加压CO2对大肠杆菌细胞膜的损伤作用

[目的]细菌细胞膜的损伤可以表现在细菌细胞内物质泄漏和细菌细胞吸收染料.与巴氏杀菌(63℃C、30 min)比较,研究加压CO2对大肠杆菌细胞膜的损伤作用,目的是分析出大肠杆菌死亡与细胞膜损伤的关系.[方法]检测大肠杆菌细胞膜通透性的改变情况,大肠杆菌内蛋白质和核酸的泄漏程度,并通过透射电镜观察大肠杆菌形态的改变情况.[结果]在研究范围内,加压CO2处理使大肠杆菌细胞膜通透性发生改变;加压CO2处理时虽然发生了胞内蛋白质泄漏,但发生泄漏的时间明显滞后于99％以上菌体死亡时间,因此并不是大肠杆菌死亡的原因,只是大肠杆菌死亡后的继发现象;大肠杆菌死亡与加压CO2处理导致的胞内核酸泄漏有关;大肠杆菌死亡与加压CO2处理导致的菌体形态改变有关.[结论]加压CO2对大肠杆菌细胞膜的损伤作用与菌体死亡有直接关系.     

莳萝蒿精油成分分析及抑菌活性机理探究

为了确定莳萝蒿精油的化学成分,并探究其抑菌活性及抑菌机理。该研究采用水蒸气蒸馏法提取莳萝蒿精油,并通过气相色谱-质谱联用法测定其化学成分。采用抑菌圈法、二倍稀释法和生长曲线法测定精油的抑菌活性,采用电导率法和扫描电镜法探究精油的抑菌机理。结果表明:(1)莳萝蒿精油的主要化学成分包括醇类(47.12%)和萜烯类(19.90%),在所有成分中桉油精(12.39%)含量最高,其次为松油醇(8.70%)。(2)精油对金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为(22.57±1.68)mm和(15.36±0.71)mm。(3)精油对金黄色葡萄球菌和大肠杆菌的最小抑菌浓度分别为3.25和7.5μL/mL,最小杀菌浓度分别为7.5和15μL/mL。(4)当精油浓度为1.625和3.25μL/mL时,其分别能够延缓金黄色葡萄球菌和大肠杆菌的生长;当精油浓度为3.25和7.5μL/mL时,其能够完全抑制金黄色葡萄球菌的生长;当精油浓度为7.5和15μL/mL时,其能够完全抑制大肠杆菌的生长。(5)经精油处理之后的细菌,其相对电导率明显增大,且随精油浓度的增加而增大,同时其细胞膜发生了萎缩和破裂的现象。研究发现,莳萝蒿精油富含醇类和萜烯类等多种活性物质,对金黄色葡萄球菌和大肠杆菌具有良好的抑菌活性,且莳萝蒿精油能够改变细胞的膜结构,导致细菌中的内溶物发生泄漏,从而抑制细菌生长。     

一种源自人α-2-巨球蛋白的抗菌肽A2M3及其对金黄色葡萄球菌的抑菌机制

【目的】在人的鼻腔中鉴定出一种源自α-2-巨球蛋白的抗菌肽(命名为A2M3),并探究其对金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用和机制。【方法】结合生物信息学技术对人类鼻液的质谱结果进行分析,并筛选潜在抗菌肽;通过微量稀释法和平板涂布法分别分析A2M3对金黄色葡萄球菌最低抑菌浓度(minimum inhibitory concentration, MIC)和时间杀伤曲线(time-kill curve);采用透射电镜、碘化丙锭(propidium iodide, PI)摄取实验、流式细胞术和核酸蛋白质泄露实验分析A2M3对金黄色葡萄球菌膜完整性、膜通透性的影响;通过凝胶阻滞实验和荧光光谱实验探究A2M3对金黄色葡萄球菌基因组DNA的影响。【结果】利用生物信息学技术筛选出源自α-2-巨球蛋白的潜在抗菌肽A2M3,其对金黄色葡萄球菌的MIC为125.0 μg/mL,且能在3 h内完全杀灭细菌。A2M3通过增加细胞膜的通透性,促使核酸和蛋白质泄漏,继而穿过细胞膜嵌入DNA的碱基对,影响细菌的基因功能,从而导致菌体死亡。【结论】A2M3对金黄色葡萄球菌的抑菌机制涉及多靶点协同作用,能够改变细菌细胞膜的通透性,影响细菌的基因功能。这一发现揭示了从人体体液中筛选和分离抗菌功能肽的潜在应用价值。     

