2023, Vol. 63
表1(Table 1)
结核分枝杆菌PE_PGRS15调控分枝杆菌细胞包膜结构与耐药性
http://dx.doi.org/10.13343/j.cnki.wsxb.20230285
中国科学院微生物研究所，中国微生物学会
中国科学院微生物研究所，中国微生物学会
文章信息
- 李武, 邓磊, 阎紫菲, 艾雪峰, 吕茜, 谢建平. 2023
- LI Wu, DENG Lei, YAN Zifei, AI Xuefeng, LÜ Xi, XIE Jianping.
- 结核分枝杆菌PE_PGRS15调控分枝杆菌细胞包膜结构与耐药性
- Mycobacterium tuberculosis PE_PGRS15 modulates the envelope structure and stress resistance of mycobacteria
- 微生物学报, 63(12): 4644-4658
- Acta Microbiologica Sinica, 63(12): 4644-4658
-
文章历史
- 收稿日期：2023-04-20
- 网络出版日期：2023-07-05
引用本文 |
李武, 邓磊, 阎紫菲, 艾雪峰, 吕茜, 谢建平. 结核分枝杆菌PE_PGRS15调控分枝杆菌细胞包膜结构与耐药性[J]. 微生物学报, 2023, 63(12): 4644-4658.
Li Wu, Deng Lei, Yan Zifei, Ai Xuefeng, Lü Xi, Xie Jianping. Mycobacterium tuberculosis PE_PGRS15 modulates the envelope structure and stress resistance of mycobacteria[J]. Acta Microbiologica Sinica, 2023, 63(12): 4644-4658.
结核分枝杆菌PE_PGRS15调控分枝杆菌细胞包膜结构与耐药性
1. 内江师范学院生命科学学院 特色农业资源研究与利用四川省高等学校重点实验室, 四川 内江 641100;
2. 西南大学生命科学学院 现代生物医药研究所 三峡库区生态环境与生物资源省部共建国家重点实验室培育基地, 重庆 400715
2. 西南大学生命科学学院 现代生物医药研究所 三峡库区生态环境与生物资源省部共建国家重点实验室培育基地, 重庆 400715
摘要：[目的] 研究结核分枝杆菌PE_PGRS15的功能。[方法] 构建过表达PE_PGRS15蛋白的重组耻垢分枝菌酸杆形菌，通过细胞分级分离实验检测其细胞定位。通过涂布实验、扫描电镜和透射电镜观察细菌菌落形态、细菌表面形态及细胞包膜(cell envelope)结构。通过杀菌曲线法及微量肉汤稀释法检测重组菌对环境压力及抗生素的耐受性。通过染料摄取实验检测重组菌细胞壁通透性，并用气相色谱-质谱联用仪检测重组菌细胞壁脂肪酸谱。通过蛋白截短及融合实验分析PE_PGRS15蛋白结构域的功能。[结果] PE_PGRS15蛋白定位于重组菌细胞壁，其表达影响重组菌菌落形态和细胞包膜结构，增强重组菌对环境压力和抗生素的耐受。PE_PGRS15的表达导致重组菌细胞包膜脂肪酸含量增加，也降低了重组菌的细胞壁通透性。PE_PGRS15蛋白的PE结构域负责将该蛋白转运到细胞表面，而PGRS结构域介导重组菌对压力条件和抗生素的耐受。[结论] PE_PGRS15蛋白可能通过调控耻垢分枝菌酸杆形菌细胞包膜的结构进而影响细菌菌落形态、细胞壁通透性及耐药性，为解析PE/PPE家族蛋白的功能奠定了一定的基础。
关键词：PE_PGRS15 耻垢分枝菌酸杆形菌 压力耐受 细胞壁通透性 过表达
Mycobacterium tuberculosis PE_PGRS15 modulates the envelope structure and stress resistance of mycobacteria
1. Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang 641100, Sichuan, China;
2. State Key Laboratory Breeding Base of Eco-environment and Bio-resource of the Three Gorges Area, Institute of Modern Biopharmaceuticals, School of Life Sciences, Southwest University, Chongqing 400715, China
2. State Key Laboratory Breeding Base of Eco-environment and Bio-resource of the Three Gorges Area, Institute of Modern Biopharmaceuticals, School of Life Sciences, Southwest University, Chongqing 400715, China
Abstract: [Objective] To reveal the function of PE_PGRS15 from Mycobacterium tuberculosis. [Methods] A recombinant Mycolicibacterium smegmatis strain heterologously expressing PE_PGRS15 (MS-PE_PGRS15) was generated. The colony morphology, cell surface morphology, and envelope structure were observed by a plating method, a scanning electron microscope, and a transmission electron microscope, respectively. The localization of PE_PGRS15 was detected by the cell fractionation assay. The resistance of the recombinant strain to environmental stresses and antibiotics was measured by the killing curve method and micro-broth dilution method. The permeability and fatty acid profile of the cell wall of the recombinant strain were determined by dye uptake assay and gas chromatography-mass spectrometry, respectively. The functions of different domains of PE_PGRS15 were analyzed by protein truncation and fusion experiments. [Results] PE_PGRS15 was located on the cell envelope of MS-PE_PGRS15. MS-PE_PGRS15 showed altered colony morphology, envelope structure, and cell wall fatty acid profile, with noticeable increase in resistance to multiple environmental stresses and antibiotics. The dye uptake experiments with ethidium bromide and Nile red suggested that the cell wall of MS-PE_PGRS15 was more impermeable than that of the control strain. The PGRS domain of PE_PGRS15 affected mycobacterial cell wall permeability and stress resistance, while the PE domain was involved in the transport of the protein to the cell surface. [Conclusion] PE_PGRS15 was present in the cell wall fraction of MS-PE_PGRS15 and influenced cell wall permeability and colony morphology, ultimately enhancing the resistance of recombinant M. smegmatis to stresses.
