgaaccgg gtccggccga ttggcaggcc gcgatcgccg tcggtgcgca acacgtcgtg 180

acgctgcccc gccaggacac cgatcttgtg gcggcgctgt ctgtcgtcga cgagggcggt 240

ggacaccgcg gaccggtggt tgcggtggtc gcggcgaaag gcggcgccgg ggcatcggtg 300

ttcgccgccg cgcttgccct gtcggcgccg ggagcgttgc tcgtcgacgc cgacccgtgg 360

agcggcggca tcgacctcgt gctcggcagt gaggatcagc cgggtctgcg gtgggcggat 420

cttgcgctgc agggcggcag gctcgggtac ggcgcgttgc gcgacgcgct gccgcgccgc 480

ggcgagatca gtgtgctctc gggtggacgc gcgggcgtcg acatcaccgc ggcggcactg 540

cacgccgtga tcgacgcggg ttgtcgcggc gcgactttgg tggtctgcga cgtcccccgg 600

cgcagcaccg acgcggccga ggccgcgctc gaggcagccg acctggtggt cgtggtcgcc 660

cgcgccgatg tgcgttcgtg tgccgcggca gcggccgccg gatcctggat cgccacgtgc 720

aatcccaaca tcggtgtggt ggtgcgcggt ccggcaccgg gaggtctgcg tgccgccgag 780

gtcgccgaca tcgtcgacct gccgctgctg gcgtcgatgc gtccgcagcc cggtctcgac 840

gaggcactcg aacgtggcgg gttgcggctc acgcgccgct ctccgctggc caccgcggcg 900

cgacgcgtgc tcggcgtgct cgcgcagcat cctgtgcggg aggcggcgtg a 951

Claims

1. A method of modulating the sensitivity of mycobacterium smegmatis to an antibiotic, comprising: the expression level of MSMEG-6171 is adjusted; the antibiotic is any one or more of vancomycin, cefotaxime, cefoxitin and cephapridine; the amino acid sequence of the MSMEG-6171 is shown in SEQ ID No. 1.

2. The method of claim 1, wherein: the inhibition of MSMEG _6171 expression can enhance the sensitivity of the mycobacterium smegmatis to antibiotics, and the promotion of MSMEG _6171 overexpression can weaken the sensitivity of the mycobacterium smegmatis to antibiotics.

3. The method of claim 1, wherein: the MSMEG _6171 expression level is the MSMEG _6171 protein expression level.

4. The application of the reagent for regulating the expression level of MSMEG-6171 in the preparation of a kit for regulating the sensitivity of mycobacterium smegmatis to antibiotics, wherein the antibiotics are any one or more of vancomycin, cefotaxime, cefoxitin and cephalosporine; the amino acid sequence of the MSMEG-6171 is shown in SEQ ID No. 1.

5. Use according to claim 4, characterized in that: a kit for enhancing the sensitivity of Mycobacterium smegmatis to antibiotics is prepared by using an agent for inhibiting the expression of MSMEG-6171, and a kit for reducing the sensitivity of Mycobacterium smegmatis to antibiotics is prepared by using an agent for promoting the overexpression of MSMEG-6171.

6. Use according to claim 4, characterized in that: the MSMEG _6171 expression level is the MSMEG _6171 protein expression level.