2023, Vol. 63中国科学院微生物研究所，中国微生物学会
文章信息
- 楼洁, 胡晓, 梁延群, 朱怡铃, 音建华. 2023
- LOU Jie, HU Xiao, LIANG Yanqun, ZHU Yiling, YIN Jianhua.
- 肽聚糖的生物合成及其调控机制研究进展
- Peptidoglycan biosynthesis and the regulatory mechanism
- 微生物学报, 63(1): 106-123
- Acta Microbiologica Sinica, 63(1): 106-123
-
文章历史
- 收稿日期：2022-05-09
- 网络出版日期：2022-07-14
引用本文 |
肽聚糖(peptidoglycan)是细菌细胞壁的重要组成部分，对于维持细胞形态、大小、渗透压稳定以及细胞存活是必需的[1-2]。同时，肽聚糖是绝大多数细菌特有的结构，很多重要的抗生素都能特异性靶向肽聚糖的生物合成过程，如β-内酰胺类和糖肽类抗生素等[3-4]。肽聚糖由N-乙酰胞壁酸(N-acetylmuramic acid, MurNAc)和N-乙酰葡糖胺(N-acetylglucosamine, GlcNAc)通过β-1, 4糖苷键交替相连的线性糖链组成，其中MurNAc上连接一个短肽，相邻短肽之间可形成交联(cross-link)，使肽聚糖最终成为网状结构。革兰氏阴性菌(如大肠杆菌Escherichia coli)和革兰氏阳性菌(如枯草芽孢杆菌Bacillus subtilis)中短肽的组成略有差异，其中前者为l-Ala-d-Glu-meso- Dap-d-Ala-d-Ala (meso-Dap为内消旋-二氨基庚二酸)，而后者中meso-Dap被l-Lys代替。
细菌细胞在生长和分裂过程中，包裹在细胞质膜外的肽聚糖层并非一成不变，而是同样需要不断地扩展和生长[1-2]。肽聚糖单体在细胞质中合成后翻转至细胞质膜外侧，然后在肽聚糖合酶(peptidoglycan synthase)的作用下聚合和交联至新生的肽聚糖链。该过程还需要肽聚糖水解酶(peptidoglycan hydrolase)的参与，它们将已有的肽聚糖链切开，从而为肽聚糖单体的插入提供空间。此外，将近一半的肽聚糖水解产物还可以经过循环(recycling)过程，重新参与肽聚糖的合成[5]。在整个肽聚糖生物合成过程中参与的酶多达几十种，它们在细胞内受到严谨的时空调控，从而保证肽聚糖生长的同时维持其完整性[1, 6]。
正是因为肽聚糖的重要性，其生物合成及调控机制一直是微生物学中重要的基础研究和前沿研究方向。早期的研究明确了细胞质中肽聚糖单体的生物合成过程，但其如何翻转至细胞质膜外侧以及如何组装至已有的肽聚糖层并不清楚。近些年，国内外研究团队在这些问题上取得了突破性研究进展，包括鉴定出转运肽聚糖单体的翻转酶、发现更为保守的肽聚糖合酶以及揭示了肽聚糖合酶的调控机制等，这些研究结果极大地丰富甚至颠覆了我们原先对于肽聚糖生物合成的认知。基于此，本文总结了肽聚糖生物合成及其调控机制的最新研究进展，尤其是重点阐述两类肽聚糖合酶及其在不同层次上的调控机制，以期为深入开展肽聚糖生物学及其生物工程应用研究提供指导。
1 肽聚糖的生物合成过程肽聚糖的生物合成过程涉及到多步反应和多种酶，根据其发生反应的位置以及所需酶的分布可以分为3个阶段(图 1，表 1)[1-3, 6]。
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| 图 1 肽聚糖的生物合成过程 Figure 1 Pathway for peptidoglycan biosynthesis. OM: Outer membrane; PG: Peptidoglycan; CM: Cytoplasmic membrane; MurA: UDP-GlcNAc enolpyruvyl transferase; MurB: UDP-MurNAc dehydrogenase; MurC: UDP-MurNAc-l-Ala ligase; MurD: UDP-MurNAc-l-Ala-d-Glu ligase; MurE: UDP-MurNAc-l- Ala-d-Glu-meso-Dap ligase; Alr and DadX: Ala racemase; DdlA: d-Ala-d-Ala ligase A; MurF: UDP-MurNAc-tripeptide-d-alanyl-d-Ala ligase; MraY: UDP-MurNAc-pentapeptide phosphotransferase; MurG: UDP-GlcNAc undecaprenoyl-pyrophosphoryl-MurNAc-pentapeptide transferase; MurJ: Lipid Ⅱ fippase; GTase: Glycosyltransferase; TPase: Transpeptidase; meso-Dap: meso-diaminopimelic acid. |
| Protein | Activity or catagory | Function |
| Peptidoglycan synthesis | ||