Keywords:
PE_PGRS15 Mycolicibacterium smegmatis stress resistance cell wall permeability overexpression
结核分枝杆菌(Mycobacterium tuberculosis, Mtb)感染导致的结核病(tuberculosis, TB)仍然是全球公共卫生的巨大威胁。特别是多重和广泛耐药Mtb及与人类免疫缺陷病毒(human immunodeficiency virus, HIV)共感染使得TB疫情愈发严峻。全球范围内，TB在2021年造成约1 060万新发病例和超过140万死亡病例[1]。因此，迫切需要通过研究Mtb的致病机理，开发新的TB防治策略。
脯氨酸-谷氨酸/脯氨酸-脯氨酸-谷氨酸(proline-glutamic acid/proline-proline-glutamic acid, PE/PPE)家族基因占Mtb基因组编码能力的10%左右[2]。多态性富含GC的重复序列(polymorphic GC-rich repetitive sequence, PE_PGRS)是PE/PPE家族的亚家族蛋白，在Mtb中有65个成员，由一个高度保守的PE结构域和一个可变的PGRS结构域组成[3]。PE_PGRS蛋白只存在于慢生型致病分枝杆菌中，如Mtb、海分枝杆菌和牛结核分枝杆菌等[4]。越来越多的证据表明该亚家族蛋白参与Mtb致病性，然而大多数成员在Mtb生理和毒力中的具体作用仍然未知[5]。因此研究PE_PGRS蛋白的功能有助于深入理解Mtb的生理功能和致病能力。
PE_PGRS亚家族蛋白的一个显著特点是各成员之间高度的序列同源性和功能重叠性[6]。比如开发针对特定PE_PGRS蛋白的抗体，会与其他成员产生大量的交叉反应[7]。因此，采用传统方法(如在致病分枝杆菌中敲除或过表达某个PE_PGRS蛋白)研究单个PE_PGRS蛋白可能会受到该亚家族其他成员的严重干扰。有研究报道Mtb的重要模式菌种——耻垢分枝菌酸杆形菌(Mycolicibacterium smegmatis，旧称耻垢分枝杆菌)中完全不存在PE_PGRS的同源蛋白[8]。因此，研究人员普遍采用在M. smegmatis中过表达PE_PGRS蛋白来研究其功能[8-10]。
本研究以过表达PE_PGRS15蛋白的重组M. smegmatis为研究对象，检测PE_PGRS15蛋白的细胞定位情况，及对重组菌细胞壁通透性、脂肪酸含量、压力耐受性和抗生素耐药性的影响，并明确各个亚基的不同功能。这些结果揭示了PE_PGRS15蛋白的具体功能，为进一步探索PE_PGRS亚家族蛋白在Mtb生理和毒力中的作用奠定了坚实的基础。
1 材料与方法 1.1 材料M. smegmatis mc2155、大肠埃希菌及pMV261质粒由本实验室保藏。Middlebrook 7H9/7H10培养基购自BD Difco公司。甘油、Tween 80及抗生素等购自生工生物工程(上海)股份有限公司。BCA Protein Assay Kit、鼠抗Myc抗体购自上海碧云天生物技术有限公司。外排泵特异性抑制剂氰氯苯腙(carbonyl cyanide 3-chlorophenylhydrazine, CCCP)购自北京索莱宝科技有限公司。限制性内切酶、连接酶及高保真DNA聚合酶购自纽英伦生物技术(北京)有限公司和宝日医生物技术(北京)有限公司。质粒提取试剂盒、胶回收试剂盒、鼠抗His抗体购自天根生化科技(北京)有限公司。引物合成和基因测序均由北京六合华大基因科技有限公司重庆分公司完成。其他实验常规试剂主要购自上海碧云天生物技术有限公司等公司。
1.2 重组质粒和菌株的构建及培养条件本研究所用引物信息见表 1，基因扩增的模板为Mtb实验室标准株H37Rv的基因组。扩增PE_PGRS15全长基因的引物为261-15FL-For和261-15FL-Rev，扩增PE_PGRS15的PE结构域(以下称15PE)的引物为261-15FL-For和261-15PE-Rev，扩增PE_PGRS15的PGRS结构域(以下称15PGRS)的引物为261-15PGRS-For和261-15FL-Rev，扩增PE_PGRS33的PE结构域(以下称33PE)的引物为261-33PE-For和261-33PE-Rev。先使用261-33PE-For和33-15overlap-Rev引物扩增33PE片段，同时使用33-15overlap-For和261-15FL-Rev引物扩增15PGRS片段；再使用重叠PCR技术构建33PE和15PGRS融合基因(以下称33PE-15PGRS)，使用的引物为261-33PE-For和261-15FL-Rev。PCR产物与pMV261质粒分别用EcoR Ⅰ和Hind Ⅲ进行双酶切后，用T4连接酶重新连接产生相应的重组质粒；再将重组质粒电转化入M. smegmatis感受态，产生相应的重组菌。M. smegmatis及其重组菌培养于7H9液体培养基，同时添加0.2% (体积分数)甘油，0.2% (质量体积分数)葡萄糖和0.05% (体积百分比)Tween 80。在培养重组菌时，添加20 μg/mL的卡那霉素防止质粒丢失。
表 1. 本研究使用的引物
Table 1. Primers used in this study
| Primers | Sequences (5′→3′) | Restriction sites |
| 261-15FL-For | GCGGAATTCATGTCGTATGTATTGGCGAC | EcoR Ⅰ |
| 261-15FL-Rev | CGCAAGCTTTCACAGATCCTCTTCAGAGATGAGTTTCTGCTCGCCGGGTTGGCCGGCG | Hind Ⅲ |
