关丽君¹, 薛云¹, 丁文文¹, 赵战勤^,1,21 河南科技大学动物科技学院兽医生物制品工程实验室,河南洛阳 471003
2 河南科技大学动物科技学院/河南省高等学校环境与 畜产品安全重点学科开放实验室,河南洛阳 471003

Advances in Mechanisms of Biosynthesis and Regulation of Pasteurella multocida Capsule

GUAN LiJun¹, XUE Yun¹, DING WenWen¹, ZHAO ZhanQin^,1,21 Laboratory of Veterinary Biologics Engineering, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, Henan
2 College of Animal Science and Technology, Henan University of Science and Technology/ Key-Disciplines Laboratory of Safety of Environment and Animal Product, Luoyang 471003, Henan

通讯作者: 赵战勤,Tel：0379-64282341;E-mail：zhaozhanqin@126.com

责任编辑: 林鉴非
收稿日期:2018-09-3接受日期:2019-11-5网络出版日期:2020-02-01

基金资助:

国家自然科学基金项目.31302106 国家自然科学基金项目. U1704117 国家自然科学基金项目.31672530

Received:2018-09-3Accepted:2019-11-5Online:2020-02-01
作者简介 About authors
关丽君,E-mail：gljguanlijun@163.com。






摘要
多杀性巴氏杆菌可广泛感染多种动物,引起出血性败血症或传染性肺炎。多杀性巴氏杆菌的细胞表面具有一层黏液样的荚膜多糖,是其重要的结构成分和毒力因子,在细菌与宿主的相互作用中起到重要作用,促进细菌粘附于宿主表面,增强细菌的毒力。多杀性巴氏杆菌荚膜的分子结构与脊椎动物的糖胺聚糖（GAG）相似,都由重复的二糖单元聚合形成线性多糖链,这是该菌在感染宿主过程中进行分子伪装、抵抗吞噬和发生免疫逃逸的重要免疫学物质基础。近年来,在多杀性巴氏杆菌荚膜的生物合成及其调控机制方面取得了一系列重要的研究进展,为多杀性巴氏杆菌荚膜的分子致病机理研究提供了一定的基础知识,为多杀性巴氏杆菌荚膜多糖疫苗的研发提供了理论依据。文章系统阐述了多杀性巴氏杆菌荚膜的生物合成途径及其表达调控机制,主要包括荚膜的血清分型、荚膜多糖的成分与结构、荚膜的生物合成基因簇与功能、荚膜多糖的分子合成机制、荚膜生物合成基因簇的表达调控机制,共5个方面。依据荚膜抗原,多杀性巴氏杆菌可分为A、B、D、E、F共5种荚膜血清型。A型荚膜GAG成分是透明质酸、D型是肝素、F型是软骨素,分别由其相应的二糖单元[β-葡糖醛酸/β-乙酰葡糖胺]、[β-葡糖醛酸/α-乙酰葡糖胺]、[β-葡糖醛酸/β-乙酰半乳糖胺]重复构成;B型荚膜多糖是由阿拉伯糖、甘露糖和半乳糖以某种结构形式聚合而成,E型荚膜多糖的成分与化学结构尚不确定。多杀性巴氏杆菌A型、B型、D型、E型和F型荚膜多糖生物合成的相关基因以基因簇的形式存在,分为3个不同的功能区,R1、R2和R3;R1区负责转运荚膜多糖,R2区负责单糖的活化和荚膜多糖的组装,R3区负责荚膜多糖的修饰（磷脂替换）;根据R2区结构和基因数量的不同又可将5种荚膜的生物合成基因簇分为两类：A型、D型、F型为I类,R2区含有4个基因;B型和E型为II类,R2区含有9个基因,且利用R2区特异性基因设计引物,可以通过PCR方法快速鉴定多杀性巴氏杆菌的荚膜血清型。多杀性巴氏杆菌的荚膜GAG在细胞质中生成,由R1区编码蛋白所形成的ABC转运体输出至细胞表面,末端糖脂通过分子间氢键与细胞壁紧密结合,形成菌体表面的粘液状荚膜;在多杀性巴氏杆菌荚膜GAG的生物合成过程中,位于R2区的糖基转移酶基因决定了活化单糖的种类和组装后荚膜多糖的类型。在多杀性巴氏杆菌荚膜的生物合成基因簇中,R1和R2区形成一个操纵子,转录方向一致,而R3转录方向与其相反,两者的启动子区域均位于R2和R3区域之间的DNA序列上;多杀性巴氏杆菌荚膜生物合成基因簇的转录过程受Fis蛋白正向调控,翻译过程主要受Hfq蛋白正向调节。
关键词： 多杀性巴氏杆菌;荚膜;糖胺聚糖;生物合成;表达调控