| MurA, MurB | Transferase and dehydrogenase, respectively | Synthesis of UDP-MurNAc from UDP-GlcNAc |
| MurC, MurD, MurE, MurF, DdlA | Amino acid ligases | Synthesis of UDP-MurNAc-pentapeptide |
| MurI, Alr, DadX | Racemases | Synthesis of d-Glu or d-Ala from l-Glu or l-Ala, respectively |
| MraY, MurG | GTases | Synthesis of Lipid Ⅰ and Lipid Ⅱ, respectively |
| MurJ | Lipid Ⅱ flippase | Translocation of Lipid Ⅱ across the cytoplasmic membrane |
| PBP1A, PBP1B, PBP1C | Bifunctional GTase-TPases (aPBPs) | PBP1A and PBP1B are major peptidoglycan synthases. The role of PBP1C remains unknown |
| PBP2, PBP3 | TPases (bPBPs) | Interact with SEDS proteins and form peptide cross-links |
| FtsW, RodA | GTases (SEDS family proteins) | Interact with bPBPs and polymerize the glycan chains |
| Peptidoglycan hydrolysis | ||
| AmiA, AmiB, AmiC, AmiD | N-acetylmuramoyl-l-Ala amidases | Hydrolyze the amide bond between l-Ala and MurNAc in the periplasm |
| Slt, MltA, MltB, MltC, MltD, MltE, MltF, MltG | Lytic transglycosylases | Cleave the β-1, 4-glycosidic bond between MurNAc and GlcNAc in the periplasm |
| PBP4, PBP4b, PBP5, PBP6a, PBP6b, PBP7, AmpH | Carboxypeptidases | Cleave the terminal d-Ala-d-Ala bond of pentapeptide |
| MepA, MepM, MepS, MepH, MepK, PBP4, PBP7 | Endopeptidases | Hydrolyze cross-links between existing glycan strands |
| Peptidoglycan recycling | ||
| AmpG | GlcNAc-anhMurNAc permease | Transports GlcNAc-anhMurNAc into cytoplasm |
| AmpD | anhMurNAc-l-Ala amidase | Cleaves the anhMurNAc-l-Ala bond in the cytoplasm |
| NagZ | β-N-acetylglucosaminidase | Cleaves the β-1, 4-glycosidic bond between MurNAc and GlcNAc in the cytoplasm |
| LdcA | ld-carboxypeptidase | Cleaves d-Ala from tetrapeptides |
| Mpl | UDP-MurNAc: l-Ala-d-Glu-meso-Dap ligase | Adds l-Ala-d-Glu-meso-Dap to UDP-MurNAc |
1.1 肽聚糖前体UDP-MurNAc-pentapeptide的合成
第一个阶段发生在细胞质(cytoplasm)中。被尿苷二磷酸(uridine diphosphate, UDP)活化的前体UDP-GlcNAc在MurA和MurB的催化下生成UDP-MurNAc，随后l-Ala、d-Glu和meso-Dap分别在特异性氨基酸连接酶MurC、MurD和MurE的催化下逐个转移到UDP-MurNAc上形成UDP-MurNAc-l-Ala-d-Glu-meso-Dap。l-Ala在丙氨酸消旋酶Alr和DadX的催化下形成d-Ala，两个d-Ala经d-Ala-d-Ala连接酶DdlA的作用下合成d-Ala-d-Ala，然后在MurF的催化下连接至UDP-MurNAc-l-Ala-d-Glu-meso- Dap，形成肽聚糖前体UDP-MurNAc-pentapeptide。