| 261-15PE-Rev | CTGAAGCTTTCACAGATCCTCTTCAGAGATGAGTTTCTGCTCGATCAGGGGGCGCCCG | Hind Ⅲ |
| 261-15PGRS-For | CGTGAATTCGGCAACGGCACCAATGGTG | EcoR Ⅰ |
| 261-33PE-For | CGCGAATTCATGTCATTTGTGGTCACG | EcoR Ⅰ |
| 261-33PE-Rev | AGTAAGCTTTCACAGATCCTCTTCAGAGATGAGTTTCTGCTCGATCAGTGGGCGCCCCA | Hind Ⅲ |
| 33-15overlap-For | GCGCCCACTGATCGGCAACGGCACCAATG | |
| 33-15overlap-Rev | CATTGGTGCCGTTGCCGATCAGTGGGCGC | |
| The underlined lines represent the restriction sites. The italics represent Myc (human c-Myc proto-oncogene) epitope tag. | ||
1.3 Western blotting检测重组蛋白的表达
离心收集对数生长期的重组M. smegmatis，洗涤并重悬于裂解缓冲液[50 mmol/L Tris-HCl (pH 8.0)、300 mmol/L NaCl、1 mmol/L PMSF及1 mmol/L DTT]中。冰上超声30 min (工作5 s，暂停5 s，30%功率)，离心收集上清液。使用BCA Protein Assay Kit测量样品蛋白浓度。取相同质量的细胞裂解液进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)分析，将样品转移至聚偏二氟乙烯(polyvinylidene fluoride, PVDF)印迹膜上，使用鼠抗Myc抗体检测重组蛋白的表达。
1.4 亚细胞分级分离实验检测蛋白定位M. smegmatis亚细胞分级分离实验参考文献[11]进行。离心收集对数生长期的重组 M. smegmatis，洗涤并重悬于裂解缓冲液，超声破碎。3 000×g离心30 min，上清即为全细胞裂解液(whole cell lysates, WCL)。将WCL在27 000×g离心力下继续离心60 min，沉淀即为细胞壁组分(cell wall fraction, CW)，剩下的上清即为细胞膜和细胞质组分(cell membrane and cytosolic fractions, CM+Cy)。所有离心步骤均在4 ℃下完成。取相同质量的各组分样品进行Western blotting分析。M. smegmatis的内源性GroEL蛋白含有一串组氨酸残基，可以被抗His标签抗体识别[12]，因此将其作为细胞质的指示蛋白。
1.5 生长曲线的绘制将重组M. smegmatis分别培养于丰富培养基(添加0.5%甘油，0.2%葡萄糖和0.05% Tween 80的7H9培养基)或基本培养基(添加0.05% Tween 80的苏通培养基)中，调整起始OD600值至约0.02。在70−80 h时间段内，每间隔6 h或12 h检测其吸光度，最后绘制生长曲线。
1.6 菌落形态的观察将相应重组菌涂布于7H10固体培养基上，37 ℃培养5−6 d。使用数码相机对菌落进行拍照。
1.7 扫描电镜观察细菌形态离心收集对数生长期的重组M. smegmatis，洗涤并重悬于2.5%戊二醛固定液中固定过夜，经浓度递增的乙醇脱水处理。超临界干燥机中干燥1 h后，固定在样品台上喷金，上镜观察拍照。
1.8 透射电镜观察细胞包膜(cell envelope)结构离心收集对数生长期的重组M. smegmatis，洗涤并重悬于2.5%戊二醛固定液中固定10 min，铜网蘸取后，自然状态晾干5 min，1%磷钨酸染色3 min，自然状态晾干15 min，上镜观察拍照。
1.9 重组菌抗压力检测离心收集对数生长期的重组M. smegmatis，洗涤并重悬于7H9培养基中，调整起始OD600值至约0.02。为检测重组菌对不同压力条件的浓度依赖的敏感性，将重组菌用不同浓度的SDS、H2O2和溶菌酶分别处理8、12和24 h，以及用不同梯度酸性条件处理6 h。为检测重组菌对不同压力条件的时间依赖的敏感性，将重组菌分别在0.075% SDS、10 mmol/L H2O2、500 μg/mL溶菌酶及pH 5.0的酸性条件下处理8−24 h。重组菌对抗生素的杀菌曲线的绘制参照文献[13]进行。
1.10 重组菌对抗生素的敏感性评价重组菌对抗生素的敏感性评价指标为最小抑菌浓度(minimal inhibitory concentration, MIC)。微量稀释法检测抗生素的MIC值参考文献[14]进行。将对数生长期(OD600约为0.8)的重组菌调整浓度至5×103 CFU/mL，分别向其中加入不同浓度的抗生素，以2倍倍比稀释，于37 ℃静置培养3 d后，经肉眼观察，孔内无明显细菌生长的浓度即为该抗生素的MIC值。
