艰难梭菌荚膜的研究

本文采用提取细菌荚膜多糖的三种方法,提取艰难梭菌(Clostridium difficile)的荚膜多糖,经鉴定证实,该菌荚膜是由多糖和蛋白质两种成份组成。这不仅证明该菌存在荚膜,而且为进一步研究该菌的生物学特性提供了有利条件。     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

艰难梭菌荚膜多糖单糖组成的气相色谱分析

本文对两株艰难梭菌（ＣｄＡ３１８和ＣｄＣＤＣ２７９）荚膜多糖进一步分析,结果证实纯化的艰难梭菌荚膜多糖为单一均匀的物质;经气相色谱分析表明,其单糖组分主要由Ｄ－葡萄糖、Ｄ－甘露糖和Ｄ－半乳糖组成,不同菌株的荚膜多糖可能有微小差异。     

产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及小鼠口服免疫保护效力的测定

摘要: 【目的】构建产气荚膜梭菌（Clostridium perfringens, C．perfringens）α 毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法。【方法】将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌（Lactobacillus casei L.casei）,获得阳性重组菌pPG1-α/ L.casei 393 乳酸乳杆菌表面表达系统和pPG2-α/ L.casei393乳酸乳杆菌分泌表达系统。重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达。将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性sIgA 抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性IgG抗体水平。并对免疫小鼠进行α毒素的腹腔攻毒实验及对获得的抗血清进行α毒素中和试验测定。【结果】重组干酪乳杆菌pPG1-α/ L.casei 393及pPG2-/ L.casei 393免疫小鼠能够产生明显的抗α毒素的sIgA 和IgG 抗体水平,其对α毒素中和试验结果为完全保护。腹腔攻毒实验结果为能抵抗3倍最小致死剂量的α毒素攻击。【结论】表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力。     

产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及其小鼠口服免疫力

[目的]构建产气荚膜梭菌(Clostridium perfringens,C.perfringents)α毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法.[方法]将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌(Lactobacillus casei L.casei),获得阳性重组菌pPG1-α/L.case/393乳酸乳杆菌表面表达系统和pPG2-α/L.casei 393乳酸乳杆菌分泌表达系统.重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达.将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性slgA抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性lgG抗体水平.并对免疫小鼠进行α毒素的腹腔攻毒试验及对获得的抗血清进行α毒素中和试验测定.[结果]重组干酪乳杆菌pPG1-α/L.casei 393及pPG2-/L.casei 393免疫小鼠能够产生明显的抗α毒素的slgA和IgG抗体水平,其对α毒素中和试验结果为完全保护.腹腔攻毒实验结果为能抵抗3倍最小致死剂量(minimum lethal dose,MLD100)的α毒素攻击.[结论]表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力.     

猪链球菌2型荚膜缺失株生物学特性及其免疫保护性

对前期构建的猪链球菌2型中国强致病株05ZYH33荚膜缺失株Δcps2B进行相关生物学特性及免疫保护性研究。通过比较野生株05Z33和荚膜缺失株Δcps2B生物学特性发现,Δcps2B株显微形态发生改变,失去与2型荚膜特异抗血清发生凝集反应的能力,突变株更易被全血清除,对上皮细胞HEP-2的粘附能力增强。动物保护性实验结果表明该荚膜缺失株Δcps2B具有良好的免疫保护作用。研究结果提示荚膜在细菌抵抗吞噬和细菌粘附过程中发挥重要作用。     

酪酸梭菌与婴儿型双歧杆菌对产气荚膜梭菌试管内生物拮抗作用的研究

用酪酸权菌（Clostridium butyricum）和婴儿型双歧杆菌（Bifidobacterium Infantis）对产气荚膜梭菌（Clostriduim perfringens）进行试管内的生物拮抗试验。将酪酸梭菌、婴儿型双歧杆菌及酪酸梭菌和婴儿型双歧杆菌分别对产气荚膜酸菌以一定的比例等量混合接种于GAM液体培养基中进行厌氧培养。实验证明酪酸梭菌和婴儿型双歧能明显抑制产气荚膜梭菌的生长,并且比各自单独培养时显示了较强的生物拮抗作用。     

