Immunochemical and partial chemical characterization of fractions of membrane-bound smooth lipopolysaccharide-protein complex from Brucella abortus

Smooth lipopolysaccharide (sLPS) of Brucella abortus was prepared and fractionated by a modification of the procedures of Moreno et al. (J. Bac. 138:361–369, 1979). Washed B. abortus cells were disrupted by 21 freeze-quick thaw cycles with ultrasonication to separate the non-membrane-bound material. Ultrasonicated bacteria were used for preparation of membrane-bound sLPS ( f5, the main crude sLPS fraction described by Moreno et al.). Phenol extraction was repeated 3 times and then washed with H₂O 10 times to remove most of the chromogen, polysaccharides and nucleic acids, eliminating the need for enzyme treatment as described previously. The membrane-bound sLPS was fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation, these fractions required only 80 ng for positive ELISA, about 0.2 ng for positive Limulus lysate tests, and reacted well with precipitating antibodies in the serum from a strain 2308 infected cow. They had marked differences in precipitin curves and chemical composition. The protein content varied from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which implies that the proteins associated with LPS may also play important roles in the complex for the immunochemical interactions and the heterogeneity of B. abortus lipopolysaccharide protein complex. As compared with previous reports, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, was obtained. Group f5A, which had a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19 and 2308). The amount of other subfractions obtained varied with batches or strains of B. abortus. These results provide a new profile of the immunochemical reactivities and the heterogeneity on B. abortus smooth membrane-bound endotoxins.Abbreviations LPS lipopolysaccharides - sLPS smooth lipopolysaccharides - cLPS crude lipopolysaccharides - AH acid hapten - KDO 3-deoxyoctulosonic acid - ELISA enzyme-linked immunosorbent assay - f5 the main crude sLPS described by Mooreno et al. [2] - LAL limulus amoebocyte lysate test - HexN Hexosamine - PS phenol sulfuric method - O orcinol method     

The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells

We have previously determined the protective efficacy of intranasal vaccination with chlamydial protease-like activity factor (CPAF) against genital chlamydial infection. Since T-helper 1 (Th1) responses are important for anti-chlamydial immunity, we examined the contribution of CD4(+) T cells in CPAF mediated immunity against intravaginal (i.vag.) Chlamydia muridarum infection in C57BL/6 mice. CPAF+IL-12 vaccination induced antigen-specific CD4(+) T cells that secreted elevated levels of IFN-gamma, and generated strong humoral responses. The protective effects of CPAF vaccination against genital chlamydial challenge were abrogated by anti-CD4 neutralizing antibody treatment. Moreover, anti-chlamydial immunity could be adoptively transferred to na?ve recipients using CPAF-specific CD4(+) T cells. Therefore, CPAF mediated anti-chlamydial immunity is highly dependent upon antigen-specific CD4(+) T cells.     

Identification of active site residues of the inverting glycosyltransferase Cgs required for the synthesis of cyclic beta-1,2-glucan, a Brucella abortus virulence factor

Brucella abortus cyclic glucan synthase (Cgs) is a 320-kDa (2868-amino acid) polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive beta-1,2-autoglucosyltransferase and has the three enzymatic activities required for the synthesis of the cyclic glucan: initiation, elongation, and cyclization. To gain further insight into the protein domains that are essential for the enzymatic activity, we have compared the Cgs sequence with other glycosyltransferases (GTs). This procedure allowed us to identify in the Cgs region (475-818) the widely spaced D, DxD, E/D, (Q/R)xxRW motif that is highly conserved in the active site of numerous GTs. By site-directed mutagenesis and in vitro and in vivo activity assays, we have demonstrated that most of the amino acid residues of this motif are essential for Cgs activity. These sequence and site-directed mutagenesis analyses also indicate that Cgs should be considered a bi-functional modular GT, with an N-terminal GT domain belonging to a new GT family related to GT-2 (GT-84) followed by a GH-94 glycoside hydrolase C-terminal domain. Furthermore, over-expression of inactive mutants results in wild-type (WT) production of cyclic glucan when bacteria co-express the mutant and the WT form, indicating that Cgs may function in the membrane as a monomeric enzyme. Together, these results are compatible with a single addition model by which Cgs acts in the membrane as a monomer and uses the identified motif to form a single center for substrate binding and glycosyl-transfer reaction.     

沙眼衣原体感染后大鼠输卵管与植物凝集素BS-1结合的糖蛋白的变化

目的研究沙眼衣原体感染后输卵管与植物凝集素BS-1结合的糖蛋白的变化.方法取25只成熟Wistar雌性大鼠随机分为实验组和对照组,每组5只.对照组接种2SP代替沙眼衣原体,其余每组大鼠均在卵巢囊接种沙眼衣原体D型株.分别于感染后3,5,7,14d处死动物,取输卵管,观察大鼠输卵管与植物凝集素BS-1结合的糖蛋白在感染后不同天数发生的改变,其变化可通过荧光显微镜观察与植物凝集素BS-1结合后的荧光强度来检测,并采用HPIAS-1000图文分析系统进行定量分析和SPSS11.0统计软件作统计分析.结果输卵管受损部位主要在粘膜层,富含N-乙酰-D-半乳糖胺的糖蛋白随感染的天数不同,其变化有所不同,尤其以输卵管峡部变化明显.图像分析结果显示:感染第7d时其分泌量最低,感染后第14d其分泌量逐渐恢复,且各组间差异有显著性意义(P<0.05).结论输卵管是精子与卵子结合的地方,也是胚胎早期发育的场所.富含N-乙酰-D-半乳糖胺的糖蛋白可通过参与精卵结合、受精卵卵裂和早期胚胎发育等方式,对受精卵及早期胚胎发挥保护作用.     

