Antigenic Variation among Bordetella: BORDETELLA BRONCHISEPTICA STRAIN MO149 EXPRESSES A NOVEL O CHAIN THAT IS POORLY IMMUNOGENIC*

The O chain polysaccharide (O PS) of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharide is a homopolymer of 2,3-diacetamido-2,3-dideoxygalacturonic acid (GalNAc3NAcA) in which some of the sugars are present as uronamides. The terminal residue contains several unusual modifications. To date, two types of modification have been characterized, and a survey of numerous strains demonstrated that each contained one of these two modification types. Host antibody responses against the O PS are directed against the terminal residue modifications, and there is little cross-reactivity between the two types. This suggests that Bordetella O PS modifications represent a means of antigenic variation. Here we report the characterization of the O PS of B. bronchiseptica strain MO149. It consists of a novel two-sugar repeating unit and a novel terminal residue modification, with the structure Me-4-α-l-GalNAc3NAcA-(4-β-d-GlcNAc3NAcA-4-α-l-GalNAc3NAcA-)_5–6-, which we propose be defined as the B. bronchiseptica O3 PS. We show that the O3 PS is very poorly immunogenic and that the MO149 strain contains a novel wbm (O PS biosynthesis) locus. Thus, there is greater diversity among Bordetella O PSs than previously recognized, which is likely to be a result of selection pressure from host immunity. We also determine experimentally, for the first time, the absolute configuration of the diacetimido-uronic acid sugars in Bordetella O PS.     

Three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide

The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (αHexN-(1,4)-αGalA) attached by an α1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-α-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase.     

MCP-induced protein 1 suppresses TNFα-induced VCAM-1 expression in human endothelial cells

Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. We here report that treatment of human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor (TNF) α substantially increased the expression of MCP-induced protein 1 (MCPIP1). Overexpression of MCPIP1 protected ECs against TNFα-induced endothelial activation, as characterized by the attenuation in the expression of the adhesion molecule VCAM-1 and monocyte adherence to ECs. Conversely, small interfering RNA-mediated knock down of MCPIP1 increased the expression of VCAM-1 and monocytic adherence to ECs. These studies identified MCPIP1 as a feedback control of cytokines-induced endothelial inflammation.     

Receptors and signaling mechanisms for B-lymphocyte activation, proliferation and differentiation - Insights from both in vivo and in vitro approaches

During the last three decades, a number of B-lymphocyte specific surface antigens have been defined some of which may also show activation/differentiation specific expression. Here, we review the various signaling events and the receptor-ligand interactions for B-cell development, activation and differentiation. Our discussion and presentation include reviewing the in vivo and in vitro mechanisms. Focus is on the experiments that give us valuable insights into the B cell signaling mechanisms in vitro. Three significant pathways in B-cell development - c-Kit, FLT-3 and IL-7 signaling pathways are elucidated upon. Both antigen dependent and antigen independent mechanisms of B cell stimulation are also reviewed.     

Lancemaside A inhibits lipopolysaccharide‐induced inflammation by targeting LPS/TLR4 complex

In our previous study, lancemaside A isolated from Codonopsis lanceolata (family Campanulaceae) ameliorated colitis in mice. In this study, the anti‐inflammatory effects of lancemaside A was investigated in lipopolysaccharide (LPS)‐stimulated mice and their peritoneal macrophage cells. Lancemaside A suppressed the production of pro‐inflammatory cytokines, TNF‐α and IL‐1β, in vitro and in vivo. Lancemaside A also down‐regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), as well as the inflammatory mediators, nitric oxide (NO), and PGE₂. Lancemaside A also inhibited the expression of IL‐1 receptor‐associated kinase‐4 (IRAK‐4), the phosphorylation of IKK‐β and IκB‐α, the nuclear translocation of NF‐κB and the activation of mitogen‐activated protein kinases in LPS‐stimulated peritoneal macrophages. Furthermore, lancemaisde A inhibited the interaction between LPS and TLR4, as well as IRAK‐4 expression in peritoneal macrophages. Based on these findings, lancemaside A expressed anti‐inflammatory effects by regulating both the binding of LPS to TLR4 on macrophages. J. Cell. Biochem. 111: 865–871, 2010. © 2010 Wiley‐Liss, Inc.     