Abstract
Pasteurella multocida can be widely infected with a variety of animals, causing hemorrhagic septicemia or infectious pneumonia. P. multocida possess a viscous capsular polysaccharide on the cell surface, which is a critical structural component and virulence factor, and plays an important role in the interaction between bacteria and the host, promoting the adhesion of bacteria to the host surface and enhancing the virulence of the bacteria. The molecular structure of the P. multocida capsule is similar to that of vertebrate glycosaminoglycan, which is polymerized by repeated disaccharide units to form a linear polysaccharide chain, which is an important immunological material basis for molecular mimicry, resistance to phagocytosis, and immune evasion during the infection of the host. In recent years, a series of important research advances have been made in the biosynthesis and regulation mechanism aspects of P. multocida capsule, providing a certain basic knowledge for the molecular pathogenesis of P. multocida capsule, and supplying a theoretical basis for the development of the capsular polysaccharide vaccine of P. multocida. This paper systematically illuminates the biosynthesis and expression regulation mechanisms of P. multocida capsule, including the serotyping of the capsule, the composition and structure of the capsular polysaccharide, the biosynthesis gene cluster and function of the capsule, the molecular synthesis mechanism of capsular polysaccharide, the expression regulation mechanism of capsular biosynthesis gene cluster, a total of five aspects. According to the capsular antigen, P. multocida is divided into five capsular serogroups of A, B, D, E, and F. The type A capsule GAG component is hyaluronic acid; the type D is heparosan; the type F is chondroitin, which is repeatedly composed of its corresponding disaccharide unit [β-GlcUA/β-GlcNAc], [β-GlcUA/α-GlcNAc], [β-GlcUA/β-GalNAc], respectively; the type B capsular polysaccharide is composed of arabinose, mannose and galactose in a certain structural form, and the composition and chemical structure of type E capsular polysaccharide are uncertain. Genes related to the biosynthesis of A, B, D, E and F capsules of P. multocida exist in the form of gene clusters and are divided into three distinct functional regions, R1, R2 and R3; the R1 region is responsible for transporting the capsular polysaccharide, the R2 region is responsible for the activation of the monosaccharide and the assembly of the capsular polysaccharide, and the R3 region is responsible for the modification of capsular polysaccharide (phospholipid replacement); according to the structure and the number of genes of the R2 region, the biosynthetic gene clusters of the five capsules can be divided into two categories: type A, D and F are Class I, and R2 contains 4 genes; types B and E are Class II, and R2 contains 9 genes, and using the specific gene in the R2 region to design primers, the capsular serotype of P. multocida can be rapidly identified by PCR. The capsular GAG of P. multocida is synthesized in the cytoplasm, and then exported to the cell surface via the ABC transporter formed by the protein encoded by the R1 region, and tightly bound to the cell surface by covalent attachment to the phospholipid; during the biosynthesis of the P. multocida capsular GAG, the glycosyltransferase gene located in the R2 region determines the type of activated monosaccharide and the type of capsular polysaccharide after assembly. In the biosynthetic gene cluster of the P. multocida capsule, the R1 and R2 regions form an operon with the same transcriptional direction, while the R3 transcription direction is opposite, and the promoter regions of both are located on the DNA sequence between the R2 and R3 regions; the transcriptional process of the P. multocida capsular biosynthesis gene cluster is positive regulated by the Fis protein, and the translation process is mainly positive regulated by Hfq protein.
Keywords：Pasteurella multocida;capsule;glycosaminoglycan;biosynthesis;expression regulation


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本文引用格式
关丽君, 薛云, 丁文文, 赵战勤. 多杀性巴氏杆菌荚膜的生物合成及其调控机制研究进展[J]. 中国农业科学, 2020, 53(3): 658-668 doi:10.3864/j.issn.0578-1752.2020.03.016
GUAN LiJun, XUE Yun, DING WenWen, ZHAO ZhanQin. Advances in Mechanisms of Biosynthesis and Regulation of Pasteurella multocida Capsule[J]. Scientia Acricultura Sinica, 2020, 53(3): 658-668 doi:10.3864/j.issn.0578-1752.2020.03.016


