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细菌粘附到宿主细胞 Bacterial Adhesion to Host Cells

细菌粘附到宿主细胞 Bacterial Adhesion to Host Cells. 赵 蔚. 部分病原菌的组织嗜性. 病原菌 组织 Neisseria meningitidis 鼻咽上皮细胞 Neisseria gonorrhoeae 尿道上皮细胞 Vibrio cholerae 肠道上皮细胞 Bordetella pertussis 呼吸道上皮细胞 Salmonella typhimurium 肠道上皮细胞 Helicobacter pylori 胃粘膜 Streptococcus pyogenes 咽上皮细胞

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细菌粘附到宿主细胞 Bacterial Adhesion to Host Cells

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  1. 细菌粘附到宿主细胞Bacterial Adhesion to Host Cells 赵 蔚

  2. 部分病原菌的组织嗜性 病原菌 组织 Neisseria meningitidis鼻咽上皮细胞 Neisseria gonorrhoeae尿道上皮细胞 Vibrio cholerae肠道上皮细胞 Bordetella pertussis呼吸道上皮细胞 Salmonella typhimurium肠道上皮细胞 Helicobacter pylori胃粘膜 Streptococcus pyogenes咽上皮细胞 Campylobacter jejuni肠道上皮细胞 Mycoplasma pneumoniae呼吸道上皮细胞

  3. 特异性粘附 物理化学力 粘附 对细菌影响 对宿主细胞影响 侵入等后续过程

  4. 与粘附有关的细菌的结构 • Pili • Capsule • Cell wall

  5. Pili and Fimbria Pili are adhesive hair-like organelles that protrude from the surface of bacteria. The term ‘‘fimbria’’ is more commonly used to describe pili, whose function is devoted to attach bacteria to a surface. TypeⅠ Pili TypeⅣ Pili P pili

  6. pyelonephritis-associated (P) pilus P pili biogenesis is the paradigm of the ‘‘chaperone/usher’’ pathway

  7. Retractile Type IV Pili • EPEC, EHEC, Salmonella enterica serovar Typhi, Pseudomonas aeruginosa, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, and Vibrio cholerae • composed of a homopolymer of a single pilin subunit • type IV pili are formed at the cytoplasmic membrane and the intact organelle is extruded across the outer membrane. • retract through the bacterial cell wall while the pilus tip remains firmly adhered to the target surface.

  8. Function • involved in bacterial adhesion to host cells • EPEC type IV bundle-forming pili: attachment to brush border cells • Neisseria spp., PilC-mediated adhesion to host cells via recognition of the complement regulator CD46 • biofilm formation • DNA uptake by natural transformation.

  9. Pili in Gram-Positive Bacteria Corynebacterium diphtheriae SpA , SpaB , SpaC generated by the sortase machinery

  10. 细菌的粘附分子——粘附素adhesin 细菌细胞表面配基, 细菌表达的黏附相关的表面蛋白 • Lectin(外源凝集素 ) • S. saprophyticus • B. pertusssis • S. typhimurium • E. coli • P. aeruginosa • N. gonorrhoeae • H. pylori • K. pneumoniae • H. influenzae • A. naeslundii • S. pneumoniae • V. cholerae

  11. Fibronectin Binding Proteins • S. pyogenes:> 12 fibronectin and collagen binding proteins • the major adhesins SfbI of S. pyogenes bind ECM-associated fibronectin and induce the clustering of fibronectin bound integrin receptors, triggering intracellular signaling

  12. Autotransporters • molecules secreted by type V secretion, contain an N-terminal leader peptide for secretion across the inner membrane, a C-terminal domain that forms a pore in the outer membrane, and a passenger domain that is autotransported through the outer membrane pore to be exposed on the bacterial surface