1.11 细菌细胞壁通透性的检测溴化乙锭(ethidium bromide)和尼罗红(Nile red)常被分别用作检测细胞壁对亲水性和疏水性化合物的通透性的指示分子[15-16]。离心收集对数生长期的重组菌，洗涤并重悬于含0.05% Tween 80和25 mmol/L葡萄糖的磷酸缓冲盐溶液(phosphate-buffered saline, PBS)中。将100 μL细菌菌悬液转入黑色的96孔荧光板中，分别向孔中添加终浓度为2 μg/mL的溴化乙锭或2 μmol/L的尼罗红。立即用多功能酶标仪检测荧光值，检测溴化乙锭时，设置激发波长为545 nm，发射波长为600 nm；检测尼罗红时，设置激发波长为540 nm，发射波长为630 nm。为确定染料的富集是否受到外排泵的影响，在检测溴化乙锭和尼罗红的富集实验中，分别加入终浓度为0.625 μg/mL的外排泵特异性抑制剂CCCP，其他实验条件不变。
1.12 脂肪酸定量分析将重组菌培养至对数生长期，离心收集样本，送至青岛科创质量检测有限公司，进行脂肪酸定量分析。
1.13 统计学分析使用GraphPad Prism 5软件进行统计学分析，多组间差异的比较采用单因素方差分析，进一步的组内两两比较采用Students’ t检验，P < 0.05为差异有统计学意义。误差棒代表标准偏差(standard deviation, SD)。
2 结果与分析 2.1 PE_PGRS15定位于细胞表面并改变重组菌的菌落形态及细胞包膜结构使用PCR方法扩增pe_pgrs15基因(长度为1 821 bp)，获得大小正确的目的基因(图 1A)。MS-PE_PGRS15重组菌表达约50 kDa的目的蛋白，而MS-pMV空载菌则没有条带(图 1B)，说明菌株构建成功。亚细胞分级分离实验检测到PE_PGRS15定位于MS-PE_PGRS15重组菌的细胞壁组分(图 1C)。有研究显示，过表达某些蛋白质可能对宿主造成负担，从而影响宿主菌的生长[17]。为了检测这一可能，将MS-pMV空载菌和MS-PE_PGRS15重组菌分别培养于丰富培养基和基本培养基中，结果显示过表达PE_PGRS15不影响重组菌在培养基中的生长(图 1D、1E)。
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| 图 1 PE_PGRS15的细胞定位及其表达对重组菌菌落形态的影响 Figure 1 PE_PGRS15, a cell wall-associated protein, alters the colony morphology of Mycolicibacterium smegmatis. A: PCR amplification of the pe_pgrs15 gene from Mtb H37Rv genome. B: Western blotting demonstrated the heterologous expression of Myc-tagged PE_PGRS15 protein in recombinant M. smegmatis. C: Proteins from whole cell lysates (WCL), cytoplasmic membrane and cytoplasm (CM+Cy), and cell wall (CW) from M. smegmatis expressing PE_PGRS15 were separated by SDS-PAGE and detected by Western blotting. Cytosolic GroEL was detected as a cytoplasmic control. D: The growth of MS-pMV and MS-PE_PGRS15 at 37 ℃ in Middlebrook 7H9 liquid medium was monitored by determining OD600 at 6 h or 12 h intervals. E: The growth of MS-pMV and MS-PE_PGRS15 at 37 ℃ in Sauton's medium was monitored by determining OD600 at 6 h or 12 h intervals. F: MS-pMV and MS-PE_PGRS15 strains were grown at 37 ℃ on 7H10 agar without Tween 80. Pictures were taken on day 5. MS-pMV and MS-PE_PGRS15 were cultured in 7H9 medium with 0.05% Tween 80, and at an OD600 of 0.6, the bacteria were harvested for scanning electron microscopy (G) and transmission electron microscopy (H). Two representative images for MS-pMV and MS-PE_PGRS15 are shown. Experiments were performed three times, and similar results were obtained. Error bars indicate the standard deviation. |
肉眼观察于7H10固体培养基上生长5 d的MS-pMV空载菌和MS-PE_PGRS15重组菌，发现二者菌落形态差异明显：前者呈现典型的分枝杆菌形态，即干燥、易碎和不规则的褶皱结构；而后者菌落湿润、光滑且粘稠(图 1F)。研究表明分枝杆菌菌落形态的变化往往与菌体表面结构相关[8, 18]。扫描电镜观察发现，二者在菌体长度、表面形态等方面并无明显差异(图 1G)。然而，透射电镜观察发现，与对照组相比，MS-PE_PGRS15重组菌具有更厚的包膜结构(图 1H)。以上结果表明，PE_PGRS15是一个细胞表面蛋白，其表达改变了M. smegmatis的菌落形态及细胞包膜结构。
