شناسایی مولکولی ژن پروتئین فعال کننده نوتروفیل هلیکوباکتر پیلوری موثر در کنترل آسم آلرژیک به روش PCR
الموضوعات :
علیرضا خالقی خرمی
1
,
ساناز مهمازی
2
,
رسول شکری
3
,
سید شمس الدین اطهاری
4
1 - گروه میکروبیولوژی، دانشکده علوم پایه، واحد زنجان، دانشگاه آزاد اسلامی، زنجان، ایران
2 - گروه میکروبیولوژی، دانشکده علوم پایه، واحد زنجان، دانشگاه آزاد اسلامی، زنجان، ایران
3 - گروه میکروبیولوژی، دانشکده علوم پایه، واحد زنجان، دانشگاه آزاد اسلامی، زنجان، ایران
4 - گروه ایمونولوژی، دانشکده پزشکی، دانشگاه علوم پزشکی زنجان، زنجان، ایران
الکلمات المفتاحية: هلیکوباکتر پیلوری, آسم آلرژیک, پروتئین فعال کننده نوتروفیل,
ملخص المقالة :
پروتئین فعال کننده نوتروفیل هلیکوباکتر پیلوری (HP-NAP) یکی از پروتئین های مهم هلیکوباکتر پیلوری (H. pylori) است HP-NAP.دارای خواص تعدیل کننده ایمنی است و پاسخ های آلرژیک را از Th2 به Th1 هدایت می کند. هدف از این مطالعه شناسایی مولکولی ژن HP-NAP بومی ایران است. سویه بومی هلیکوباکتر پیلوری از انستیتو پاستور خریداری شد. آزمایش اوره آز اختصاصی، رنگ‌آمیزی گرم و مشاهده میکروسکوپی انجام شد. برای شناسایی HP-NAP در باکتری های بومی از روش PCR استفاده شد. در نهایت، محصول PCR برای تشخیص ژن HP-NAP الکتروفورز شد. پس از رنگ‌آمیزی باکتریایی و نیز مشاهده میکروسکوپی، باکتری‌های گرم منفی خمیده مشاهده شدند. آزمایش اوره آز تغییر در معرف اوره (فنل قرمز) را نشان داد که نشان دهنده هلیکوباکتر پیلوری است. آزمایش PCR و الکتروفورز یک باند 260 جفت باز مربوط به ژن HP-NAP را در مقایسه با نردبان (لدر) 100 نشان داد. با توجه به مطالعه انجام شده و تایید وجود این پروتئین در ساختار باکتری های بومی و همچنین مطالعات قبلی در در مناطق مختلف جهان، با مشاهده اثرات این پروتئین به عنوان تعدیل کننده ایمنی سیستم ایمنی، می توان امیدوار بود که در آینده از این پروتئین به عنوان وسیله ای بالقوه برای کنترل و درمان بیماری های آلرژیک به ویژه آسم آلرژیک استفاده شود.
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Molecular identification gene of the Helicobacter pylori neutrophil activating protein effective in controlling allergic asthma by PCR method
Abstract
Objective: Helicobacter pylori neutrophil activating protein (HP-NAP) is one of the important proteins of Helicobacter pylori (H.pylori). HP-NAP has immunomodulatory properties and directs allergic responses from Th2 to Th1. The aim of this study is the molecular identification of HP-NAP gene native to Iran.
Method: The native strain of H.pylori was purchased from the Pasteur Institute, then specific urease test was performed, as well as gram staining and observation under the microscope, and in order to identify HP-NAP in native bacteria, this was done by PCR method. Finally, the PCR product should be electrophoresed to detect the HP-NAP gene.
Results: After bacterial staining and microscopic observation, bent gram-negative bacteria were observed. Also, the result of the urease test was seen with a change in urea reagent (phenol red), which indicated H.pylori. The result of the PCR test and perform electrophoresis, the 260 bp band related it was the HP-NAP gene was seen in comparison with ladder 100.
Conclusion: According to the study conducted and the confirmation of the presence of this protein in the structure of native bacteria, as well as previous studies in different regions of the world and observing the effects of this protein as an immunomodulatory of the immune system, we can hope that in the future this protein will be used as a It has the potential to control and treat allergic diseases, especially allergic Asthma.
