結核分枝桿菌疫苗候選物蛋白UgpB結構與功能研究
發(fā)布時間：2018-12-08 20:38
【摘要】：結核病仍是目前威脅人類健康的重大傳染病之一,而TB/HIV雙重感染和結核耐藥菌株的出現(xiàn),使得全球范圍內(nèi)結核病防控工作更加困難。卡介苗BCG是當前唯一的結核疫苗,但僅對新生兒效果良好。目前結核病標準療程需要3到4種藥混合使用,治療周期長達6個月,治療過程中患者依從度較差。同時,抗藥結核患者數(shù)量持續(xù)增加,甚至已有完全抗藥結核病例報道。因此,亟待新的抗結核藥物和疫苗來控制結核病的全球肆虐。針對結核分枝桿菌蛋白質組中藥物靶點蛋白和疫苗候選物的三維結構研究將推動新型抗結核藥物的發(fā)現(xiàn)與開發(fā),以及促進抗結核藥理學和生物學行為背后的生化機制描述。 ABC轉運體超家族是膜整合蛋白,大部分家族成員是耦合ATP水解能量來跨膜轉運溶質的轉運蛋白。根據(jù)溶質運輸方向,分為內(nèi)向轉運體和外向轉運體。原核生物內(nèi)向轉運蛋白介導了細胞生存和生長相關營養(yǎng)物質的跨膜攝取。目前結核分枝桿菌在人宿主體內(nèi)的關鍵營養(yǎng)物質和相應的轉運體仍未知。UgpABCE是結核分枝桿菌中5個可辨別碳水化合物輸入轉運體之一,基因組分析認為它是sn-G3P轉運體。相關研究表明其周質結合蛋白UgpB對于結核分枝桿菌體外最優(yōu)生長非常關鍵,也被認為是一個潛在的抗結核疫苗候選物。 本研究中,我們解析了結核分枝桿菌UgpB的三維高分辨率結構(1.5A),并基于結構進行了相關生化實驗分析。有趣的是,ITC實驗結果顯示,結核UgpB并不結合sn-G3P,而是結合GPC,暗示了GPC是結核分枝桿菌在宿主體內(nèi)利用的磷源和碳源之一。結構比對分析表明,大腸桿菌UgpB中對于底物G3P結合至關重要的Trp169在結核UgpB中被Leu205代替。此外,大腸UgpB中其它一些與G3P相互作用的氨基酸殘基在結核UgpB中也不保守。因此結核UgpB喪失了對G3P的親和力。針對Leu205的突變實驗也證實了它對于UgpB底物結合的重要性。ITC實驗還表明,G2P和麥芽糖不是結核UgpB結合的底物。本課題研究不僅有助于理解結核分枝桿菌在宿主體內(nèi)對碳源和磷源的利用,也將為抗結核藥物或疫苗的開發(fā)提供結構基礎,進而促進結核病化學療法的改進。
[Abstract]:Tuberculosis is still one of the major infectious diseases threatening human health at present, and the emergence of TB/HIV double infection and TB resistant strains make it more difficult to prevent and control tuberculosis worldwide. BCG is the only TB vaccine at present, but it only works well in newborns. The current standard course of treatment for tuberculosis requires a combination of 3 to 4 drugs for a period of 6 months with poor compliance. At the same time, the number of drug-resistant TB patients continues to increase, even drug-resistant TB cases have been reported. Therefore, new anti-TB drugs and vaccines are urgently needed to control the global spread of tuberculosis. The research on the three-dimensional structure of drug target protein and vaccine candidate in proteome of Mycobacterium tuberculosis will promote the discovery and development of new anti-tuberculosis drugs and the description of biochemical mechanism behind the pharmacological and biological behaviors of anti-tuberculosis drugs. The ABC transporter superfamily is a membrane integrin, and most of the family members are transporters coupled with ATP hydrolysis energy to transport solute across the membrane. According to the transport direction of solute, it can be divided into inward transporter and extroverted transporter. Prokaryotic inward transporter mediates the transmembrane uptake of nutrients associated with cell survival and growth. At present, the key nutrients and corresponding transporters of Mycobacterium tuberculosis in human host are unknown. UgpABCE is one of the five identifiable carbohydrate transporters in Mycobacterium tuberculosis, which is considered to be a sn-G3P transporter by genomic analysis. Related studies have shown that its periplasmic binding protein UgpB is critical to the optimal growth of Mycobacterium tuberculosis in vitro and is also considered as a potential candidate for anti-tuberculosis vaccine. In this study, we analyzed the three-dimensional high-resolution structure (1.5A) of Mycobacterium tuberculosis UgpB, and analyzed the related biochemical experiments based on the structure. Interestingly, ITC results show that TB UgpB does not bind to sn-G3P, but GPC, suggests that GPC is one of the phosphorus and carbon sources used by Mycobacterium tuberculosis in the host. Structural comparison analysis showed that Trp169, which is important for substrate G3P binding in Escherichia coli UgpB, was replaced by Leu205 in tuberculous UgpB. In addition, some other amino acid residues interacting with G3P in colorectal UgpB are not conserved in tuberculous UgpB. Therefore, TB UgpB lost its affinity to G 3 P. The mutation test for Leu205 also confirmed the importance of G2P and maltose for the binding of UgpB substrates. The ITC experiment also showed that G2P and maltose were not the substrates of UgpB binding of tuberculosis. This study will not only help to understand the utilization of carbon and phosphorus sources by Mycobacterium tuberculosis in the host, but also provide a structural basis for the development of anti-tuberculosis drugs or vaccines, and promote the improvement of TB chemotherapy.
