Characterization of Two Pore-Forming Proteins Isolated from the Outer Membrane of Synechococcus PCC 6301

Two major proteins, A and B, were isolated and purified from outer membranes of the unicellular cyanobacterium Synechococcus PCC 6301 by gel filtration, anion-exchange chromatography, and preparative SDS-PAGE. Protein A revealed a single-channel conductance of 0.4 nanoSiemens (nS) in 1 M KCl, whereas preparations containing both proteins showed two different conductance maxima of 0.4 and 0.9 nS, suggesting that B also forms pores. The apparent molecular mass of the two closely migrating proteins was determined as 52 kDa, whereas native porin extracts revealed a relative molecular mass of ca. 140 kDa, indicating trimeric pore-forming units. Partial sequences of both proteins were obtained by N-terminal sequencing of tryptic peptides, and the C-terminal amino acid sequences were derived from the complete proteins. These sequences were aligned to protein sequences available in the databases. The results are discussed. Received: 22 August 1997 / Accepted: 1 October 1997     

Outer Membrane Proteins of Escherichia coli IV. Differences in Outer Membrane Proteins Due To Strain and Cultural Differences

When the 42,000-dalton major outer membrane protein of Escherichia coli O111 is examined on alkaline polyacrylamide gels containing sodium dodecyl sulfate, it is resolved into three distinct bands designated as proteins 1, 2, and 3. Band 3 consists of two distinct polypeptides, proteins 3a and 3b. E. coli K-12 does not make any protein 2, but makes proteins similar to 1, 3a, and 3b as indicated by comparison of cyanogen bromide peptide patterns. Several Shigella species and most other strains of E. coli resemble E. coli K-12 in that they lack protein 2, whereas Salmonella typhimurium is more similar to E. coli O111. In addition to these species and strain differences, cultural differences resulted in differences in the outer membrane protein profiles. Under conditions of catabolite repression, the level of protein 2 in E. coli O111 decreased while the level of protein 1 increased. An enterotoxin-producing strain similar to E. coli O111 produced no protein 1 and an elevated level of protein 2 under conditions of low catabolite repression. The levels of proteins 1 and 3 are also different in different phases of the growth curve, with protein 1 being the major species in the exponential-phase cells and protein 3 being the major species in stationary-phase cells. A multiply phage-resistant mutant of E. coli K-12 with no obvious cell wall defects produced no protein 1 or 2, but made increased amounts of protein 3. Thus, the major outer membrane proteins of E. coli and related species may vary considerably without affecting outer membrane integrity.     

Outer Membrane Proteins and Serosubtyping with Outer Membrane Vesicles from Clinical Isolates of Neisseria meningitidis

The currently practiced protocol for routine serosubtyping of Neisseria meningitidis relies on reactivity of whole cells to monoclonal antibodies against the class 1 outer membrane protein (OMP) in ELISAs or dot-blots. This procedure, however, failed to yield serosubtyping information in 28% (48/174) of clinical isolates (1993–1994) in the province of Québec, Canada. These 48 strains were characterized by OMP profiles and ELISAs with outer membrane vesicles (OMVs). Forty out of the 48 strains expressed class 1 OMP, indicating that the inability to assign a serosubtype was not owing to the absence of the class 1 OMP. Of these, 15 (38%) were serosubtypable in ELISAs with outer membrane vesicles. Thus, 81% (141/174) of all meningococcal strains were serosubtypable with ELISAs using whole-cells or OMVs. Because the routinely used procedure for serosubtyping of meningococci is limited in providing serosubtype information, alternate procedures are proposed to obtain comprehensive information for epidemiological identification of this bacterium. Received: 11 June 1996 / Accepted: 5 July 1996     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.     

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.     

Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

Summary: Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens.     

Physical Properties of Bacterial Outer Membrane Models: Neutron Reflectometry & Molecular Simulation

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM.     

利用分散量理论辨识外膜蛋白

利用分散量的数学理论,提出了基于最小分散增量的蛋白质序列辨识方法.通过多种特征联合对蛋白质序列进行编码,并建立基于最小分散增量的分类器MID_OMP,应用于革兰氏阴性细菌外膜蛋白序列辨识.在数据集上的Jackknife测试中,MID_OMP辨识外膜蛋白和α螺旋跨膜蛋白的准确率达到95.7%,辨识外膜蛋白和球状蛋白的准确率达到91.0%;在14个细菌基因组内挖掘结果显示,MID_OMP具有较高的敏感性和特异性,预测结果的可信度明显优于另外一种OMPs挖掘工具TMBETA-GENOME.     

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.     