1.2 肽聚糖前体的跨膜翻转第二个阶段发生在细胞质膜(cytoplasmic membrane)上，在细胞质中合成的肽聚糖前体需要跨过细胞质膜到膜外侧才能进行下一步反应。首先，肽聚糖前体UDP-MurNAc-pentapeptide在磷酸转移酶MraY的作用下与细胞质膜内侧的十一烷基焦磷酸脂质载体(undecaprenyl pyrophosphate)相连，形成Lipid Ⅰ；然后，转移酶MurG将UDP-GluNAc连接至Lipid Ⅰ，形成与细胞质膜锚定的肽聚糖单体GlcNAc- MurNAc-pentapeptide，即Lipid Ⅱ；最后，在翻转酶的作用下Lipid Ⅱ跨膜翻转至细胞质膜外侧。
Lipid Ⅱ的翻转由何种酶执行长期以来备受争议[7]，最初发现SEDS (shape elongation division and sporulation)家族蛋白成员FtsW在体外实验中具有翻转酶活性，故认为该家族蛋白(包括FtsW、RodA和SpoVE)是Lipid Ⅱ翻转酶[8-9]。2014年，SHAM等建立了在体内测定Lipid Ⅱ翻转酶活性的实验方法，发现SEDS家族蛋白并没有翻转酶活性，而MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide)转运蛋白超家族成员MurJ才是真正的Lipid Ⅱ翻转酶[10]。MurJ由14个跨膜区组成，在质子动力势的驱动下，通过构象的内外开合来实现Lipid Ⅱ的跨膜翻转[11-13]。枯草芽孢杆菌中除MurJ外，Amj蛋白也具有翻转酶活性，MurJ和Amj之间缺少序列和结构同源性，但二者在功能上可以相互补偿[14]。
1.3 肽聚糖链的聚合和交联第三阶段发生在周质空间(periplasm)中。翻转至细胞质膜外侧的Lipid Ⅱ在转糖基酶(glycosyltransferase, GTase)的作用下聚合至新生的肽聚糖链；与此同时，作为类脂载体的十一烷基焦磷酸得以释放，并被转运至细胞质中，重新参与Lipid Ⅱ的翻转。随后，转肽酶(transpeptidase, TPase)催化相邻的肽聚糖链短肽之间形成交联，最终形成网状的肽聚糖。周质空间中肽聚糖的聚合和交联主要由青霉素结合蛋白(penicillin-binding protein, PBP)介导[15]。其中，A类PBPs (aPBPs)为双功能酶，同时具有转糖基酶和转肽酶活性，长期以来被认为是主要的肽聚糖合酶；而B类PBPs (bPBPs)为单功能酶，仅具有转肽酶活性，最新的研究表明bPBPs与具有转糖基酶活性的SEDS家族蛋白在肽聚糖聚合和交联过程中起主导作用(详见下文)[16-19]。
2 肽聚糖的循环再利用肽聚糖不仅能以UDP-GlcNAc和3种氨基酸为原材料进行从头合成，还能经肽聚糖循环过程重新再利用。肽聚糖循环是指肽聚糖经水解酶作用后产生的肽聚糖水解片段，进入细胞质中重新参与肽聚糖合成的过程[5, 20]。肽聚糖水解酶主要包括肽聚糖酰胺酶(N-acetylmuramoyl-l-Ala amidase)、裂解性转糖基酶(lytic transglycosylase, LT)、羧肽酶(carboxypeptidase)和内肽酶(endopeptidase)四大类(表 1)[5, 21-23]。其中，肽聚糖酰胺酶水解MurNAc和短肽之间的酰胺键；LTs水解MurNAc和GlcNAc之间的β-1, 4糖苷键，产生GlcNAc-1, 6-anhMurNAc-peptides；羧肽酶和内肽酶分别切割短肽末端以及相邻短肽之间的酰胺键，负责肽聚糖链的修饰及交联程度。这些肽聚糖水解酶对于肽聚糖生物合成也很关键，它们将已有的肽聚糖糖链切开，然后插入新合成的肽聚糖。通常情况下，肽聚糖水解酶并不是细菌生长所必需的，并且细菌中每一类肽聚糖水解酶的数目都有多个，这种冗余的特性限制了肽聚糖水解酶功能的研究，但最新的研究表明这些最初被认为功能冗余的肽聚糖水解酶可能在不同的环境中发挥作用[22, 24]。
在正常的生长过程中，上述肽聚糖水解酶作用后产生的肽聚糖水解产物以GlcNAc-1, 6-anhMurNAc-tetrapeptide为主，经位于细胞质膜中的通透酶(permease) AmpG进入细胞质，在肽聚糖酰胺酶AmpD和β-乙酰氨基葡糖糖苷酶NagZ的共同作用下生成GlcNAc、anhMurNAc和游离四肽l-Ala-d-Glu-meso-Dap-d-Ala。ld-羧肽酶LdcA可以将游离四肽末端的d-Ala切掉，生成的游离三肽l-Ala-d-Glu-meso-Dap经连接酶Mpl的作用连接至UDP-MurNAc，形成UDP-MurNAc-l-Ala-d-Glu-meso-Dap，从而进入肽聚糖生物合成过程(图 2，表 1)[5, 20]。GlcNAc和anhMurNAc则在一系列酶促反应后生成UDP-GlcNAc重新参与肽聚糖或脂多糖的合成。此外，游离三肽l-Ala-d-Glu-meso-Dap还可以经酰胺酶等作用后成为单个氨基酸进行回收利用。