2.2 MS-PE_PGRS15重组菌对环境压力更加耐受分枝杆菌独特而高度复杂的细胞包膜结构在该属细菌抵抗巨噬细胞内各种杀菌因子如表面活性剂、活性氮/氧中间产物、抗菌肽及酸性条件等的过程中发挥了关键作用[19]。本研究已经证明PE_PGRS15是一个细胞表面相关蛋白，有可能影响重组菌对抗各种环境压力。因此，首先检测MS-pMV空载菌和MS-PE_PGRS15重组菌对SDS、H2O2、溶菌酶及酸性条件的敏感性。浓度依赖性压力测试实验结果显示，和MS-pMV空载菌相比，MS-PE_PGRS15重组菌分别对0.050%和0.075%的SDS (图 2A)、5 mmol/L和10 mmol/L的H2O2 (图 2C)、250 μg/mL和500 μg/mL的溶菌酶(图 2E)及pH 5.0和pH 6.0的酸性条件(图 2G)更加耐受。进一步的时间依赖性压力测试实验结果也佐证了MS-PE_PGRS15重组菌比MS-pMV空载菌更加耐受环境压力(图 2B、2D、2F、2H)。以上结果表明，PE_PGRS15赋予了重组菌更加出色的环境压力耐受能力。
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| 图 2 PE_PGRS15改变重组菌对环境压力的耐受能力 Figure 2 MS-PE_PGRS15 cells are more resistant to stress conditions. The mid-log-phase cultures of MS-pMV and MS-PE_PGRS15 were either treated with medium alone or subjected to SDS for 8 h (A), H2O2 for 12 h (C), and lysosome for 24 h (E) at the indicated concentrations or treated with different pH gradients for 6 h (G). The mid-log-phase cultures of MS-pMV and MS-PE_PGRS15 were incubated in 7H9 supplemented with 0.075% (W/V) SDS (B), 10 mmol/L H2O2 (D), 500 μg/mL lysosome (F) and low pH (pH 5.0) (H) for the indicated time. Then, the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution, and the bacterial CFUs were counted after 3−4 d of cultivation. Data are x ± s of technical triplicate from one representative of three or more independent experiments.*: P < 0.05; **: P < 0.01. |
2.3 MS-PE_PGRS15重组菌对抗生素更加耐受
分枝杆菌致密的细胞包膜结构(包括细胞膜、细胞壁和荚膜)是一道天然屏障，能够极大地抑制抗生素等药物向细菌胞内的运输[20-21]。为进一步研究PE_PGRS15的表达是否影响重组菌对抗生素的抗性，使用3种亲水性抗生素(异烟肼、万古霉素和庆大霉素)和3种疏水性抗生素(红霉素、诺氟沙星和利福平)处理重组菌和空载菌，并测定每种抗生素对重组菌的MIC值。结果显示(表 2)，在6种受试抗生素中，MS-PE_PGRS15重组菌的耐药性都优于MS-pMV。为进一步验证以上结果，分别绘制以上6种抗生素对细菌的杀菌曲线。结果显示(图 3A−3F)，MS-PE_PGRS15重组菌的抗生素耐药性均强于MS-pMV空载菌。
表 2. 不同抗生素对MS-PE_PGRS15重组菌和MS-pMV空载菌的MIC值
Table 2. MICs of antibiotics for the MS-pMV and MS-PE_PGRS15 strains
| Strains | MIC (μg/mL) | |||||
| INH | VAN | GEN | ERY | NOR | RIP | |
| MS-pMV | 8 | 2 | 2 | 8 | 0.5 | 4 |
| MS-15PE | 8 | 2 | 2 | 4−8 | 0.5 | 4 |
| MS-15PGRS | 4−8 | 2 | 2 | 8 | 0.5 | 2−4 |
| MS-PE_PGRS15 | 32 | 4−8 | 8 | 16 | 2.0 | 8 |
| MS-33PE | 8 | 2 | 2 | 8 | 0.5 | 4 |
| MS-33PE_15PGRS | 32−64 | 4 | 8 | 16 | 2.0−4.0 | 8 |
| INH: Isoniazid; VAN: Vancomycin; GEN: Gentamicin; ERY: Erythromycin; NOR: Norfloxacin; RIF: Rifampicin. | ||||||