Analysis of Surface-Exposed Outer Membrane Proteins in Helicobacter pylori

More than 50 Helicobacter pylori genes are predicted to encode outer membrane proteins (OMPs), but there has been relatively little experimental investigation of the H. pylori cell surface proteome. In this study, we used selective biotinylation to label proteins localized to the surface of H. pylori, along with differential detergent extraction procedures to isolate proteins localized to the outer membrane. Proteins that met multiple criteria for surface-exposed outer membrane localization included known adhesins, as well as Cag proteins required for activity of the cag type IV secretion system, putative lipoproteins, and other proteins not previously recognized as cell surface components. We identified sites of nontryptic cleavage consistent with signal sequence cleavage, as well as C-terminal motifs that may be important for protein localization. A subset of surface-exposed proteins were highly susceptible to proteolysis when intact bacteria were treated with proteinase K. Most Hop and Hom OMPs were susceptible to proteolysis, whereas Hor and Hof proteins were relatively resistant. Most of the protease-susceptible OMPs contain a large protease-susceptible extracellular domain exported beyond the outer membrane and a protease-resistant domain at the C terminus with a predicted β-barrel structure. These features suggest that, similar to the secretion of the VacA passenger domain, the N-terminal domains of protease-susceptible OMPs are exported through an autotransporter pathway. Collectively, these results provide new insights into the repertoire of surface-exposed H. pylori proteins that may mediate bacterium-host interactions, as well as the cell surface topology of these proteins.     

IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.     

细菌外膜囊泡(OMV)研究进展

外膜囊泡(outer membrane vesicles,OMV)是在细菌生命活动中不断从细菌细胞表面脱离而形成的功能性囊泡,其内部含有蛋白质、脂质和核酸等成分,具有多种特殊的生物学功能,在细菌-细菌和细菌-宿主相互作用中起着关键作用.虽然大多数OMV的研究都是关于动物病原菌,但最近OMV在植物-细菌相作领域的作用已逐...     

Characterization of Serologically Dominant Outer Membrane Proteins of Neisseria gonorrhoeae

The proteins of the outer membrane of Neisseria gonorrhoeae play an important role in the serotyping system defined by K. H. Johnston et al. (J. Exp. Med. 143:741–758, 1976). This study attempted to delineate the molecular arrangement of the major proteins of the outer membrane of the gonococcus by using three approaches. First, natural protein-protein relationships were demonstrated by symmetrical, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, proteins exposed on the surface of outer membrane vesicles were cross-linked by using the bifunctional reagents dimethyl-3,3′-dithiobispropionimidate and dithiobis[succinimidyl propionate]. Third, specific antigen-antibody interactions on the surface of membrane vesicles were analyzed by radioautographic techniques. The major proteins of the outer membrane of the gonococcus were defined, and a nomenclature was devised to take into account the effects of heat and reducing agents on the resolution of these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of cross-linking experiments strongly suggest that two of the major proteins of the gonococcal outer membrane (proteins 1 and 3) form a hydrophobically associated trimeric unit in situ which can be stabilized by selective cross-linking reagents. Results substantiated that these proteins are responsible for imparting serotypic specificity.     

细菌外膜囊泡在抗肿瘤治疗方面的研究进展*

细菌外膜囊泡(outer membrane vesicles,OMVs)是在细菌生长过程中分泌出的一种直径为20~300 nm的膜性小泡。由磷脂、脂多糖、蛋白质、RNA或DNA等组成。OMVs包含大量细菌抗原,通过启动信号转导通路增强细胞因子和共刺激分子的表达,促进抗原呈递,有效激活免疫系统。OMVs中的毒力因子可以传递给宿主细胞,刺激细菌-宿主细胞之间的相互作用,具有内在的抗肿瘤活性。另外OMVs有利于进行工程设计,还可作为高效的药物运载体,实现免疫治疗和化疗-光疗的结合,从而提高药物的抗癌能力。OMVs在肿瘤免疫、肿瘤工程疫苗和载药等方面具有良好前景,被认为是抗肿瘤治疗的新型手段。从OMVs的结构组分、产生机制和抗肿瘤机制等方面概述了OMVs在肿瘤治疗中的研究进展,为将来OMVs的深入研究和临床应用提供参考。     

The Fluidity of the Bacterial Outer Membrane Is Species Specific

The outer membrane (OM) is an essential barrier that guards Gram-negative bacteria from diverse environmental insults. Besides functioning as a chemical gatekeeper, the OM also contributes towards the strength and stiffness of cells and allows them to sustain mechanical stress. Largely influenced by studies of Escherichia coli, the OM is viewed as a rigid barrier where OM proteins and lipopolysaccharides display restricted mobility. Here the discussion is extended to other bacterial species, with a focus on Myxococcus xanthus. In contrast to the rigid OM paradigm, myxobacteria possess a relatively fluid OM. It is concluded that the fluidity of the OM varies across environmental species, which is likely linked to their evolution and adaptation to specific ecological niches. Importantly, a fluid OM can endow bacteria with distinct functions for cell-cell and cell-environment interactions.     

细菌外膜囊泡应用于肿瘤治疗

细菌外膜囊泡（outer membrane vesicles,OMVs）是由革兰氏阴性菌分泌的纳米囊泡,主要由细菌外膜和周质成分组成,因此表面富集的病原体相关分子模式（PAMPs）使OMVs能激起强烈的免疫反应。在抗肿瘤研究中,OMVs主要被用于抗肿瘤药物的递送,不仅能增加药物的肿瘤富集还能激活免疫反应协同杀伤肿瘤;同时,OMVs也用于开发肿瘤疫苗的佐剂,可显著提高免疫响应的能力。本综述主要概括了OMVs的生物发生机理、OMVs对宿主免疫系统的影响及其在肿瘤治疗中的研究进展。