  13. Tir (translocated intimin receptor) • The bacterially encoded cellular receptor of EPEC and EHEC

  14. 与粘附有关的细胞表面结构和分子 • 宿主细胞的细胞膜 • 脂质双层：脂类、蛋白质 • 细胞表面粘附受体 • 整合素，钙粘蛋白，免疫球蛋白超家族，选择素 • ECM(extracellular matrix) • 由细胞产生和释放的大量大分子构成细胞外复杂的结构网，包括纤连蛋白、纤维蛋白原、胶原质、蛋白聚糖和粘多糖等。ECM不仅具有结构功能，也影响大量的细胞活动，也提供了细菌粘附到宿主细胞的大量受体

  15. 细菌与ECM的粘附

  16. 细菌的特异性粘附机制 • 直接粘附 • 间接粘附－经由ECM蛋白的粘附 • EPEC粘附

  17. 细菌 细胞表面 直接粘附和间接粘附(桥机制) Type I pili --FimH monomannose- and trimannose-containing glycoprotein commensal E. coli FimH variants: bind with high affinity to trimannose residues uropathogenic bacteria FimH molecules that exhibit a higher affinity for monomannose residues enriched within the urinary tract.

  18. 病原菌 配体 受体 直接互作 革兰阴性菌 菌毛 糖基（ECM、整合素、Ig超家族） 百日咳鲍特菌 FHA CR3 FimD RGD结合整合素 百日咳毒素 P-选择素和E-选择素 脑膜炎奈瑟菌 荚膜/polysialic acid NCAM 淋病奈瑟菌 Opa 蛋白聚糖，CD66 桥机制   葡萄球菌 LTA 纤连蛋白 链球菌 LTA,M 蛋白，FnBP 纤连蛋白 耶尔森菌 YadA 纤连蛋白，胶原质 奈瑟菌 Opc 玻璃体结合蛋白，纤连蛋白 分枝杆菌 纤连蛋白

  19. EPEC的粘附 活化Ⅲ型分泌系统 基座形成 intimin BFP 骨架蛋白重排 +EspA+EspB Tir 活化信号传导途径

  20. 粘附对细菌的影响 • 对生长的调节： 抑制：大肠埃希菌-尿道上皮细胞 促进：淋病奈瑟菌-HeLa细胞 大肠埃希菌-肠上皮细胞 • 产生或释放与侵入和粘附有关结构 伤寒沙门菌：invasome EPEC: microcolony 福氏志贺菌：IpaB、IpaC、IpaD 假结核耶氏菌：yopE • 载铁体的产生

  21. 伤寒沙门菌侵袭结构的产生和消失 invG、invC突变：不能合成侵袭体 invA、invE突变：能合成侵袭体，但不能收回 InvJ

  22. 黏附对细胞的影响 • 上皮细胞 • 成纤维细胞 • 吞噬细胞

  23. 细菌粘附对上皮细胞的影响 • 没有可见的影响： • 多数正常菌群 • 病原菌 （仅就粘附行为本身） • 形态上的改变：EPEC、H.pylori、S.pyogenes • 诱导细胞因子的释放：Oral streptococci、 H.pylori， Uropathogenic E.coli • 侵入：Salmonella、Neisseria

  24. 形态上的改变 EPEC的粘附 ‘‘attaching and effacing’’ (A/E) 1 the local effacement of microvilli 2 The formation of pedestal-like structures

  25. EPEC

  26. 诱导细胞因子的产生 • 口腔链球菌：IL-8 • E.coli： • IL-6、IL-8、IL-1α和IL-1β • 粘附诱导胞间粘附分子的表达 • 幽门螺杆菌 IL-6、IL-8 • 肺炎衣原体 ICAM-1上调

  27. 对成纤维细胞的作用 • 齿垢密螺旋体 • HGF细胞改变：伪足的回收和膜状泡的形成；丝状肌动蛋白网络重排；细胞从基质脱离；细胞死亡。 • 杜氏嗜血杆菌 • HFF：分裂细胞单层，改变细胞形态，最后使细胞死亡。 • 牙龈卟啉单胞菌 • 围绕细菌簇的长的微绒毛形成

