Protein-translocating outer membrane porins of Gram-negative bacteria

Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins.     

Omptin proteins: an expanding family of outer membrane proteases in Gram-negative Enterobacteriaceae (Review)

The Escherichia coli K-12 outer membrane protein OmpT is a prototype of a unique family of bacterial endopeptidases known as the omptins. This family includes OmpT and OmpP of E. coli, SopA of Shigella flexneri, PgtE of Salmonella enterica, and Pla of Yersinia pestis. Despite their sequence similarities, the omptins vary in their reported functions. The OmpT protease is characterized by narrow cleavage specificity defined by the extracellular loops of the β-barrel protruding above the lipid bilayer. It employs a distinct proteolytic mechanism that involves a histidine and an aspartate residue. Most of the omptin proteins have been implicated in bacterial pathogenesis. As a result, the omptins are potential targets for antimicrobial drug and vaccine development. This review summarizes recent developments in omptins structure and function, emphasizes their role in pathogenesis, proposes evolutionary relation among the existing omptins, and offers possible directions for future research.     

革兰氏阴性菌脂多糖运输系统的构成及作用机制

革兰氏阴性菌包含有两层组分不同的膜结构——内膜和外膜,对大多数革兰氏阴性菌而言,脂多糖(lipopolysaccharides,LPS)是其外膜上最主要的脂质成分,锚定在外膜小叶(the outer leaflet of the OM)上,是革兰氏阴性菌固有免疫的重要组成部分。脂多糖运输系统(lipopolysaccharide transport system,Lpt)将胞内装配完整的LPS正确装配到外膜,使得与脂多糖相关的阻渗、有机溶剂耐受性、疏水性抗生素耐受性、膜通透性等功能得以实现。该运输系统的正确作用主要依赖7个不同的脂多糖运输蛋白(Lpt ABCDEFG)协同完成,整个系统贯穿细菌内膜至外膜,由内膜上ABC转运体复合物Lpt B2FG、胞质内转运协同蛋白Lpt A/C及被许多学者称作脂多糖运输的"命门"的外膜蛋白复合物Lpt DE共同构成。本文就革兰氏阴性菌脂多糖的具体结构功能进行简介,进而综述脂多糖运输系统的7个蛋白的构成和作用机制,以期为进一步研究该系统中每个蛋白的功能提供理论基础及参考。     

部分革兰氏阴性菌脂多糖运输蛋白LptD的结构及功能研究进展

在革兰氏阴性菌中,脂多糖是外膜的重要组成部分,并参与构成细菌的固有免疫。而在大多数革兰氏阴性菌中,Lpt系统都是运输脂多糖的唯一途径,在该系统中LptD作为一个跨膜的外膜蛋白,也是脂多糖输出的最后一步,因此被许多学者称作脂多糖运输的"命门"。LptD参与多种重要的生物学功能,包括有机溶剂耐受性、疏水性抗生素耐受性、膜通透性等。但近来的研究表明,LptD最重要的功能是参与了脂多糖的运输,也因为其参与脂多糖运输而具有了多种功能。本文重点介绍部分革兰氏阴性菌LptD的蛋白结构及其功能研究进程,以期为进一步研究其它革兰氏阴性菌脂多糖运输通路(Lpt通路)及该通路上各蛋白间的相互作用机制提供参考。     

Outer membrane protein biogenesis in Gram-negative bacteria

Gram-negative bacteria contain a double membrane which serves for both protection and for providing nutrients for viability. The outermost of these membranes is called the outer membrane (OM), and it contains a host of fully integrated membrane proteins which serve essential functions for the cell, including nutrient uptake, cell adhesion, cell signalling and waste export. For pathogenic strains, many of these outer membrane proteins (OMPs) also serve as virulence factors for nutrient scavenging and evasion of host defence mechanisms. OMPs are unique membrane proteins in that they have a β-barrel fold and can range in size from 8 to 26 strands, yet can still serve many different functions for the cell. Despite their essential roles in cell survival and virulence, the exact mechanism for the biogenesis of these OMPs into the OM has remained largely unknown. However, the past decade has witnessed significant progress towards unravelling the pathways and mechanisms necessary for moulding a nascent polypeptide into a functional OMP within the OM. Here, we will review some of these recent discoveries that have advanced our understanding of the biogenesis of OMPs in Gram-negative bacteria, starting with synthesis in the cytoplasm to folding and insertion into the OM.     

Buffering capacity and H+ membrane conductance of Gram-negative bacteria

Abstract Buffering capacity and membrane H⁺ conductance were examined in seven Gram-negative species: Aquaspirillum serpens, Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli, Salmonella typhimurium, Proteus mirabilis and Aeromonas hydrophila . All strains of Enterobacteriaceae studied here showed a decrease in both parameters as the external pH increased, over the pH range studied. The other four species presented an increase in buffering capacity and membrane conductance to protons as the external pH increased from 5.5 to 7.0.     

