Type VI secretion delivers bacteriolytic effectors to target cells

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.     

Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli

The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.     

红茶菌形态及菌液抑菌作用的研究

对红茶菌形态进行初步研究，采用纸片法测试红茶菌对细菌的(G^ ，G^-)抑制作用．结果表明：它由细菌A，B和酵母菌Y形成共生菌，其菌液对大肠杆菌(E．coli)、枯草芽孢杆菌(B．subtilis)和金黄色葡萄球菌(S．aureus)有明显的生长抑制作用。     

贝类分泌液保护作用的实验研究

探讨贝类分泌液对烧伤伤口保护作用的机理。实验采用背角无齿蚌、圆顶珠蚌、河蚬、中华圆田螺、铜锈环棱螺、耳河螺的分泌液制成四种不同浓度的药敏纸片和离心得到可溶性及不溶性组分,分别对金葡菌、表皮葡萄球菌、乙型链球菌、甲型副伤寒杆菌、福氏痢疾杆菌、大肠杆菌、枯草杆菌、变形杆菌、绿脓杆菌进行了药敏和生长影响试验。结果表明,其机理不是抑菌,而是生物保护膜的作用。从而,为贝类分泌液的临床应用提供了重要的科学依据。     

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.     

微量量热法对细菌最低生长温度的研究

用微量量热计测定了大肠杆菌,金黄色葡萄球菌,白色葡萄球菌和枯草杆菌,在牛肉膏汤液体中不同温度下的产热曲线,按限制性条件下微生物生长模型进行了处理,求得了细菌的生长速率常数k,衰减速度常数β和在实验条件下细菌增长的最大倍数M和细菌的最大数目K,建立了细菌生长速率常数k和细菌生长温度T的定量关系,得到k=0时的温度T0,即细菌的最低生长温度。     

Chaperone release and unfolding of substrates in type III secretion

Type III protein secretion systems are essential virulence factors of many bacteria pathogenic to humans, animals and plants. These systems mediate the transfer of bacterial virulence proteins directly into the host cell cytoplasm. Proteins are thought to travel this pathway in a largely unfolded manner, and a family of customized cytoplasmic chaperones, which specifically bind cognate secreted proteins, are essential for secretion. Here we show that InvC, an ATPase associated with a Salmonella enterica type III secretion system, has a critical function in substrate recognition. Furthermore, InvC induces chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner. Our results show a similarity between the mechanisms of substrate recognition by type III protein secretion systems and AAA + ATPase disassembly machines.     

Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells

Insulin is produced and secreted by the B cells in the endocrine pancreas. In vivo, insulin secretion is under the control of a number of metabolic, neural and hormonal substances. It is now clear that stimulation of insulin release by fuel secretagogues, such as glucose, involves the closure of K+ channels that are sensitive to the intracellular ATP concentration (KATP channels). This leads to membrane depolarization and the generation of Ca2(+)-dependent action potentials. The mechanisms whereby hormones and neurotransmitters such as adrenaline, galanin and somatostatin, which are released by intraislet nerve endings and the pancreatic D cells, produce inhibition of insulin secretion are not clear. Here we show that adrenaline suppresses B-cell electrical activity (and thus insulin secretion) by a G protein-dependent mechanism, which culminates in the activation of a sulphonylurea-insensitive low-conductance K+ channel distinct from the KATP channel.     

绒白乳菇发酵液提取物的抑茵活性及稳定性研究

研究了绒白乳菇发酵液及其提取物对常见食品污染微生物和杨树叶枯病菌的抑制作用,并探讨了各种因素对提取物抑制叶枯病茵活性的影响,结果表明,该提取物对供试细菌的抑制能力强于真菌,对金黄色葡萄球菌的抑制能力最强,其次为枯草杆茵与大肠杆茵,对酵母茵抑制作用微弱;对黑根霉、黑曲霉均不具有抑制作用.提取物对叶枯病茵生长具有较强的抑制...     

杜仲提取物对鱼类病原菌作用的研究

采用二次水浸提法提取杜仲有效成分进行杯碟法抑菌试验.结果表明,杜仲提取物对嗜水气单胞菌、鳗弧菌、温和气单胞菌具有较好的抑菌效果,而对大肠杆菌的抑菌效果不明显.通过SDS-聚丙烯酰胺凝胶电泳分析病原菌在杜仲提取物作用下蛋白的差异表达,结果发现,药物抑菌效果越明显的菌种,其蛋白表达的差异性就越大,且受抑制的蛋白和诱导表达的特异蛋白的分布比较广;而抑菌效果较差的菌种,其蛋白表达差异性较小.     