  28. 对吞噬细胞的作用 • 释放细胞因子，诱导调亡等

  29. 研究前景 • 新抗菌策略: 干扰病原菌与宿主细胞的粘附 • 疫苗

  30. Bacterial Entry into Host Cells

  31. 细胞骨架Cytoskeleton • 既是细胞形态学的框架，同时也是发生信号传导和膜结构变化等细胞内反应的场所。所有细胞内外信号刺激引起的信号传递过程中，细胞骨架都参与其中，通过迅速重组改变局部或整个细胞的形态。不同细胞的细胞骨架发挥各自不同的作用，例如巨噬细胞和中性粒细胞凭借细胞骨架运动并穿过组织，吞噬入侵的细菌；而上皮细胞则靠肌动蛋白结构维持与相邻细胞以及细胞下结构的牢固粘附和连结。 • 改变细胞骨架 侵入细胞

  32. 细菌侵袭的机制 • 拉链机制 zipper mechanism • 细菌接触宿主细胞后，与宿主相应受体结合，激活宿主细胞信号转导系统，引发肌动蛋白细胞骨架重排。随后宿主膜包裹于菌体周围，细菌似陷入其中，此过程称为拉链机制。

  33. InlA binds to E-cadherin（species-specific） • recruitment of two catenins: β-catenin and a-catenin. • Actin polymerization in the InlA-dependent pathway relies on RhoGTPase Rac1, cortactin and Arp2/3 • myosin VIIA and its ligand vezatin are also • required, probably generating the tension required for internalization of engulfed bacteria . traverse three human barriers—the intestinal barrier, the blood-brain barrier, and the fetoplacental barrier—leading to several clinical manifestations including gastroenteritis, meningitis, or abortion

  34. Invasion of epithelial cells In the InlB-dependent pathway of Listeria, the loosely cell-wall-attached bacterial protein InlB interacts with the molecule gC1qR, and with the signaling receptor Met, which recruits several molecular adaptors, which will perform several functions including the recruitment of a PI3K (involved in the activation of the RhoGTPase Rac1 and the polymerization of actin), and also the ubiquitination of Met and the endocytosis of the receptor via a clathrin-dependent mechanisms. A balance between actin polymerization and actin depolymerization required for efficient bacterial entry is controlled by regulation of the activities of the Lim kinase and the actin depolymerizing factor cofilin

  35. The Yersinia outer membrane invasin interacts with β1 integrin receptors. • The short cytoplasmic tail of integrins has no enzymatic activity but recruits components that serve as linking or docking proteins for cytoskeletal-associated elements. FAK or the Src family of kinases are often associated with physiological integrin-mediated signaling, and Y. pseudotuberculosis subverts the function of these proteins to invade nonphagocytic cells. • Integrin engagement by invasin leads also to activation of several small GTPases, including Rac1, and Arf6, these proteins promote the recruitment to the bacterial entry site of the phosphatidylinositol-phosphate-5-kinase, inducing the local production of phosphatidylinositol-4,5-biphosphate, an important second messenger affecting the subcellular localization and activation of actin-regulating molecules to induce actin rearrangements at the site of bacterial entry, promoting invasion.

  36. 触发机制 trigger mechanism • 细菌与宿主细胞表面接触后引起快速、大范围的细胞骨架反应，胞浆膜下皱突的肌动蛋白丝聚合物伸出大型片状或伪足样结构，这种类似伪足的结构折返并与细胞表面融合，将膜包裹的细胞外物质卷入细胞内

  37. (B) Salmonella translocates several effectors into target cells, some of them allowing the initial uptake of the bacterium: SipC is part of the TTSS and drives actin polymerization and actin-filament bundling; SopE activates Rho GTPases, fostering actin polimerization and membrane ruffle formation; SopB modulates inositol-polyphosphate metabolism, activating indirectly the same Rho GTPases as SopE; and SipA blocks the actin depolymerization factor cofilin, favoring also membrane ruffle formation. SptP plays a role once the internalization has taken place, inactivating the Rho GTPases, inhibiting actin polymerization, and helping the closure of the plasma membrane over internalized bacteria.