革兰阴性细菌耐药特性的双聚类分析

目的通过双聚类分析了解临床分离的革兰阴性菌的耐药特征。方法采用K-B法对临床分离的113株细菌进行药物敏感试验,用软件WHONET 5.6和MATLAB对药敏试验结果进行统计分析。结果传统耐药分析方法表明113株细菌总体耐药率较低,其中大肠埃希菌、铜绿假单胞菌和鲍曼不动杆菌的耐药率则较高,而肺炎克雷伯菌耐药率较低。通过双聚类分析,所有菌株被聚为三大类,I类菌株占23.0%,耐药率最高,几乎对18种药物都耐药,细菌种类以肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌为主;Ⅱ类菌株占56.6%,耐药率普遍较低,以肺炎克雷伯菌为主;Ⅲ类菌株占20.4%,菌株的耐药率介于I和Ⅱ类之间,包括肺炎克雷伯菌、铜绿假单胞菌和大肠埃希菌。其中Ⅱ大类,根据所耐抗生素的不同又分为Ⅱ-A、Ⅱ-B、Ⅱ-C三个亚类,每一亚类都具有相似的耐药特点。结论本实验收集的革兰阴性菌株根据耐药特征被聚为Ⅰ、Ⅱ、Ⅲ类,耐药程度为IⅢⅡ,双聚类分析法有利于快速找到具有相同耐药特征的菌株以及不同菌株之间的耐药差别。     

重庆地区儿童2000年至2004年首位革兰阴性细菌和阳性细菌的耐药性监测

目的了解重庆地区儿童感染的分离至临床标本的首位革兰阴性细菌和阳性细菌对常用抗生素的耐药趋势,指导临床合理使用抗生素。方法常规方法分离、培养细菌,应用美国德灵公司WalkAway-40细菌鉴定仪对2000年至2004年我院细菌室分离至临床标本的首位革兰阴性细菌和阳性细菌共2854株进行细菌鉴定及药敏试验。结果2000年至2004年检出的首位革兰阴性细菌和阳性细菌分别为大肠埃希菌和金黄色葡萄球菌。2000年至2004年前5位革兰阴性菌5777株,革兰阳性菌1565株,其中大肠埃希菌2090株,金黄色葡萄球菌764株,分别占36.2%和48.8%;5年间大肠埃希菌对氨苄西林、头孢吡肟、头孢西丁、庆大霉素、亚胺培南、环丙沙星、头孢噻肟、头孢他啶的总耐药率分别为80.9%、37.5%、15.4%、54.0%、0.8%、34.0%、46.6%、46.2%;金黄色葡萄球菌对青霉素、红霉素、复方新诺明、万古霉素、阿莫西林/克拉维酸的总耐药率分别为95.6%、63.4%、5.8%、0%、11.0%。结论通过细菌耐药监测发现：大肠埃希菌对常用抗生素的总耐药率变化不大,金黄色葡萄球菌对常用抗生素的总耐药率有下降趋势,应引起临床医生重视。     

Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6–8°C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the β-structural proteins (they have 59–96% total β structures and 0–17% α helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl β-D-glucoside for SDS: the content of β structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.     

抗生素诱导革兰阴性菌外膜囊泡产生及发挥生理作用的机制

外膜囊泡是革兰阴性菌分泌的一种球形纳米颗粒,由外膜及其所含成分组成,是细菌在外界压力条件下分泌的具有生理活性的特殊结构。外界压力如抗生素、缺氧等可触发细菌释放外膜囊泡,甚至在正常生长周期中,一些革兰阴性菌也会释放囊泡。外膜囊泡与细菌的多种生理过程相关,如应激反应、毒素传递、致病、细胞间通讯、免疫调节、基因水平转移及维持微生物群稳态等。在使用抗生素治疗过程中,尤其是当人体微生物群处于低剂量抗生素环境时,细菌会大量分泌外膜囊泡。在肠道中,外膜囊泡释放后会通过多种机制刺激肠道而引发多种炎症。本文综述了外膜囊泡的产生、结构及生理作用,提出抗生素治疗不但会破坏人体正常菌群而导致菌群失调,还会诱导细菌产生大量外膜囊泡而引发慢性炎症。噬菌体治疗不破坏正常菌群,特异性杀灭细菌时也不引起外膜囊泡的产生,因此开发使用噬菌体靶向治疗细菌感染将大大减少不良反应。     

Osmoregulatory expression of porin genes in Escherichia coli: a comparative study on strains B and K-12

Escherichia coli K-12 produces both the OmpF and OmpC porins, the relative amounts of which in the outer membrane are affected in a reciprocal manner by the osmolarity of the growth medium. In contrast, E. coli B produces only the OmpF porin, regardless of the medium osmolarity. In this study, it was revealed that there is an extensive deletion within the ompC locus of the E. coli B chromosome. Cloning and nucleotide sequencing of the regulatory gene, ompR , of E. coli B revealed that there are two amino acid alterations (Lys-6 to Asn and Ala-130 to Thr) in the amino acid sequence of the OmpR protein, as compared with that of E. coli K-12. It is suggested that these particular amino acid alterations are responsible for the constitutive expression of the ompF gene observed in E. coli B.     

革兰氏阴性菌血红素载体蛋白Hemophore的结构及作用机制

血红素作为宿主体内最丰富的铁离子来源,是致病菌营养竞争的主要目标,尤其对于血红素自身合成途径部分丧失的细菌。革兰氏阴性菌血红素转运系统由血红素载体蛋白(Hemophore)、外膜血红素受体、TonB-ExbB-ExbD复合物、ABC转运体等组成。Hemophore是存在于细菌细胞膜上或分泌到胞外环境中的一种蛋白。它能从宿主血红素结合蛋白中捕获血红素并将其传递给外膜受体。目前,在不同革兰氏阴性菌中已发现3种类型的Hemophore,分别是HasA、HxuA和HmuY型。本文将详细描述这3种Hemophore捕获血红素及与外膜受体相互作用的机制,以期为进一步研究其他细菌血红素载体蛋白的功能及作用机制奠定基础。     

Characterization of reduced susceptibility to chlorhexidine among Gram-negative bacteria

Chlorhexidine gluconate (CHG) is one of the most commonly used antiseptic, acting against Gram-negative, Gram-positive bacteria, yeast and fungi. However, over use may lead to reduced susceptibility of different bacteria to CHG. This study aimed to characterize the CHG susceptibility among Gram-negative strains in Israel, to evaluate factors that may affect this susceptibility, and to compare CHG susceptibility between ESBLs bacteria to strains without these enzymes. Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella spp, Escherichia coli, and Acinetobacter baumannii were isolated from clinical samples of 193 patients hospitalized at Padeh–Poriya Medical Center. Phenotypic CHG susceptibility was assessed by determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The highest CHG MIC was found among P. mirabilis. The differences between the CHG MIC values among the different strains were statistically significant (p < 0.001). ESBL-positive strains had higher MIC values as compared to ESBL-negative strains (p = 0.030). A significant association was found between CHG susceptibility and sample source (p = 0.015). In conclusion, the information gathered here significantly improves our knowledge on the reduced susceptibility to CHG among Gram-negative bacteria in Israel. Moreover, ESBL-positive bacteria are less susceptible to CHG and finally, bacteria in sputum, wounds, and body fluids are less CHG-susceptible.     

Lipid trafficking to the outer membrane of Gram-negative bacteria

The envelope of Gram-negative bacteria is composed of two distinct lipid membranes: an inner membrane and outer membrane. The outer membrane is an asymmetric bilayer with an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharide. Most of the steps of lipid synthesis occur within the cytoplasmic compartment of the cell. Lipids must then be transported across the inner membrane and delivered to the outer membrane. These topological features combined with the ability to apply the tools of biochemistry and genetics make the Gram-negative envelope a fascinating model for the study of lipid trafficking. In addition, as lipopolysaccharide is essential for growth of most strains and is a potent inducer of the mammalian innate immune response via activation of Toll-like receptors, Gram-negative lipid transport is also a promising target for the development of novel antibacterial and anti-inflammatory compounds. This review focuses on recent developments in our understanding of lipid transport across the inner membrane and to the outer membrane of Gram-negative bacteria.     

常见革兰阴性菌耐药性分析与合理用药研究

目的 分析芜湖市第一人民医院革兰阴性病原菌分布情况及对常见抗菌药物的耐药程度,为临床抗菌药物的选择及其合理使用提供参考。方法 收集2015年1月至2017年12月我院临床科室送检的细菌培养标本,采用法国生物梅里埃公司生产VITEK2 compact全自动微生物分析系统及其配套试剂盒进行细菌鉴定及药敏试验,数据采用WHONET 5.6软件进行统计分析。对年度耐药株数使用R语言3.4.0软件进行分析。结果 2015—2017年我院共检出病原菌3 728株,其中革兰阳性菌1 055株,占28.3%;革兰阴性菌2 673株,占71.7%。革兰阴性菌中主要致病菌为大肠埃希菌、肺炎克雷伯菌、铜绿假单胞杆菌、鲍曼不动杆菌,对青霉素类和第一代头孢菌素类抗菌药物高水平耐药。2017年这4种细菌对头孢唑林的耐药率均已达100.0%。鲍曼不动杆菌对头孢曲松的耐药率逐年上升,对头孢替坦连续3年耐药率均为100.0%,对氨苄西林/舒巴坦、氟喹诺酮类以及头孢吡肟耐药率未逐年上升,对亚胺培南耐药率变化显著,差异均有统计学意义（P<0.05）。铜绿假单胞杆菌对氨曲南耐药率逐年上升,对氨基糖苷类及头孢他啶耐药率逐年下降,差异均有统计学意义（P<0.05）。大肠埃希菌和肺炎克雷伯菌对头孢吡肟耐药率逐年下降,差异有统计学意义（P<0.05）。结论 我院住院患者病原菌以革兰阴性菌为主,不同病原菌对药物的敏感性各异,临床医生应结合患者情况和药敏结果综合分析,合理选用抗菌药物。     