痢疾结合疫苗LPS制备条件的优化研究

通过对痢疾杆菌LPS提取过程中主要制备环节的优化和改进,确立最佳提取条件和纯化过程,并应用优化后的工艺路线分别制备八批次福氏2 a痢疾杆菌和宋内氏痢疾杆菌LPS;福氏2 a痢疾杆菌LPS批平均产量为1.633g,宋内氏痢疾杆菌LPS批产量平均为1.251g,批产量相对稳定,平均产量比优化改进前提高20%以上。LPS经过酸水解、柱层析纯化获得目标O-SP,福氏2 a痢疾杆菌和宋内氏痢疾杆菌O-SP的得率分别为20%和28%。检测结果证明,LPS和O-SP各项指标均符合规程(草案)相关要求。实验为今后痢疾结合疫苗大规模制备工艺的改进和提高打下了基础。     

SARS冠状病毒PLpro蛋白酶的结构与功能

SARS冠状病毒基因组编码2种病毒蛋白酶,即木瓜样蛋白酶（PLpro）和3C样蛋白酶(3CLpro).其中,PLpro蛋白酶结构与功能研究是近年来冠状病毒分子生物学研究的热点之一. PLpro蛋白酶参与SARS冠状病毒1a(1ab)复制酶多聚蛋白N端部分的切割加工,是SARS冠状病毒复制酶复合体(RC)形成的重要调节蛋白分子;最新研究表明,SARS冠状病毒PLpro蛋白酶是一种病毒编码的去泛素化酶（DUB）,对细胞蛋白具有明显去泛素化作用;而且对泛素(Ub)和泛素样分子ISG15均具有活性. PLpro蛋白酶对宿主抗病毒天然免疫反应具有负调节作用,是SARS冠状病毒的一种重要干扰素拮抗分子.PLpro蛋白酶是一种多功能病毒蛋白酶.本文结合作者课题组研究工作,对SARS冠状病毒PLpro蛋白酶结构和功能研究最新进展进行综述.     

分泌型卷曲相关蛋白家族在Wnt信号通路中的双向调节作用

Wnt信号通过直接参与细胞的增殖、极化和命运特化,控制胚胎发育和成体稳态,其信号异常不仅会造成发育缺陷,而且与多种癌症和代谢性疾病的发生密切相关。分泌型卷曲相关蛋白(secreted frizzled-related proteins,sFRPs)是一种可溶性蛋白质,因其结构与Wnt信号的卷曲蛋白(frizzled,Fz)受体高度同源而被认为是一类Wnt通路拮抗剂。但随着对sFRPs家族的深入研究,发现sFRPs在Wnt信号通路传导过程中并不局限于作为一种拮抗因子,还发挥激活因子的功能。最新研究还发现,sFRPs不仅作为经典的胞外因子发挥作用,在一些肿瘤干细胞中还可进入细胞核双向调节(拮抗或激活)Wnt信号传导。本文结合最新研究,全面综述了sFRPs家族蛋白在Wnt信号传导中的双向调节作用,这有助于理解sFRPs蛋白在生物体器官发育和疾病发生中的作用。     

Autophagy-dependent PELI3 degradation inhibits proinflammatory IL1B expression

Lipopolysaccharide (LPS)-induced activation of TLR4 (toll-like receptor 4) is followed by a subsequent overwhelming inflammatory response, a hallmark of the first phase of sepsis. Therefore, counteracting excessive innate immunity by autophagy is important to contribute to the termination of inflammation. However, the exact molecular details of this interplay are only poorly understood. Here, we show that PELI3/Pellino3 (pellino E3 ubiquitin protein ligase family member 3), which is an E3 ubiquitin ligase and scaffold protein in TLR4-signaling, is impacted by autophagy in macrophages (MΦ) after LPS stimulation. We noticed an attenuated mRNA expression of proinflammatory Il1b (interleukin 1, β) in Peli3 knockdown murine MΦ in response to LPS treatment. The autophagy adaptor protein SQSTM1/p62 (sequestosome 1) emerged as a potential PELI3 binding partner in TLR4-signaling. siRNA targeting Sqstm1 and Atg7 (autophagy related 7), pharmacological inhibition of autophagy by wortmannin as well as blocking the lysosomal vacuolar-type H⁺-ATPase by bafilomycin A₁ augmented PELI3 protein levels, while inhibition of the proteasome had no effect. Consistently, treatment to induce autophagy by MTOR (mechanistic target of rapamycin (serine/threonine kinase)) inhibition or starvation enhanced PELI3 degradation and reduced proinflammatory Il1b expression. PELI3 was found to be ubiquitinated upon LPS stimulation and point mutation of PELI3-lysine residue 316 (Lys316Arg) attenuated Torin2-dependent degradation of PELI3. Immunofluorescence analysis revealed that PELI3 colocalized with the typical autophagy markers MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) and LAMP2 (lysosomal-associated membrane protein 2). Our observations suggest that autophagy causes PELI3 degradation during TLR4-signaling, thereby impairing the hyperinflammatory phase during sepsis.