某些细菌在生命活动过程中,可产生多糖并在细胞表面以共价键聚合,形成一层包围整个菌体的黏液样物质,称为荚膜（capsule）^[1]。荚膜紧附于细菌细胞壁外,有一定的形状和轮廓,能与周围环境明显区分,有较一致的密度,是细菌构造的一部分^[2]。还有一些细菌能分泌一层很疏松且与周围外界不明显,易与菌体脱离的黏液样物质,称为黏液层。细菌产生荚膜或黏液层,可使液体培养基具有黏性。有荚膜的细菌在固体培养基上形成表面湿润、有光泽的光滑型或黏液型菌落,失去荚膜的细菌则形成粗糙型菌落^[3]。

荚膜的化学成分因菌种而异,多数细菌荚膜由糖胺聚糖（glycosaminoglycan,GAG;曾称粘多糖、氨基多糖或酸性多糖）组成,如多杀性巴氏杆菌（Pasteurella multocida）、链球菌（Streptococcus）;少数细菌荚膜由多肽组成,如炭疽杆菌（Bacillus anthraci）;也有极少数细菌两者兼有,如巨大芽孢杆菌（Bacillus megaterium）^[4]。荚膜具有抗原性,称为荚膜抗原或K抗原。各种细菌荚膜的具体组成及分子结构都是不同的,具有种和型的特异性,可用于细菌的鉴定。荚膜多糖在细菌与环境之间的相互作用中扮演者重要角色^[1],例如：①维持细胞形态和结构的完整性;②当处于营养贫乏的环境时,可作为营养储备加以利用;③维持电荷和离子平衡;④增强细菌抵抗不良环境的能力,如干燥、金属离子、抗生素等。同时,荚膜多糖是细菌重要的毒力因子^[5,6,7],在细菌与宿主之间的相互作用中具有多种生物学功能^[8],使其充分发挥致病作用,例如：⑤增强细菌抵抗宿主某些抑菌（杀菌）物质的能力,如胆盐、溶菌酶、胃和胰酶、乙醇等;⑥可抵抗免疫细胞的吞噬和补体的杀伤作用,从而使其对宿主具有侵袭能力;⑦可使细菌彼此相连,在黏膜细胞表面形成生物被膜,增强细菌耐药性^[9],这是影响细菌侵袭力的重要因素。

多杀性巴氏杆菌为巴氏杆菌科巴氏杆菌属的一种革兰氏阴性小杆菌,可引起多种动物的巴氏杆菌病（Pasteurellosis）,主要症状为出血性败血症或传染性肺炎^{[10,11,12,13,14]}。多杀性巴氏杆菌是一种典型的产荚膜细菌,荚膜是其重要的结构成分和致病因子。本文以多杀性巴氏杆菌为代表重点综述了荚膜的血清分型、成分与结构、基因簇与功能,及荚膜的分子生物合成与表达调控机理方面的研究进展,为系统了解多杀性巴氏杆菌荚膜GAG的糖化学和分子生物学提供参考。

1 多杀性巴氏杆菌的荚膜及其血清分型

多杀性巴氏杆菌荚膜的主要成分为多糖,具有抗原性,并具有种和型特异性,是CARTER等对多杀性巴氏杆菌进行荚膜分型的抗原基础。1952年,CARTER等用被动血凝试验对多杀性巴氏杆菌荚膜抗原（K抗原）进行分类,鉴定出A、B、D和E共4种血清型（Carter分型）^[15]。1987年,RIMLER和RHOADES等又从火鸡中分离并鉴定出了F型多杀性巴氏杆菌^[16]。1953年,CARTER等鉴定出多杀性巴氏杆菌A型荚膜的主要成分是透明质酸（hyaluronic acid, HA）;1993年,PANDIT和SMITH等通过提纯、化学和免疫学分析对其进行了系统的验证^[17];2002年,DEANGELIS等鉴定出D型荚膜的主要成分是肝素,F型荚膜成分是软骨素^[18]。B型荚膜的化学结构尚未被确定,但1993年,MUNIANDY等对B型荚膜进行水解后,鉴定出其单糖成分主要为阿拉伯糖,甘露糖和半乳糖,说明其应为这3种单糖以某种结构形式形成的聚合物^[19],E型荚膜的成分尚未鉴定。多杀性巴氏杆菌病的病型、宿主特异性、地方流行性、致病性、免疫性等与荚膜血清型有一定的关联^[20,21,22]。如A型可引起禽霍乱、猪肺疫;B型可引起牛和猪等动物的败血症;D型和E型可引起猪、牛、兔、羊等动物的肺炎和败血症;F型主要发生于火鸡,其致病作用目前不清楚。在我国猪群中曾以A型为主,其次是B型,D型少见。但是我们的研究最新发现A型和D型为主要流行血清型,而B型罕见。另外,发现A型菌株的毒力普遍强于D型,这表明多杀性巴氏杆菌的血清型在一定程度上可作为其毒力的标志。相同血型的不同菌株,其毒力也存在较大差异^{[23,24,25,26]}。

