生物膜状态变异链球菌临床株合成胞外多糖能力的研究

目的:了解不同致龋力的变异链球菌临床分离株(593号高致龋力临床株和18号低致龋力临床株)在生物膜状态不同时间段合成胞外多糖的能力差异.方法:①在聚苯乙烯塑料片上分别形成3、12、20 h变异链球菌生物膜标本,用异硫氰酸荧光素标记的伴刀豆球蛋白A对其中的胞外多糖进行染色,激光共聚焦扫描显微镜观察.②采用静置法培养形成粘...     

激光共聚焦扫描显微镜观察变异链球菌高致龋力临床株与标准株合成胞外多糖能力差异

目的了解变异链球菌(简称变链菌)593号高致龋力临床分离株在生物膜状态不同时期与标准株ATCC25175(均为血清型c)合成胞外多糖的能力差异。方法在聚苯乙烯塑料片上分别形成3、12、20 h变链菌生物膜标本,采用异硫氰酸荧光素标记的伴刀豆球蛋白A对合成的胞外多糖进行染色,用激光共聚焦扫描显微镜(CLSM)观察。采用静置法培养形成黏附生长的细菌,蒽酮法测定3～24 h其合成胞外多糖的量。结果胞外多糖与染料结合后发出绿色荧光,各时间段593号临床株绿色荧光的分布较ATCC 25175标准株更致密和广泛。3～20 h 593号临床株合成水溶性葡聚糖能力强于标准株(P<0.05);3～16 h 593号临床株合成水不溶性葡聚糖能力强于标准株(P<0.05);其他时间两者合成胞外多糖的能力差异无统计学意义(P>0.05)。结论在生物膜形成过程中两菌株合成胞外多糖的模式相同,均随时间延长而合成量增加,但在生物膜形成早期(3～16 h)高致龋力临床株表现出不同于标准株的胞外多糖合成模式,其更强的合成胞外多糖能力可能是其具高致龋力的原因之一,提示研究致龋机制时使用临床株作为研究样本较标准株更为敏感。     

Inhibitory effect of Lactobacillus salivarius on Streptococcus mutans biofilm formation

Dental caries arises from an imbalance of metabolic activities in dental biofilms developed primarily by Streptococcus mutans. This study was conducted to isolate potential oral probiotics with antagonistic activities against S. mutans biofilm formation from Lactobacillus salivarius, frequently found in human saliva. We analysed 64 L. salivarius strains and found that two, K35 and K43, significantly inhibited S. mutans biofilm formation with inhibitory activities more pronounced than those of Lactobacillus rhamnosus GG (LGG), a prototypical probiotic that shows anti‐caries activity. Scanning electron microscopy showed that co‐culture of S. mutans with K35 or K43 resulted in significantly reduced amounts of attached bacteria and network‐like structures, typically comprising exopolysaccharides. Spot assay for S. mutans indicated that K35 and K43 strains possessed a stronger bactericidal activity against S. mutans than LGG. Moreover, quantitative real‐time polymerase chain reaction showed that the expression of genes encoding glucosyltransferases, gtfB, gtfC, and gtfD was reduced when S. mutans were co‐cultured with K35 or K43. However, LGG activated the expression of gtfB and gtfC, but did not influence the expression of gtfD in the co‐culture. A transwell‐based biofilm assay indicated that these lactobacilli inhibited S. mutans biofilm formation in a contact‐independent manner. In conclusion, we identified two L. salivarius strains with inhibitory activities on the growth and expression of S. mutans virulence genes to reduce its biofilm formation. This is not a general characteristic of the species, so presents a potential strategy for in vivo alteration of plaque biofilm and caries.     

Structural and functional analysis of de‐N‐acetylase PgaB from periodontopathogen Aggregatibacter actinomycetemcomitans

The oral pathogen Aggregatibacter actinomycetemcomitans uses pga gene locus for the production of an exopolysaccharide made up of a linear homopolymer of β‐1,6‐N‐acetyl‐d ‐glucosamine (PGA). An enzyme encoded by the pgaB of the pga operon in A. actinomycetemcomitans is a de‐N‐acetylase, which is used to alter the PGA. The full length enzyme (AaPgaB) and the N‐terminal catalytic domain (residues 25–290, AaPgaBN) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the AaPgaB enzymes were determined using 7‐acetoxycoumarin‐3‐carboxylic acid as the substrate. The AaPgaB enzymes displayed significantly lower de‐N‐acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis, a member of the CE4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of AaPgaBN was determined. The AaPgaBN structure has two metal ions in the active site instead of one found in other CE4 enzymes. Based on the crystal structure comparisons among the various CE4 enzymes, two residues, Q51 and R271, were identified in AaPgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with AaPgaB, validating the requirement that an activating aspartate residue in the active site is essential for higher activity. In summary, our study provides the first structural evidence for a di‐nuclear metal site at the active site of a member of the CE4 family of enzymes, evidence that AaPgaBN is catalytically active and that mutant Q51E exhibits higher de‐N‐acetylase activity.     

