【作者】 史一博；

【导师】 孙建和；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2012， 博士

【摘要】 猪链球菌（Streptococcussuis）2型因致多起人严重感染和死亡事件而引发关注，临床分离菌株大多呈现对抗生素严重耐药和多重耐药，抗生素应用面临挑战，而基于噬菌体的抗菌策略则再现潜力。目前，国内外对猪链球菌噬菌体裂解的生物化学基础和裂解机制鲜有研究，故本论文预测并验证了穿孔素基因（HolSMP）的存在，分析了基因序列或功能域与其生物学功能的相关性，确认了噬菌体SMP存在“穿孔素-裂解酶”裂菌系统，揭示了穿孔素的胞外裂菌作用，为应用噬菌体裂解系统防控猪链球菌感染奠定了理论基础。为了挖掘猪链球菌噬菌体基因组上的穿孔素基因，基于生物信息学预测和分析发现，穿孔素基因（HolSMP）定位于裂解酶基因（LySMP）上游，位于基因组7267~7695位碱基，全长429bp，与链球菌（尤其是肺炎链球菌）噬菌体的穿孔素蛋白序列具有同源性。鉴于穿孔素基因序列的特殊性和编码产物的复杂性，将基因HolSMP可能编码的所有蛋白统称为HolSMP；而该基因全长编码的分子量约15.7kDa的膜蛋白命名为HolSMP(142)。HolSMP(142)包含穿孔素第四超家族保守序列，具有Ⅰ型穿孔素蛋白结构特征。为了分析HolSMP在噬菌体感染宿主菌后的转录与表达特征，本论文在优化噬菌体悬液制备和猪链球菌细胞膜提取方案的基础上，基于噬菌体SMP的一步生长曲线，提取噬菌体SMP感染后不同时间点的猪链球菌2型菌株SS2-H的总RNA和膜蛋白，通过反转录PCR和荧光定量PCR检测发现，在噬菌体感染20min时，HolSMP基因开始转录，60min时转录水平达到峰值。用蛋白质免疫杂交分析HolSMP的表达水平，发现在感染20min后可检测到HolSMP蛋白，60min时蛋白表达量达到峰值。结果表明，在SMP感染宿主菌后HolSMP基因被转录，其编码的蛋白HolSMP分布于细胞膜上，提示基因HolSMP具有穿孔素基因的转录、表达和亚细胞定位特征。为了揭示HolSMP基因的功能，选择合适的评价系统非常关键。鉴于穿孔素蛋白发挥功能无种属特异性，故大肠杆菌也可作为研究革兰氏阳性菌噬菌体穿孔素蛋白功能的理想系统。本论文构建了携带HolMSP基因的重组质粒pEXH1，并在大肠杆菌的背景下证实HoLSMP是SMP的穿孔素基因：HolSMP基因的表达抑制了重组菌BL21(DE3)的生长、导致了BL21(DE3)pLysS的快速裂解；HolSMP基因的表达产物定位于细菌的细胞膜上；能量毒素氰化钾可以提前触发HolSMP基因表达产物形成跨膜通道并引起菌体裂解，符合穿孔素判定的重要标准；HolSMP基因和LySMP基因的共表达可以迅速触发细菌裂解，说明二者可能在噬菌体SMP释放过程中发挥协同胞内裂菌作用；HolSMP基因可以补偿穿孔素基因缺失型λ噬菌体的相应功能，并使之形成噬菌斑。这些结果表明HolSMP基因具有编码穿孔素的功能。为了解析穿孔素基因HolSMP的结构与功能的相关性，通过对该基因的分析发现，其编码的全长蛋白HolSMP(142)含有10个由“ATG”编码的甲硫氨酸，分别以这10个“ATG”为起始密码子，扩增穿孔素HolSMP基因片段，其表达产物呈现不同的胞内裂菌表型。以前5个“ATG”为起始密码子的HolSMP基因片段的表达均导致宿主菌裂解，其中以第五个“ATG”为起始密码子，其裂菌时间点较表达HolSMP基因全长提前20min，而该密码子发生突变后HolSMPa43c基因可导致裂菌时间延迟15min。据此，初步推测穿孔素基因HolSMP可能具有与λ噬菌体穿孔素基因S相似的“双起始”基序。另外，以第6个“ATG”为起始密码子的HolSMP(103)对宿主菌无毒性，适用于制备HolSMP抗体。研究表明穿孔素的生物学活性与其功能域密切相关，通过缺失突变发现，缺失任一跨膜区均会导致穿孔素的生物学活性降低甚至丧失。同时，研究显示HolSMP基因两端分别或同时添加组氨酸标签编码序列，均会引起穿孔素介导的宿主菌裂解时间的改变和裂菌强度的下降。为了大量获得保持生物学活性的猪链球菌噬菌体穿孔素，本论文对穿孔素基因的表达策略进行了优化。比较了大肠杆菌、乳酸乳球菌、哺乳动物细胞和酵母等表达系统对HolSMP基因的表达效果，最终确定大肠杆菌BL21(DE3)pLysS是目前最理想的穿孔素表达系统，建立的浓缩对数生长期菌液再进行IPTG诱导的表达工艺凸显优势。研究发现在保持穿孔素生物学活性的前提下，HolSMP(142)的Gly139残基是可以插入组氨酸标签的唯一位点。基于该标签，表达纯化HolSMP蛋白，测定其胞外裂菌特性，结果显示，该蛋白在37℃、pH5.2和添加0.8%β-巯基乙醇的条件下裂菌活性最高。平板裂解实验结果显示，该蛋白可裂解金黄色葡萄球菌和枯草芽孢杆菌；与LySMP联合应用时，能裂解分别对HolSMP和LySMP不敏感的7株猪链球菌2型菌株和1株沙门氏菌，表明噬菌体SMP的穿孔素能增强裂解酶的裂菌活性，具有一定的开发应用前景。综上所述，位于裂解酶LySMP基因上游的穿孔素基因HolSMP所编码的穿孔素蛋白与同源裂解酶可协同发挥裂菌作用，构成猪链球菌噬菌体SMP的“穿孔素-裂解酶”二元裂解系统，具有抗菌应用前景；噬菌体SMP穿孔素蛋白的生物学功能与其氨基酸残基密切相关，因此，深入揭示穿孔素的拓扑结构与功能以及触发裂菌的分子机制，是研发基于噬菌体抗菌制剂的基础和未来方向。更多还原

【Abstract】 Streptococcus suis（S. suis） type2is an important zoonotic pathogen that leads to economic losses and severe invasive diseases in humans, and the increasing prevalence of antibiotics-resistant SS2strains became one of the greatest challenges worldwide. Bacteriophage has been examined as potential agent for SS2control, but its biochemistry and bacterial lysis mechanism is still open. Holin-lysin two-step lysis system typically existing in the double-stranded