Localization of the receptor-binding protein adhesin at the tip of the bacterial pilus

Strains of the bacterium Escherichia coli that cause infections of the human urinary tract produce so-called Pap-pili, which are hair-like appendages consisting of about 10(3) helically arranged subunits of the protein PapA. These pili mediate binding to digalactoside-containing glycolipids present on the epithelial cells which line the urinary tract. Recently, it has been suggested that three proteins, PapE, PapF and PapG, are responsible for this binding. In the absence of PapA, non-piliated bacteria are formed which nonetheless exhibit binding, showing that the bulk of the pilus is not essential for binding. Although pili can form without PapF and PapG, such pili are unable to bind to the digalactoside. The protein PapG mediates binding specificity in trans-complementation experiments, so this protein is the digalactoside-specific adhesin. Using immuno-electron microscopy we have found that Pap-pili are heteropolymers composed of the major pilin, PapA, the minor pilins, PapE and PapF, and the adhesin, PapG. The last three proteins are located at the tip of the pilus.     

Identification and characterization of E. coli type-1 pilus tip adhesion protein

The type-1 pilus of Escherichia coli is the prototype of this class of hair-like, multimeric adhesive organelles. This pilus mediates adherence to mannose-containing receptors on mucosal epithelia and other cells. The type-1 pilus, in one of several serological variants, is expressed by nearly all E. coli strains, and its promotion of colonization by pathogenic bacteria and the protective effects of purified pilus vaccines suggest that it is important as a bacterial virulence factor. Both the adhesive function and the serological variation of the type-1 pilus have been attributed to the thousand or so pilin protein monomers making up the pilus rods. This idea has been contradicted by our earlier observations on an E. coli strain expressing adhesion-defective pili. More recent genetic evidence also indicates that auxiliary pilus proteins are required for adhesive function. We report here the identification of three previously undetected integral minor proteins on the type-1 pilus, and show that one of them is the receptor-binding adhesin. This protein is antigenically conserved among strains with different pilin serotypes and is located at the pilus tip.     

Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli

Gram-negative bacteria are known to produce two types of surface organelles: flagella, which are required for motility and chemotaxis, and pili (fimbriae), which play a part in the interaction of bacteria with other bacteria and with eukaryotic host cells. Here we report a third class of E. coli surface organelles for which we propose the name curli. Curli are coiled surface structures composed of a single type of subunit, the curlin, which differs from all known pilin proteins and is synthesized in the absence of a cleavable signal peptide. Although the gene encoding this structural subunit, crl, is present and transcribed in most natural isolates of E. coli, only certain strains are able to assemble the subunit protein into curli. This assembly process occurs preferentially at growth temperatures below 37 degrees C. The ability of curli to mediate binding to fibronectin may be a virulence-associated property for wound colonization and for the colonization of fibronectin-coated surfaces.     

The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination.

Tuberculosis remains the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, with 3 million deaths and 10 million new cases per year. The infection initiates in the lungs and can then spread rapidly to other tissues. The availability of the entire M. tuberculosis genome sequence and advances in gene disruption technologies have led to the identification of several mycobacterial determinants involved in virulence. However, no virulence factor specifically involved in the extrapulmonary dissemination of M. tuberculosis has been identified to date. Here we show that the disruption of the M. tuberculosis or Mycobacterium bovis Bacille Calmette-Guérin (BCG) hbhA gene encoding the heparin-binding haemagglutinin adhesin (HBHA) markedly affects mycobacterial interactions with epithelial cells, but not with macrophage-like cells. When nasally administered to mice, the mutant strains were severely impaired in spleen colonization, but not in lung colonization. Coating wild-type mycobacteria with anti-HBHA antibodies also impaired dissemination after intranasal infection. These results provide evidence that adhesins such as HBHA are required for extrapulmonary dissemination, and that interactions with non-phagocytic cells have an important role in the pathogenesis of tuberculosis. They also suggest that antibody responses to HBHA may add to immune protection against tuberculosis.     

Conservation of the D-mannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae

A variety of genera and species of the family Enterobacteriaceae bear surface fimbriae that enable them to bind to D-mannose residues on eukaryotic cells. Until recently, it was thought that the D-mannose binding site was located in the major structural subunit (FimA), of relative molecular mass (Mr) 17,000 (17 K), of these organelles in Escherichia coli. New evidence indicates that this binding site resides instead in a minor protein Mr 28-31 K (FimH) located at the tips and at long intervals along the length of the fimbriae, and is reminiscent of the minor tip adhesion proteins of pyelonephritis-associated pili (Pap) and S fimbriae. In contrast to the antigenic heterogeneity of the major FimA subunit, the antigenic structure of FimH is conserved among different strains of E. coli. Here, we report an even broader conservation of this minor adhesion protein extending to other genera and species of type 1 fimbriated Enterobacteriaceae. Our results may have implications for the development of broadly protective vaccines against Gram-negative bacillary infections in animals and perhaps in man.     

