HVEM signalling at mucosal barriers provides host defence against pathogenic bacteria

The herpes virus entry mediator (HVEM), a member of the tumour-necrosis factor receptor family, has diverse functions, augmenting or inhibiting the immune response. HVEM was recently reported as a colitis risk locus in patients, and in a mouse model of colitis we demonstrated an anti-inflammatory role for HVEM, but its mechanism of action in the mucosal immune system was unknown. Here we report an important role for epithelial HVEM in innate mucosal defence against pathogenic bacteria. HVEM enhances immune responses by NF-κB-inducing kinase-dependent Stat3 activation, which promotes the epithelial expression of genes important for immunity. During intestinal Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli infection, Hvem?/? mice showed decreased Stat3 activation, impaired responses in the colon, higher bacterial burdens and increased mortality. We identified the immunoglobulin superfamily molecule CD160 (refs 7 and 8), expressed predominantly by innate-like intraepithelial lymphocytes, as the ligand engaging epithelial HVEM for host protection. Likewise, in pulmonary Streptococcus pneumoniae infection, HVEM is also required for host defence. Our results pinpoint HVEM as an important orchestrator of mucosal immunity, integrating signals from innate lymphocytes to induce optimal epithelial Stat3 activation, which indicates that targeting HVEM with agonists could improve host defence.     

Plasmids from Staphylococcus aureus replicate in yeast Saccharomyces cerevisiae

It is known that some plasmids, such as RP4, can replicate in many Gram-negative bacteria. Certain small Staphylococcus aureus plasmids have an even broader host range, being able to replicate in not only phylogenetically distant Gram-positive bacteria such as Bacillus subtilis or Streptococcus pneumoniae, but also in the Gram-negative bacterium Escherichia coli. Here we have examined whether these plasmids can also replicate in a lower eukaryote, the yeast Saccharomyces cerevisiae. For this purpose we constructed hybrids between a S. aureus plasmid pC194 and an E. coli plasmid YIp5, which carries a ura-3 gene easy to select for in yeast but cannot replicate in this host. We found that the hybrids transformed yeast with high efficiency (as did hybrids between YIp5 and three other S. aureus plasmids); were maintained extrachromosomally in yeast; and were not modified during residence in yeast. We conclude from this evidence that S. aureus plasmids can replicate in yeast, which raises the questions of whether the replication signals used by prokaryotes and eukaryotes are similar, and how far up the phylogenetic tree the organisms still able to be hosts to S. aureus plasmids may be.     

A transposon, Tn732, encoding gentamicin/tobramycin resistance.

Gentamicin and tobramycin are important antibiotics in the treatment of hospital infections because of their activity against a wide range of bacterial genera. With their increasing use, bacteria resistant to these drugs have appeared, the resistance being frequently plasmid determined. The resistance genes determine various enzymes that modify and inactivate the drugs and there is association between particular gentamicin/tobramycin resistance genes and plasmids of particular groups, implying that acquisition of such a gene by any plasmid is a rare event. We now report the identification of a transposon or 'jumping gene' encoding the gentamicin/tobramycin adenylylating enzyme, ANT(2"), on a plasmid of incompatiblity group FII (IncFII).     

Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator

The polymorphonuclear leukocyte (PMN), or neutrophil, is the major host defence cell protecting the body against invasion by bacteria and fungi. Products of oxidative metabolism mediate PMN microbicidal and tumoricidal activity but the mechanisms by which these pathways become activated are not well understood. We have previously described a human granulocyte-macrophage colony-stimulating factor (GM-CSF) of relative molecular mass (Mr) 22,000 that also inhibits neutrophil motility (NIF-T activity). Because of its direct action on granulocytes, this lymphokine is a candidate for a neutrophil-activating factor. We have studied the effect of GM-CSF/NIF-T on superoxide anion generation in response to the bacterial chemo-attractant N-formylmethionyl-leucylphenylalanine (f-MLP), and report here that PMNs preincubated with either purified natural GM-CSF or biosynthetic (recombinant) GM-CSF showed increased (as much as fourfold) superoxide anion production in response to f-MLP. These results indicate that human GM-CSF is a neutrophil-activating factor.     