  38. (C) Shigella also translocates several TTSS effectors into target cells to induce invasion: the translocon component IpaC nucleates the formation of actin filaments; VirA indirectly stimulates the RhoGTPase Rac1 favoring actin polymerization (the host tyrosine kinases Abl/Arg also activate indirectly Cdc42 and Rac1) and inhibits microtubule polymerization; IpgD affects phosphoinositide metabolism and promotes the extension of membrane ruffles by decreasing the interactions between the plasma membrane and the actin cytoskeleton; IpaA activates the host protein vinculin, inducing actin depolymerization and recovery of the plasma membrane architecture once the bacteria are internalized.

  39. 共同点 • 均要在细胞外细菌刺激信号跨膜传递后采能引起局部肌动蛋白聚合 • 信号激活宿主细胞内已有的信号级联传递模式 • 均需细菌表达的特殊毒力因子参与 • 能量均来自宿主细胞

  40. 细菌在细胞内的动力及播散 少数一些致病菌在宿主细胞溶解之前设法在细胞间进行播散。这些细菌分泌膜降解酶离开吞噬泡，在宿主细胞质中利用细胞骨架四处活动。这样，细菌可在细胞内运动和分裂，而包含细菌的膜延伸结构也可被邻近细胞吞入。致病菌通过这种独特的方式在宿主上皮细胞之间播散而始终处于细胞质中，从而逃避宿主抗体的作用。 • 李斯特菌，志贺菌，立克次体

  41. 产单核细胞李氏菌 • 细菌表面肌动蛋白丝的聚合和延伸是受细菌表面蛋白调控的，但细菌尾状结构的组成及活动力则是由宿主细胞控制的。 • 细菌表面蛋白 ActA • 促使肌动蛋白丝在细菌表面聚合 • 肌动蛋白丝重排形成一个极化的尾状结构以维持单一运动 • 凭借其特异序列加速细菌运动。 • 宿主蛋白 • Arp2/3复合物 • Profilin • VASP • α-辅肌动蛋白 • 维持尾状结构中的肌动蛋白微丝的交互连结，维持尾状结构的机械稳定从而推动细菌运动。

  42. 福氏志贺菌 • 志贺菌和李斯特菌的细胞内生活方式非常接近。形成与产单核细胞李氏菌非常相似的富含肌动蛋白的尾状结构 • IcsA（intercellular spread A）或VirG。 • 120Kda，具N端信号序列可传递信号至胞浆周区域，C端344个氨基酸序列（β结构域）可将蛋白运送至宿主细胞外膜并固着其上。其余706个氨基酸即α结构域与延伸至细胞外的蛋白N端结合，此结构域可能在与宿主细胞蛋白相互作用中发挥作用。α结构域具有5个富含甘氨酸的重复序列，可能与参与肌动蛋白丝聚合形成尾状结构的那些蛋白的结合密切相关。 • IcsA结合蛋白 • 肌动蛋白结合蛋白纽带蛋白 • 神经元Wiskott-Aldrich综合症蛋白(N-WASP)。

  43. 动力产生过程 • 细菌表面蛋白与宿主因子结合引起肌动蛋白聚合（李斯特菌Arp2/3复合物，志贺菌N-WASP和其它一些不明因子） • 肌动蛋白“云雾”在细菌一端形成尾状结构， • 在宿主细胞另一套因子作用下，肌动蛋白丝在细菌表面加速延伸，最终使细菌运动速度全面提高。 细菌动力所需能量均来自宿主细胞而非细菌本身，细菌凭借基于肌动蛋白的动力系统可到达宿主细胞表面，并在细胞之间播散

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