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| 图 2 肽聚糖循环过程 Figure 2 Pathway for peptidoglycan recycling. OM: Outer membrane; PG: Peptidoglycan; CM: Cytoplasmic membrane; AmpG: GlcNAc-anhMurNAc permease; AmpD: AnhMurNAc-l-Ala amidase; LdcA: ld-carboxypeptidase; NagZ: β-N-acetylglucosaminidase; Mpl: UDP-MurNAc: l-Ala-d-Glu-meso-Dap ligase; MurF: UDP-MurNAc-tripeptide-d-alanyl-d-Ala ligase; MraY: UDP-MurNAc-pentapeptide phosphotransferase; MurG: UDP-GlcNAc undecaprenoyl-pyrophosphoryl-MurNAc-pentapeptide transferase; meso-DAP: meso-diaminopimelic acid. |
尽管在实验室条件中肽聚糖循环并不是细菌生长所必需的，但是当细菌面临碳源饥饿等不利环境时，肽聚糖循环过程产生的中间产物可以快速合成肽聚糖，确保细菌能够正常完成一轮分裂从而帮助细菌度过不利环境[5]。近年来的研究发现，革兰氏阳性菌中肽聚糖循环对于细菌的长期存活至关重要[25]。有趣的是，越来越多的研究表明肽聚糖循环中间产物还能作为信号分子参与多种生理过程的调控，如芽孢萌发、形态转变、细菌与宿主之间相互作用以及诱导抗生素耐药性等[26-28]。我们在革兰氏阴性菌奥奈达希瓦氏菌(Shewanella oneidensis)中的研究也表明，肽聚糖水解产物能够作为信号分子诱导β-内酰胺酶基因的表达从而对β-内酰胺类产生耐药性[29-31]。
3 肽聚糖合酶在细胞周质空间中，肽聚糖的生物合成主要由两类肽聚糖合酶负责：一是同时具有转糖基酶活性和转肽酶活性的aPBPs；二是由具有转糖基酶活性的SEDS家族蛋白和具有转肽酶活性的bPBPs组成的复合体。
3.1 aPBPsaPBPs广泛分布于各种革兰氏阴性菌和革兰氏阳性菌中[32]，在SEDS家族蛋白发现之前被认为是细菌中起主导作用的肽聚糖合酶。不同细菌中aPBPs的数量不同，如大肠杆菌中含有3个aPBPs (PBP1A、PBP1B和PBP1C)[22]，而枯草芽孢杆菌中含有4个aPBPs (PBP1、PBP4、PBP2c和PBP2d)[33]。大肠杆菌中，PBP1A和PBP1B单独缺失不影响细菌的正常生存，但两个同时缺失则会引起细菌裂解，表明这两个aPBPs在参与肽聚糖合成过程中可以相互替代[34]；而PBP1C在肽聚糖合成中的作用目前尚不清晰，且过表达PBP1C不能补偿PBP1A和PBP1B同时缺失引起的致死表型[35]。
尽管PBP1A和PBP1B在很大程度上功能冗余，但缺少单个aPBP的菌株表型并不完全相同。大肠杆菌中PBP1B缺失的菌株对β-内酰胺类抗生素更加敏感[34]、在稳定期的存活能力下降[36]以及生物膜形成受损[37]；另外，经溶菌酶处理后的细菌原生质球(spheroplasts)重新合成肽聚糖依赖于PBP1B，而非PBP1A[38]，表明大肠杆菌中PBP1B在肽聚糖合成中的作用比PBP1A大。与此相反的是，霍乱弧菌(Vibrio cholerae)中PBP1A的作用更大，其缺失后在基本培养基中的生长迟缓并对胆汁酸和β-内酰胺类抗生素更加敏感[39]，在稳定期的细胞形态也由杆状转变为球形[40]。近期的研究表明，大肠杆菌中PBP1A和PBP1B分别在碱性和酸性环境中发挥作用，二者的共同存在确保细菌在多变的环境中维持正常生长以及完整的细胞壁[41]。
我们对希瓦氏菌的研究发现，该菌中也含有3个aPBPs (PBP1A、PBP1B和PBP1C)，其中PBP1A和PBP1B不可同时缺失[42]。与大肠杆菌不同的是，希瓦氏菌中PBP1A在肽聚糖合成中的作用比PBP1B大，PBP1A缺失后细菌表现出诸多生理缺陷，如细胞形态异常(包括球形和分枝状等)、对低渗环境敏感、外膜通透性提高以及对β-内酰胺类抗生素耐药性增强等；而PBP1B缺失后不会引起明显的表型缺陷[42-43]。进一步的研究发现，PBP1B和大肠杆菌PBP1A的适度表达可以补偿由PBP1A缺失引起的生理功能缺陷[44]。此外，PBP1A中的转糖基酶和转肽酶活性对其发挥正常的生理功能必不可少，在野生型菌株中过表达任一酶活突变的PBP1A均能引起细胞形态的改变[44]。由此可见，细胞中aPBPs表达量以及酶活性的变化都能显著影响肽聚糖正常的生理功能。
3.2 SEDS-bPBPs在枯草芽孢杆菌中，缺失所有已知aPBPs的菌株虽生长变慢但仍能够存活并具有正常的肽聚糖以及杆状形态，表明该菌中还存在其他未知的具有转糖基酶活性的蛋白负责肽聚糖糖链的聚合。2016年，多个团队联合鉴定出这种未知的蛋白，即前文所述的SEDS家族蛋白成员RodA，RodA的过表达能够恢复由aPBPs缺失引起的表型缺陷[17, 45]。随后的研究发现，最初被认为能够翻转Lipid Ⅱ的SEDS家族蛋白另一个成员FtsW也具有转糖基酶活性[18]。在大肠杆菌等革兰氏阴性菌中，SEDS家族蛋白同样具有转糖基酶活性[16]。SEDS家族蛋白需要与具有转肽酶活性的bPBPs共同作用才能实现肽聚糖的合成，其中RodA与PBP2形成复合体，而FtsW与PBP3形成复合体，分别在细胞延伸(elongation)和分裂(division)过程中合成肽聚糖[16-19]。
