抗菌肽OH-CATH对大肠杆菌Escherichia coli的作用

OH-CATH是眼镜王蛇中新发现的cathelicidin家族抗菌肽.它在1%NaCI存在的条件下对多种细菌都有较强的抗菌活性,同时,在高浓度下对人红细胞无溶血活性.OH-CATH足开发新型抗菌药物的优良模板.蜊明OH-CATH的作用机理及其对微生物的选择性,对研发以OH-CATH为先导结构的药物研发有十分重要的意义.本文利用扫描电镜以及透射电镜对OH-CATH与革兰氏阴性菌一大肠杆菌ATCC 25922相互作用的效应研究.结果揭示:OH-CATH对大肠杆菌的作用涉及到3个步骤.首先,OH-CATH借助其带正电的氨基酸残基附着到细菌带负电荷的细胞壁:然后,附着的OH-CATH在达剑一定浓度后发生聚集,以孔道彤成的方式破坏细菌的膜结构;最终,由十细菌膜的损坏,膜的渗透性被破坏,胞内内含物释放造成细菌死亡.     

抗菌药物靶标丙氨酸消旋酶及其抑制剂研究进展

抗生素的滥用和人口的大量流动使得病原菌耐药性增强并与其他病原体产生共感染等问题,严重威胁人类的生命安全,因此,研发新型抗菌药物成为人类亟待解决的问题。丙氨酸消旋酶是以磷酸吡哆醛为辅酶催化L-丙氨酸与D-丙氨酸旋光结构互换的一类异构酶,其消旋产物D-丙氨酸对细菌细胞壁的形成具有决定性作用,与细菌性疾病密切相关。抑制丙氨酸消旋酶的活性会影响细菌的生存,近年来成为设计抗菌药物的一个理想靶位,其抑制剂的开发已成为抗菌药物研发的热点。本文从丙氨酸消旋酶的来源、结构、功能、应用及抑制剂等方面进行系统阐述,并对丙氨酸消旋酶的研究提出新的策略,为进一步研究丙氨酸消旋酶与致病菌的关系及抗菌药物候选靶标的研究提供理论基础。     

2009-2011年胆石症患者胆汁肠球菌菌群分布及菌种耐药性变迁

目的 了解2009年1月年2011年12月胆石症患者胆汁中肠球菌的分布及菌种耐药性变迁,指导临床合理应用抗菌药物.方法 采用法国生物梅里埃公司生产的VITEK-2细菌分析仪对胆汁标本中分离的肠球菌做鉴定及药敏试验.结果 2009年1月至2010年6月共分离到74株肠球菌;2010年7月至2011年12月共分离到115株肠球菌,排在前两位的均为粪肠球菌和屎肠球菌.药敏结果显示粪肠球菌对除红霉素和喹努普汀/达福普汀外的其他抗菌药物具有较高的抑菌活性;屎肠球菌对除喹努普汀/达福普汀、利奈唑胺、万古霉素外的其他抗菌药物具有较高的耐药率;首次检测到耐利奈唑胺的肠球菌.结论 胆石症患者胆汁中肠球菌感染以粪肠球菌和屎肠球菌为主,且多种细菌对抗菌药物均有不同程度的耐药,因此加强细菌耐药监测对临床合理使用抗菌药物有重要的参考意义.     