Ecology drives a global network of gene exchange connecting the human microbiome

Horizontal gene transfer (HGT), the acquisition of genetic material from non-parental lineages, is known to be important in bacterial evolution. In particular, HGT provides rapid access to genetic innovations, allowing traits such as virulence, antibiotic resistance and xenobiotic metabolism to spread through the human microbiome. Recent anecdotal studies providing snapshots of active gene flow on the human body have highlighted the need to determine the frequency of such recent transfers and the forces that govern these events. Here we report the discovery and characterization of a vast, human-associated network of gene exchange, large enough to directly compare the principal forces shaping HGT. We show that this network of 10,770 unique, recently transferred (more than 99% nucleotide identity) genes found in 2,235 full bacterial genomes, is shaped principally by ecology rather than geography or phylogeny, with most gene exchange occurring between isolates from ecologically similar, but geographically separated, environments. For example, we observe 25-fold more HGT between human-associated bacteria than among ecologically diverse non-human isolates (P = 3.0 × 10(-270)). We show that within the human microbiome this ecological architecture continues across multiple spatial scales, functional classes and ecological niches with transfer further enriched among bacteria that inhabit the same body site, have the same oxygen tolerance or have the same ability to cause disease. This structure offers a window into the molecular traits that define ecological niches, insight that we use to uncover sources of antibiotic resistance and identify genes associated with the pathology of meningitis and other diseases.     

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.     

农杆菌质粒DNA高效提取方法研究

以EHA105菌株为实验材料,通过优化质粒扩增条件、加大菌液使用量、改良提取纯化过程中所用试剂等,简化了操作流程,给出了一种高效提取农杆菌中质粒DNA的方法。结果表明,选择LB＋KANA为培养基、抗生素质量浓度为50 mg/L,细菌培养至OD600=0.634～0.714时,以5 mL/次用量取菌液裂解;以改进方法提取试剂,得率与常规方法相当。该方法在满足PCR检测条件的前提下既减少了酚、氯仿等有毒试剂的用量,又省时、经济、方便,为植物转基因工程中目的物DNA的检测提供了技术支持。     

植物类黄酮的生理功能与抗菌机制

类黄酮是植物产生于不同部位的一大类次生代谢小分子,在植物各器官履行多种生理功能;对人类健康有广泛的药理和有益作用,包括抗氧化活性、自由基清除能力、预防冠心病、抗动脉粥样硬化、保肝、抗炎和抗癌活性,已获得医药及保健业的高度关注;研究表明:类黄酮还能通过破坏细菌细胞膜、抑制细菌脂肪酸、粘肽层、核酸与电子传递链和ATP合成、抑制细菌金属酶活性等,发挥抗菌抑菌作用;在细胞水平上可阻止细菌粘附到宿主受体,抑制细菌生物膜形成,不仅选择性地针对细菌细胞,也抑制毒性因子以及其他形式的微生物威胁;一些植物类黄酮能明显逆转抗生素的抗药性,提高其药效;开发和应用类黄酮药物,对抗生素耐药感染可能是一有前途的方法。     

Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200?mV to -300?mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.     

Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota

The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth. Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development, with implications ranging from enhanced crop productivity to phytoremediation. Endophytic colonization represents an apparent paradox of plant innate immunity because plant cells can detect an array of microbe-associated molecular patterns (also known as MAMPs) to initiate immune responses to terminate microbial multiplication. Several studies attempted to describe the structure of bacterial root endophytes; however, different sampling protocols and low-resolution profiling methods make it difficult to infer general principles. Here we describe methodology to characterize and compare soil- and root-inhabiting bacterial communities, which reveals not only a function for metabolically active plant cells but also for inert cell-wall features in the selection of soil bacteria for host colonization. We show that the roots of Arabidopsis thaliana, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil-type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell-wall features of other tested plant species seem to provide a sufficient cue for the assembly of approximately 40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found on any plant root or plant debris in the tested soils. By contrast, colonization of Arabidopsis roots by members of the Actinobacteria depends on other cues from metabolically active host cells.     

Direct role of the himA gene product in phage lambda integration

The integration of phage lambda into the Escherichia coli chromosome is accomplished by a site-specific recombination between two unique DNA sequences (attB on the bacterial genome and attP on the phage; reviewed in refs 2, 3) and requires proteins encoded by both the bacterium and the phage. Genetic and biochemical studies have shown that bacterial strains mutant in the himA gene, located at 38 min on the E. coli map, are defective in the activity of the host-encoded component. They are, moreover, defective for the growth of bacteriophage Mu, for precise excision of transposable antibiotic resistance determinants and for the synthesis of the lambda int gene product. We now show that the himA gene product (phimA) is not solely a regulator of genes involved in integration but is one of two host polypeptides required for integrative recombination.     

