A medium for screening of fusarium inhibitors and use thereof

Abstract

The application discloses the role of proline metabolism in Fusarium proliferation and pathogenicity for the first time, proves that the proline component plays an important role in the corneal infection caused by Fusarium in the human corneal internal environment, and provides a culture medium which can be used for screening of Fusarium inhibitors, the culture medium is helpful for screening of drugs which can effectively inhibit the proliferation and pathogenicity of Fusarium, and greatly promotes the treatment and drug development of keratitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug screening, and more particularly, the present application relates to a culture medium for screening fusarium inhibitors and application thereof. BACKGROUND

[0002] Fungal infection of cornea can cause serious blindness, and is one of the most harmful eye infections. In the past few decades, fungal infection of cornea has shown a significant upward trend. This is mainly because the widespread use of corneal contact lenses and the change of agricultural work mode increase the opportunity of corneal trauma and contact with fungi, and the abuse of broad-spectrum antibiotics and glucocorticoids leads to conjunctival sac bacterial flora imbalance and low immunity of ocular surface. Fusarium is the most common pathogenic bacteria of fungal keratitis.

[0003] At present, the treatment effect of fungal keratitis is very unsatisfactory. Different fungal genera have different morphological structures, growth and reproduction, variation, resistance, pathogenicity, and growth and infiltration methods in tissues, and sensitivity to antifungal drugs. Because of the similarity between fungi and mammals in biological and biochemical levels, and the weak penetration of drugs to the cornea, the efficiency of eye local drug is low, the toxicity of systemic drug is large, the drug acting site is limited, the drug type is less, and drug-resistant bacteria are more common. This brings great difficulty to the early control of infection and the prevention of fungal infection recurrence after surgery of fungal keratitis. Therefore, it is urgent to develop a method for effectively screening corneal antibacterial drugs suitable for clinical use. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides a culture medium for screening fusarium inhibitors and application thereof. Specifically, the present application provides the following technical solutions:

[0005] In a first aspect of the present application, a culture medium for screening fusarium inhibitors is provided, and the culture medium comprises the following components:

[0006] Sucrose 20-40 g / L

[0007] KH2PO4 0.1-2 g / L

[0008] KCl 0.1-2 g / L

[0009] MgSO4·7H2O 0.1-2 g / L

[0010] Trace element 0.1-1 ml / L

[0011] FeSO4·7H2O 5-15 ml / L

[0012] Proline 2-10 mM,

[0013] wherein the Trace element is formulated as:

[0014] Citric acid 2-8 g / 100 mL

[0015] MnSO4 25-75 mg / 100 mL

[0016] ZnSO4.6H2O 2-8 g / 100 mL

[0017] H3BO3 25-75 mg / 100 mL

[0018] Na2MoO4.2H2O 25-75 mg / 100 mL

[0019] CuSO4.5H2O 100-500 mg / 100 mL.

[0020] As a preferred embodiment, the components of the medium are:

[0021] Sucrose 25-35 g / L

[0022] KH2PO4 0.5-1.5 g / L

[0023] KCl 0.2-0.8 g / L

[0024] MgSO4.7H2O 0.2-0.8 g / L

[0025] Trace element 0.1-0.5 ml / L

[0026] FeSO4.7H2O 8-12 ml / L

[0027] Proline 3-8 mM,

[0028] wherein the Trace element is formulated as:

[0029] Citric acid 3-6 g / 100 mL

[0030] MnSO4 40-60 mg / 100 mL

[0031] ZnSO4.6H2O 3-6 g / 100 mL

[0032] H3BO3 40-60 mg / 100 mL

[0033] Na2MoO4.2H2O 40-60 mg / 100 mL

[0034] CuSO4.5H2O 200-300 mg / 100 mL.

[0035] As a preferred embodiment, the components of the culture medium are:

[0036] Sucrose 30g / L

[0037] KH2PO4 1g / L

[0038] KCl 0.5g / L

[0039] MgSO4·7H2O 0.5g / L

[0040] Trace element 0.2ml / L

[0041] FeSO4·7H2O 10ml / L

[0042] Proline 5mM,

[0043] wherein the formula of the Trace element is:

[0044] Citric acid 5g / 100mL

[0045] MnSO4 50mg / 100mL

[0046] ZnSO4·6H2O 5g / 100mL

[0047] H3BO3 50mg / 100mL

[0048] Na2MoO4·2H2O 50mg / 100mL

[0049] CuSO4·5H2O 250mg / 100mL.

[0050] In a second aspect of the present application, there is provided a use of the above culture medium in screening Fusarium inhibitors.

[0051] In a third aspect of the present application, there is provided a use of the above culture medium in screening keratitis drugs.

[0052] In one embodiment, the use is to culture Fusarium using the above culture medium, to add a candidate drug to the culture medium, and to investigate the inhibitory effect of the candidate drug on the pathogenicity or proliferation of Fusarium.

[0053] Compared with the prior art, the present application has the following advantages:

[0054] The application discloses the role of proline metabolism in Fusarium proliferation and pathogenicity for the first time, proves that the proline component plays an important role in the corneal infection caused by Fusarium in the human corneal internal environment, and provides a culture medium which can be used for screening Fusarium inhibitors, the culture medium helps to screen drugs which can effectively inhibit the proliferation and pathogenicity of Fusarium, greatly promotes the treatment and drug development of keratitis. BRIEF DESCRIPTION OF DRAWINGS

[0055] The accompanying drawings are included to provide a further understanding of the application, and constitute a part of the specification, illustrate the application together with the embodiments thereof, and explain the principles of the application, and do not constitute a limitation of the application. In the drawings:

[0056] Figure 1 Expression of P5C dehydrogenase (one of proline metabolism enzymes) of clinical isolated Fusarium strain P5C: transcriptome analysis is carried out on standard strains of Fusarium, primary infection strains from clinical sources and recurrent infection strains from clinical sources, it is found that the expression of P5C dehydrogenase (one of proline metabolism enzymes) has obvious difference, wherein the expression of the clinical strains is higher than that of the standard strains, and the expression of the strains of recurrent cases is higher. It is suggested that proline metabolism and P5C dehydrogenase play a role in the pathogenicity and recurrence of keratitis caused by Fusarium infection;

[0057] Figure 2 Inhibition of the growth of Fusarium in the medium with proline as nitrogen source after adding different concentrations of THFA: different concentrations of THFA (0, 1.25mM, 2.5mM, 5mM, 10mM, 20mM) are added in the medium with proline as nitrogen source, and the growth of Fusarium is observed for 3 days, it can be seen that the growth of Fusarium is inhibited after adding THFA, and the inhibition is related to the concentration of THFA, the higher the concentration, the more obvious the inhibition of the growth of Fusarium;

[0058] Figure 3 Detection of the proportion of amino acids in corneas of different species: the proportion of amino acids in corneas of different species is detected. A-D are the proportion of various amino acids in human corneas, rat corneas, pig corneas and rabbit corneas, respectively. Proline is contained in the corneas of each species, but the proportion is different;

[0059] Figure 4 Grouping diagram of mixed amino acid culture medium (the left column of each group is the non-inhibitor group, and the right column is the inhibitor group): the culture medium with proline, proline combined with taurine, proline combined with glutamine, proline combined with glycine and proline combined with the above three kinds of amino acids as nitrogen source is prepared respectively, and the growth of Fusarium after adding 10mM THFA is compared;

[0060] Figure 5: Respectively prepare the culture medium with proline, proline combined with taurine, proline combined with glutamine, proline combined with glycine and proline combined with the above three amino acids as nitrogen source, and then add 10 mM THFA and observe the growth of Fusarium oxysporum for 3.5 days. A: 0.5 days, no difference in each culture medium. B (1.5 days), C (2.5 days), D (3.5 days), THFA has growth inhibition effect on Fusarium oxysporum in each culture medium. It is proved that in the case of various amino acids as nitrogen source, the proline metabolic enzyme inhibitor THFA can still play a key inhibitory role on the growth of Fusarium oxysporum;