蛇毒抗菌肽OH-CATH对大肠杆菌引起家兔泌尿系感染的保护作用

为探讨蛇毒抗菌肽OH-CATH对大肠杆菌标准株和临床耐药株引起家兔泌尿系感染的保护作用,该文利用大肠杆菌标准株(E.coli ATCC 25922)和耐头孢菌素大肠杆菌临床耐药株建立雄性家兔泌尿系感染模型.通过膀胱直接给药的方式,分别给予动物感染模型生理盐水、蛇毒抗菌肽OH-CATH及头孢哌酮舒巴坦,并于给药后第1、5、l0及14天留取家兔中段尿用于尿液培养.将动物膀胱标本行H-E染色石蜡切片及透射电镜观察.结果显示:使用蛇毒抗菌肽OH-CATH的大肠杆菌标准株和临床耐药株不同组别中段尿培养阳性率明显降低;给予头孢哌酮舒巴坦可降低大肠杆菌标准株(E.coli ATCC 25922)引起感染的阳性率,但对耐头孢菌素大肠杆菌引起的感染的阳性率则无明显作用(P＜0.05);使用蛇毒抗菌肽OH-CATH组炎症细胞浸润、坏死及钙化组织较其他组为少.该结果提示蛇毒抗菌肽OH-CATH对大肠杆菌标准株(E.coli ATCC 25922)及耐头孢菌素大肠埃希菌临床耐药株有稳定活性,对其引起的家兔泌尿系感染有较好的保护作用,并为临床日益严重的耐药病原菌感染的治疗提供了新的思路和方向.     

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...     

湛江沿海潮间带产胞外抗菌活性海洋细菌的分离鉴定及生物多样性分析

从硇洲岛和徐闻珊瑚礁自然保护区潮间带采集海水和沉积物标本,采用纯培养的方法分离其中的海洋细菌;以金黄色葡萄球菌、枯草芽抱杆菌和大肠埃希菌等为指示菌,以氨苄青霉素、青霉素-链霉素为阳性对照,采用琼脂扩散法筛选抗菌活性菌株;采用基于16S rDNA序列比对及其系统发育分析对分离培养的阳性海洋细菌进行分类鉴定和生物多样性分析;为进一步从海洋细菌资源中发掘新型抗菌药物奠定基础。结果从106株海洋细菌中筛选出了44株抗细菌活性菌株,阳性率为41.5%。其中,11株具有抗金黄色葡萄球菌作用,31株有抗枯草芽孢杆菌作用,13株有抗大肠埃希菌作用。抗菌活性菌株分布于31属,优势属为芽孢杆菌属(Bacillus)和弧菌属(Vibrio)。这表明分离自硇洲岛和徐闻珊瑚礁自然保护区潮间带的海洋细菌中的抗菌活性菌株具有丰富的生物多样性。     

根除细菌生物被膜的新视角：分散

细菌生物被膜(biofilm,BF)是细菌为对抗外界压力形成的一种自我保护结构,对于抗菌药物具有极高的耐受性,在临床上极易引发难治性慢性感染。BF分散是指在BF形成周期中,膜内细胞主动逸出,恢复浮游生长模式,寻找新定植位点的过程。由于细菌在浮游状态下,更易受到抗菌药与免疫反应的作用,诱导BF分散是控制BF相关感染(biofilm-associated infections, BAI)的一条富有前景的策略。本文从BF分散的方式和信号分子等角度,对BF分散的调控机制进行分析;归纳能影响BF分散的物质,并对BF分散后可能带来的危害及未来的研究思路进行简述,以期为研发新型分散剂和深入研究药物作用的靶点提供理论参考。     

红树内生细菌CⅢ-1菌株鉴定及其胞外抗菌蛋白性质研究

用对峙生长法从422株红树内生细菌中筛选到1株对青枯雷尔氏菌(Ralstonia solanacearum)、金黄色葡萄球菌(Staphylococcus aureus)、溶藻弧菌(Vibrio alginolyticus)、辣椒疫霉菌(Phytophthora capsici)、香蕉枯萎病菌(Fusarium oxysporum f. sp. cubense)等动植物病原细菌和真菌均具有较强的拮抗作用的海洋细菌CⅢ-1菌株, 经形态和生理生化特性及16S rRNA序列分析, 鉴定该菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。用33%硫酸铵从菌株的培养滤液中盐析获得对供试病原菌均具有较强拮抗作用的抗菌活性蛋白, 性质测定发现, 该抗菌活性蛋白对热不稳定, 60?C和100?C下处理10 min, 其抗菌活性分别下降了62.5%和完全丧失, 在pH 5.0?10.0范围内均具有抑菌作用, 以pH 7.0时抗菌活性最强。     