大黄素对金黄色葡萄球菌的抑菌作用机制

以金黄色葡萄球菌为供试菌,通过测定大黄素对其细胞膜的通透性、可溶性蛋白质和呼吸代谢的影响,来阐述大黄素的抑菌作用机制. 利用电导率、生物大分子分析、呼吸代谢抑制检测等方法,验证大黄素的药效作用. 实验结果显示,大黄素作用金黄色葡萄球菌后,培养基溶液中电导率比对照组增加了2.23%,DNA和RNA大分子的含量比对照组增加了67.36%,大黄素作用金黄色葡萄球菌16 h后,菌体可溶性蛋白总量比对照组减少了28.3%;大黄素能抑制金黄色葡萄球菌物质代谢中的2种关键酶的活性,其中琥珀酸脱氢酶活性抑制率为53.8%,苹果酸脱氢酶的活性抑制率为25.5%.上述结果表明,大黄素可以破坏细菌细胞膜的通透性,抑制菌体内的蛋白质合成,通过抑制代谢关键酶的活性发挥杀菌作用.     

红谷霉素对金黄色葡萄球菌抑菌机制的研究

红谷霉素是一种抗细菌的新型抗生素。为探讨红谷霉素对金黄色葡萄球菌的抑菌机制,本文采用浓度为EC50和EC90的红谷霉素对试验菌株进行处理,并设空白对照,测定供试菌株的胞外多糖、细胞膜渗透性和生物大分子(DNA、RNA、蛋白质和胞外酶活性)。结果显示:红谷霉素对金黄色葡萄球菌的抑制作用首先表现在对核酸合成的抑制,由于核酸的合成受阻,引起菌体内的蛋白质和其它的生物大分子的合成受阻,细菌细胞膜通透性改变。透视电镜照片显示:红谷霉素处理后金黄色葡萄球菌菌体内部的原生质体明显变稀。     

冷冻干燥对乳酸菌细胞膜通透性的影响

对细胞膜通透性变化的研究是认识冷冻干燥过程对乳酸菌损伤机理的途径之一。用荧光探针检测冻干过程前后细胞内H+和Ca2+浓度的变化, 可以精确的表征细胞膜通透性的改变。利用荧光探针BCECF-AM和Fluo3-AM对德氏乳杆菌保加利亚亚种在冻干前后的细胞膜通透性进行研究, 并对比菌种在冻干过程中的活力损失, 发现细胞膜在冻干前后通透性有显著增加, 并与活力的损失成反相关关系。说明在冻干过程中细胞受到了生理性损伤, 细胞膜通透性的改变可能是导致乳酸菌在冻干过程中致死和失活的原因之一。     

荔枝皮黄酮抑菌性能及其作用机理研究

采用荧光分析法测定荔枝皮中总黄酮含量,并研究了荔枝皮黄酮对常见4种微生物的抑菌活性及机理。结果表明,其黄酮纯度与得率分别为48%和13.61%;同时,采用牛津杯法测得荔枝皮黄酮对金黄色葡萄球菌、大肠杆菌和酵母菌的抑菌圈直径分别为15.1、14.0和13.8 mm;采用菌饼法测得对黑曲霉的抑菌率为28.75%。采用平板稀释法测得金黄色葡萄球菌和大肠杆菌的最低抑菌浓度MIC为2.5 mg/mL,最低杀菌浓度MBC为5mg/mL,而酵母菌和黑曲霉的MIC为5 mg/mL,没有杀菌能力。扫描电镜实验结果表明,荔枝黄酮的抑菌性和杀菌功能与其对金黄色葡萄球菌细胞和细胞壁结构的破坏直接相关。     