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| 图 3 PE_PGRS15影响重组菌对抗生素的耐受能力 Figure 3 Expression of PE_PGRS15 in Mycolicibacterium smegmatis enhanced bacterial survival following exposure to various antibiotics. The control strain (MS-pMV) and recombinant strain (MS-PE_PGRS15) were diluted in 7H9 broth and then treated with isoniazid for 6 h (A), vancomycin for 10 h (B), gentamicin for 6 h (C), erythromycin for 22 h (D), norfloxacin for 6 h (E), and rifampicin for 6 h (F) at the indicated concentrations. Then, a ten-fold dilution of the bacteria was spotted on 7H10 supplemented with kanamycin, and the MS-pMV and MS-PE_PGRS15 bacteria were counted after 3−4 d of cultivation. Data are x ± s of technical triplicate from one representative of three or more independent experiments. *: P < 0.05; **: P < 0.01. |
2.4 PE_PGRS15的表达影响重组菌细胞壁通透性及脂肪酸组成
鉴于MS-PE_PGRS15重组菌对所有已测环境压力和抗生素都耐受，推测其潜在的耐受机制具有普遍性。本研究采用染料富集法检测以上结果是否与细菌细胞壁通透性有关。结果显示(图 4A、4B)，MS-PE_PGRS15重组菌对亲水性化合物和疏水性化合物的富集作用都明显弱于MS-pMV空载菌；而外排泵特异性抑制剂CCCP并没有改变染料在2株菌中的富集趋势(图 4C、4D)。这说明PE_PGRS15通过降低重组菌细胞壁通透性，而不是增加药物外排作用，来增加重组菌对环境压力的耐受和抗生素的抗性。
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| 图 4 PE_PGRS15的表达影响重组菌细胞壁通透性及脂肪酸含量 Figure 4 Modulation of cell wall permeability and fatty acid composition of MS-PE_PGRS15 cells. A: Mid-log-phase cultures of MS-pMV and MS-PE_PGRS15 were incubated in PBS with 25 mmol/L glucose and 2 μg/mL ethidium bromide for the indicated time. B: Mid-log-phase cultures of MS-pMV and MS-PE_PGRS15 were incubated in PBS containing 25 mmol/L glucose and 2 μmol/L Nile red stain. C: Mid-log-phase cultures of MS-pMV and MS-PE_PGRS15 were incubated in PBS with 25 mmol/L glucose and 2 μg/mL ethidium bromide and co-incubated with 0.625 μg/mL CCCP. D: Mid-log-phase cultures of MS-pMV and MS-PE_PGRS15 were incubated in PBS containing 25 mmol/L glucose and 2 μmol/L Nile red stain and co-incubated with 0.625 μg/mL CCCP. The assay of accumulation of ethidium bromide and Nile red over time in Mycolicibacterium smegmatis was conducted at 37 ℃. E: Fatty acid composition (%) of MS-pMV and MS-PE_PGRS15 estimated by GC/MS analysis. F: Quantification of total FAMEs extracted from the MS-pMV and MS-PE_PGRS15. G: The increased amount (%) of FAMEs in the recombinant MS-PE_PGRS15 strain compared with the MS-pMV control strain. Data are x ± s of technical triplicate from one representative of three or more independent experiments. *: P < 0.05; **: P < 0.01; ***: P < 0.001. |
分枝杆菌包膜结构中的细胞壁是由结构复杂的脂质如分枝菌酸等组成[14]。分枝菌酸是一种高度饱和的长链脂肪酸，它的存在导致分枝杆菌细胞壁极低的流动性和不透性，最终使得分枝杆菌具有卓越的环境压力耐受性和抗生素耐药性[22]。为进一步探索MS-PE_PGRS15重组菌的耐受机制，本研究利用气相色谱-质谱联用仪比较了2株菌的脂肪酸种类和含量。结果显示(图 4E)，MS-PE_PGRS15重组菌和MS-pMV空载菌中脂肪酸种类并未发生变化，都拥有32种脂肪酸(从C8−C24)。然而，MS-PE_PGRS15重组菌中总脂肪酸含量明显高于MS-pMV空载菌(图 4F)。具体表现为MS-PE_PGRS15重组菌中C11:0 (十一烷酸)、C15:0 (十五烷酸)、C18:2n6c (亚油酸)、C20:2 (附子脂酸)和C24:0 (木腊酸)等脂肪酸的含量明显高于MS-pMV空载菌(图 4G)。以上结果表明，PE_PGRS15可能通过增加细菌细胞壁脂肪酸含量，导致细胞壁通透性进一步降低，减少药物进入细胞内，最终导致重组菌耐药性增加。
2.5 PE结构域负责PE_PGRS15的细胞定位，而PGRS结构域负责其耐药机制Mtb的PE_PGRS亚家族蛋白拥有类似的分子结构，即由PE结构域和PGRS结构域组成[3]。为研究PE_PGRS15的结构对于功能的影响，本研究分别单独表达其PE结构域和PGRS结构域(图 5)。和全长蛋白一样，15PE和15PGRS的表达也未影响重组菌的生长(图 6B、6C)。令人意外的是，15PE和15PGRS都不能重现PE_PGRS15在M. smegmatis中的耐药表型(表 2，图 6D−6H)。通过蛋白定位实验发现，全长PE_PGRS15和15PE可以定位于细胞壁组分，但15PGRS却不能(图 1C、图 6A)。根据以上结果推测，PE_PGRS15的PE结构域负责其亚细胞定位；而PGRS结构域负责耐药等功能，前提是该结构域能被转运到细菌细胞表面。
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| 图 5 本研究中构建的质粒及重组菌图示 Figure 5 Schematic representation of the recombinant constructs used in this study. aa: Amino acids. |
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| 图 6 PE结构域影响PE_PGRS15的细胞定位，而PGRS结构域影响其耐药机制 Figure 6 PGRS domain of the PE_PGRS15 protein affects mycobacterial cell wall permeability and stress resistance. A: Cell fractionation experiments were performed to determine the sub-cellular localization of the indicated proteins. WCL represents whole cell lysates, CM+Cy represents cytoplasmic membrane and cytoplasm, and CW represents cell wall. Cytosolic GroEL was detected as a cytoplasmic control. B: The growth of the indicated strains at 37 ℃ in Middlebrook 7H9 liquid medium was monitored by determining OD600 at 6 h or 12 h intervals. C: The growth of the indicated strains at 37 ℃ in Sauton's medium was monitored by determining OD600 at 6 h or 12 h intervals. D: Mid-log-phase cultures of the indicated strains were incubated in PBS with 25 mmol/L glucose and 2 μg/mL ethidium bromide for the indicated time. The assay of accumulation of ethidium bromide over time in M. smegmatis was conducted at 37 ℃. The mid-log-phase cultures of the indicated strains were either treated with medium alone or subjected to SDS for 8 h (E), H2O2 for 12 h (F), and lysosome for 24 h (G) at the indicated concentrations or treated with different pH gradients for 6 h (H). Then, the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution, and the bacterial CFUs were counted after 3−4 d of cultivation. Data are x ± s of technical triplicate from one representative of three or more independent experiments with similar results. Significant differences (*: P < 0.05; **: P < 0.01) are compared with MS-pMV (two-tailed Student's t-test). |