kpsE和kpsD双基因缺失显著降低肠道外致病性大肠杆菌的毒力

【目的】探究荚膜对肠道外致病性大肠杆菌致病作用的影响。【方法】选取负责荚膜多糖转运的基因kpsE和kpsD,利用λRed重组系统构建APEC E058和UPEC U17荚膜缺失株E058ΔkpsED和U17ΔkpsED,并通过一系列的体内及体外试验对其生物学特性及致病性进行研究。【结果】双基因缺失株的生长速度较野生株没有明显差异,但缺失株抗血清补体杀菌能力和抗鸡巨噬细胞HD-11细胞吞噬能力显著下降。1日龄雏鸡LD50致病性试验结果显示,缺失株E058ΔkpsED和U17ΔkpsED对鸡失去致病力,而回复株毒力恢复至野生株水平;35日龄SPF鸡体内动态分布和竞争试验显示ΔkpsED缺失株在鸡体内定殖能力和竞争性生长能力显著下降,表明kpsED双基因的缺失能显著降低APEC E058和UPEC U17的致病力。【结论】荚膜与肠道外致病性大肠杆菌的致病性相关,是其重要的毒力因子。     

假想的脂蛋白连接酶对肺炎链球菌毒力的影响

【目的】探索假想脂蛋白连接酶(putative lipoate-protein ligase,LPL)对肺炎链球菌毒力的影响。【方法】采用长臂同源多聚酶链式反应(LFH-PCR)的方法失活lpl基因,通过PCR、测序鉴定缺陷菌株,采用细胞实验比较缺陷菌和野生菌对宿主细胞的粘附能力,并通过动物实验观察lpl基因缺陷后菌株毒力的变化。【结果】小鼠毒力实验表明野生菌株和缺陷株半数致死时间均为12h,两者比较无统计学差异;缺陷菌在对宿主细胞的粘附能力明显高于野生菌株(P0.01);体外荚膜染色实验表明,野生菌和缺陷菌均有荚膜。【结论】实验结果提示lpl基因对细菌粘附宿主细胞有抑制作用,但不影响其腹腔感染小鼠的能力。     

对引入仓鼠菌群的C_3H无菌小鼠拮抗艰难梭菌定居实验模型的研究

正常成年仓鼠肠道菌群可拮抗艰难梭菌在该动物肠道中定居。将该菌群转移到无菌小鼠并经过抗生素和热(70℃,10min)简化处理后,获得一组由严格厌氧菌组成的菌群,仍有拮抗艰难梭菌的能力,由此建立了一研究拮抗艰难梭菌在肠道中定居的实验动物模型。作者研究了屏障菌群与艰难梭菌相互作用的可能机制,并对与艰难梭菌有关的伪膜性结肠炎的病原学进行了讨论。     

The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site

Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd), attL, attR, attTn, and attB(Bs)(3) with relative binding affinities attTn approximately attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.     

Pathogenicity of Bacteroides fragilis group in rat intra-abdominal abscesses.

Attempts were made to study the pathogenicity of some strains of Bacteroides fragilis group in the rat intra-abdominal abscess model. Multiple intraabdominal abscesses were produced in 50 to 70% of animals when an inoculum containing 10(9) CFU/ml of any of the five species of Bacteroides fragilis group was injected. Rising homologous antibody titers determined by indirect fluorescent antibody test were observed till the 3rd week when tested last, indirectly confirming the multiplication of the organisms as also evident by viable count of bacteria in the abscesses. In some cases in addition to inoculated organisms some intestinal bacteria like Escherichia coli, Proteus mirabilis and Streptococcus spp. were also recovered from the abscess pus. Studies with the electron microscope showed presence of capsular polysaccharide only in Bacteroides fragilis and Bacteroides thetaiotaomicron. It was doubtful in Bacteroides distasonis and absent in Bacteroides ovatus and Bacteroides vulgatus, suggesting that virulence factor beside the capsular polysaccharide may be playing a role. Further studies are required to investigate the virulence factor responsible for the pathogenicity of noncapsulated species.     

细菌荚膜多糖

随着分子生物学、糖化学和免疫学的发展,细菌荚膜多糖的研究逐步深入.不仅对其特性及组成有了进一步的了解,而且对其多糖合成相关基因、合成调节和致病性进行了更为细致的研究.本文概括了细菌荚膜多糖的化学结构、合成相关基因、多样性产生机制、合成调节、功能与致病性和应用前景,总结其研究热点,以期为荚膜多糖的研究和应用提供理论依据和思路.     

Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface

Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants.     

