Application of escherichia coli combined tryptophan in activation of lysosome function

Abstract

The invention discloses application of escherichia coli combined tryptophan in activation of lysosome functions, and belongs to the technical field of microorganisms and application thereof. The invention finds that the lysosome function of caenorhabditis elegans and mammalian cells can be enhanced by combined use of escherichia coli and tryptophan. Therefore, the escherichia coli and the tryptophan can be combined for preparing the medicine for activating the lysosome.

Description

Technical Field

[0001] The invention relates to application of Escherichia coli combined with tryptophan in activating lysosome function, belonging to the technical field of microorganisms and their applications. Background Art

[0002] Lysosomes are dynamic organelles present in all eukaryotic cells and play a vital role in various processes that maintain cellular and tissue homeostasis. Lysosomes are composed of a single bilayer phospholipid membrane, and the lumen contains more than 50 hydrolases, including proteases, nucleases, and lipases. The lysosomal lumen is acidic, with a pH range of 4.6 to 5.2, which is required for the activity of hydrolases. Lysosomes play an important role in maintaining cellular homeostasis by degrading macromolecules, including various intracellular and extracellular substances (such as proteins, glycans, and lipids) produced by autophagy, through acidic hydrolases. In addition, lysosomes interact with different intracellular organelles and participate in a wide range of cellular processes, including calcium homeostasis, lipid transfer, cholesterol homeostasis, exocytosis, and plasma membrane repair.

[0003] Lysosomes are crucial for catabolic function at the systemic level, and multiple studies have indicated that lysosomal dysregulation may contribute to a wide range of diseases. For example, genetic manipulation of genes involved in autophagy and lysosomal proteolysis results in embryonic lethality in mice; mutational inactivation of genes encoding lysosomal hydrolases and transporters leads to a range of catabolic diseases, known as lysosomal storage disorders; and lysosomal dysfunction is associated with numerous age-related pathologies. More importantly, regulation of lysosomal biogenesis and function is crucial for lipid catabolism in the liver, where fat accumulation can cause diseases such as fatty liver disease.

[0004] Given the role of lysosomes in catabolic turnover and metabolic signaling, regulating lysosomal function is of great significance for maintaining normal cellular metabolic activity and preventing microbial infection. Studies have demonstrated the effectiveness of many lysosomal function inhibitors, such as chloroquine ammonium chloride, which can disrupt lysosomal pH and block autophagy and lysosomal pathways, and BafA1, which inhibits V-ATPase activity. At the same time, researchers have also identified that gene knockouts such as ATG5 or ATG7 block the degradation of ATTEC (autophagosome-bound compounds). Studies have shown that transcription factor EB promotes lysosomal biogenesis; however, there are few reports on small molecule activators of lysosomes. Therefore, the development of lysosomal function activators and natural products that induce lysosomal-specific degradation of large molecules is a new strategy for discovering innovative drugs. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an application of Escherichia coli combined with tryptophan in activating lysosomal function.

[0006] Preferably, Escherichia coli is combined with tryptophan to prepare a lysosome activator.

[0007] Preferably, tryptophan is added to the culture medium to obtain a culture medium containing tryptophan.

[0008] Preferably, a single clone of E. coli is cultured overnight at 36-37.5° C. for 12-16 hours in a culture medium containing tryptophan to obtain an effective E. coli bacterial solution.

[0009] Technical effects of the present invention:

[0010] (1) The present invention first discovered that Escherichia coli combined with tryptophan can effectively enhance the function of lysosomes with significant effects. Therefore, Escherichia coli combined with tryptophan can be used to prepare related drugs for activating lysosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 elegans transcriptome sequencing sample processing schematic diagram in Example 1 and the clustering analysis results of differentially expressed genes in the sequencing results.

[0012] Figure 2 Figure 1 shows the results of an acidity detection experiment on the epidermal lysosomes of the two groups of nematodes in Example 1 using reporter nematodes. Figure A is a schematic diagram of the experimental principle, Figure B is a fluorescent image of the nematode epidermal lysosomes, and Figure C on the right is a statistical graph of the relative fluorescence intensity of lysosomal pHTomato.

[0013] Figure 3 In order to use the reporter nematode NUC-1::mcherry, the nematode lysosomal protein degradation activity in Example 1 was detected by cleavage of mcherry, wherein Figure A is a schematic diagram of the experimental principle and Figure B is the Western Blot result.

[0014] Figure 4 The results of the experiment using Magic Red dye to detect lysosomal cathepsin B activity in Caenorhabditis elegans in Example 1 are shown. Figure A is a fluorescent imaging image of the nematode after Magic Red staining; Figure B is the statistics of the average fluorescence intensity of Magic Red.

