Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation

The zoonotic pathogen Bartonella henselae ( Bh ) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh . Spheroids generated from Bh -infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh -induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella - induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.     

霍乱弧菌Ⅵ型分泌系统的效应蛋白对细菌细胞壁的降解机制

【背景】肽聚糖(Peptidoglycan,PG)是细菌细胞壁的重要组成部分,而霍乱弧菌Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)可以分泌具有肽聚糖水解酶活性的效应蛋白到受体细菌中杀死细胞,这类水解酶的作用机制尚未研究清楚。【目的】通过对细菌细胞壁的PG成分进行研究,建立细胞壁PG成分分析方法,并对霍乱弧菌T6SS分泌的2个破坏细胞壁的效应蛋白TseH和VgrG3的作用机制进行解析。【方法】使用显微镜观察TseH和VgrG3异位表达对宿主细菌生长的影响;纯化大肠杆菌细胞壁,使用透射电子显微镜(Transmission Electron Microscope,TEM)观察提纯的细胞壁形态;使用纯化的TseH和VgrG3分解消化PG,利用超高效液相色谱-飞行时间质谱(Ultra-Performance LiquidChromatography-Time-of-FlightMassSpectrometry,UPLC-TOFMS)分析鉴定消化后的产物成分;通过分析结果推导结构。【结果】通过透射电子显微镜观察,发现提纯的PG呈现半透明的薄膜泡状;通过UPLC-TOFMS的分析以及逆向推导,得到了提纯的PG被VgrG3水解酶降解之后的3种主要产物,分别是二糖二肽(Disaccharide,Di)、二糖三肽(Disaccharide Tripeptide,Tri)和二糖四肽(Disaccharide Tetrapeptide,Tetra)。【结论】建立了提纯PG和UPLC-TOFMS分析PG成分的方法,揭示了效应蛋白VgrG3而非TseH可以降解PG多糖链N-乙酰葡糖胺和N-乙酰胞壁酸之间的β(1-4)糖苷键的功能。由于攻击细胞壁的效应蛋白在革兰氏阴性细菌中广泛存在,本研究不仅为鉴定这类重要效应蛋白的功能提供了有效的方法,而且对研究靶向细胞壁的新型抗生素也有重要的指导作用。     

Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1-11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.     

Deoxyribonucleic Acid of Anaplasma marginale

Deoxyribonucleic acid from isolated marginal bodies and calf erythrocytes infected with Anaplasma marginale is found to be double stranded and to contain 51 moles per cent guanine plus cytosine.     

Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium of granulocytes. A. phagocytophilum specifically induces tyrosine phosphorylation of a 160 kDa protein (P160) in host cells. However, identity of P160, kinases involved, and effects of tyrosine phosphorylation on bacterial infection remain largely unknown. Here, we demonstrated through proteomic analysis that P160, an abundant and rapidly tyrosine-phosphorylated protein throughout infection, was AnkA of bacterial origin. Differential centrifugation and confocal microscopy revealed that AnkA was rarely retained within A. phagocytophilum or its inclusion, but localized mainly in the cytoplasm of infected cells. Using Cre recombinase reporter assay of Agrobacterium tumefaciens, we proved that AnkA could be secreted by VirB/D4-dependent type IV secretion (T4S) system. Yeast two-hybrid and coimmunoprecipitation analyses demonstrated that AnkA could bind to Abl-interactor 1 (Abi-1), an adaptor protein that interacts with Abl-1 tyrosine kinase, thus mediating AnkA phosphorylation. AnkA and Abl-1 were critical for bacterial infection, as infection was inhibited upon host cytoplasmic delivery of anti-AnkA antibody, Abl-1 knockdown with targeted siRNA, or treatment with a specific pharmacological inhibitor of Abl-1. These data establish AnkA as the first proven T4S substrate in members of obligate intracellular alpha-proteobacteria; furthermore, it demonstrated that AnkA plays an important role in facilitating intracellular infection by activating Abl-1 signalling pathway, and suggest a novel approach to treatment of human granulocytic anaplasmosis through inhibition of host cell signalling pathways.     