悉生小鼠体内大肠杆菌和青春型双歧杆菌对艰难梭菌的拮抗作用

本文应用悉生小鼠做模型,研究了大肠杆菌(E.coli)和青春型双歧杆菌(Bifidobacterium adolescentis)对艰难梭菌(Clostridium diffi-cile)的拮抗作用。E.coli和B.adolescentis预先接种无菌SSB小鼠,再用C.difficile攻击。结果表明,E.coli和E.coli B.adolescentis对小鼠均有保护作用,保护平分别为87.5%(7/8)和100%(8/8)。B.adolescentis定值后数量达10~(10.28)CFU/g,且对E.coli数量和小鼠本身无影响。E.coli和B.adolescentis联合比E.coli单独抑制C.difficile在肠道中繁殖的作用更强(0.02>P>0.01),但对其毒素产生和粘附力的作用无明显差异。C.difficile攻击后的1～14天,小鼠粪便中C.difficile菌数在10~4至10~8CFU/g内变化,细胞毒素为10~3CFU/g,A毒素滴度为10~2/g,B.adolescentis也一度下降10~2CFU/g。接种C.difficile后,小鼠虽无明显的腹泻症状,但组织学仍可观察到肠粘膜有充血和分泌增加等轻度损害。扫描电镜和普通光镜均发现E.coli单独或与B.adolescentis共同吸附在肠粘膜微绒毛表面,未见有C.difficile吸附。     

肺炎链球菌pcsB组成型表达的yycF缺陷菌株的构建及其致病能力

【背景】YycFG双组分系统是肺炎链球菌(Streptococcus pneumoniae,S. pn)应对外界环境的重要信息传递系统,其中表达反应调节子YycF的编码基因是肺炎链球菌生长的必需基因,但其是否调控细菌毒力尚不清楚。【目的】构建肺炎链球菌pcsB组成型表达及yycF缺陷菌,分析YycF对肺炎链球菌生物学特征和毒力的影响。【方法】采用Janus cassette (JC)反选的方法构建pcsB组成型表达菌株(Pc-PcsB~+),从该菌株出发用替代失活的方法构建yycF缺陷菌株(Pc-PcsB~+DyycF),比较野生株D39rpsl41、pcsB组成型表达株及yycF缺陷株的生长特性、荚膜多糖(capsular polysaccharide,CPS)含量、粘附侵袭能力和致病性的差异。【结果】成功构建pcsB组成型表达的yycF缺陷菌株(Pc-PcsB+DyycF);yycF缺陷导致细菌生长缓慢、分裂异常、胞内荚膜多糖和小分子荚膜多糖增多;体外实验结果显示,yycF缺陷菌株粘附能力较Pc-PcsB~+菌株减弱(P=0.006)。体内毒力实验显示,感染野生菌的小鼠全部死亡,感染Pc-PcsB+和Pc-PcsB~+DyycF菌株的小鼠死亡率分别为91.7%、75%,二者没有统计学差异(P=0.183),但Pc-PcsB~+DyycF菌株感染组有降低趋势;定殖结果显示,yycF缺陷菌株感染组的肺匀浆菌载量显著低于对照组(P=0.033)。【结论】成功构建yycF缺陷菌株,并初步证明yycF基因会影响肺炎链球菌的生物性状和致病能力,为后续探讨YycFG双组分系统对肺炎链球菌致病能力调控机制的研究奠定了基础。     

Evidence that Clostridium difficile TcdC is a membrane-associated protein

Clostridium difficile produces two toxins, A and B, which act together to cause pseudomembraneous colitis. The genes encoding these toxins, tcdA and tcdB, are part of the pathogenicity locus, which also includes tcdC, a putative negative regulator of the toxin genes. In this study, we demonstrate that TcdC is a membrane-associated protein in C. difficile.     

Phosphorylation of the Synthetic Hexasaccharide Repeating Unit Is Essential for the Induction of Antibodies to Clostridium difficile PSII Cell Wall Polysaccharide

Clostridium difficile is emerging worldwide as a major cause of nosocomial infections. The negatively charged PSII polysaccharide has been found in different strains of C. difficile and, thereby, represents an important target molecule for a possible carbohydrate-based vaccine. In order to identify a synthetic fragment that after conjugation to a protein carrier could be able to induce anti-PSII antibodies, we exploited a combination of chemical synthesis with immunochemistry, confocal immunofluorescence microscopy, and solid state NMR. We demonstrate that the phosphate group is crucial in synthetic glycans to mimic the native PSII polysaccharide; both native PSII and a phosphorylated synthetic hexasaccharide repeating unit conjugated to CRM(197) elicit comparable immunogenic responses in mice. This finding can aid design and selection of carbohydrate antigens to be explored as vaccine candidates.