与aPBPs相比，SEDS家族成员和bPBPs在细菌中的保守性更高[17, 46]。尤为特别的是，衣原体门(Chlamydia)和浮霉菌门(Planctomycetes)等细菌缺少aPBPs，但它们仍然能够合成肽聚糖[47-49]。这些细菌中都含有至少一个SEDS家族蛋白和一个bPBP蛋白，暗示它们的肽聚糖由SEDS-bPBPs合成[17, 49]。最新的研究表明，SEDS-bPBPs在肽聚糖装配过程中起核心作用，而aPBPs主要负责肽聚糖的修复和成熟[50]。尽管如此，两类肽聚糖合酶各自的功能以及相互之间的关系还有待进一步探究，目前已有多篇综述对此问题进行深入阐述[6, 51-53]。
4 肽聚糖生物合成的调控机制在细菌的生长和分裂过程中，肽聚糖水解酶不断将已有的肽聚糖链切开，然后插入新合成的肽聚糖。因此，肽聚糖合成和水解相关的酶必然受到严谨的时空调控，从而保证肽聚糖生长的同时维持其完整性[1]。目前关于肽聚糖生物合成的调控研究主要集中在两个层面：一是在翻译后水平通过蛋白相互作用对酶活性进行调控；二是在转录水平对基因的表达进行调控(表 2)。
4.1 翻译后水平的调控肽聚糖生物合成相关的酶以多酶复合体的形式发挥作用，蛋白质之间的相互作用可以在翻译后水平快速调节肽聚糖合酶的活性[1-2, 83]。复合体中既包括锚定在外膜的脂蛋白，也包括定位在细胞内的原核生物细胞骨架蛋白，分别实现对肽聚糖生物合成“由外向内”和“由内向外”的调控。
4.1.1 外膜脂蛋白对aPBPs的调控通过对大肠杆菌的研究发现，aPBPs的酶活性严格受外膜脂蛋白调控。两个外膜脂蛋白LpoA和LpoB分别作为辅因子与PBP1A和PBP1B形成复合体，进而促进aPBPs的转糖基酶和转肽酶活性[32, 34, 55]。外膜脂蛋白失活后与其对应的aPBP活性丧失，因此2个Lpo蛋白或与其非对应的aPBP蛋白同时缺失也能导致细胞裂解[32, 34]。两个复合体在细胞中的定位具有偏好性，其中PBP1A/LpoA主要定位于侧壁(lateral wall)，而PBP1B/LpoB主要定位于隔膜(septum)[32, 34]。除大肠杆菌外，现已发现霍乱弧菌[39]和希瓦氏菌[42]等其他革兰氏阴性菌中aPBPs的功能也需要外膜脂蛋白参与。铜绿假单胞菌(Pseudomonas aeruginosa)中虽不含有LpoB同源蛋白，但含有与LpoB同功能的外膜脂蛋白LpoP[61, 84]。
外膜脂蛋白锚定在细菌外膜并跨越周质空间与aPBPs中的非催化结构域(PBP1A的ODD结构域和PBP1B的UB2H结构域)相互作用[85-86]。体外生化实验结果表明，两个外膜脂蛋白对aPBPs的调控机制不同，其中LpoA直接促进转肽酶活性而LpoB直接促进转糖基酶活性[55]；然而，最新的体内研究发现LpoA对PBP1A的转糖基酶和转肽酶活性都有增强作用[87]。由此可见，细胞内两个外膜脂蛋白通过相似的方式调控aPBPs的酶活性。
4.1.2 多酶复合体中肽聚糖合成的调控在大肠杆菌等杆状细菌中，单个细胞的生长包含细胞延伸和分裂两个过程，因此肽聚糖的合成包括细胞延伸时沿侧壁的合成和分裂时隔膜处的合成。大量的研究表明，肽聚糖生物合成相关的酶组装成多酶复合体发挥作用，包括细胞延伸复合体elongasome (也称Rod复合体)和细胞分裂复合体divisome (表 2和图 3)[1-2, 6, 83, 88-89]。两个多酶复合体中的蛋白不仅包括SEDS-bPBPs和aPBP-Lpo等肽聚糖合酶复合体，还包括肽聚糖水解酶、细胞骨架蛋白以及相关的调控蛋白等。其中，elongasome复合体主要由RodA、PBP2、PBP1A、LpoA、RodZ、MreB、MreC和MreD构成；而divisome复合体主要由FtsW、PBP3 (FtsI)、PBP1B、LpoB、FtsA、FtsK、FtsQLB、ZipA和FtsZ构成(图 3和表 2)。
| Protein | Category | Role | References |
| Elongasome complex | |||
| RodA | SEDS family protein | GTase, polymerizes the glycan chains | [17] |
| PBP2 | bPBP | TPase, forms peptide cross-links | [54] |
| PBP1A | aPBP | GTase and TPase, peptidoglycan synthase | [55] |
| LpoA | Outer membrane-anchored lipopotein | Essential for the PBP1A function | [32, 34] |
| MreB | Actin-like cytoskeleton protein; cell shape determinant | Maintains the cylindrical rod shape | [56] |
| MreC | Cell shape determinant | Interacts with MreB, MreD and PBP2 and regulates peptidoglycan crosslinking activity | [57] |
| MreD | Cell shape determinant | Balances the activity of PBP2 with MreC | [58] |