[0015] Figure 5 Schematic diagram of the sample processing of the human liver cancer cell line Huh7 in Example 2.

[0016] Figure 6 Figure 1 is a graph showing the results of detecting the pH of cell lysosomes using Lysosenor Green (DND-189) dye, wherein Figure A is a fluorescence imaging image of the pH in the lysosomes of the human liver cell line detected by LysoSenor Green in Example 2, and Figure B is a statistical graph of the average fluorescence intensity of LysoSenor Green.

[0017] Figure 7 The results of using Magic Red to detect the activity of cathepsin B in the lysosomes of the human liver cell line in Example 2, wherein Figure A is a cell fluorescence imaging image, and Figure B is a statistical graph of the average fluorescence intensity of Magic Red.

[0018] Figure 8 This is the experimental result of Western Blot detection of the amount of the precursor and mature forms of cathepsin CTSD in lysosomes in the cell line of Example 2.

[0019] Figure 9 These are the experimental results of using Oil Red O to detect the mouse primary hepatocyte line in Example 2, wherein Figure A is the result of Oil Red O staining, and Figure B is a statistical graph of Oil Red staining. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0021] The experimental methods used in the present invention, such as lysosomal acidity detection, Magic Red staining, Western Blot detection, LysoSenor Green detection and Oil Red O detection, are all conventional experimental methods well known to those skilled in the art. They are only used to verify the effect of Escherichia coli combined with tryptophan and do not belong to the protection content of the present invention.

[0022] PcedNUC-1::mcherry is a reporter nematode that indicates lysosomes. NUC-1 is deoxyribonuclease II in lysosomes. Gene fusion expression of mcherry can detect the lysosome's ability to cut this protein through different forms of mcherry protein.

[0023] The Escherichia coli used in the examples was Escherichia coli K12 strain, purchased from Dharmacon.

[0024] Example 1

[0025] The model organism Caenorhabditis elegans was used to verify the effect of Escherichia coli combined with tryptophan in enhancing lysosomal function.

[0026] A single clone of Escherichia coli K12 strain was inoculated into 10 ml of liquid LB and cultured with shaking at 37° C. overnight for 14 h to obtain an Escherichia coli bacterial liquid.

[0027] In this example, two different experiments were conducted. In the first group, 350 μl of bacterial solution was plated in 60 mm NGM standard nematode culture medium and allowed to stand at room temperature for 12 hours before feeding wild-type Caenorhabditis elegans. In the second group, 350 μl of bacterial solution was plated in 60 mm NGM nematode culture medium (tryptophan was added to the culture medium to a final concentration of 10 mM) and allowed to stand at room temperature for 12 hours before feeding wild-type Caenorhabditis elegans. Figure 1 As shown in A, the nematodes were fed to the L4 stage and two groups of nematodes were collected for transcriptome sequencing. Figure 1 B shows that there are significant differences in the expression of transcriptional levels between the two groups of nematodes. Clustering of differentially expressed genes revealed that bacterial tryptophan activates genes related to the nematode lysosomal pathway.

[0028] After 96 hours of feeding, the pH of lysosomes of nematodes was measured on the second day of adult nematodes. The experimental principle is as follows Figure 2 As shown in A, Figure 2 B is a fluorescence image of nematode epidermal lysosomes. Figure 2 C is the statistical diagram of the relative fluorescence intensity of lysosomal pHTomato. Figure 2 B and Figure 2 The results of C showed that the use of Escherichia coli combined with tryptophan enhanced the acidity of Caenorhabditis elegans lysosomes. As an acid-dependent degradative organelle, the increase in acidity will maintain better degradation activity of lysosomes.

[0029] After about 60 hours of feeding, the protein degradation capacity of the lysosomes of the nematodes on the first day of adulthood was tested using the reporter nematode strain PcedNUC-1::mcherry, which expresses a fusion of deoxyribonuclease II NUC-1 and the red fluorescent protein mcherry in the lysosomes. The degradation capacity of the lysosomes was measured by measuring the ratio of the amount of cleaved mcherry to the amount of synthesized NUC-1::mcherry. Figure 3 A is a schematic diagram of the experimental principle. Figure 3 B is the Western Blot result, which shows that the use of Escherichia coli combined with tryptophan enhances the degradation activity of Caenorhabditis elegans lysosomes.