连翘对金黄色葡萄球菌的抑菌作用及机制的试验研究

探讨连翘对金黄色葡萄球菌的抑菌作用及机制。根据2,3,5-氯化三苯基四氮唑(TTC)染色法测定连翘对金黄色葡萄球菌的最低抑菌浓度(MIC)。用连翘的MIC浓度的处理金黄色葡萄球菌,分别在0、4 h、8h、12 h、16 h、20 h、24 h、28 h、32 h取样测定光密度值(OD值),绘制其生长曲线。用MIC的连翘处理金黄色葡萄球菌24 h,检测琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)活性。用MIC的连翘作用于金黄色葡萄球菌,电导仪检测0、2 h、4 h、6 h、8 h、10 h时上清液稀释20倍的电导率,紫外分光光度计检测DNA、RNA等大分子物质的外渗水平。连翘对金黄色葡萄球菌的MIC为0.12 mg/m L。连翘作用后的金黄色葡萄球菌生长明显受到抑制并且提前4 h进入衰亡期。连翘作用后的金黄色葡萄球菌中SDH和MDH活性显著下降,而培养基上清的电导率明显升高,DNA、RNA等大分子物质外渗水平明显增多。连翘能抑制金黄色葡萄球菌的生长,作用机制可能与细胞膜通透性及SDH和MDH代谢酶活性有关。     

5-氯水杨醛缩2,4-二羟基苯酰肼的抗菌抗氧化活性研究

目的考察5-氯水杨醛缩2,4-二羟基苯酰肼对金黄色葡萄球菌的抗菌活性及体外抗氧化活性。方法采用试管法和平板法测定其对金黄色葡萄球菌最小抑菌浓度(MIC)、最小杀菌浓度(MBC),绘制杀菌曲线,并通过扫描电镜研究其作用机制,采用DPPH法测定抗氧化活性。结果该化合物对金黄色葡萄球菌的MIC为2.5 mg/m L,MBC为5 mg/m L。杀菌曲线结果表明该化合物对金黄色葡萄球菌的杀菌作用表现出明显的浓度依赖性。扫描电镜结果表明该化合物的作用机制可能与破坏菌体细胞壁、改变其通透性有关。同时该化合物具有较强的清除自由基的能力。结论 5-氯水杨醛缩2,4-二羟基苯酰肼具有显著的抗金黄色葡萄球菌作用,同时具有较好的体外抗氧化活性。     

紫穗槐种子提取物的抑菌活性研究

目的研究紫穗槐种子提取物的抑菌活性。方法将紫穗槐种子乙醇提取物分别通过石油醚、乙酸乙酯和正丁醇萃取,选择金黄色葡萄球菌和肺炎克雷伯杆菌为供试菌,采用试管二倍稀释法测定紫穗槐种子提取物的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）,涂布平板法绘制杀菌曲线,电镜下观察药物对细菌超微结构的影响。结果紫穗槐种子提取物经乙酸乙酯萃取后对供试菌抑制作用较强,其中对金黄色葡萄球菌的MIC和MBC分别为2．5、5．0mg／mL;对肺炎克雷伯杆菌的MIC和MBC分别为5．0、10．0mg／mL;杀菌曲线结果表明,药物对供试菌的抑制作用存在浓度和时间依赖性;电镜结果说明,药物的作用可能与破坏菌体细胞壁、改变细胞膜通透性有关。结论紫穗槐种子提取物具有显著的抗菌活性。     

Antimicrobial activity of tertiary amine covalently bonded to a polystyrene fiber.

Tertiary amine was covalently bonded to a polystyrene fiber and examined for antibacterial activity. The tertiary amine covalently bonded to a polystyrene fiber (TAF) showed a high antimicrobial activity against Escherichia coli. TAF exhibited a stronger antibacterial activity against gram-negative bacteria (E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens) than against gram-positive bacteria (Staphylococcus aureus and Streptococcus faecalis) or Candida albicans. This activity against E. coli was accentuated by 0.1% deoxycholate or 10 mg of actinomycin D per ml, to which E. coli is normally not susceptible. This implies that TAF causes an increase of the bacterial outer membrane permeability. On the other hand, the antimicrobial activity was inhibited by adding Mg2+ or by lowering the pH. This suggest an electrostatic interaction between the bacterial cell wall and TAF. Scanning electron microscopy showed that E. coli cells were initially attached to TAF, with many projections on the cell surface, but then were apparently lysed after contact for 4 h. Taken together, these results imply that bacteria initially interact with TAF by an electrostatic force between the anionic bacterial outer membrane and the cationic tertiary amine residues of TAF and that longer contact with TAF damages the bacterial outer membrane structure and increases its permeability.     