| RodZ | Morphogenic protein | Functions as an adaptor between MreB and peptidoglycan synthases |
[59] |
| Divisome complex | |||
| FtsW | SEDS family protein | GTase, polymerizes the glycan chains | [18] |
| PBP3 (FtsI) | bPBP | TPase, forms peptide cross-links | [60] |
| PBP1B | aPBP | GTase and TPase, peptidoglycan synthase | [55] |
| LpoB | Outer membrane-anchored lipopotein | Essential for the PBP1B function | [32, 34] |
| LpoP | Outer membrane-anchored lipopotein | Essential for PBP1B function in P. aeruginosa | [61] |
| FtsZ | Tublin-like cytoskeleton protein | Forms a dynamic cytoplasmic ring at midcell | [62] |
| FtsA | Z ring membrane anchor | Tethers FtsZ filaments to the membrane | [63] |
| ZipA | Z ring membrane anchor | Tethers FtsZ filaments to the membrane | [64] |
| FtsK | Cell division protein | Interacts with numerous divisome components | [65] |
| FtsN | Cell division protein | Interacts with PBP3, FtsW and PBP1B, and regulates peptidoglycan activity of PBP1B | [66] |
| FtsEX | Cell division proteins | Interacts with FtsA to regulate cell division. | [67-68] |
| FtsQLB | Cell division proteins | Recruits and regulates the peptidoglycan synthases FtsW-PBP3 | [69] |
| Transcriptional regulators | |||
| σM | Alternative sigma factor | Upregulates the core biosynthesis pathways for assembly of cell wall | [70-71] |
| σI | Alternative sigma factor | Upregulates the expression of MreBH and LytE autolysin | [72] |
| σD | Alternative sigma factor | Modulates the expression of l, d-transpeptidase in Corynebacterium glutamicum | [73] |
| SspA | RNAP-associated regulatory protein | Modulates the expression of both aPBPs and SDES-bPBPs | [74] |
| WalKR | Two-component system | Controls cell wall metabolism | [75] |
| VrxAB (WigKR) | Two-component system | Activates multiple steps of the peptidoglycan synthesis | [76] |
| CpxAB | Two-component system | Modifies peptidoglycan cross-linking via the l, d-transpeptidase LdtD | [77-78] |
| BolA | Transcriptional regulator | Regulates the transcription of mreBCD and several peptidoglycan hydrolases | [79-80] |
| DdlR | GntR-family transcription regulator | Activates transcription of d-Ala-d-Ala ligase gene | [81-82] |
|