Natural polymorphisms in C. elegans HECW-1 E3 ligase affect pathogen avoidance behaviour

Heritable variation in behavioural traits generally has a complex genetic basis, and thus naturally occurring polymorphisms that influence behaviour have been defined only in rare instances. The isolation of wild strains of Caenorhabditis elegans has facilitated the study of natural genetic variation in this species and provided insights into its diverse microbial ecology. C. elegans responds to bacterial infection with conserved innate immune responses and, although lacking the immunological memory of vertebrate adaptive immunity, shows an aversive learning response to pathogenic bacteria. Here, we report the molecular characterization of naturally occurring coding polymorphisms in a C. elegans gene encoding a conserved HECT domain-containing E3 ubiquitin ligase, HECW-1. We show that two distinct polymorphisms in neighbouring residues of HECW-1 each affect C. elegans behavioural avoidance of a lawn of Pseudomonas aeruginosa. Neuron-specific rescue and ablation experiments and genetic interaction analysis indicate that HECW-1 functions in a pair of sensory neurons to inhibit P. aeruginosa lawn avoidance behaviour through inhibition of the neuropeptide receptor NPR-1 (ref. 10), which we have previously shown promotes P. aeruginosa lawn avoidance behaviour. Our data establish a molecular basis for natural variation in a C. elegans behaviour that may undergo adaptive changes in response to microbial pathogens.     

Signal sequence directs localized secretion of bacterial surface proteins

All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.     

一株高产丝氨酸羟甲基转移酶基因工程菌的构建及发酵产酶条件优化

以E.coli AB90054基因组中DNA为模板,采用PCR方法扩增得到glyA基因,将该基因克隆到表达载体pET28a(+)中,转化表达菌株BL21(DE3)后筛选阳性重组子,并对筛选所得高表达基因工程菌G16进行培养条件和表达条件的优化。结果表明,当培养条件为38℃、pH为7.0、转速为150r/min时,基因工程菌的生长速度和菌液浓度最佳;当表达条件的诱导温度为30℃、诱导剂浓度为0.5mmol/L、诱导时间为5h时,基因工程菌诱导目的蛋白表达效果最好。通过SDS-PAGE分析和酶活测定后发现,在优化条件下基因工程菌G16发酵产丝氨酸羟甲基转移酶能力占菌体总蛋白的60%左右,且蛋白活性正常。     

Protein delivery into eukaryotic cells by type III secretion machines

Bacteria that have sustained long-standing close associations with eukaryotic hosts have evolved specific adaptations to survive and replicate in this environment. Perhaps one of the most remarkable of those adaptations is the type III secretion system (T3SS)--a bacterial organelle that has specifically evolved to deliver bacterial proteins into eukaryotic cells. Although originally identified in a handful of pathogenic bacteria, T3SSs are encoded by a large number of bacterial species that are symbiotic or pathogenic for humans, other animals including insects or nematodes, and plants. The study of these systems is leading to unique insights into not only organelle assembly and protein secretion but also mechanisms of symbiosis and pathogenesis.     

两种培养基对蒲公英体外抑菌效果观察

对蒲公英全草水提液与醇提液分别在普通营养培养基和M-H琼脂培养基上进行了大肠杆菌、金黄色葡萄球菌抑茵作用的观察，比较两种培养基对中药体外抑菌效果，结果表明，M-H琼脂培养基对其抑菌作用的检测效果比普通营养培养基灵敏。     

Nitration of a peptide phytotoxin by bacterial nitric oxide synthase

Nitric oxide (NO) is a potent intercellular signal in mammals that mediates key aspects of blood pressure, hormone release, nerve transmission and the immune response of higher organisms. Proteins homologous to full-length mammalian nitric oxide synthases (NOSs) are found in lower multicellular organisms. Recently, genome sequencing has shown that some bacteria contain genes coding for truncated NOS proteins; this is consistent with reports of NOS-like activities in bacterial extracts. Biological functions for bacterial NOSs are unknown, but have been presumed to be analogous to their role in mammals. Here we describe a gene in the plant pathogen Streptomyces turgidiscabies that encodes a NOS homologue, and we reveal its role in nitrating a dipeptide phytotoxin required for plant pathogenicity. High similarity between bacterial NOSs indicates a general function in biosynthetic nitration; thus, bacterial NOSs constitute a new class of enzymes. Here we show that the primary function of Streptomyces NOS is radically different from that of mammalian NOS. Surprisingly, mammalian NO signalling and bacterial biosynthetic nitration share an evolutionary origin.     

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.