细菌的IV型分泌系统

细菌的分泌系统与细菌的生存及致病性密切相关。细菌的分泌系统包括I-VI型,其中,IV型分泌系统是与细菌接合机制有关的一类分泌系统。IV型分泌系统不但可以转运DNA,还可以转运蛋白质及核糖核蛋白复合物等大分子物质,这点区别于其他几种分泌系统。IV型分泌系统介导基因水平转移,通过细菌间接合作用,传递抗性基因和毒力基因,有利于细菌进化;另一方面,IV型分泌系统转运效应蛋白质分子到宿主细胞,参与细菌致病。本文着重从IV型分泌系统几种主要类型的分泌机制等方面对IV型分泌系统进行概述。     

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N‐terminal Fic domain and a C‐terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS‐mediated translocation into host cells. A proteolysis resistant fragment (residues 10–302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α‐[³²P]‐ATP. Its crystal structure, determined to 2.9‐Å resolution by the SeMet‐SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β‐rich domain at the C‐terminus. On crystal soaking with ATP/Mg²⁺, additional electron density indicated the presence of a PP_i/Mg²⁺ moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg²⁺ and target tyrosine. The model is consistent with an in‐line nucleophilic attack of the deprotonated side‐chain hydroxyl group onto the α‐phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence‐independent mechanism of target positioning through antiparallel β‐strand interactions between enzyme and target is suggested.     

Evidence of Lactate Dehydrogenase in Anaplasma marginale

Extracts from preparations of partially purified Anaplasma marginale revealed low levels of lactate dehydrogenase (LDH). Enzyme inhibition by immune sera further indicated that A. marginale possesses a protein moiety the same as that of the normal red blood cell (RBC), although data suggested an alteration of LDH(1) from that observed in normal RBC. Bimodal isozyme distribution was detected after electrophoresis of the extracts. One isozyme approached the cathode and the other the anode, and both appeared to be nicotinamide adenine dinucleotide-dependent. Heterogeneity of parasite and host cell isozymes was established on the basis of zone electrophoresis on cellulose acetate strips.     

Identification of Pseudomonas syringae pv. syringae 61 type III secretion system Hrp proteins that can travel the type III pathway and contribute to the translocation of effector proteins into plant cells

Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.     

Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale

Monoclonal antibodies were raised against the vaccine strain of Anaplasma centrale used in Australia. A monoclonal antibody that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test that will be useful for confirming species identity in patent infections. Another monoclonal antibody that reacted with a 116 kDa antigen was used to develop an A. centrale-specific competitive inhibition enzyme-linked immunosorbent assay (ELISA) for the serological identification of vaccinated cattle. The sensitivity of the ELISA was 100% in cattle experimentally infected with A. centrale, 97.1% in a vaccinated beef herd and 98.3% in a vaccinated dairy herd. The specificity of the ELISA was 98.6% in non-vaccinated cattle outside the Anaplasma marginale-endemic area, 97.9% in non-vaccinated cattle within the A. marginale-endemic area and 100% in cattle experimentally infected with A. marginale. The ELISA detected antibodies to A. centrale in cattle up to 9 years after vaccination with no apparent decrease in sensitivity. The assay has proved extremely valuable in Australia for investigating reported failures of multivalent live vaccines used to protect cattle against anaplasmosis and babesiosis, and should be similarly useful elsewhere in the world where these types of vaccines are used, e.g. Israel and South America.     

A colorimetric assay for studying effector secretion through the bacterial type III secretion system

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.     

Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system

Type IV secretion systems (T4SS) are utilized by a wide range of Gram negative bacteria to deliver protein and DNA substrates to recipient cells. The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/Icm systems found in the pathogens Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Here we report the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. Based on biochemical and genetic analysis, we discerned the existence of a critical five-protein subassembly that spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7-10 subcomplex, suggesting a remarkable conservation of the core subassembly in these evolutionarily distant type IV secretion machines.     

A binary effector module secreted by a type VI secretion system

Gram‐negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring cells. Cargo effectors are secreted by binding noncovalently to the T6SS apparatus. Occasionally, effector secretion is assisted by an adaptor protein, although the adaptor itself is not secreted. Here, we report a new T6SS secretion mechanism, in which an effector and a co‐effector are secreted together. Specifically, we identify a novel periplasm‐targeting effector that is secreted together with its co‐effector, which contains a MIX (marker for type sIX effector) domain previously reported only in polymorphic toxins. The effector and co‐effector directly interact, and they are dependent on each other for secretion. We term this new secretion mechanism “a binary effector module,” and we show that it is widely distributed in marine bacteria.     

Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram‐negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.     

Bartonella type IV secretion effector BepC induces stress fiber formation through activation of GEF-H1

Bartonella T4SS effector BepC was reported to mediate internalization of big Bartonella aggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentation induced by c-AMP) domain and C-terminus BID (Bartonella intracellular delivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway.