CN120860190A - Application of lyase LySMP in removal of S.mis and prevention and treatment of chronic obstructive pulmonary disease - Google Patents

Abstract

This invention discloses a drug/disinfectant for eliminating S. mitis, comprising the lyase LySMP. Compared with other lyases, LySMP exhibits unique inhibitory and killing effects on S. mitis without affecting other bacterial flora. Because LySMP has a specific clearance effect on S. mitis, a biomarker of chronic obstructive pulmonary disease (COPD), this invention also discloses the application of LySMP in the preparation of drugs for the prevention or treatment of COPD.

Description

Application of lyase LySMP in removal of S.mis and prevention and treatment of chronic obstructive pulmonary disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of lyase LySMP in removing S.mis and preventing and treating chronic obstructive pulmonary disease.

Background

Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death worldwide and is characterized by irreversible decline in lung function due to lower airway inflammation, airway narrowing, and alveolar destruction (emphysema). Since COPD has evolved over the years, early discovery of high risk groups can take preventive measures before onset of disease, thus preventing progression of disease.

Recent studies by the inventors on bronchoalveolar lavage (BALF) samples from early COPD patients showed reduced bacterial diversity in early COPD patients compared to the smoker control, enrichment of opportunistic pathogens such as streptococcus and haemophilus, with streptococcus showing the strongest discrimination. Streptococcus abundance is associated with decreased lung function and increased local inflammation, suggesting a role in the early pathogenesis of COPD. Furthermore, the mouse cigarette smoke model of early COPD showed that s.mis promotes progression of early COPD through AT2 cell and macrophage dysfunction and dysregulated macrophage-AT 2 crosstalk. In our study data, the s.mis ratio (3.89%) was significantly higher in the early COPD group than in the smoking control group (1.83%), s.mis was also a significant biomarker in the early COPD group, LDA score 3.9978. Notably, the abundance of s.mis is associated with decreased lung function and increased inflammation, suggesting that s.mis promotes the progression of early COPD. Thus, targeting s.mitis bacteria in the lower respiratory tract in combination with smoking cessation may be a potential treatment for early COPD.

Streptococcus mitis (Streptococcus mitis, S.mitis) is a gram-positive bacterium of the genus Streptococcus. It belongs to one of the normal flora of the oral cavity and respiratory tract, but has a certain pathogenicity, especially when the immune function is low, it can cause various infections. It has genetic correlation with Streptococcus pneumoniae, and part of S.mis strain contains virulence factor genes of Streptococcus pneumoniae, which can gradually increase pathogenicity. It is a rich oral symbiotic bacteria, and can escape from the oral ecological niche to cause infectious endocarditis, meningitis, bacteremia, pneumonia and other invasive diseases. In the case of smoke exposure, s.mitis combined with other common oral symbiotic bacteria, virella parvula (v.parvula) and propidium melanogenesis (p.melaninogenica), can cause inflammatory lesions in mild to moderate chronic obstructive pulmonary disease.

To prevent the high risk group from developing COPD and also to better treat COPD, clearance of s.mis is a potential means and how to efficiently clear s.mis is a research hotspot in the art.

Disclosure of Invention

Based on the above-mentioned problems in the prior art, the present inventors have found through research and comparison that lyase LySMP has a unique killing effect on streptococcus mitis (s.mis), and thus the present invention provides a drug/disinfectant for removing s.mis, including lyase LySMP.

Preferably, the working concentration of the lyase LySMP is 30-120. Mu.g/mL, more preferably 50-100. Mu.g/mL.

Preferably, the medicament is for the eradication of s.mis infection of the lung and/or lower respiratory tract.

Further, the present invention provides a method for removing s.mis, which uses a lyase LySMP to act on s.mis.

Preferably, S.mis is subjected to action for more than 30 minutes, more preferably 30 to 60 minutes, using a disinfectant containing a cleavage enzyme LySMP at a working concentration of 30 to 120. Mu.g/mL.

Further, the invention provides the use of a lyase LySMP in the preparation of a s.mis-clearing medicament/disinfectant.

Further, since s.mis is a biomarker for early chronic obstructive pulmonary disease, the present invention provides an application of lyase LySMP in the preparation of a medicament for preventing or treating chronic obstructive pulmonary disease.

The invention also provides a medicament for treating or preventing chronic obstructive pulmonary disease, which comprises lyase

LySMP。

Preferably, the concentration of lyase LySMP in the drug is 30-120 μg/mL, more preferably 50-100 μg/mL.

Preferably, the drug is administered by intratracheal injection or aerosol inhalation.

Preferably, the medicament further comprises other pharmaceutically acceptable carriers.

According to the invention, researches show that the lyase LySMP has unique inhibition and killing effects on S.mis compared with other lyase, hardly causes interference on other microbial communities in the microbial communities, and is an ideal choice for targeted removal of S.mis. In addition, alveolar damage in mice is alleviated following intratracheal administration of lyase LySMP to s.mis-infected early COPD mice. The result shows that the lyase therapy has potential application value in the chronic respiratory diseases requiring accurate regulation and control of microbiota, can be used for preparing medicines for preventing or treating chronic obstructive pulmonary disease, prevents high-risk groups from developing COPD, and is also beneficial to the treatment of COPD patients.

The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a graph showing the statistical results of bacterial turbidity ratios before and after 30 minutes of action of three lyases on Streptococcus mitis ATCC 49456;

FIG. 2 is a graph showing the statistical results of bacterial turbidity ratios before and after 30 minutes of action of various concentrations of lyase LySMP on Streptococcus mitis ATCC 49456;

FIG. 3 is a graph showing the sterilization profile of the action time of a 2MIC concentration of lyase LySMP on Streptococcus mitis ATCC 49456;

FIG. 4 is a graph showing the statistical results of bacterial turbidity ratios before and after 30 minutes of action of lyase LySMP on different species;

FIG. 5 is a schematic representation of a 22 week experimental design involving microbiota removal (weeks 1-2), colonization (weeks 3-6), flushing (weeks 7-10) and disease model establishment (weeks 11-22);

fig. 6 is a fluorescence staining result of FISH detection of DNA from s.mis in mouse lung tissue sections at the end of 6 and 10 weeks, respectively, blue DAPI, green s.mis, scale bar = 50 microns;

fig. 7 is an image of representative H & E stained lung sections of mice of different groups on the 22 nd weekend of example 5, scale bar = 50 microns;

Fig. 8 is a graph of lung histopathological scoring of H & E stained lung section images measured by MLI, MAD and MAA in example 5, n=9/group, three replicates, one-way anova for statistical analysis, P <0.001, P <0.0001;

Fig. 9 is the results of lung function measurements by FEV100/FVC (%), cdyn, IC and FVC in example 5, n=9/group, three replicates were run for statistical analysis, single factor anova with P <0.001, P <0.0001;

FIG. 10 is a schematic of a 20 week experimental design involving microbiota removal (weeks 1-2), colonization (weeks 3-6), sterilization period (weeks 7-8) and disease model establishment (weeks 9-20);

FIG. 11 is a graph showing the results of culturing S.mitis colonized mice of pulmonary origin with S.mitis smeared on TSA plates with LySMP and without LySMP in example 6;

fig. 12 is an image of representative H & E stained lung sections of mice of different groups on the 20 th weekend of example 6, scale bar = 50 microns

Fig. 13 shows the results of lung histopathological scoring by MLI, MAD and MAA measurements on H & E stained lung sections of example 6, n=9/group, three replicates, one-way anova for statistical analysis, P <0.001, P <0.0001.

Detailed Description

The S.mis strain used in the invention is Streptococcus mitis ATCC 49456 purchased from China general microbiological culture Collection center.

EXAMPLE 1 screening of lyase

1. Experimental method

Streptococcus mitis ATCC 49456 was picked up and inoculated into BHI medium for overnight incubation at 37 ℃ with shaking. The following day, the bacterial liquid is transferred to fresh BHI culture according to the proportion of 1% and shake-cultured to logarithmic phase (OD 600 = 0.6-0.8), the bacterial liquid is collected by centrifugation and washed three times by PBS, the bacterial body is resuspended by PBS, the bacterial liquid concentration is adjusted to about OD600 = 1.0, and the prepared bacterial liquid is reserved at 4 ℃. To the prepared bacterial solutions, lyase LySMP (50. Mu.g/mL), ly7917 (50. Mu.g/mL) and Ly5218 (50. Mu.g/mL) were added, respectively, and the control group was added with an equal volume of PBS, and after 30 minutes of stationary culture at 37℃the OD600 was measured, and the experiment was repeated three times.

2. Experimental results

As shown in FIG. 1, only lyase LySMP significantly reduced the turbidity of Streptococcus mitis ATCC 49456 bacteria by more than 50% in 30 minutes, whereas Ly7917 and Ly5218 had no bactericidal effect on Streptococcus mitis ATCC 49456, and thus lyase LySMP was selected as a follow-up study object.

Example 2 MIC determination of Streptococcus lyase LySMP on ATCC 49456

1. Experimental method

The preparation method of the bacterial liquid is the same as that described above. The OD600 was determined after 30min of stationary culture at 37℃with equal volumes of the cleavage enzyme LySMP at different concentrations (from 0-240. Mu.g/mL) and the experiment was repeated three times.

Note that the minimum enzyme concentration that reduced bacterial OD600 by 50% within 30 minutes was defined as MIC (minimum inhibitory concentration) of lyase.

2. Experimental results

As shown in FIG. 2, when the action concentration of the lyase LySMP was less than 15. Mu.g/mL, there was no significant bactericidal effect on Streptococcus mitis ATCC 49456. When the action concentration of the lyase LySMP was 30. Mu.g/mL, the turbidity of Streptococcus mitis ATCC 49456 was reduced by about 50% in 30 minutes, and thus the minimum inhibitory concentration of the lyase LySMP against ATCC 49456 was determined to be 30. Mu.g/mL. The cleavage effect is better when the action concentration of the cleavage enzyme LySMP is not less than 2MIC (60. Mu.g/mL), but no obvious concentration dependence exists.

Example 3 sterilizing time Curve of lyase LySMP

1. Experimental method

The preparation method of the bacterial liquid is the same as that described above. Based on the measurement results of MIC values, the action concentration of the lyase LySMP on the in vitro sterilization curve was determined to be 2MIC (60. Mu.g/mL). The prepared bacterial liquid is added with the lyase LySMP (60 mug/mL), the control group is added with the PBS with the same volume, the mixture is subjected to static culture at 37 ℃, the OD600 value is measured according to the designated time point, and the experiment is repeated three times.

2. Experimental results

As shown in fig. 3, when the action concentration of the lyase LySMP is 60 μg/mL, the turbidity of the streptococcus mitis ATCC 49456 bacteria can be reduced by about 80% in 60 minutes (from od600=1 to od600=0.2 or so), and then the bacteria tend to be stable.

Example 4 sterilizing specificity of Streptococcus lyase LySMP

1. Experimental method

And respectively selecting clinical isolates of klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, stenotrophomonas maltophilia, enterococcus and staphylococcus aureus, and verifying the sterilization specificity of the lyase LySMP by two strains. The bacteria are respectively inoculated into LB culture, shake culture is carried out at 37 ℃ until the bacterial liquid reaches the logarithmic growth phase (OD 600 = 0.6-0.8), the bacterial liquid is collected by centrifugation and is subjected to resuspension washing three times by PBS, the bacterial liquid concentration is adjusted to be about OD600 = 1, and the prepared bacterial liquid is ready for use. To the prepared bacterial liquid, lyase LySMP (60. Mu.g/mL) was added, and a sample to which no lyase LySMP was added was used as a negative control, and Streptococcus mitis ATCC 49456+LySMP (60. Mu.g/mL) was used as a positive control. After 30 minutes of stationary culture at 37 ℃, OD600 was measured and the test was repeated three times.

2. Experimental results

As shown in FIG. 4, the lyase LySMP has no bactericidal activity against Klebsiella Pneumoniae (KP), acinetobacter Baumannii (AB), pseudomonas Aeruginosa (PA), escherichia Coli (EC), pseudomonas Maltophilia (PMA), enterococcus (EF) and Staphylococcus Aureus (SA).

EXAMPLE 5S mice model for early COPD progression induced by mis

1. Experimental method

Healthy male C57BL/6 broad mice (4-5 weeks old) were used for this experiment. To deplete the intestinal and pulmonary microbiota prior to the start of the experiment, mice were dosed with the combination antibiotic ABX (0.5 mg/mL ampicillin, 0.25mg/mL vancomycin, 0.5mg/mL metronidazole, 0.5mg/mL neomycin and 0.5mg/mL gentamicin) in drinking water for 2 consecutive weeks while being dosed intranasally (50 μl) with the same concentration of combination antibiotic ABX for 5 consecutive days, 1 time per day, within week 2.

To investigate the role of s.mis in COPD, 5 x 10 ⁵ Colony Forming Units (CFU) s.mis (streptococcus mitis ATCC 49456) or negative control gingival carbon dioxide-philic bacteria (Capnocytophaga gingivalis, cg) were inoculated into each 3 weather tubes within 4 weeks prior to the establishment of a Cigarette Smoke (CS) induced COPD model, each group consisting of 10 mice. Four-week clearing period is arranged after field planting, and no operation is performed. Mouse lung tissue was harvested at week 6 and week 10, respectively, and the distribution of s.mis was detected using Fluorescence In Situ Hybridization (FISH) with s.mis specific probes.

Construction of a cigarette smoke-induced mouse COPD model mice were placed in a 35 cm by 20 cm smoke box and exposed to eight cigarettes producing smoke twice daily for 5 days a week for 12 weeks. Control animals were exposed to normal room air for 12 weeks for a total of 22 weeks of experimental period (as shown in fig. 5). Mice were tested for lung function by forced pulmonary motor system after 22 weeks, after which the mice were sacrificed. Mice lung tissue was collected for H & E staining and lung histopathological scoring.

2. Experimental results

We observed an increase in s.mis load in the lungs of s.mis vaccinated mice compared to control mice, both at the end of the 4 week vaccination period and at the end of the other 4 week washout period, as shown by FISH results (fig. 6).

Those colonized with s.mis exhibited increased emphysema destruction of lung parenchyma compared to those colonized with Cg and non-colonized mice (fig. 7). Specifically, the average linear intercept (MLI), average alveolar diameter (MAD), and average alveolar area (MAA) of abx+cs+s.mis group were significantly larger than other groups (fig. 8). Although the structure was compromised, no immediate decline in lung function was observed, 1 second after bronchodilators with forced expiratory volume/forced vital capacity (FEV 100/FVC (%)), dynamic lung compliance (Cdyn), inspiratory Capacity (IC), and Forced Vital Capacity (FVC) (fig. 9). These results indicate that s.mis promotes early COPD progression by inducing alveolar lesions.

Example 6LySMP reduction of alveolar injury in S.mis-induced early COPD mice

1. Experimental method

As shown in FIG. 10, chronic airway colonization by S.mis was repeated for the first 4 weeks and 50. Mu.L (weeks 7-8) of LySMP PBS solution was administered intratracheally at 60. Mu.g/mL every three days prior to CS exposure (weeks 9-20). At the end of the 2-week sterilization period (end of week 8), lung tissue from mice colonized with s.mis, which received LySMP and did not receive LySMP, was ground and plated with TSA for 4 days, after which the mice were observed for s.mis consumption in lung tissue. Mice lung tissue was harvested at the end of 20 weeks for H & E staining and lung histopathological scoring.

2. Experimental results

Results of TSA culture after grinding of mouse lung tissue collected on the 8 th weekend showed significant S.mis depletion in mouse lung tissue received LySMP (FIG. 11), H & E staining of mouse lung tissue after 20 weeks of end and pulmonary histopathological scoring showed that LySMP treatment reduced emphysema destruction of S.mis lung parenchyma in early COPD mice colonization (FIGS. 12 and 13). These results establish LySMP as a potential targeted therapy for early COPD.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims (10)

1. A drug/disinfectant for removing s.mis comprising a lyase LySMP.

2. The s.mis-clearing drug/disinfectant of claim 1, wherein the working concentration of the lyase LySMP is 30-120 μg/mL.

3. A s.mis clearing medicament as in claim 1 for clearing s.mis infection of the lung and/or lower respiratory tract.

4. A method for removing s.mis, wherein the s.mis is acted upon by a lyase LySMP.

5. The method of claim 4, wherein the S.mis is exposed to the disinfectant containing the lyase LySMP at a working concentration of 30-120 μg/mL for more than 30 minutes.

6. Use of lyase LySMP in the preparation of a medicament/disinfectant for the removal of s.

7. Use of lyase LySMP in the manufacture of a medicament for the prevention or treatment of chronic obstructive pulmonary disease.

8. A medicament for preventing or treating chronic obstructive pulmonary disease, comprising lyase LySMP.

9. A medicament for the prophylaxis or treatment of chronic obstructive pulmonary disease according to claim 7, wherein the medicament is administered by intratracheal injection or aerosol inhalation.

10. A medicament for the prophylaxis or treatment of chronic obstructive pulmonary disease according to claim 7, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.