红树内生细菌CⅢ-1菌株鉴定及其胞外抗菌蛋白性质

用对峙生长法从422株红树内生细菌中筛选到1株对青枯雷尔氏菌(Ralstonia solanacea-rum)、金黄色葡萄球菌(Staphylococcus aureus)、溶藻弧菌(Vibrio alginolyticus)、辣椒疫霉菌(Phytophthora capsici)、香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)等动植物病原细菌和真菌均具有较强的拮抗作用的海洋细菌CⅢ-1菌株,经形态和生理生化特性及16S rRNA序列分析,鉴定该菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。用33%硫酸铵从菌株的培养滤液中盐析获得对供试病原菌均具有较强拮抗作用的抗菌活性蛋白,性质测定发现,该抗菌活性蛋白对热不稳定,60°C和100°C下处理10min,其抗菌活性分别下降了62.5%和完全丧失,在pH5.0?10.0范围内均具有抑菌作用,以pH7.0时抗菌活性最强。     

乳铁蛋白抗菌机理研究进展

乳铁蛋白(Lactoferrin,LF)是发现于哺乳动物初乳中具有多种生物活性的单体糖蛋白,对幼体初期免疫具有重要作用。前人研究发现LF蛋白的酶解后其抗菌活性得到增强,是由于酶解后产生了比其本身抗菌能力更强的肽段。目前关于LF蛋白抗菌活性的报道很多,但其抗菌的具体分子机制尚不清楚。以LF蛋白与细菌细胞膜的结合能力和LF蛋白酶解产物的抗菌活性为切入点,揭示了LF蛋白行使抗菌功能的过程,认为LF蛋白与细菌表面相结合而发生结构变化,进而暴露出敏感的酶切位点,经酶解而释放抗菌活性肽,这些抗菌肽破坏细菌的细胞膜结构,达到抑菌或杀菌的目的。对LF蛋白抗菌机理的阐释将为研制抗菌能力更强的活性蛋白提供理论依据。     

King cobra peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant isolates

Cationic antimicrobial peptides(AMPs) are considered as important candidate therapeutic agents, which exert potent microbicidal properties against bacteria, fungi and some viruses. Based on our previous findings king cobra cathelicidin(OH-CATH) is a 34-amino acid peptide that exerts strong antibacterial and weak hemolytic activity. The aim of this research is to evaluate the efficacy of both OH-CATH30 and its analog D-OH-CATH30 against clinical isolates comparing with routinely utilized antibiotics in vitro.In this study, 584 clinical isolates were tested(spanning 2013-2016) and the efficacy of the candidate peptides and antibiotics were determined by a broth microdilution method according to the CLSI guidelines. Among the 584 clinical isolates, 85% were susceptible to OH-CATH30 and its analogs. Both L-and D-OH-CATH30 showed higher efficacy against(toward) Gram-positive bacteria and stronger antibacterial activity against nearly all Gram-negative bacteria tested compare with antibiotics. The highest bactericidal activity was detected against Acinetobacter spp., including multi-drug-resistant Acinetobacter baumannii(MRAB)and methicillin-resistant Staphylococcus aureus(MRSA). The overall efficacy of OH-CATH30 and its analogs was higher than that of the 9 routinely used antibiotics. OH-CATH30 is a promising candidate drug for the treatment of a wide variety of bacterial infections which are resistant to many routinely used antimicrobial agents.     

Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576bp and coded for 191 amino acid residue protein precursors. Each of the deduced elapid cathelicidin has a 22 amino acid residue signal peptide, a conserved cathelin domain of 135 amino acid residues and a mature antimicrobial peptide of 34 amino acid residues. Unlike the highly divergent cathelicidins in mammals, the nucleotide and deduced protein sequences of the three cloned elapid cathelicidins were remarkably conserved. All the elapid mature cathelicidins were predicted to be cleaved at Valine157 by elastase. OH-CATH, the deduced mature cathelicidin from king cobra, was chemically synthesized and it showed strong antibacterial activity against various bacteria with minimal inhibitory concentration of 1-20microg/ml in the presence of 1% NaCl. Meanwhile, the synthetic peptide showed no haemolytic activity toward human red blood cells even at a high dose of 200microg/ml. Phylogenetic analysis of cathelicidins from vertebrate suggested that elapid and viperid cathelicidins were grouped together in the tree. Snake cathelicidins were evolutionary closely related to the neutrophilic granule proteins (NGPs) from mouse, rat and rabbit. Snake cathelicidins also showed a close relationship with avian fowlicidins (1-3) and chicken myeloid antimicrobial peptide 27. Elapid cathelicidins might be used as models for the development of novel therapeutic drugs.     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Genome-wide mining of microsatellites in king cobra (Ophiophagus hannah) and cross-species development of tetranucleotide SSR markers in Chinese cobra (Naja atra)

Molecular Biology Reports - The complete genome sequence provides the opportunity for genome-wide and coding region analysis of SSRs in the king cobra and for cross-species identification of...     

眼镜王蛇神经毒素CM—11核磁共振氢谱谱峰的完全归属

眼镜王蛇抽提物ＣＭ－１１为含７２个残基的长链神经毒素,对其进行了ＤＱＦ－ＣＯＳＹ,ＴＯＣＳＹ和ＮＯＥＳＹ等一系列２Ｄ－ＮＭＲ谱测定,借助序列专一归属法完成了ＣＭ－１１ＮＭＲ氢谱的完整归属。     

Antimicrobial activity of the Naja atra cathelicidin and related small peptides

We have identified an 11-residue pattern (KR(F/A)KKFFKK(L/P)K), which we have named the ATRA motif, within the sequence of the Chinese cobra (Naja atra) cathelicidin. A series of 11-residue peptides (ATRA-1, -2, -1A and -1P) were designed to probe the significance of the conserved residues within the ATRA motif, and their contributions to antimicrobial performance. The antimicrobial activities of the peptides were assessed against Escherichia coli K12 strain and Aggregatibacter actinomycetemcomitans Y4. ATRA-1 and -1A, demonstrated potencies comparable to that of N. atra cathelicidin. Structural examination by circular dichroism of the four short peptides suggested the significance of specific amino acid positions within the motif by their contribution to helicity. The results of these studies indicate that short peptides derived from the repeated ATRA motif from the N. atra cathelicidin can demonstrate both low toxicity against host cells and high antimicrobial activity against the gram-negative bacteria used in this study. They constitute novel, effective antimicrobial peptides that are much shorter (and thus less expensive to produce) than the natural cathelicidins, and they may represent new templates for therapeutic drug development.     

Substrate specificity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase

1. Substrate specificity of purified king cobra (Ophiophagus hannah) venom L-amino acid oxidase was investigated. 2. The enzyme was highly specific for the L-enantiomer of amino acid. Effective oxidation of L-amino acid by the enzyme requires the presence of a free primary alpha-amino group but the alpha-carboxylate group is not as critical for the catalysis. 3. The enzyme was very active against L-Lys, L-Phe, L-Leu, L-Tyr, L-Tryp, L-Arg, L-Met, L-ornithine, L-norleucine and L-norvaline and moderately active against L-His, L-cystine and L-Ileu. Other L-amino acids were oxidized slowly or not oxidized. 4. The data suggest the presence of a side chain binding site in the enzyme, and that the binding site comprises at least five 'subsites': the hydrophobic subsites a, b and c; and the two 'amino' binding subsites d and e. Subsite b appears to be able to accommodate two methylene/methyl carbons.