| 图 3 细胞延伸和分裂过程中的肽聚糖合成多酶复合体 Figure 3 Multienzyme peptidoglycan-synthesizing complexes during cell elongation and division. A great number of enzymes involved in peptidoglycan biosynthesis are organized into the two multienzyme complexes, divisome (left) and elongasome (right), which are responsible for the assembly of peptidoglycan during cell division and elongation, respectively. For simplicity, peptidoglycan hydrolases associated with both complexes are not shown. The activity and role of these proteins are listed in the Table 2. |
两个多酶复合体中肽聚糖合酶的活性依赖于蛋白之间的相互作用。除前文所述的aPBPs与外膜脂蛋白以及SEDS家族蛋白与bPBPs存在相互作用外，两类肽聚糖合酶之间也存在相互作用。例如，PBP1A与PBP2相互作用，并且PBP2促进PBP1A的转糖基酶活性[54]；而FtsW、PBP3和PBP1B形成三元复合物，PBP3缺失时FtsW与Lipid Ⅱ相互作用并阻止PBP1B的转糖基酶活性，PBP3存在时促进Lipid Ⅱ的释放以及PBP1B的酶活性[60, 90]。原核生物细胞骨架蛋白同样能够影响肽聚糖合酶的活性[1, 7]。延伸复合体elongasome中肌动蛋白类细胞骨架蛋白(actin-like cytoskeleton protein) MreB在细胞内沿侧壁呈螺旋式排列，通过膜蛋白RodZ、MreC和MreD调控RodA-PBP2的酶活性，从而决定细胞的杆状形态[91-92]；分裂复合体divisome中微管蛋白类细胞骨架蛋白(tublin-like cytoskeleton protein) FtsZ通过FtsA和ZipA在隔膜处形成细胞分裂环(Z环)，然后募集FtsK、FtsQLB、FtsW、PBP3和FtsN等其他蛋白，从而调控PBP3和PBP1B的酶活性，最终决定细胞的分裂[88]。正是因为多酶复合体中不同蛋白的协调作用和严谨调控，才使得细胞以合适的速率在正确的位置精准合成肽聚糖。
4.2 转录水平的调控与翻译后水平的调控机制相比，目前对于肽聚糖生物合成基因在转录水平的调控了解不多，尤其是在革兰氏阴性菌中。
4.2.1 RNA聚合酶结合蛋白介导的调控革兰氏阳性菌中影响RNA聚合酶(RNA polymerase, RNAP)转录活性的蛋白能够调控肽聚糖生物合成相关基因的表达(表 2)[70-71]。枯草芽孢杆菌中编码17个选择性σ因子(alternative σ factor)，它们通过替换RNAP全酶中的σA改变转录活性，如σM、σI、σW、σX和σV等。σM的主要作用是维持细胞壁的完整性，其调控肽聚糖生物合成过程中一系列基因的表达，包括肽聚糖前体的合成(Ddl、MurB和MurF)、肽聚糖装配和修饰(PBP1和PbpX)以及肽聚糖合成复合体关键组分(MreBCD、RodA、DivIB、DivIC和MinCD)[17, 71]。有趣的是，σM还能调控Lipid Ⅱ翻转酶Amj的表达[71]。由此可见，σM在革兰氏阳性菌肽聚糖生物合成过程中起核心调控作用。σI是一种在热激等不利条件下发挥作用的选择性σ因子，研究发现σI缺失的菌株细胞形态由杆状变为畸形，可能的原因是σI缺失后显著下调elongasome复合体蛋白MreBH和肽聚糖水解酶LytE的表达[93-95]。在aPBPs缺失时，σI可以通过上调elongasome复合体的活性实现对σM的功能补偿[72]。此外，其他选择性σ因子(如σD、σW、σX和σV)也能调控少数肽聚糖合成酶或水解酶基因的表达[71, 73]。
在革兰氏阴性菌中，目前尚无关于σ因子调控肽聚糖生物合成基因的报道。近年来，笔者团队以希瓦氏菌为研究对象，发现与RNAP结合的严谨饥饿蛋白SspA (stringent starvation protein A)能够同时调控两类肽聚糖合酶基因的表达[74]。SspA在革兰氏阴性菌中保守存在，其在细菌进入稳定期以及营养饥饿时诱导表达，通过与σ70-RNAP复合体相互作用调控基因的转录[96-97]。当sspA基因缺失后，PBP1B和SEDS-bPBPs家族蛋白基因的表达显著上调，从而补偿由PBP1A缺失引起的肽聚糖损伤[74]。进一步研究发现，大肠杆菌中SspA也能调控两类肽聚糖合酶基因的表达，暗示SspA对肽聚糖合酶基因表达的转录抑制是革兰氏阴性菌中保守的调控机制。
4.2.2 双组分系统介导的调控双组分系统(two-component system)是细菌感知并响应外界复杂环境最为重要的信号转导系统，一般由组氨酸激酶(histidine kinase)和应答调控蛋白(response regulator)二元组分构成。组氨酸激酶感知外界信号后自磷酸化，然后将位于细胞内的应答调控蛋白磷酸化，从而激活或抑制靶基因的表达，使细菌适应不同的环境变化。革兰氏阳性菌中的双组分系统WalKR (也称为YycGF)对肽聚糖代谢的调控已研究较为深入，该系统调控多个肽聚糖水解酶基因(如yocH、cwlO和lytE等)以及细胞分裂相关基因(如ftsAZ操纵子)的表达[75, 98-100]。经序列分析发现，应答调控蛋白WalR结合位点的保守基序为5′-TGTWAH-N5-TGTWAH-3′[99]，枯草芽孢杆菌中WalKR对靶基因的调控作用取决于该结合位点与靶基因启动子之间的相对位置[75]。
革兰氏阴性菌中，现已发现霍乱弧菌中的双组分系统VxrAB (也称为WigKR)能够调控肽聚糖的生物合成，过表达模拟磷酸化的应答调控蛋白VxrBD78E上调一系列肽聚糖合成酶基因的表达，包括肽聚糖前体合成(MurA−E、DdlA、MraY和MurG)、Lipid Ⅱ翻转(MurJ)以及肽聚糖聚合和交联(FtsW、PBP1A和PBP1B)[76]。VxrAB能够被β-内酰胺类抗生素(如青霉素)激活，通过上调整个肽聚糖生物合成通路中基因的表达来提高细菌对抗生素的耐受性。有意思的是，VxrAB还能被D-环丝氨酸和磷霉素等肽聚糖靶向抗生素以及在肽聚糖内肽酶ShyA过表达时激活，表明该双组分系统可以感知和应答不同因素引起的肽聚糖损伤[76]。尽管VxrB能够与编码PBP1A和MurJ的基因启动子区结合，但这些VxrB结合位点不具有保守性，推测VxrB与靶基因启动子区的结合可能需要其他未知因子的参与[101]。除调控肽聚糖生物合成外，VxrAB还能调控细菌毒力因子、VI型分泌系统以及生物膜形成等众多生理过程[76, 101-103]。VxrAB仅在弧菌科细菌中保守，其在副溶血弧菌(Vibrio parahaemolyticus)中的同源双组分系统VbrKR能够诱导β-内酰胺酶基因的表达进而对β-内酰胺类产生耐药性[104]，但是否调控肽聚糖的生物合成尚不清楚。此外，由于革兰氏阴性菌的肽聚糖层被包裹在细菌胞膜(cell envelope)中，多个与细菌胞膜胁迫感知和应答相关的双组分系统(如Cpx和Rcs等)也能够调控肽聚糖生物合成相关基因的表达。例如，大肠杆菌中Cpx系统在肽聚糖损伤时激活，通过上调ld-转肽酶基因ldtA的表达致使肽聚糖组成发生变化，最终保护细胞免受裂解[77-78]。
相较于调控的靶基因，目前对于这些双组分系统中组氨酸激酶感知何种信号尚不清楚。尽管现有的研究暗示WalK和VxrA感知的信号很有可能是某种未知的肽聚糖组分，但仍缺少直接的证据。
4.2.3 其他转录因子介导的调控除σ因子和双组分系统外，还有一些其他转录因子参与肽聚糖生物合成的调控(表 2)。大肠杆菌中的BolA是一种胁迫应答蛋白，当其过表达时细胞形态由杆状变成球形[105-106]。研究发现，BolA不仅能直接抑制mreBCD操纵子的转录，还能上调一系列肽聚糖水解酶基因(dacA、dacC和ampC)的表达，这些基因表达的变化导致细胞形态的改变[79-80]。此外，革兰氏阳性菌中GntR家族转录因子DdlR通过直接与启动子区结合调控d-Ala-d-Ala连接酶基因ddl的表达[81-82]。
5 总结与展望作为绝大多数细菌中最重要的结构之一，肽聚糖与细菌细胞的形态、大小、生长和分裂等基本生命过程密切相关。结合生物化学、遗传学和结构生物学等研究方法，近些年国内外研究团队在肽聚糖生物合成及其调控机制方面取得了许多突破性研究进展，包括鉴定出Lipid Ⅱ翻转酶MurJ和第二类肽聚糖合酶SEDS-bPBPs以及从不同层次阐明肽聚糖生物合成的调控机制等，这些结果极大地加深了我们对细菌如何合成肽聚糖以及维持肽聚糖稳态等重要基础科学问题的理解。然而，由于肽聚糖与众多基本生命过程密切相关，并且涉及到的蛋白非常多，目前对于整个肽聚糖生物学的认识可能只是“冰山一角”。
结合已有研究我们提出未来研究方向包括：(1) 探究不同细菌中肽聚糖的生物合成及其调控机制。目前已有的研究大多局限在少数模式菌株如大肠杆菌和枯草芽孢杆菌中，未来需要对一些具有代表性的非模式菌株(如生长极快的需钠弧菌Vibrio natriegens)以及特殊生境的极端微生物进行深入研究，从而揭示肽聚糖生物合成及其调控机制的多样性。(2) 探究肽聚糖合酶基因在转录水平的调控机制。目前对于肽聚糖生物合成调控的研究主要集中在翻译后水平，而在转录水平的调控机制了解较少，尤其是在革兰氏阴性菌中鲜有报道。通过对转录水平调控机制的研究有助于回答细菌如何协调肽聚糖生物合成与其他细胞结构生物合成保持一致。(3) 阐明细菌在不同生长和胁迫条件下维持肽聚糖稳态的分子机制。细菌中通常编码多套具有冗余特性的肽聚糖合酶以及水解酶，它们在实验室条件下可以相互替代，但近年来研究发现这些冗余的酶实际上在不同环境中发挥作用[22, 24]。接下来可以在不同培养条件下研究肽聚糖合酶以及水解酶的功能，从而丰富对肽聚糖稳态维持机制的认识。(4) 开发靶向Lipid Ⅱ翻转酶MurJ和SEDS家族蛋白的新型抗菌药物。这些蛋白对于细菌的生长不可或缺，并且这些蛋白在细菌中广泛存在，因此可作为潜在的靶点开发新型抗菌药物，从而应对日益严重的抗生素耐药性问题。目前针对这些靶点的抗菌药物已逐步开展[45]。(5) 加强肽聚糖生物学基础研究向应用研究转化。近年来兴起的微生物形态工程正是基于对肽聚糖的深入理解发展起来的一种新兴生物工程技术[107]。利用合成生物学理念对微生物细胞形态和大小等细胞固有特性进行人工设计和改造，可以实现细胞形态和大小的精准控制，从而构建高效微生物细胞工厂。
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