为了验证15PGRS需转运到细胞表面才能行使其功能这一猜想，本研究构建了MS-33PE-15PGRS重组菌：能表达PE_PGRS33的PE结构域(33PE)和15PGRS的融合蛋白(图 5)。大量研究表明PE_PGRS33定位于分枝杆菌细胞壁，33PE负责该蛋白的定位，而且也能帮助其他融合蛋白定位于细菌细胞表面[8, 23-24]。因此，本研究构建33PE与15PGRS的融合蛋白，以帮助15PGRS定位于细胞表面。与预期一致，在重组菌的细胞壁组分中检测到了33PE-15PGRS (图 6A)。结果显示(表 2，图 6D−6H)，33PE-15PGRS的表达能够重现重组菌对环境压力和抗生素的耐受性以及细胞壁通透性的改变。以上结果证明，PE结构域负责PE_PGRS15的细胞定位，而PGRS结构域负责其耐药机制等生理功能。
3 讨论与结论PE_PGRS亚家族蛋白在Mtb生理和毒力方面具有重要作用，然而单个成员的具体功能仍然有待研究[2, 4-5]。组学及感染实验发现PE_PGRS15在Mtb感染巨噬细胞和动物模型时，表达上调[25-27]。而pe_pgrs15基因缺失的Mtb在感染非人灵长类动物的肺部时，毒力明显变弱[28]。在体外模拟Mtb感染的环境压力条件下，也会诱导pe_pgrs15基因的上调表达。比如，芯片分析缺氧条件[29]、酸性条件[30]及SDS[31]处理的Mtb的转录组时，都发现pe_pgrs15基因的上调表达。以上数据均暗示PE_PGRS15在协助Mtb适应胞内感染或休眠环境时的重要作用。
Mtb基因组编码65个pe_pgrs基因，其中至少有51基因能够表达有功能的蛋白质[3]。然而在Mtb中研究单个PE_PGRS蛋白充满挑战，主要原因在于该家族蛋白序列高度同源且功能冗余[8, 32]。因此，本研究在Mtb的经典替代模式菌——M. smegmatis中过表达PE_PGRS15来探索其生物学功能。由于M. smegmatis中不含有PE_PGRS的同源蛋白，因此结果不会受到过多干扰；且本研究同时构建了多种截短和融合蛋白，最大限度排除蛋白质过表达可能造成的假象，从而使本研究更加严谨。当然，PE_PGRS15在Mtb的生理和毒力中具体扮演何种角色，还需在Mtb中进一步验证。然而，如何避开该亚家族其他成员的干扰，是这类研究首要考虑的问题。
本研究证明PE_PGRS15蛋白定位于细胞壁，并显著改变了重组菌的菌落形态。而菌落形态的变化通常与细菌毒力和抗生素敏感性密切相关[18]。环境压力及抗生素敏感实验证明，过表达PE_PGRS15可以提高M. smegmatis的耐受性。重组菌能够耐受众多环境压力和抗生素，暗示其耐受机制应该是广谱的，而不是针对某个特定的环境压力或抗生素。染料富集实验显示，MS-PE_PGRS15重组菌对疏水性和亲水性染料的富集作用都比对照菌弱。外排泵抑制剂CCCP的加入，不会改变2株菌对染料的富集趋势，这说明PE_PGRS15是通过降低细胞壁的通透性，而不是提高药物外排作用来增加耐受性。
分枝杆菌具有独特而复杂的细胞包膜(cell envelope)结构，由内向外依次为细胞质膜、细胞壁和荚膜[21]。其中，细胞壁和荚膜富含致密且疏水的脂质层，厚度达50 nm，可占细菌宽度的十分之一[19]。这造成整个包膜结构错综复杂，流动性和通透性极差，是分枝杆菌抵御环境压力的天然屏障[19]。大量研究表明分枝杆菌菌落形态改变和耐药性增加与细胞壁脂质成分的改变有密切联系[18, 33]。因此，PE_PGRS15可能影响了重组菌细胞壁脂质的合成。脂肪酸定量分析结果显示，PE_PGRS15的表达的确增加了重组菌细胞壁的脂质总含量，具体表现在十一烷酸、十五烷酸、亚油酸、附子脂酸和木腊酸含量的增加。因此，PE_PGRS15可能通过影响脂肪酸代谢，加速分枝菌酸等脂质的合成，造成重组菌包膜进一步增厚；增厚的细菌包膜更加致密，通透性更差，从而增加重组菌对环境压力和抗生素的抵抗。
PE_PGRS亚家族蛋白拥有类似的分子结构：由1个PE结构域和1个PGRS结构域组成[3]。大量研究表明，PE结构域可能作为信号肽，负责PE_PGRS蛋白定位于细胞表面[8, 24]。亚细胞分级分离实验证实PE_PGRS15的PE结构域也负责该蛋白的转运，这进一步证明了该亚家族蛋白PE结构域的普遍功能。单独在M. smegmatis中过表达PE_PGRS15的PGRS结构域无法重现全长蛋白的表型，且该结构域也不能转运至细胞表面。而与PE_PGRS33的PE结构域融合表达，既能实现15PGRS的细胞定位，又能重现PE_PGRS15全长蛋白的表型。这些结果表明，PE_PGRS15的PE结构域作为信号肽负责蛋白质的定位，而PGRS结构域负责其具体的生物学功能。
总而言之，本研究利用M. smegmatis替代Mtb对PE_PGRS15的功能进行了细致研究。通过亚细胞定位分析、菌落形态观察、抗性检测和脂肪酸定量分析等，证实了定位于细胞壁上的PE_PGRS15可能通过影响脂质代谢，造成细菌菌落形态、细胞壁通透性的改变，从而影响细菌对环境压力及抗生素的敏感性。本研究深化了PE_PGRS亚家族参与分枝杆菌耐药性的理解，为研发新型抗痨药物及相关疫苗提供了新思路。
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