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.     

TREM-1 amplifies inflammation and is a crucial mediator of septic shock

Host innate responses to bacterial infections are primarily mediated by neutrophils and monocytes/macrophages. These cells express pattern recognition receptors (PRRs) that bind conserved molecular structures shared by groups of microorganisms. Stimulation of PRR signalling pathways initiates secretion of proinflammatory mediators, which promote the elimination of infectious agents and the induction of tissue repair. Excessive inflammation owing to bacterial infections can lead to tissue damage and septic shock. Here we show that inflammatory responses to microbial products are amplified by a pathway mediated by triggering receptor expressed on myeloid cells (TREM)-1. TREM-1 is an activating receptor expressed at high levels on neutrophils and monocytes that infiltrate human tissues infected with bacteria. Furthermore, it is upregulated on peritoneal neutrophils of patients with microbial sepsis and mice with experimental lipopolysaccaride (LPS)-induced shock. Notably, blockade of TREM-1 protects mice against LPS-induced shock, as well as microbial sepsis caused by live Escherichia coli or caecal ligation and puncture. These results demonstrate a critical function of TREM-1 in acute inflammatory responses to bacteria and implicate TREM-1 as a potential therapeutic target for septic shock.     

Structure of the E. coli signal recognition particle bound to a translating ribosome

The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane. It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 A structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.     

Immune recognition. A new receptor for beta-glucans.

The carbohydrate polymers known as beta-1,3-d-glucans exert potent effects on the immune system - stimulating antitumour and antimicrobial activity, for example - by binding to receptors on macrophages and other white blood cells and activating them. Although beta-glucans are known to bind to receptors, such as complement receptor 3 (ref. 1), there is evidence that another beta-glucan receptor is present on macrophages. Here we identify this unknown receptor as dectin-1 (ref. 2), a finding that provides new insights into the innate immune recognition of beta-glucans.     

Metabolite-enabled eradication of bacterial persisters by aminoglycosides

Bacterial persistence is a state in which a sub-population of dormant cells, or 'persisters', tolerates antibiotic treatment. Bacterial persisters have been implicated in biofilms and in chronic and recurrent infections. Despite this clinical relevance, there are currently no viable means for eradicating persisters. Here we show that specific metabolic stimuli enable the killing of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) persisters with aminoglycosides. This potentiation is aminoglycoside-specific, it does not rely on growth resumption and it is effective in both aerobic and anaerobic conditions. It proceeds by the generation of a proton-motive force which facilitates aminoglycoside uptake. Our results demonstrate that persisters, although dormant, are primed for metabolite uptake, central metabolism and respiration. We show that aminoglycosides can be used in combination with specific metabolites to treat E. coli and S. aureus biofilms. Furthermore, we demonstrate that this approach can improve the treatment of chronic infections in a mouse urinary tract infection model. This work establishes a strategy for eradicating bacterial persisters that is based on metabolism, and highlights the importance of the metabolic environment to antibiotic treatment.     

人肿瘤坏死因子可溶性受体I cDNA的克隆及其在原核和真核细胞中的表达

以人的胎盘总ＲＮＡ为模板,用ＲＴ－ＰＣＲ的方法,获得人肿瘤坏死因子可溶性受体Ｉ（ｓｈＴＮＦＲ－Ｉ）的ｃＤＮＡ,并对其进行全序列分析,在此基础上构建了原核及真核的表达载体,进行真核瞬间表达及活性测定和原核表达及产物初步纯化     

Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex

Intimin and its translocated intimin receptor (Tir) are bacterial proteins that mediate adhesion between mammalian cells and attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli (EPEC) causes significant paediatric morbidity and mortality world-wide. A related A/E pathogen, enterohaemorrhagic E. coli (EHEC; O157:H7) is one of the most important food-borne pathogens in North America, Europe and Japan. A unique and essential feature of A/E bacterial pathogens is the formation of actin-rich pedestals beneath the intimately adherent bacteria and localized destruction of the intestinal brush border. The bacterial outer membrane adhesin, intimin, is necessary for the production of the A/E lesion and diarrhoea. The A/E bacteria translocate their own receptor for intimin, Tir, into the membrane of mammalian cells using the type III secretion system. The translocated Tir triggers additional host signalling events and actin nucleation, which are essential for lesion formation. Here we describe the the crystal structures of an EPEC intimin carboxy-terminal fragment alone and in complex with the EPEC Tir intimin-binding domain, giving insight into the molecular mechanisms of adhesion of A/E pathogens.     