Curative Effect of Crude Exopolysaccharides of Some Macrofungi on Alcohol-induced Liver Damage

Abstract

The authors investigate the curative effects of crude exopolysaccharides (EPS) produced by four Basidomycetes strains on the symptoms of alcoholic liver injury. EPSs were administered to experimental groups at levels of 100?mg per kg body weight per day for 7 days using an oral zonde needle after the onset of the disease. Serum levels of alanine transaminase (ALT), lactate dehydrogenase (LDH), DNA fragmentation, caspase-3 activities, and mitochondrial outer membrane integrity were determined following sacrifice of the rats. Light and transmission electron microscope (TEM) studies were performed on liver sections for histopathological and cytological evaluations. EPS that was obtained from Coprinus comatus OBCC 1014 decreased serum ALT level and increased outer membrane integrity, and allowed for the regaining of histologically and cytologically normal hepatocyte and tissue views. As a result, based on the obtained data, it can be argued that among all studied mushroom strains crude exopolysaccharides from Coprinus comatus OBCC 1014 strain have therapeutic potential for alcoholic liver injury according to control groups.     

Exploring two contrasting surface‐active exopolysaccharides from a single strain of Ochrobactrum utilizing different hydrocarbon substrates

During production and characterization of exopolysaccharides (EPS) of Ochrobactrum pseudintermedium C1, it was observed that an experimental change in the basic hydrocarbon type of substrate for bacterial utilization led to elicitation of different surface‐active properties in the EPS produced. In the sugar substrate, it elicited surfactant property, while in oil substrates it elicited emulsifying property, which indicated that the EPS might be different. Consequently, attention was focused on a detailed analysis of this substrate‐specific EPS. Utilizing waste sugar, edible, and mineral oil substrates, EPS produced in each situation was characterized. Besides estimating surface activity and thermostability, each substrate‐specific EPS was analyzed by Fourier‐transform infrared spectroscopy, gas chromatography‐mass spectroscopy, ¹H‐nuclear magnetic resonance, and matrix‐assisted laser desorption/ionization—time of flight mass spectroscopy to find any structural difference. The results were significantly contrasting although the similarity in molecular mass suggested a basic similarity in polysaccharide structure. Morphological differences were also evident both macroscopically and microscopically with scanning electron microscopy. As the surface‐active property of EPS was dependent on the substrate utilized, their structural differences might account for it. These diverse surface activities of EPS produced by a single bacterial strain simply by changing the nature of substrate would also augment their bioapplications. Moreover, utilization of waste and easily available substrates should make such applications convenient, ecofriendly, and cost‐worthy.     

Lactobacillus casei胞外多糖对Balb/c小鼠FKN和MIP-3α及其骨髓来源树突状细胞受体的影响

目的探讨干酪乳杆菌(Lactobacillus casei,L.casei)胞外多糖影响树突状细胞迁移的机制。方法利用细胞因子诱导的方法获得骨髓来源树突状细胞(BMDCs)。体外实验为干酪乳杆菌胞外多糖(EPS)处理DCs 24 h后,采用双抗体夹心ELISA法检测细胞培养上清液中趋化因子受体CCR6和CX3CR1的数量;体内实验为胞外多糖腹腔注射Balb/c小鼠12 h后用采用双抗体夹心ELISA法检测小鼠血液中趋化因子FKN和MIP-3α的分泌量。结果体外实验中,经胞外多糖处理的DCs受体CCR6和CX3CR1数量增加极显著(P<0.01),并且与胞外多糖的浓度呈剂量依赖性;体内实验中,经胞外多糖处理的小鼠血液中FKN和MIP-3α含量增加极显著(P<0.01),并且在50 mg/kg注射量时达到最大。结论胞外多糖能够增加血清中FKN和MIP-3α的含量,并能增加BMDCs受体CCR6和CX3CR1的数量。     

桑黄液体发酵生产多糖工艺研究

目的 考察不同培养条件对桑黄菌液体发酵生产多糖的影响.方法 通过正交试验设计,对不同培养基种类、起始pH值、发酵温度和摇床转速进行优化,在此工艺基础上,采用5L发酵罐进行发酵放大试验.结果 采用添加0.3 9/L维生素B1和0.3 g/L维生素B2的PD培养基,起始pH值5,发酵温度28℃,摇床转速180 r/min条件下,桑黄菌摇瓶发酵液中胞内多糖和胞外多糖分别为5.80、2.37 g/L;5 L发酵罐发酵液中桑黄胞内多糖和胞外多糖分别为5.85、2.30 g/L.结论 桑黄液体发酵可获得桑黄多糖,本实验结果为桑黄液体发酵工业化生产提供参考.     

D-甲硫氨酸通过降低环二鸟苷酸清除牙龈卟啉单胞菌生物膜

目的探讨D-甲硫氨酸(D-methionine,D-Met)通过调控环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)清除牙龈卟啉单胞菌生物膜的作用机制。方法通过细胞活性、最小抑菌浓度(minimum inhibitory concentration,MIC)和最小杀菌浓度(minimum bactericidal concentration,MBC)实验确定D-Met的有效浓度。分别在牙龈卟啉单胞菌生物膜形成抑制实验和成熟生物膜裂解实验中加入不同浓度的D-Met,检测生物膜生物量、胞外多糖量、生物膜形态、细胞膜完整性以及c-di-GMP的含量变化。结果 D-Met <40 mmol/L为生物安全浓度。在生物膜形成抑制和成熟生物膜裂解实验中,D-Met≥20 mmol/L降低了生物膜生物量和胞外多糖量。扫描电镜结果显示,D-Met≥20 mmol/L时,细胞外基质和细菌密度显著降低。透射电镜结果表明,35 mmol/L D-Met在生物膜形成过程中导致了细胞膜破裂,并增加了成熟生物膜裂解时细胞膜的通透性。Cdi-GMP水平随着D-Met浓度的增加而降低,呈浓度依赖性。结论 D-Met≥20 mmol/L可通过抑制c-di-GMP水平清除牙龈卟啉单胞菌生物膜。