Antimicrobial action of novel chitin derivative

Aminoethyl-chitin (AEC) was synthesized in an attempt to both increase solubility of chitin in water and biological activity. AEC was obtained by grafting 2-chloroethylamino hydrochloride onto chitin at C-6 position. The structure of AEC was elucidated FT-IR and (1)H NMR spectroscopy, and its antimicrobial activity was investigated using three Gram-positive and Gram-negative bacteria. The integrity of the cell membranes of the representatives E. coli and S. aureus was investigated by determining the release of intracellular components of cells. When treated with AEC, release of 260 nm absorbing materials quickly increased both E. coli and S. aureus, but absorbance value was different due to the difference in cell structures. For detailed study, outer membrane (OM) and inner membrane (IM) permeabilization assay were performed using the fluorescent probe 1-N-phenylnaphthylamine (NPN) and the release of cytoplasmic beta-galactosidase activity. The results showed that AEC rapidly increased NPN uptake and the release of cytoplasmic beta-galactosidase via increasing the permeability of OM and IM. In addition, cytotoxic effect of AEC was assessed using human lung fibroblast (MRC-5) cell line, and AEC showed less toxic against MRC-5.     

Effects of Pro --> peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide

To investigate the effect of Pro --> peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --> Nphe (TPf) or Pro --> Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --> Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --> Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.     

SERCA activity is required for timely progression through G1/S

Changes in intracellular Ca2+ correlate with specific events in the cell cycle. Here we investigated the role of Ca2+ in the G1 phase. HEK 293 cells were arrested in mitosis and subjected to short-term treatments that alter Ca2+ homeostasis prior to their release into G1. Treatment with thapsigargin (TG), an irreversible inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) lengthened the G1 phase. Moreover, TG treatment also resulted in a dramatic alteration in cellular morphology and attachment and in the reduction of MAPK activity and lower levels of cyclin D1 and cyclin E proteins. Treatments with reagents that transiently increase or decrease cytosolic Ca2+ or that temporarily inactivate SERCA did not alter any of the above parameters. Cells expressing a TG-resistant form of SERCA progressed normally through the G1/S transition after TG treatment. These results suggest that long-term SERCA inactivation affects cell cycle-dependent events and compromises progression through G1/S.     

Interaction of wasp venom mastoparan with biomembranes

Mastoparan-induced changes in the K+ permeability of rat peritoneal mast cells, human erythrocytes, Staphylococcus aureus and Escherichia coli were examined. Mastoparan did not efficiently increase the K+ permeability of cells except for S. aureus. The release of membrane phospholipids was also observed from S. aureus cells in the concentration range of the permeability enhancement. Mastoparan stimulated histamine release from mast cells, independently of a small efflux of K+. Mastoparan became markedly effective to E. coli cells whose outer membrane structure was chemically disrupted beforehand, showing that the peptide can enhance the permeability of the cytoplasmic membranes of both Gram-positive and -negative bacteria. In experiments using liposomes, mastoparan increased the permeability of the liposomes composed of egg phosphatidylethanolamine and egg phosphatidylglycerol, which are the lipid constituents of the cytoplasmic membrane of E. coli cells, while it showed a weak activity to the liposomes composed of egg phosphatidylcholine and cholesterol. The latter result related closely to the fact that this peptide acted weakly on erythrocytes and mast cells in which acidic lipids constitute a minor portion. Mastoparan decreased the phase transition temperature of dipalmitoylphosphatidylglycerol liposomes, but it did not affect that of dipalmitoylphosphatidylcholine liposomes. These results indicate that mastoparan penetrated into membranes mainly containing acidic phospholipids and disrupted the membrane structure to increase the permeability. The action of the wasp venom mastoparan was compared with that of a bee venom melittin.     

The antibacterial effect of attacins from the silk moth Hyalophora cecropia is directed against the outer membrane of Escherichia coli.