DNA phage is effective and specific. By accumulating and forming lesions in the cytoplasmic membrane, holins control the access of phage-encoded endolysins to the peptidoglycan and thereby trigger the lysis of the host cell at a precise time point. S. suis serotype2（SS2） lytic bacteriophage SMP is a double-stranded DNA phage. LySMP was identified to be the lysin gene and a putative holin-lysin lysis system was supposed to exist in SMP. In this work, a putative holin gene, HolSMP, was predicted and identified. The relationship of gene sequence and its biological function was analyzed. Moreover, the antibacterial activity of holin from SMP and the synergistic antimicrobial activity of holin and lysin against S suis were tested.To find and locate the putative holin gene of phage SMP, DNA and protein sequence homology alignments were performed using BLAST tools on NCBI. TMHMM, SOSUI and PredictProtein servers were used to predict the transmembrane helices. The results showed that HolSMP, which is located upstream of LySMP gene, was a putative holin gene of SMP. HolSMP is429bp long and shows similarity to the sequence of holin gene from Streptococcus phage. HolSMP（142）, a putative Class I holin with three transmambrane domains encoded by HolSMP, shows similarity to holin protein of Streptococcus phage and exhibits the characteristics of phage-holin₄superfamily.To characterize the transcription and expression of the HolSMP gene in SMP-infected S. suis, the protocols for phage collection and membrane sample extraction were optimized. Based on one-step growth curve of SMP, the total RNA samples and membrane samples were prepared at a series time points during phage infection. The results of reverse transcript PCR, real-time fluorescence quantitative PCR and western blotting assay revealed that HolSMP was transcribed and expressed at15-20min. Accumulation of HolSMP in the cytoplasmic membrane was found before60min. With a reduction of HolSMP transcripts and destruction of host cells, the amount of HolSMP in the membrane also reduced. All the results show that HolSMP is a membrane protein and HolSMP is a late stage gene in conformity with holin gene.To determine the function of HolSMP gene, it is crucial to develop an evaluating system. Holin-dependent membrane lesions are non-specific, and this enables testing of HolSMP in Escherichia coli（E. coli）. Plasmid pEXH1harboring gene HolSMP was transformed into E. coli. Expression of HolSMP inhibited growth of BL21（DE3） and resulted in collapse and death of BL21（DE3）pLysS. Production of HolSMP accumulated in membrane is sensitive to energy toxin potassium cyanide, which is a very important character to identify a new holin family member. Significant cell lysis in the presence of LySMP suggested their putative synergistic