2 多杀性巴氏杆菌荚膜的成分及其结构

多杀性巴氏杆菌荚膜主要由GAG组成,呈黏液状包围在细菌细胞壁外。GAG属于杂多糖,为不分支的长链聚合物,由含己糖醛酸和己糖胺成分的重复二糖单元构成。二糖单元的通式为：[己糖醛酸-己糖胺]_n。二糖单位中至少有一个单糖残基为带有负电荷的羧基,因此GAG是呈酸性的阴离子多糖链。不同多杀性巴氏杆菌荚膜的GAG各有差异,A型荚膜的透明质酸是由二糖单元β-D-葡糖醛酸（β-D-GlcUA）和β-D-N-乙酰葡糖胺（β-D-N-GlcNAc）通过1,3-糖苷键连接而成,相邻二糖单位之间通过1,4-糖苷键聚合^[27,28];D型荚膜的肝素是由二糖单元β-D-GlcUA和α-D-N-GlcNAc通过1,4-糖苷键连接而成,相邻二糖单位之间也通过1,4-糖苷键聚合^[18];F型荚膜的软骨素是由二糖单元β-D-GlcUA和β-D-N-乙酰半乳糖胺（β-D-N-GalNAc）通过1,3-糖苷键连接而成,相邻二糖单位之间通过1,4-糖苷键聚合。

值得注意的是,哺乳动物细胞中存在与A型、D型和F型多杀性巴氏杆菌荚膜结构相同或相似的GAG^[29],且是细胞外基质的重要成分^[30]。其中,哺乳动物HA的结构与多杀性巴氏杆菌完全相同,都不发生分子结构的差向异构化和硫酸化,而哺乳动物肝素或软骨素中的D-GlcUA会发生差向异构化,变为L-艾杜糖醛酸（L-IdoUA）结构,并进一步发生硫酸化。因此,后两者在多杀性巴氏杆菌中以硫酸乙酰肝素前体和硫酸软骨素前体的形式存在,而哺乳动物细胞中是以成熟的形式存在。多杀性巴氏杆菌GAG荚膜与宿主糖胺聚糖的相似性^[31],是GAG荚膜抵抗吞噬、逃避宿主防御系统的重要免疫学基础。

3 多杀性巴氏杆菌荚膜生物合成的基因簇及其功能

多杀性巴氏杆菌荚膜合成的相关基因以基因簇的形式存在^[32,33],分为3个不同的功能区,R1、R2和R3。在不同血清型多杀性巴氏杆菌中,R1和R3区域均高度保守,但R2区域具有明显不同,并依此将多杀性巴氏杆菌荚膜分为两类^[34]。A、D、F型为I类^{[33, 35-36]},其荚膜的生物合成基因簇约为16 kb^[33],含有10个ORFs（GenBank accession nos. AF067175.2、CP003313^[35]、AF302467）,如表1所示。B和E型为II类^[35,36],其荚膜的生物合成基因簇约为21 kb,含有15个ORFs（GenBank accession nos. AF169324、AF302466）,如表2所示。多杀性巴氏杆菌荚膜I类（II类）基因簇的R1区包含4个基因hexABCD（或cexABCD）,编码形成的4个蛋白质分子能形成一种复合体,其功能是将胞内合成的GAG转运到细胞表面^[34]（表1, 2）,R3区包含2个基因phyAB（或lipAB）,编码形成的蛋白质负责GAG的磷脂替换^[33],使其锚定到细胞表面上^[37]。

Table 1
表1
表1I类荚膜生物合成基因簇及编码蛋白的功能
Table 1Biosynthesis gene clusters and protein-coding function of class I capsule