The role of parasites in sympatric and allopatric host diversification

Exploiters (parasites and predators) are thought to play a significant role in diversification, and ultimately speciation, of their hosts or prey. Exploiters may drive sympatric (within-population) diversification if there are a variety of exploiter-resistance strategies or fitness costs associated with exploiter resistance. Exploiters may also drive allopatric (between-population) diversification by creating different selection pressures and increasing the rate of random divergence. We examined the effect of a virulent viral parasite (phage) on the diversification of the bacterium Pseudomonas fluorescens in spatially structured microcosms. Here we show that in the absence of phages, bacteria rapidly diversified into spatial niche specialists with similar patterns of diversity across replicate populations. In the presence of phages, sympatric diversity was greatly reduced, as a result of phage-imposed reductions in host density decreasing competition for resources. In contrast, allopatric diversity was greatly increased as a result of phage-imposed selection for resistance, which caused populations to follow divergent evolutionary trajectories. These results show that exploiters can drive diversification between populations, but may inhibit diversification within populations by opposing diversifying selection that arises from resource competition.     

Mealybug beta-proteobacterial endosymbionts contain gamma-proteobacterial symbionts

Some insects have cultivated intimate relationships with mutualistic bacteria since their early evolutionary history. Most ancient 'primary' endosymbionts live within the cytoplasm of large, polyploid host cells of a specialized organ (bacteriome). Within their large, ovoid bacteriomes, mealybugs (Pseudococcidae) package the intracellular endosymbionts into 'mucus-filled' spheres, which surround the host cell nucleus and occupy most of the cytoplasm. The genesis of symbiotic spheres has not been determined, and they are structurally unlike eukaryotic cell vesicles. Recent molecular phylogenetic and fluorescent in situ hybridization (FISH) studies suggested that two unrelated bacterial species may share individual host cells, and that bacteria within spheres comprise these two species. Here we show that mealybug host cells do indeed harbour both beta- and gamma-subdivision Proteobacteria, but they are not co-inhabitants of the spheres. Rather, we show that the symbiotic spheres themselves are beta-proteobacterial cells. Thus, gamma-Proteobacteria live symbiotically inside beta-Proteobacteria. This is the first report, to our knowledge, of an intracellular symbiosis involving two species of bacteria.     

氰污染环境中细菌质粒的研究

三大氰污染环境中所分离得到的６６株有代表性的优势菌进行了质粒检测和一些带质粒菌的分类学研究。结果为：所选择的三大氰污染样区的细菌质粒检出率均高于非污染样区，且假单胞菌在氰污染中占绝对优势。选择了高效降氰菌株Ｎ４０进行质粒消除的研究，结果表明所得到的高效降氰菌株Ｎ４０的质粒较难被吖啶橙的ＳＤＳ消除。     

Construction of ice nucleation active <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> for control of insect pests

Ice nucleation active(INA)bacteria are the most potent heterogeneous ice nuclei in nature,which have become an important biological resource for diverse applications.Many researches have proved that INA bacteria can raise the supercooling points(SCPs)of insect pests,then reduce their cold hardiness.However,INA bacteria‘s inefficient colonization on the surface or in the guts of insects,and the high incidence of frost injury induced by their release hampered the application of INA bacteria in controlling insect pests in agricultural fields.In this study,we constructed a recombinant plasmid mob-Tn5-iceA with the ability of broad-host-range conjugal mobilization and integration of the ina gene of icaA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition.In addition,Ent.cloacae strains stably carrying iceA and expressing high ice nucleation activity(INA),even in the absence of antibiotic pressure,were constructed through conjugal mobilization and Tn5 transposition.Ent,cloacae strains have benn reported to be able to efficently colonize in the guts of insects,but have weak plant epiphytic ability.Therefore,these transgenic Ent.Cloacae may be promising candidates for control of insect pests in agricultural fields.     

High-frequency transformation of the fission yeast Schizosaccharomyces pombe

The fission yeast, Schizosaccharomyces pombe, has been used extensively for genetic studies but until now it has not been utilized as a host organism for DNA cloning. Here we describe a method for high-frequency transformation fo a leu 1(-) strain of this yeast with hybrid plasmids containing the Saccharomyces cerevisiae LEu 2(+) gene, a bacterial plasmid and either the S. cerevisiae 2 μm plasmid or autonomously replicating sequences (ars)(1) derived from S. pombe DNA. Some of the plasmids contain unique restriction sites which make them suitable for the isolation of S. pombe genes, and they can also be used for the exchange of DNA between S. pombe and S. cerevisiae.     

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase

Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.     

Molecular mechanisms that confer antibacterial drug resistance

Antibiotics--compounds that are literally 'against life'--are typically antibacterial drugs, interfering with some structure or process that is essential to bacterial growth or survival without harm to the eukaryotic host harbouring the infecting bacteria. We live in an era when antibiotic resistance has spread at an alarming rate and when dire predictions concerning the lack of effective antibacterial drugs occur with increasing frequency. In this context it is apposite to ask a few simple questions about these life-saving molecules. What are antibiotics? Where do they come from? How do they work? Why do they stop being effective? How do we find new antibiotics? And can we slow down the development of antibiotic-resistant superbugs?     

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 ? crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.