【學位授予單位】：南開大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R52
，
本文編號：2368966
[Abstract]:Tuberculosis is still one of the major infectious diseases threatening human health at present, and the emergence of TB/HIV double infection and TB resistant strains make it more difficult to prevent and control tuberculosis worldwide. BCG is the only TB vaccine at present, but it only works well in newborns. The current standard course of treatment for tuberculosis requires a combination of 3 to 4 drugs for a period of 6 months with poor compliance. At the same time, the number of drug-resistant TB patients continues to increase, even drug-resistant TB cases have been reported. Therefore, new anti-TB drugs and vaccines are urgently needed to control the global spread of tuberculosis. The research on the three-dimensional structure of drug target protein and vaccine candidate in proteome of Mycobacterium tuberculosis will promote the discovery and development of new anti-tuberculosis drugs and the description of biochemical mechanism behind the pharmacological and biological behaviors of anti-tuberculosis drugs. The ABC transporter superfamily is a membrane integrin, and most of the family members are transporters coupled with ATP hydrolysis energy to transport solute across the membrane. According to the transport direction of solute, it can be divided into inward transporter and extroverted transporter. Prokaryotic inward transporter mediates the transmembrane uptake of nutrients associated with cell survival and growth. At present, the key nutrients and corresponding transporters of Mycobacterium tuberculosis in human host are unknown. UgpABCE is one of the five identifiable carbohydrate transporters in Mycobacterium tuberculosis, which is considered to be a sn-G3P transporter by genomic analysis. Related studies have shown that its periplasmic binding protein UgpB is critical to the optimal growth of Mycobacterium tuberculosis in vitro and is also considered as a potential candidate for anti-tuberculosis vaccine. In this study, we analyzed the three-dimensional high-resolution structure (1.5A) of Mycobacterium tuberculosis UgpB, and analyzed the related biochemical experiments based on the structure. Interestingly, ITC results show that TB UgpB does not bind to sn-G3P, but GPC, suggests that GPC is one of the phosphorus and carbon sources used by Mycobacterium tuberculosis in the host. Structural comparison analysis showed that Trp169, which is important for substrate G3P binding in Escherichia coli UgpB, was replaced by Leu205 in tuberculous UgpB. In addition, some other amino acid residues interacting with G3P in colorectal UgpB are not conserved in tuberculous UgpB. Therefore, TB UgpB lost its affinity to G 3 P. The mutation test for Leu205 also confirmed the importance of G2P and maltose for the binding of UgpB substrates. The ITC experiment also showed that G2P and maltose were not the substrates of UgpB binding of tuberculosis. This study will not only help to understand the utilization of carbon and phosphorus sources by Mycobacterium tuberculosis in the host, but also provide a structural basis for the development of anti-tuberculosis drugs or vaccines, and promote the improvement of TB chemotherapy.
【學位授予單位】：南開大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R52
，
本文編號：2368966
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