功能区 Region	开放阅读框（ORF）			A型/D型/F型编码的蛋白质 Protein encoded by type A/D/F	功能 Function
	A型 Type A	D型 Type B	F型 Type D
R1	hexA	hexA	hexA	HexA	ATP结合蛋白 ATP-binding protein
	hexB	hexB	hexB	HexB	内膜蛋白 Inner membrane protein
	hexC	hexC	hexC	HexC	具有周质结构域的内膜蛋白 Inner membrane protein with a periplasmic domain
	hexD	hexD	hexD	HexD	外膜蛋白（脂蛋白） Outer membrane protein (lipoprotein)
R2	hyaB	dcbB	fcbB	HyaB/DcbB/FcbB	糖基转移酶 Glycosyltransferase
	hyaC	dcbC	fcbC	HyaC/DcbC/FcbC	UDP-葡萄糖脱氢酶,催化UDP-葡萄糖转化为UDP-葡糖醛酸 UDP-glucose dehydrogenase, catalyzing the conversion of UDP-glucose to UDP-glucuronic acid
	hyaD	dcbF	fcbD	HyaD/DcbF/FcbD	透明质酸合成酶/肝素合成酶/软骨素合成酶 PmHAS/PmHS/PmCS
	hyaE	dcbE	fcbE	HyaE/ DcbE / FcbE	起始酶,将重复单元的第一个残基转移至 Poly-KDO linker Initiates enzyme, which transfers from the first residue of the repeat-unit to the poly-KDO linker
R3	phyA	phyA	phyA	PhyA	β-KDO糖基转移酶 β-Kdo glycosyltransferase
	phyB	phyB	phyB	PhyB	β-KDO糖基转移酶 β-Kdo glycosyltransferase

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Table 2
表2
表2II类荚膜生物合成基因簇及编码蛋白的功能
Table 2Biosynthesis gene clusters and protein-coding function of class II capsule

功能区 Region	开放阅读框（ORF）		B型/E型编码的蛋白质 Protein encoded by type B/E	功能 Function
	B型 Type B	E型 Type F
R1	cexA	cexA	CexA	ATP结合蛋白 ATP-binding protein
	cexB	cexB	CexB	内膜蛋白 Inner membrane protein
	cexC	cexC	CexC	具有周质结构域的内膜蛋白 Inner membrane protein with a periplasmic domain
	cexD	cexD	CexD	外膜蛋白 Outer membrane protein
R2	bcbA	ecbA	BcbA/EcbA	UDP-N-乙酰葡糖胺2-差向异构酶 UDP-N-acetylglucosamine 2-epimerase
	bcbB	ecbB	BcbB/EcbB	UDP-N-乙酰-D-甘露糖胺糖醛酸脱氢酶 UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase
	bcbC	ecbK	BcbC/EcbK	假定的糖基转移酶,可能参与活化单糖的聚合 Putative glycosyltransferase, which likely involved in polymerization of the activated sugar
	bcbDEFGI	ecbDEFGI	BcbDEFGI/EcbDEFGI	未知 Unknown
	bcbH	ecbJ	BcbH/EcbJ	未知,是B型或E型的独有基因 Unknown, unique to serogroup B or E
R3	lipA	lipA	LipA	参与多糖的磷脂替换 Involved in phospholipid substitution of GAG
	lipB	lipB	LipB	参与多糖的磷脂替换 Involved in phospholipid substitution of GAG

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多杀性巴氏杆菌I类（A、D、F型）和II类（B、E型）荚膜生物合成基因簇的主要区别位于R2区域,该区域的主要功能是单糖的活化和GAG的组装。I类基因簇的R2区域含有4个基因,A型为hyaBCDE,D型为dcbBCFE,F型为fcbBCDE（表1）。其中,hyaBCE,dcbBCE和fcbBCE中对应基因编码的均是同源产物,在GAG的合成中均具有特定的生物学功能（表1）;而hyaD、dcbF和fcbD基因编码产物不同,三者分别编码多杀性巴氏杆菌的透明质酸合成酶（P. multocida hyaluronic acid synthase, PmHAS）^[38]、肝素合成酶（P. multocida heparosan synthase, PmHS）^[39]和软骨素合成酶（P. multocida chondroitin synthase, PmCS）^[40],这三种酶的主要功能就是进行A、D、F型荚膜GAG的分子合成。II类基因簇的R2区域含有9个基因,B型为bcbABCDEFGHI,E型为ecbABJKDEFGI。B型bcbABDEFGI和E型ecbABDEFGI对应基因编码的均是同源产物^{[32, 36]},但是除了bcbAB和ecbAB,其他5个基因（bcbDEFGI或ecbDEFGI）的功能均不清楚（表1）。另外,bcbC和ecbK均是假定的糖基转移酶,编码产物的C末端具有55%相似性;bcbH和ecbJ不具有相似性,是B型或E型独有的基因,且功能尚不清楚（表1）。TOWNSEND等利用多杀性巴氏杆菌荚膜生物合成基因簇R2区域中的特异性基因（A型hayD,B型bcbD,D型dcbF,F型fcbD和E型ecbJ）设计引物,建立了鉴定5种荚膜血清型多杀性巴氏杆菌的PCR方法^[36],该方法具有快速、简便、准确等优点,得到广泛应用,已基本取代传统的荚膜血清因子鉴定方法。笔者实验室利用此方法对2009—2015年分离的296株猪源多杀性巴氏杆菌进行了PCR分型,发现其主导血清型是A型（占49.3%）和D型（占47.6%）菌株^[23]。