[0061] Figure 6 : A: The growth curve is summarized, and it can be seen that the growth of Fusarium oxysporum in different amino acid culture media is different; B-F: the growth curve of Fusarium oxysporum in various amino acid culture media after adding 10 mM THFA, wherein Figure 6 B-F are culture media with proline (B), proline combined with taurine (C), proline combined with glutamine (D), proline combined with glycine (E) and proline combined with the above three amino acids (F) as nitrogen source. DETAILED DESCRIPTION

[0062] The preferred embodiments of the present application are described below in conjunction with the accompanying drawings, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0063] Example 1: Inhibition of Fusarium Pathway

[0064] In the inventors' previous studies, it was found that the Fusarium isolated from the ocular surface of keratitis patients had obvious proline metabolic enzyme expression, and the expression of these enzymes was more in the recurrent cases. According to the visible standard strain, the expression of TPM was 0, while the clinical isolated bacteria expressed significantly activated, and the expression of the recurrent infection strain was higher, which suggested that P5C dehydrogenase was involved in the process of Fusarium infection of cornea, and it was very likely related to its pathogenicity. The above all suggest that proline metabolism plays an important role in the pathogenic process of Fusarium oxysporum Figure 1 ). Therefore, we consider whether we can inhibit the growth and pathogenicity of Fusarium oxysporum by inhibiting the proline metabolism of Fusarium oxysporum, and then further whether we can screen antibacterial drugs through the proline metabolism pathway of Fusarium oxysporum. Based on the above consideration, we selected a compound with proline metabolic enzyme inhibition function predicted according to the previous experimental results of our research group, tetrahydrofolic acid (abbreviation: THFA, commercially available pure product, purity ≥95%) to verify the possibility of realizing drug screening as follows. The specific method is as follows:

[0065] 1. Preparation of proline as the only nitrogen source culture medium (abbreviation: pro-culture medium):

[0066] The pro-medium formula is as follows:

[0067]

[0068] 1 Filter sterilization

[0069] The formula of the Trace element is as follows:

[0070]

[0071] 2. Fusarium was cultured in the pro-medium (purchased from China General Microbiological Culture Collection Center; No. CGMCC 3.5840), and different concentrations of THFA were added to intervene in the proline metabolic process of Fusarium, so as to screen the lowest concentration that can achieve a better inhibitory effect on Fusarium.

[0072] The results are shown in Figure 2 After adding THFA, the growth of Fusarium was obviously inhibited, and the inhibition degree had obvious correlation with the concentration of THFA: the colony diameter after 5 mM THFA treatment was significantly lower than that of the 2.5 mM, 1.25 mM and 0 mM groups, the 10 mM group and the 20 mM group were significantly lower than the 5 mM group, and the growth inhibition effect of the 20 mM group was more obvious. Meanwhile, the Fusarium was sent for transcriptome detection to observe the expression inhibition of proline metabolic enzymes. It was found that with the increase of THFA concentration (2.5 mmol / L, 5 mmol / L, 10 mmol / L, 20 mmol / L), the important life activities such as growth and metabolism of Fusarium were severely damaged.

[0073] Unlike the expected change in oxidative stress-related processes, the main manifestation was a severe growth inhibition state caused by nitrogen deficiency.

[0074] However, the above results have shown that the growth and pathogenicity of Fusarium can be inhibited by inhibiting the proline metabolic process of Fusarium.

[0075] Example 2 verifies the possibility of screening of human corneal bacteriostatic drugs

[0076] Because there are more than one kind of amino acid in the cornea, in order to simulate the real state of the cornea, we need to prove that in the case of mixed amino acids, inhibiting the growth of Fusarium by inhibiting the proline metabolic pathway is still effective. For this purpose, we compared the proline ratios of corneas of multiple species (rat, human, pig, rabbit, etc.) to find out the types of nitrogen sources that Fusarium may use after infecting the cornea, and screened the species close to the proline ratio of human cornea. The results are shown in Figure 3As shown: the proportion of proline in human cornea is about 1.08%, proline is not the highest proportion of amino acid in human cornea, the highest proportion is taurine, followed by phenylalanine, lysine, then glutamine and tyrosine Figure 3 A). The proportion of proline in rat cornea is 2.59%, the top few amino acids are taurine, glutamine Figure 3 B). The proportion of proline in pig cornea is 2.97%, the top few amino acids are taurine, glutamine Figure 3 C). The proportion of proline in rabbit cornea is 4.04%, the top few amino acids are taurine, glutamine Figure 3 D).

[0077] The above results show that the proportion of amino acids in the cornea of commonly used experimental animals such as rats, mice, pigs and rabbits does not completely coincide with that of human cornea. In terms of proline, the cornea of rats and pigs is most similar to that of humans, but the proportion of glutamine is higher than that of human cornea, which has a great influence on the selection of the dominant nitrogen source of fungi.

[0078] According to the above obtained amino acid species in human cornea, the proline in the above pro culture medium is replaced by the combination of different amino acids in human cornea (combination 1: taurine, glutamine, glutamic acid and proline four amino acids mixed; combination 2: taurine + proline; combination 3: glutamine + proline; combination 4: glutamic acid + proline) as nitrogen source medium, so as to simulate the environment of amino acid mixture in real cornea, and 10mM THFA Figure 4 ) is added in each group of medium, to investigate whether the blockage of proline can inhibit the growth and pathogenicity of fungi under the condition that various amino acids can provide nutrients for fusarium. Cultivate fusarium and observe its growth.

[0079] The results are shown in Figure 5 There is no significant growth change in the culture dish mixed with taurine and proline compared with the single proline culture dish. However, from 1.5 days later, the culture dish added with glutamine and glycine has enhanced colony pigment compared with the single proline culture dish Figure 5 B), which shows that different amino acids have different effects on the growth and pathogenicity of fusarium, and glutamine and glycine have a promoting effect on the pathogenicity of fusarium. However The addition of 10 mM THFA to the mixed amino acid medium inhibited the growth of F. oxysporum, and significantly reduced pigmentation Figure 5 B-D) .

[0080] The growth curve result suggests that the growth of fusarium in the four kinds of amino acid mixed medium is the best, the proline and glutamine mixed is the second, and its effect is very close to the four kinds of amino acid mixed group Figure 6A), the mixed culture of glycine and proline was better than the single proline culture, while the mixed culture of taurine and proline was lower than the single proline culture. The growth of F. oxysporum was significantly decreased in all groups with the addition of 10 mM THFA Figure 6 B-F) .

[0081] The above results show that in the human corneal environment, the inhibition of proline metabolic pathway is sufficient to inhibit the growth of Fusarium, and thus the culture medium designed by the present application with proline as the only nitrogen source can be applied to the screening of Fusarium inhibitors.

[0082] It should be noted that the above examples only illustrate some specific embodiments of the present application, and one or more technical features described in one or more technical solutions can be combined with any one or more technical solutions, and the combined technical solutions are also within the protection scope of the present application, just like the combined technical solutions have been specifically described in the disclosure of the present application.

Claims

1. A culture medium for screening Fusarium inhibitors, characterized in that, Each liter of culture medium consists of the following components: 30g sucrose, 1g potassium dihydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate heptahydrate, 10mL ferrous sulfate heptahydrate, 0.2mL trace element solution, 5mM proline, 5mM taurine, 5mM glutamine, and 5mM glycine; the trace element solution consists of the following components per 100mL: 5g citric acid, 50mg manganese sulfate, 5g zinc sulfate hexahydrate, 50mg boric acid, 50mg sodium molybdate dihydrate, and 250mg copper sulfate pentahydrate.

2. The application of the culture medium as described in claim 1 in screening Fusarium inhibitors.

3. The use of the culture medium as described in claim 1 in screening drugs for the treatment of keratitis.

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