The attacins are antibacterial proteins which accumulate in the hemolymph of the giant silk moth, Hyalophora cecropia, in response to a bacterial infection. Here we show that the permeability barrier function of the outer membrane is affected shortly after addition of attacin to growing cultures of Escherichia coli. Specifically, the penetration through the outer membrane of beta-lactam antibiotics, chicken egg white lysozyme and the detergent Triton X-100 was found to be facilitated. The sensitivity of E. coli to cecropin B, another antibacterial protein present in the hemolymph of H. cecropia, was also found to be increased after treatment with attacin. The results suggest that the target of the attacins in E. coli is the outer membrane. Other effects of the attacins which have been observed are likely to be indirect consequences of the alteration in the properties of the outer membrane. These effects include changes in the cell shape, irregular patterns of cell division and lysis. The minimal concentration at which the attacins affected the growth of E. coli was 1 and 0.5 microM for the neutral (pI 7) and basic (pI 9) attacins, respectively, which corresponds to less than 2% of the concentration of the attacins in the hemolymph of infected pupae.     

家蝇幼虫抗菌肽MDL-2对细菌细胞渗透性及代谢功能影响

研究了家蝇幼虫抗菌肽MDL-2与细菌相互作用时,抗菌肤MDL-2对细菌细胞壁的溶解作用、细胞膜渗透性和代谢的影响.抗菌肽MDL-2在抗菌过程中首先与细菌的细胞壁相互作用,使其破裂,抗菌肽对革兰氏阴性细菌大肠杆菌细胞壁的作用有浓度依赖性,而对革兰氏阳性细菌金黄色葡萄球菌MDL-2在较低的浓度时即可发生细胞壁破坏作用;抗菌...     

Different modes in antibiotic action of tritrpticin analogs, cathelicidin-derived Trp-rich and Pro/Arg-rich peptides

The cathelicidin-derived antimicrobial tritrpticin could be classified as either Trp-rich or Pro/Arg-rich peptide. We recently found that the sequence modification of tritrpticin focused on Trp and Pro residues led to considerable change in structure and antimicrobial potency and selectivity, but their mechanisms of microbial killing action were still unclear. Here, to better understand the bactericidal mechanisms of tritrpticin and its two analogs, TPA and TWF, we studied their effect on the viability of Gram-positive S. aureus and Gram-negative E. coli in relation to their membrane depolarization. Although TWF more effectively inhibited growth of S. aureus and E. coli than TPA, only a 30 min exposure to TPA was sufficient to kill both bacteria and TWF required a lag period of about 3-6 h for bactericidal activity. Their different bactericidal kinetics was associated with membrane permeabilization, i.e., TWF showed negligible ability to depolarize the cytoplasmic membrane potential of target cell membrane, whereas we observed significant membrane depolarization for TPA. In addition, while TPA caused rapid and large dye leakage from negatively charged model vesicles, TWF showed very little membrane-disrupting activity. Interestingly, we have looked for a synergism among the three peptides against E. coli, supporting that they are working with different modes of action. Collectively, our results suggest that TPA disrupts the ion gradients across the membrane, causing depolarization and a loss of microbial viability. By contrast, TWF more likely translocates across the cytoplasmic membrane without depolarization and then acts against one or more intracellular targets. Tritrpticin exhibits intermediate properties and appears to act via membrane depolarization coupled to secondary intracellular targeting.     

Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli

The antibacterial effect and mechanism of action of a silver ion solution that was electrically generated were investigated for Staphylococcus aureus and Escherichia coli by analyzing the growth, morphology, and ultrastructure of the bacterial cells following treatment with the silver ion solution. Bacteria were exposed to the silver ion solution for various lengths of time, and the antibacterial effect of the solution was tested using the conventional plate count method and flow cytometric (FC) analysis. Reductions of more than 5 log(10) CFU/ml of both S. aureus and E. coli bacteria were confirmed after 90 min of treatment with the silver ion solution. Significant reduction of S. aureus and E. coli cells was also observed by FC analysis; however, the reduction rate determined by FC analysis was less than that determined by the conventional plate count method. These differences may be attributed to the presence of bacteria in an active but nonculturable (ABNC) state after treatment with the silver ion solution. Transmission electron microscopy showed considerable changes in the bacterial cell membranes upon silver ion treatment, which might be the cause or consequence of cell death. In conclusion, the results of the present study suggest that silver ions may cause S. aureus and E. coli bacteria to reach an ABNC state and eventually die.