4 多杀性巴氏杆菌荚膜的分子合成机制

多杀性巴氏杆菌的荚膜GAG在细胞质中生成,经ABC转运体^{[1, 41]}（ATP-binding cassette transporter）输出,末端糖脂通过分子间氢键与细胞壁紧密结合,形成致病菌表面的黏液状荚膜。其合成过程主要包括：（1）GAG合成的起始;（2）GAG二糖单元的延伸;（3）GAG的输出。

4.1 荚膜GAG合成的起始

多杀性巴氏杆菌荚膜GAG生物合成的起始反应是GAG糖脂末端的形成。具体过程可概括为3步：首先,在细胞质中,糖基转移酶PhyB（表1）将第一个β-2-酮-3-脱氧辛糖酸残基（β-3-deoxy-D-manno- oct-2-ulosonic acid, β-KDO）添加至溶血甘油磷脂（lysophosphatidylglycerol, lyso-PG）受体上^[42];然后,糖基转移酶PhyA继续添加多个β-KDO残基,形成多聚β-KDO链;最后,起始糖基转移酶HyaE（表1）将活化的单糖即尿苷二磷酸-单糖（UDP-单糖）添加至多聚β-KDO链上,形成GAG糖脂末端的第一个单糖残基;后续即进入GAG二糖单元的延伸环节。其中,β-KDO是一种结构独特的八碳糖,起桥梁作用,使细菌的表面多糖结合到相应的脂质上^[43]。

4.2 荚膜GAG合成的延伸

多杀性巴氏杆菌A型、D型和F型荚膜GAG二糖单元的延伸及聚合方式基本一致^{[28, 38-40, 44]}。以A型为例,由PmHAS将活化的单糖依次添加到与KDO相连的糖基受体末端^[45],延伸GAG糖链,并释放UDP,聚合形成透明质酸,即A型荚膜GAG。在此过程中,PmHAS作为一种双功能酶,具有两个糖基转移酶活性位点,分别具有转移UDP-β-D-GlcUA和UDP-β-D-N-GlcNAc的功能（即形成A型荚膜GAG的二糖单元）^[46]。D型和F型荚膜GAG二糖单元的延伸方式与A型相似^[43,47-49],其糖基转移酶分别是PmHS和PmCS。GAG聚合的反应式可概括如下^[29]：n UDP-GlcUA+n UDP-HexNAc→2n UDP+ [GlcUA- HexNAc]_n,其中HexNAc为GlcNAc或GalNAc,n为二糖单元聚合程度,A型荚膜中n=10³- 10⁴,D型和F型荚膜中n=20-100^[40]。

4.3 荚膜GAG合成的输出

多杀性巴氏杆菌荚膜GAG从胞质转运至细胞表面主要依赖于其ABC转运系统^[50]。在多杀性巴氏杆菌中,其荚膜生物合成基因簇R1区域包含4个基因hexABCD（或cexABCD;表1）,编码的4个蛋白质分子能形成一种复合体,即ABC转运系统。该系统中,HexA是一种ATP结合蛋白,提供转运GAG的能量。HexB是一种内膜蛋白,HexC是一种具有周质结构域的内膜蛋白,HexD是一种外膜蛋白（脂蛋白）,这三种蛋白形成转运GAG的跨膜通道,在ATP结合蛋白HexA的作用下,GAG糖链通过HexBCD形成的跨膜通道,依次穿过细胞内膜、周质空间和细胞外膜,并通过磷脂共价结合于细胞表面^[37]。总结相关文献,我们绘制了多杀性巴氏杆菌荚膜生物合成的分子机制路径,如图1所示。