Key words: Helicobacter pylori, allergic asthma, neutrophil activating protein.
Graphical abstract
Introduction
H.pylori is a spiral-shaped, microaerophilic gram-negative bacterium, and according to studies conducted since 50,000 years ago, it has evolved with humans and can be considered an important risk to the health of the human population (1, 2, and 3). This bacterium infects the stomach of about 45% of the world's population and can cause stomach ulcers, gastric lymphoma and stomach cancer (4-7). Also, H.pylori infection in children can be one of the causes of iron and vitamin B12 deficiency. According to the findings, the infection with H.pylori in people who live in developing countries is more than people who live in developed countries, which can be due to the difference in the level of health in these countries (8-10).Also, the findings show that infection with H.pylori can be related to the development of bronchitis and lung cancer, however, the direct effect of H.pylori on respiratory diseases has not been fully determined to date (10-12). HP-NAP is one of the important proteins of H.pylori that during the growth of the bacterium, HP-NAP molecules are released from the bacterium and some of it is on the surface of the outer membrane of the bacterium in the form of remain connected. This protein is considered as an immunomodulatory. According to its characteristics, if the neutrophil activating factor can activate and increase the efficiency of macrophages, it can be considered as a key factor for the prevention and treatment of infection (13). This protein can strongly stimulate neutrophils, monocytes and dendritic cells and increase the production of IFN-γ and decrease IL-4, thereby directing the Th2 immune response to the Th1 response. Therefore, it is possible that HP-NAP can reduce the incidence of allergic asthma in people infected with Helicobacter pylori (14-15-17). Allergic asthma is a chronic inflammatory disease of the bronchi that is diagnosed with cough and shortness of breath and wheezing. Allergic asthma is caused by an excessive Th2 response to environmental allergens. In allergic asthma, Th2 cytokines cause the production and release of cytokines (IL-4, IL-5 and IL-13) and stimulate B cells to produce IgE, resulting in eosinophilic inflammation in the bronchi. On the other hand, they inhibit the secretion (IL-12, TNF-α, and INF-γ) related to Th1, which eventually leads to asthma attacks when observing the symptoms of atopy (22-21-20-19-18).
Recognizing, controlling, preventing and treating asthma is inevitable. An asthma attack is the result of the immune system's reaction against allergens. According to studies, some infectious diseases can affect the development of allergic disorders (23). Some bacteria can direct allergic responses from Th2 to Th1 responses by producing IFN-γ and IL-12 (25, 24). Therefore, by using some bacteria or their components that induce Th1 response, the development of allergic asthma can be prevented (26). In allergic asthma patients, stimulation of a Th1-mediated immune response can suppress Th2 responses, while if the Th1 response is insufficient, the Th2 response is high, which can ultimately lead to allergic asthma in the bronchi (19). Microbial products can be useful for maintaining the balance between Th1 and Th2 responses. Therefore, bacteria are effective in controlling and treating of asthma and can be used as potential therapeutic approach in harnessing of allergic asthma (9, 19, and 27). Recent studies have shown that an association between the presence of H.pylori containing HP-NAP and a reduced risk of asthma (28-30).
The aim of this study is to identify the molecular gene of HP-NAP effective in controlling allergic asthma by PCR method. The native bacteria of Iran should be prepared, then molecular tests PCR should be performed to confirm the presence of this peptide in native H.pylori. So that if the presence of this peptide is proven, it can be extracted for further research and for clinical research as a potential therapeutic strategy and as a New tools for the prevention and treatment of allergic asthma were investigated and used.
Method
Preparation of bacteria
Since this study was not done on native bacteria, it was decided to order and purchase the native strain of Helicobacter pylori from the Pasteur Institute of Iran. After the preparation of the bacteria, it was transferred to the laboratory for further studies by an anaerobic jar and gas pack type C.
Diagnostic tests
First, we use warm staining to observe and identify the bacteria under the microscope, then to be sure, I culture the bacteria in urea agar medium, so that if we observe the fermentation of urea by the urease enzyme of this bacteria, and as a result, the color of the medium changes from yellow to pink. Let's start the next steps clearly.