2013～2017年医院尿路感染住院患者抗菌药物应用分析

目的 通过调查医院2013~2017年尿路感染住院患者抗菌药物使用情况,为评价临床药师参与临床用药中发挥的作用提供依据。方法 检索宜春市人民医院2013~2017年456例尿路感染住院患者病例资料,分析比较2013~2015与2016~2017年抗菌药物的使用与患者治疗情况。包括用药频度（DDDs）、平均使用品种、联合用药率、换药率、选药不合理率、剂量不合理率、疗程不合理率、平均住院天数及治愈率。结果 2013~2015年氟喹诺酮类药物DDDS排序均为第一;2016~2017年头孢菌素类药物DDDS排序均为第一;单类品种中,左氧氟沙星DDDs排序在2013~2016年期间为第一;2017年时氨曲南DDDS排序位居首位而左氧氟沙星位居第五;2013~2017年患者平均用药品种数与疗程不合理率无显著变化,联合用药率、换药率于2016~2017年较以往有明显降低P<0.01,P<0.05,但平均住院天数较往年有明显升高P<0.05。剂量不合理率、选药不合理率较往年降低而治愈率较往年有所提高。结论 在临床药师的参与下,尿路感染患者抗菌药物用药逐渐走向更加规范合理,尿路感染患者的抗菌药物应用得到有效控制。     

用酵母双杂合系统筛选与人血小板生成素受体c-Mpl相互作用的蛋白质的初报

血小板生成素（ｔｈｒｏｍ ｂｏｐｏｉｅｔｉｎ, ＴＰＯ）是调节血小板生成最主要的细胞因子,其生物学效应由其受体ｃ－Ｍｐｌ介导．利用酵母双杂合系统（ｔｗ ｏ－ｈｙｂｒｉｄ ｓｙｓｔｅｍ ）筛选与ｃ－Ｍｐｌ相互作用的蛋白质因子,以Ｇａｌ４ ＢＤ融合ｃ－Ｍｐｌ膜内部分ｃＤＮＡ 的ｐＡＳＭＭ 为靶蛋白质粒,筛选了人胎盘ｃＤＮＡ 文库,分离到人波形纤维蛋白（ｖｉｍ ｅｎｔｉｎ）的部分编码序列,首次检测到波形纤维蛋白与ＴＰＯ 受体之间的相互作用,这提示细胞骨架蛋白可能在ＴＰＯ 的信号转导过程中起着重要的作用     

尿路感染病原菌及耐药性分析

目的：探讨尿路感染的病原菌构成及耐药性,指导临床合理使用抗菌药物。方法：收集我院2006年1月至2007年12月尿路感染患者清洁中段尿培养阳性588株细菌进行鉴定,并用K—B纸片法作药敏分析。结果：尿路感染病原菌以革兰阴性杆菌为主（63．8％）,前4位分别是大肠埃希菌（45．7％）、凝固酶阴性葡萄球菌（11.6％）、假丝酵母菌（11．1％）、肠球菌（7．8％）。大肠埃希菌和克雷伯菌超广谱β-内酰胺胺酶（ESBLS）检出率分别为29％和32.1％,甲氧西林耐药凝固酶阴性葡萄球菌（MRCNS）检出率为41．2％。结论：革兰阴性杆菌是尿路感染的主要病原菌,对常规抗菌药物的耐药性呈上升趋势,细菌分离培养鉴定及药敏试验对指导临床合理使用抗菌药物具有重要意义。     

A synthetic peptide that is a bombesin receptor antagonist

The tetradecapeptide bombesin was originally isolated from frog skin. Bombesin-like peptides have since been detected in mammalian gastrointestinal tract, brain and lung. These peptides have potent pharmacological effects on the central nervous system; they cause contraction of intestinal, uterine and urinary tract smooth muscle; and stimulate the release of other peptides including gastrin, cholecystokinin, motilin, pancreatic polypeptide, neurotensin, insulin, enteroglucagon, prolactin and growth hormone. Specific plasma membrane receptors for bombesin have been demonstrated on pancreatic acinar cells, brain membranes and pituitary cells. Studies defining the physiological importance of bombesin have been impeded by the lack of a bombesin receptor antagonist. Here we describe experiments which demonstrate that a peptide originally described as a substance P receptor antagonist, [D-Arg, D-Pro, D-Trp, Leu ]substance P, is also a bombesin receptor antagonist. This peptide competitively inhibits the ability of bombesin to stimulate enzyme secretion from dispersed pancreatic acini, and also inhibits the action of other peptides that interact with the bombesin receptor.     

Crystal structure of chaperone protein PapD reveals an immunoglobulin fold

The chaperone protein PapD mediates assembly of pili in Escherichia coli. Its polypeptide chain folds into two immunoglobulin-type domains that are homologous in sequence to the human lymphocyte differentiation antigen Leu-1/CD5.