革兰氏阴性细菌蛋白分泌系统研究进展

革兰氏阴性细菌由于具有复杂的双层膜结构,其蛋白质分泌能力较差.这使得革兰氏阴性细菌的典型菌株——大肠杆菌作为最常用的受体细胞在生物制药工程和其他生物技术产品生产中受到一定的限制.因此,革兰氏阴性细菌蛋白分泌系统的研究具有重要意义.本文详细地归纳了革兰氏阴性细菌已知的蛋白分泌系统,分别从分泌系统的分泌过程、分泌蛋白类别、...     

革兰氏阴性菌TonB依赖性受体的功能与结构研究进展*

为维持生长所需,革兰氏阴性菌需要从外界摄取多种营养物质。分子量小于600 Da的分子可以通过自由扩散的方式通过革兰氏阴性菌的外膜,而大分子物质则需要特殊的转运系统才能将其转运至革兰氏阴性菌的胞内。革兰氏阴性菌对大分子营养物质的识别和转运主要由TonB依赖性受体负责完成。所有革兰氏阴性菌中均有TonB依赖性受体的存在,然而不同种类的革兰氏阴性菌拥有TonB依赖性受体的数量不同且功能各异。最近研究表明,TonB依赖性受体不仅参与了铁、血红素、锰、锌、镍、维生素、碳水化合物等多种营养物质的摄取,而且参与了蛋白酶的分泌。为对TonB依赖性受体提供更为深入和系统的理解,详细介绍了目前已知的TonB依赖性受体的功能及结构,以期为更进一步探知TonB依赖性受体未知功能提供可参考依据。     

The beta-barrel finder (BBF) program,allowing identification of outer membrane beta-barrel proteins encoded within prokaryotic genomes

Many outer membrane proteins (OMPs) in Gram-negative bacteria possess known beta-barrel three-dimensional (3D) structures. These proteins, including channel-forming transmembrane porins, are diverse in sequence but exhibit common structural features. We here report computational analyses of six outer membrane proteins of known 3D structures with respect to (1) secondary structure, (2) hydropathy, and (3) amphipathicity. Using these characteristics, as well as the presence of an N-terminal targeting sequence, a program was developed allowing prediction of integral membrane beta-barrel proteins encoded within any completely sequenced prokaryotic genome. This program, termed the beta-barrel finder (BBF) program, was used to analyze the proteins encoded within the Escherichia coli genome. Out of 4290 sequences examined, 118 (2.8%) were retrieved. Of these, almost all known outer membrane proteins with established beta-barrel structures as well as many probable outer membrane proteins were identified. This program should be useful for predicting the occurrence of outer membrane proteins in bacteria with completely sequenced genomes.     

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

高龄患者血液感染常见革兰阴性杆菌及其耐药性分析

目的分析本院80~100岁高龄患者血液感染常见革兰阴性杆菌的种类及其耐药状况,为本院合理使用抗生素提供依据。方法采用BacT/Alert 3D血培养仪对血液标本进行阳性鉴定;VITEK-2Compact全自动微生物鉴定仪进行鉴定;K-B纸片扩散法对抗菌药物进行敏感性测定;使用WHONET 5.4分析软件分析数据。结果本院高龄患者血液培养阳性标本中共分离出革兰阴性杆菌108株,以肠杆菌科细菌为主,其次为非发酵菌,前者主要为大肠埃希菌52株(48.15%)和肺炎克雷伯菌37株(34.26%),后者主要包括铜绿假单胞菌10株(9.26%)和鲍曼不动杆菌8株(7.41%)。其中大肠埃希菌和肺炎克雷伯菌对亚胺培南的耐药率分别为1.92%和13.51%,两者对氨苄西林、氨苄西林/舒巴坦、头孢唑林、头孢呋辛的耐药率均高于50.00%;铜绿假单胞菌和鲍曼不动杆菌对亚胺培南的耐药率分别为20.00%和25.00%,后者对其他抗菌药物的耐药率均高于前者。结论碳青酶烯类抗生素可作为本院高龄患者常见革兰阴性杆菌所致血液感染的首选药物;但在治疗中应考虑细菌的耐药特点及患者的代谢特点合理选择抗生素。