Identification of HP-NAP by PCR method
In order to determine the HP-NAP in indigenous bacteria, this work was done by PCR method. For this purpose, the genome sequence of H.pylori containing the HP-NAP gene is available under registration number AF227081.1 (from the World Gene Bank. in NCBI database) and primer design was done with the help of Gene Runner software. The sequence of reverse primers designed is as follows.
Forward primer 5'GTTTGCGGACATGTTTGACG3'
Reverse primer 5'GCAACTTGGCCAATTGATCG3'
These primers were received from Pishgam after the order. In order to perform the PCR test, DNA extraction had to be done first. For this purpose, a sufficient amount of the bacterial colony was removed by a sterile swab and placed in a micro tube containing sterile distilled water with a comparative turbidity of half. McFarland was transferred. Then DNA extraction was done by boiling method and to check the success of the genome extraction process, the Nano drop device made by ABI, USA was used, and the appropriate Dna concentration 700nanograms was measured. In the following, the DNA was extracted to determine the HP-NAP gene. It was PCR by a thermos cycler made by Gradient Analytica Gina. The PCR reaction was carried out in a final volume of 25 microliters containing 2.5microliter Master mix 2x (pishgam,Tehran,Iran) and 1 microliter of a solution containing 1 microliter primer (10 picogram) and 1.4 ng DNA template. The temperature program included an initial temperature 95C for 5 minutes, followed by 33 repeated steps including denaturation at 94°C for one minute, annealing at 58°C for one minute, and extension at 72°C for 30 seconds, and finally, one-step final elongation at 72°C for 10 minutes was performed.
PCR product electrophoresis
The product obtained from PCR should be electrophoresed to detect the HP-NAP gene, which was done by horizontal electrophoresis with One percent agarose gel. The structure of these gels is porous and the diameter of the pores is about 100 to 500 nm, and this causes molecules with sizes so, first we prepare a One percent agarose gel to make a One percent concentration gel, one gram of agarose powder is dissolved in 100 ml of TBE (Tris/Borate/EDTA) buffer, and this mixture is then It turns into a clear and uniform liquid after heating. Heat dissolves all the agarose particles in the buffer. Then we fill the electrophoresis tank with the same buffer that we used to prepare the gel. After preparing the gel and creating suitable holes with a special comb, add 2 microliters of the PCR product into the gel and after connecting the electrodes, we turn on the device, since the DNA molecules have a negative charge, when they are placed on the gel and under an electric current, they move towards the positive electrode. In this situation, the separation of DNA molecules is done only based on their size. The movement of short DNA strands is faster than longer molecules, so it is easier to separate DNA molecules of different sizes. As we mentioned before, the tracking of samples is done by adding fluorescent dyes in the gel well. Used Safe stain (pishgam,Tehran,Iran) for this purpose.
After the electrophoresis is finished and the electric current is cut off, a DNA marker or ladder is used as a standard to measure the DNA band resulting from PCR. bp was related to HP-NAP, we look for the size of the band obtained from the PCR product on the gel, for this purpose we use the 100-2000 bp ladder (pishgam,Tehran,Iran), and observe the result using the Gel documentation machine and take a picture of it and search for the desired size we do.
Result
At first, transferred a small amount from the plate containing the desired bacteria to a microscope slide to stain the bacteria and look for gram-negative bacteria, the result of which can be seen in) Figure1 (. After staining and observing the absence of contamination of the plate with other bacteria, we used a specific urease test inside the test tube. These bacteria convert the urea in the culture medium into ammonia, carbon dioxide and water. Ammonia resulting from the decomposition of urea causes the alkalinity of the environment, which is shown by the change in the urea reagent (phenol red) and the change in the color of the environment from yellow to light pink, the result of which can be seen in (Figure 2).
Molecular test: The main goal of this research is to determine the HP-NAP gene using the PCR technique. After performing this technique and electrophoresis of the product obtained from the PCR, the image obtained from the gel documentation machine was analyzed and considering that after the design of the primer band 260 bp was related to HP-NAP gene, the appropriate laser should be used to detect DNA bands. As a result, it is better to use a 100 to 2000 bp ladder, which is capable of detecting 100 bp DNA fragments. In the figure below, the 260 bp band related to the HP-NAP gene can be seen quite clearly compared to the 200 to 300 bp ladder band, so as a result, this protein it was also detected in the native bacteria of Iran for further studies (Figure 3).