Structure and Function of the Autolysin SagA in the Type IV Secretion System of Brucella abortus

A recent genetic study with Brucella abortus revealed the secretion activator gene A (SagA) as an autolysin component creating pores in the peptidoglycan (PGN) layer for the type IV secretion system (T4SS) and peptidoglycan hydrolase inhibitor A (PhiA) as an inhibitor of SagA. In this study, we determined the crystal structures of both SagA and PhiA. Notably, the SagA structure contained a PGN fragment in a space between the N- and C-terminal domains, showing the substrate-dependent hinge motion of the domains. The purified SagA fully hydrolyzed the meso-diaminopimelic acid (DAP)-type PGN, showing a higher activity than hen egg-white lysozyme. The PhiA protein exhibiting tetrameric assembly failed to inhibit SagA activity in our experiments. Our findings provide implications for the molecular basis of the SagA-PhiA system of B. abortus. The development of inhibitors of SagA would further contribute to controlling brucellosis by attenuating the function of T4SS, the major virulence factor of Brucella.     

马流产沙门菌重组鞭毛蛋白FliC和FljB免疫原性比较

为评价与比较马流产沙门菌(Salmonella abortus equi)2种不同鞭毛蛋白FliC(Flagellin C)和FljB(Flagellin B)的免疫原性,为进一步利用这两种蛋白奠定实验基础,本研究诱导表达及纯化FliC和FljB蛋白,将纯化后的蛋白分别免疫小鼠,对免疫后小鼠的抗体水平、滴度及抗体亚型进行检测,攻击小鼠后进行免疫相关受体检测及病理组织学观察。结果显示,成功诱导表达了重组蛋白FliC和FljB,纯化后蛋白的相对分子量分别为52 kDa和42 kDa。两种蛋白分别免疫小鼠,产生较高水平的特异性抗体IgG,FljB免疫组抗体水平高于FliC免疫组,且抗体亚型以IgG1为主。FljB免疫组攻击保护率为87.5%,高于FliC免疫组的75%,病理组织学及脏器荷菌数检测结果显示,FljB免疫组效果优于FliC免疫组;FljB免疫组诱导TLR2、TLR4、MHC-I、TCR(T细胞抗原受体)的水平均高于FliC免疫组。该结果表明,FljB免疫组诱导小鼠免疫应答的水平高于FliC免疫组。     

肥大细胞在大鼠输卵管急性沙眼衣原体感染中的作用

目的　为研究肥大细胞在大鼠输卵管急性沙眼衣原体 (Chlamydialtrachomatis,CT)感染中的作用。方法　选择成年雌性SD大鼠 6 0只 ,通过手术从一侧卵巢囊接种沙眼衣原体D型株 ,对照组接种 2 -SPA缓冲液。分别于感染后第 1/ 2d、第 7d、第 14d将大鼠处死 ,取手术侧的输卵管常规固定、脱水、包埋。结果　S -P法显示 :输卵管局部的CD4 + T细胞和血管内皮细胞粘附分子 (VCAM - 1)的表达均较对照组明显增强 (P <0 0 1)。改良的甲苯胺蓝染色法显示 :感染组肥大细胞较对照组数量有显著性增高 (P <0 0 5 ) ,并且其变化趋势与CD4 + T细胞和VCAM - 1表达的变化趋势一致。结论　可以推测 ,沙眼衣原体感染引起急性输卵管炎时 ,肥大细胞通过促进炎症局部小静脉内皮细胞上VCAM -1的表达 ,诱导CD4 + T细胞的浸润 ;然后分泌IL - 4等细胞因子促进CD4 + T细胞向TH2 细胞方向转化 ,不利于机体清除沙眼衣原体 ,从而使发生局部输卵管病理损伤的可能性增加。     

Detection of a specifically amplified DNAfragment in Brucella abortus by arbitrarilyprimed polymerase chain reaction

Randomly amplified polymorphic DNA (RAPD) profiles of Brucella and non-Brucella DNA were established after polymerase chain reaction (PCR) amplification. Five arbitrary oligonucleotide primers were screened to generate Brucella-specific DNA fingerprints. The arbitrary primer OPB-01 (5-GTTTCGCTCC-3) produced DNA bands specific to Brucella. Amplification conditions must be optimized for reproductibility. Accordingly, we optimized and established the conditions, which included Mg²⁺, enzyme (DNA polymerase), primer, template and deoxyribonucleoside triphosphate (dNTP) concentrations as well as the optimum number of thermal cycles to produce OPB-01 directed Brucella DNA fingerprints.The optimized RAPD method can produce a 1.3 kb DNA fragment specific to Brucella. This DNA fragment was common to eight biovars of B. abortus and one biovar of B. melitensis. The fragment was not detected in genetically related species such as Ochrobactrum anthropi and other non-Brucella organisms associated with farm animals. We anticipate the use of this fragment as a possible probe for the detection of Brucella organisms.