图1

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图1多杀性巴氏杆菌荚膜生物合成的分子机制

Fig. 1Molecular biosynthesis pathways of P. multocida capsule

5 多杀性巴氏杆菌荚膜生物合成基因簇的表达调控机制

5.1 多杀性巴氏杆菌荚膜生物合成基因簇的表达调控序列

在多杀性巴氏杆菌荚膜的生物合成基因簇中,R1和R2区形成一个操纵子,转录方向一致,而R3转录方向与其相反,两者的启动子区域均位于R2和R3区域之间的DNA序列上^[32,33]。以A型荚膜的生物合成基因簇为例,其R2和R3区的启动子位于hyaE和phyA基因的间隔区,多杀性巴氏杆菌RNA聚合酶中负责识别该启动子并启动转录的σ亚基为σ⁷⁰因子^[33],启动子-35区与-10区间隔17 bp。R1和R2区（两者位于同一个操纵子上）的转录起始点（+1）位于hyaE起始密码子上游37 bp处^[51],核糖体结合位点（ribosome binding site, RBS）位于其上游8 bp处,终止子位于hexA终止密码子下游4 bp处^[33]。根据相关文献与A型荚膜基因簇核苷酸序列（GenBank accession nos. AF067175.2）,得到其R2和R3基因间隔区的转录调控序列（图2）。关于荚膜生物合成基因簇的R3区,目前只鉴定出其转录方向与R1和R2区相反,且该启动子不是由RNA聚合酶的σ⁷⁰因子所识别,转录及其相关调控序列均不是十分清楚^[33]。

图2

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图2多杀性巴氏杆菌A型荚膜生物合成基因簇R2和R3基因间隔区的转录调控序列

Fig. 2Transcriptional regulatory sequences of the intergenic region between the R2 and R3 of the capsular biosynthesis loci in type A P. multocida

5.2 多杀性巴氏杆菌荚膜生物合成基因簇的转录调控机制

多杀性巴氏杆菌荚膜生物合成基因簇的转录过程主要由调节因子（factor for inversion stimulation, Fis）蛋白调控^[51,52,53]。Fis蛋白是一种核相关蛋白（nucleoid -associated proteins, NAPs）,含有99个氨基酸,在第73—94氨基酸残基之间存在一个HTH DNA结合区（helix-turn-helix (HTH) DNA binding motif）,是与荚膜合成基因簇启动子区域结合的部位,正向调控荚膜GAG基因的转录。在多杀性巴氏杆菌荚膜生物合成基因簇的启动子区域,存在2个Fis蛋白结合位点（图5）。多杀性巴氏杆菌荚膜的表达量决定于Fis蛋白调控下的基因转录水平,fis的突变可导致其丧失调控功能;此时,即使荚膜核苷酸序列完整存在,荚膜也不被表达,从而产生无荚膜的菌株^{[51,52,53,54]}。

多杀性巴氏杆菌全基因序列约为2.4 Mb^[55],fis与荚膜生物合成基因簇相距较远。例如,A型HB01株（GenBank accession nos. CP006976）,其fis位于110 760—111 059 bp,荚膜生物合成基因簇位于868 702—883 642 bp处。事实上,Fis蛋白是一种整体转录调控子（global transcriptional regulator）,还参与其他多种巴氏杆菌毒力基因的转录调节,如脂多糖（Lipopolysaccharide, LPS）生物合成的基因簇、丝状血凝素B2（Pasteurella filamentous hemagglutinin B2, pfhB2）基因、巴氏杆菌脂蛋白E（Pasteurella lipoprotein E, plpE）基因等,是多杀性巴氏杆菌重要的毒力基因调节因子^[51]。

