沙门氏菌噬菌体长尾丝蛋白远端亚基介导的宽裂解谱机制研究

Bacteriophages have now become a significant tool to reduce or eliminate antibiotic resistance threats. To address the defects of host specificity in application, there is a critical need to clarify the lysis mechanism of phages to their hosts. Many studies have demonstrated that the specific adsorption of a phage to its hosts is vital to determine its host range. Furthermore, the broad host-range T4 phage can specifically adsorb to host receptors via the long tail fiber protein, and both its long tail fiber protein distal subunit and its host receptors are diverse in characteristics. Therefore, we propose that the broad-host-range T4 phage specifically recognizes and adsorbs to host receptors mediated by the long tail fiber protein distal subunit. In order to explain the mechanism of the broad host-range mediated by the long tail fiber protein distal subunit of Salmonella phage, this research takes the broad host-range Salmonella phage STP4-a, a T4-like phage, as the model organism to (1) investigate the diversity of host receptors via high-throughput sequencing and CRISPR/Cas9 seamless knockout techniques, combined with screening multi-receptor hosts using Salmonella typing phages; (2) explore the preference to different host receptors of broad host-range phages by constructing receptor-deficient strains and determining phage adsorption and hydrolysis rates; and (3) dig out recognition and adsorption activities and action sites of different domains in the long tail distal subunit by constructing a series of long tail distal subunit truncations, and finally work out mechanisms on the broad host-range of Salmonella phage mediated by its long tail fiber protein distal subunit.

噬菌体已经成为解决细菌耐药性问题的重要手段。为解决噬菌体特异性带来的应用缺陷，亟需洞悉噬菌体对宿主菌的裂解机制。研究表明，噬菌体对宿主菌特异性的吸附结合作用，是决定裂解谱的关键。宽谱T4型噬菌体通过长尾丝蛋白与宿主菌受体发生特异性结合作用，且长尾丝蛋白远端亚基与宿主菌受体均存在多样性特征。据此提出：宽谱T4型噬菌体通过长尾丝蛋白远端亚基高可变区介导与受体的特异性识别与结合作用。本研究拟以宽谱沙门氏菌噬菌体STP4-a为模式生物，与沙门氏菌分型噬菌体联用进行多受体宿主菌筛选，结合高通量测序技术和CRISPR无痕敲除技术进行受体多样性鉴定；通过受体缺陷型菌株构建及噬菌体吸附裂解试验分析宽谱噬菌体对不同受体的选择偏好性；构建系列长尾丝蛋白远端亚基截短体，分析长尾丝蛋白远端亚基不同结构域对识别结合作用的影响并最终确定活性位点。从而揭示沙门氏菌噬菌体长尾丝蛋白远端亚基介导的宽裂解谱相关机制。

噬菌体被认为是解决细菌耐药性的有效工具，但是宿主谱的高度特异性给其应用造成了一定的困难。本课题以一株宽裂解谱沙门氏菌噬菌体STP4-a为对象，研究了其宽谱识别特性相关机制，发现该噬菌体STP4-a能够以外膜蛋白OmpA和LPS外核心多糖为初级受体，以LPS内核心多糖为次级受体，以实现对沙门氏菌的宽谱识别。基因gp37和gp38编码序列共同构成STP4-a长尾丝蛋白远端亚基，利用结构预测、非完全变性电泳、免疫印记及抗体阻断噬菌体侵染实验等分析显示P3738蛋白为多聚体大分子结构；gp38的C末端编码多个富含甘氨酸区域，是噬菌体发挥宽谱识别特性的关键区域；后续进行尾丝蛋白替换，显示经改造后的噬菌体能够显著拓宽宿主谱。基于STP4-a尾丝蛋白的宽谱识别特性，研究还建立了一种以长尾丝蛋白远端亚基为识别探针的超灵敏沙门氏菌快速检测方法，以及利用重组长尾丝蛋白远端亚基与LPS的特异性结合，高效降低炎症反应损伤。相关研究成果为解决噬菌体宿主谱特异性难题提供了一定的理论支撑，为噬菌体资源开发利用提供了新的研究思路。