effect in physiological situation. Gene HolSMP complemented the defective λ S allele in a non-suppressing E. coli strain to produce phage plaques. These results indicated that HolSMP was the holin gene of SMP. To understand the correlation of structure and function of HolSMP, the start codons of the gene were analyzed. There are ten methionines in HolSMP（142）. These production of sequences started from different "ATG" resulted in various lysis phenotypes. The sequence HolSMP（387） leaded by the fifth "ATG" showed an early-dominance phenotype, starting at about20min ahead of normal lysis time point, while a mutant HolSMP at the fifth "ATG", HolSMPa43c, exhibited an delayed-onset lytic profile, at about55min after induction. These results supported an inference that a dual started model of holin gene Sfrom lambda phage probably existed in gene HolSMP. HolSMP（142） encoded by HolSMP might be the antiholin, while the HolSMP（128） encoded by HolSMP（387） might be the real holin protein. The avirulence protein HolSMP（103） was expressed for preparation of polyclonal antibody against HolSMP.It is also found that the activity of HolSMP is closely related to the encoding sequence, especially to that of N-terminal sequence and integrity of the first transmembrane domain. The addition of an oligohistidine tag at one terminus fundamentally changed the character of HolSMP, altered the triggering time and weakened its lesion-forming activity. Insertion of an oligohistidine tag after codon139yielded HolSMP that showed essentially normal timing and lysis profiles, which might be helpful for preparation and application of HolSMP in the future.To prepare the holin for the purpose in large scale, the expression systems were constructed and characterized. Compared with Lactococcus lactis, mammalian cells and yeast, E. coli strain BL21（DE3）pLysS was the optimum expression system. The expressed HolSMP139t exerted efficient activity at37℃, pH5.2, with addition of0.8%β-mercaptoethanol. Lytic spectra of purified HolSMP, LySMP or mixture of HolSMP and LySMP were investigated. The results showed that HolSMP, exhibiting a narrow lytic spectrum, was effective against Staphylococcus aureus and B. subtilis, which were insensitive to LySMP. However, the simultaneous administration of HolSMP and LySMP has a synergistic effect when applied exogenously.Taken together, this work focused on identification and characterization of holin gene from phage SMP, and a holin-lysin two step lysis system was determined as well. The combined application of HolSMP and LySMP proved that holin and lysin had a synergistic effect when administered exogenously. Further investigation of the topological structure and function of HolSMP and a candidate combined strategy for solution of drug resistance is now warranted.更多还原