Fig 1. Gram staining of bacteria and observation under the microscope with x400 magnification
Fig 2: Urease test: the left tube of the negative sample and the right tube of the positive sample (H.pylori)
Fig 3. The result of HP-NAP electrophoresis on one percent agarose gel.
Discussion
Helicobacter pylori, as an old bacterium, often settles in the human stomach and sticks to its cells. Helicobacter pylori is probably one of the microflora of human stomach. With the improvement of people's lifestyle and health conditions and the eradication of Helicobacter pylori, its prevalence has decreased drastically in developed countries, while the incidence of asthma and allergic diseases has increased greatly (31-32). To confirm the "disappearing microbiota" theory, Blaser states the loss of certain bacterial species from the old microbiota that can change the immune system and its cognitive functions, which should be developed early in our lives, and this change can lead to increased sensitivity to some diseases.Also, the loss of these microbiota, which is observed in most parts of the world, can lead to the emergence of modern diseases. These cases encourage us to understand the reasons for the loss of microbiota (5-33). Although eradicating this bacterium reduces the incidence of indigestion, stomach ulcers and stomach malignancies. However, there are also concerns for the use of its complete eradication, including resistance to foreign microbes and the increased prevalence of disorders that are inversely related to H.pylori infection, including obesity, asthma, GERD, and Barrett's esophagus (34,35). Completely eradicating this bacterium has advantages and disadvantages. Therefore, identifying and removing only the malignant strains of Helicobacter pylori is very important for treatment and eradication (36). Epidemiological studies and experiments show that exposure to Helicobacter pylori, especially in childhood, can prevent allergic asthma (37). However, several recent in vivo studies have shown that live bacteria do not play a role in inducing this protective role. Thus, the administration of Helicobacter pylori extract in newborns was able to prevent airway inflammation and goblet cell metaplasia. In this way, the injection of Helicobacter pylori extract can inhibit DCs that are involved in the processing of allergens in the mediastinal lymph nodes of the lungs. And these results show that Helicobacter pylori extract can effectively prevent allergic airway disorders after sensitization (38). H.pylori targets DCs and relies on highly suppressed T-regs. Since HP-NAP is considered as a possible regulator of T-regs and can inhibit the allergic inflammation of asthma, HP-NAP could be developed as an efficient H.pylori -specific vaccine for the treatment of allergic asthma (39, 40, and 41). Van Wyk et al have shown that Helicobacter pylori extract can effectively reduce mucus production and multiple features of inflammation in mice re-challenged by house dust mites (42). Maternal exposure to H.pylori can reduce allergic airway inflammation in offspring via T-regs and also provide new insights for interventional asthma treatment (43). Helicobacter pylori and humans have evolved together for about 50,000 years. This bacterium is the main cause of gastric ulcer, gastric lymphoma and gastric adenocarcinoma. It seems that the complete disappearance of this bacteria has consequences for humans, so that there is now a lot of information about the advantages and disadvantages of this change on the health or disease of the digestive system. However, there is a lot of evidence about its extra intestinal effects, including on allergic asthma. Similar to the inverse association between H.pylori and childhood asthma, H.pylori neutrophil-activating protein (HP-NAP) has the ability to shift allergen-specific T cell responses in vitro from Th-2 responses to Th-1 responses. Also, HP-NAP in the body of mice led to the inhibition of bronchial inflammation in line with Th-2 responses of allergic asthma. (37) Therefore, overall theories support the view that increased prevalence and severity of asthma and allergy in Western countries, it may be related, at least in part, to the reduction of H.pylori infection, which can cause long-term induction of Th1 background and leads to prevention and treatment of asthma and allergic diseases. Since in this research, we have identified the gene of this protein in native bacteria by molecular PCR method, and there is a hope that by extracting and purifying of this protein, it will be used in clinical cases of allergic asthma. Using of this peptide in case with a positive effects in the control and prevention of allergic asthma, the possibility of using it as medicine or vaccine for target patients will be initiated..
Conflict of interest statement
There is no conflict of interest.
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