5.3 多杀性巴氏杆菌荚膜生物合成基因簇的翻译调控机制

多杀性巴氏杆菌荚膜生物合成基因簇的翻译主要受Hfq蛋白（host factor for Qβ, Hfq）控制^[56]。Hfq蛋白最初发现于大肠杆菌,被认为是大肠杆菌噬菌体Qβ进行有效复制所必需的一种宿主因子,其名字Hfq也由此得来^[57]。在多杀性巴氏杆菌中,Hfq是一种RNA伴侣蛋白^[58],通过辅助小RNA（small RNA, sRNA）分子GcvB与荚膜合成相关基因的mRNA相互作用来正向调节该基因的表达,其作用方式是：首先Hfq分子与sRNA分子GcvB结合形成Hfq-GcvB复合体,该复合体可与mRNA上特定的seed序列（seed region）结合并打开靶标mRNA的5′端非翻译区（untranslated region,UTR）上的二级结构,从而暴露核糖体结合位点（ribosome binding site, RBS）和翻译起始密码子（AUG）,然后核糖体与RBS和AUG区域结合,激活被抑制的mRNA,最终使翻译开始启动^[59,60]。其中,小RNA分子GcvB与靶标mRNA上结合的seed序列为5′-AACACAACAU-3′,而GcvB上对应的互补序列为5′-AUGUUGUGUU-3′,两者均是AU富集的单链区域。

6 总结与展望

GAG的重要生理功能及其与重大疾病的密切关系近年来受到国内外科学家的广泛关注^{[61,62,63,64,65]},荚膜多糖是多杀性巴氏杆菌重要的毒力因子和保护性抗原,其血清分型、成分与结构、生物合成的研究,有利于系统了解多杀性巴氏杆菌荚膜GAG的分子生物学,并为其致病机理的研究和多糖疫苗的开发提供新思路。

20世纪30年代,人们已经开始研发细菌荚膜多糖,希望通过诱导多糖特异性抗体以保护致病菌^[66,67]。目前已有多种重要的人用荚膜多糖多价疫苗研发成功,主要包括23价肺炎链球菌荚膜多糖疫苗^[68]、4价脑膜炎球菌荚膜多糖疫苗^[69]和B型流感嗜血杆菌荚膜多糖疫苗^[70],这些疫苗对预防和控制疾病发挥了巨大的作用,降低了相关疾病的发病率,在全世界各地得到了广泛使用。为进一步提高荚膜多糖对2岁以下儿童和老年人的免疫反应,荚膜多糖与载体蛋白耦联的荚膜多糖结合疫苗研究逐渐兴起,如肺炎链球菌荚膜多糖与白喉无毒突变体（CRM₁₉₇）联合的13价肺炎球菌结合疫苗（PCV-13）^[71]、脑膜炎球菌A群、C群、Y群、W-135群与CRM₁₉₇联合的4价脑膜炎球菌结合疫苗（Menveo）^[72]、B型流感嗜血杆菌荚膜多糖与CRM197联合的结合疫苗（PRP-CRM）或与破伤风类毒素联合的（PRP-T）结合疫苗^[70]。兽用荚膜多糖疫苗的研发起步较晚,目前的研究主要集中于奶牛乳房炎疫苗的开发^[73,74],现已经研发出金黄色葡萄球菌荚膜多糖与绿脓杆菌外毒素A耦联的结合疫苗（StaphVAX）^[75]或与破伤风类毒素耦联的结合疫苗^[76,77]。

多杀性巴氏杆菌分为A型、B型、D型、E型和F型5种荚膜血清型,因此,以荚膜多糖为抗原,研发其多价疫苗值得尝试。由于GAG荚膜与人细胞的结构成分相似,需对其抗原性作深入研究^[78]。多杀性巴氏杆菌A型荚膜多糖的成分是透明质酸,其可被宿主细胞上表达的CD44蛋白识别^[79],以此可进一步研究多杀性巴氏杆菌荚膜透明质酸的结合疫苗。了解荚膜多糖的分子合成和基因表达的机制至关重要,有助于进一步分析分子致病机制和免疫机理。同时,对于未来开发特异性的靶标识别药物具有重要意义。即在不破坏宿主细胞GAG的前提下,能特异性鉴别细菌荚膜或抑制细菌荚膜形成的药物。

关于多杀性巴氏杆菌荚膜GAG的生物合成与调控研究方面,多杀性巴氏杆菌A型、D型和F型的荚膜GAG的分子合成机制已经基本明确,但是尚需对B型和E型荚膜GAG的化学结构进行解析。R1和R2区转录与翻译的调控序列已经基本清楚,但是R3区转录与翻译的调控序列仍需进一步研究。在多杀性巴氏杆菌荚膜GAG的生物合成过程中,GAG糖基转移酶揭示了荚膜多糖生物合成的本质：GAG荚膜不同于自然界中广泛存在的同多糖（如淀粉、糖原、纤维素等）,荚膜多糖是由双功能糖基转移酶控制的二糖聚合物,不含支链,尚需对GAG糖基转移酶的三维分子结构及其详细的分子作用机制进行阐释。

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