[0030] After feeding for about 60 hours, the two groups of wild-type nematodes were stained with Magic Red dye. Magic Red, a substrate analog of the cathepsin CTSB, emits fluorescence after being cleaved by cathepsin B in the lysosome. Figure 4 A is a fluorescent imaging image of nematodes after being stained with magic red; Figure 4 B is the statistics of the average fluorescence intensity of Magic Red, such as Figure 4 The results showed that the fluorescence level of Magic Red staining was enhanced by combining Escherichia coli with tryptophan, indicating that the activity of cathepsin B in the lysosome of Caenorhabditis elegans was enhanced, further demonstrating the enhanced function of lysosomes.

[0031] Example 2

[0032] Escherichia coli-tryptophan combination enhances lysosomal function using mammalian hepatocytes.

[0033] like Figure 5 As shown in this example, three treatments were performed on the human liver cell line Huh7: the first group was treated with LB medium as a blank control; the second group was treated with the supernatant of an Escherichia coli K12 suspension; and the third group was treated with the supernatant of an Escherichia coli K12 suspension containing tryptophan. The specific treatment process is as follows:

[0034] A single clone of Escherichia coli K12 strain was cultured in 10 ml of liquid LB overnight with shaking at 37°C for 14 h, and 100 μL of the bacterial solution was inoculated into 10 ml of standard LB liquid. The culture was shaken overnight at 37°C for 14 h, and the supernatant was collected by centrifugation at 8000 g for 5 min to obtain the bacterial supernatant.

[0035] A single clone of Escherichia coli K12 strain was cultured in 10 ml of liquid LB at 37°C overnight with shaking for 14 hours. 100 μL of the bacterial solution was then inoculated into LB liquid containing tryptophan to a final tryptophan concentration of 50 mM. The culture was shaken at 37°C overnight for 14 hours, and the supernatant was collected by centrifugation at 8000 g for 5 minutes to obtain the bacterial supernatant containing tryptophan.

[0036] When the cells grow to a density of about 60%-70%, stimulate the cells with LB medium, bacterial supernatant and bacterial supernatant containing tryptophan for 24 hours. The volume ratio of LB medium, bacterial supernatant or bacterial supernatant containing tryptophan to cell culture medium is 1:10.

[0037] Lysosenor Green (DND-189) dye was used to detect the pH of cell lysosomes. Figure 6 As shown: the lysosomal acidity of cells treated with tryptophan and E. coli supernatant was significantly increased compared with that of cells treated with the control group. Figure 6 A is the fluorescence imaging diagram. Figure 6 Below B is a statistical graph of the average fluorescence intensity of Lysosenor Green.

[0038] Magic Red dye was used to detect the activity of cathepsin B in cell lysosomes. Figure 7 As shown: The relative fluorescence intensity of magic red in cells treated with tryptophan and E. coli supernatant was significantly increased compared with that in cells treated with the control group, indicating that the activity of lysosomal cathepsin B was enhanced, further demonstrating that the function of lysosomes was increased. Figure 7 A on the left is the fluorescence imaging image. Figure 7 B is a statistical graph of the relative fluorescence intensity of Magic Red.

[0039] After 24 hours, Western Blot was performed to detect the precursor and mature forms of cathepsin D (CTSD) in the cell lysosome. The precursor form of CTSD will be further processed into the mature form of CTSD protein in the lysosome. By detecting the amount of the mature form of CTSD, the function of the lysosome can be indicated, such as Figure 8 The results showed that the combined use of E. coli and tryptophan enhanced the maturation of the cathepsin CTSD within lysosomes, implying enhanced lysosomal function.

[0040] To further test whether the lysosome's ability to degrade fat is enhanced, a model of fat accumulation in mouse hepatocytes was induced using palmitic acid at a final concentration of 400uM. Mouse hepatocytes were stimulated with 400uM palmitic acid and LB medium (control group), bacterial supernatant, or bacterial supernatant containing tryptophan for 24 hours (the treatment method was the same as that used for the human liver cell line Huh7) and then stained with Oil Red O. The results are shown in Figure 2. Figure 9 As shown in A and B: Compared with the control group and cells treated with bacterial supernatant, the fat accumulation of cells treated with bacterial supernatant containing tryptophan was significantly reduced, indicating that the combined use of E. coli and tryptophan in mammalian cell lines can activate lysosomes to decompose lipids.

[0041] In summary, the E. coli combined with tryptophan of the present invention can be used to prepare a lysosome activator.

Claims

1. An application of Escherichia coli combined with tryptophan in activating lysosomal function.

2. The use according to claim 1, characterized in that: Application of Escherichia coli combined with tryptophan in the preparation of lysosomal activators.

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