Polymorphic secreted kinases are key virulence factors in toxoplasmosis

The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F(1) progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.     

Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation

Yersinia species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor kappaB (NFkappaB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl-coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.     

Plague bacteria target immune cells during infection

The plague is caused by the bacterium Yersinia pestis. Plague bacteria are thought to inject effector Yop proteins into host cells via the type III pathway. The identity of the host cells targeted for injection during plague infection is unknown. We found, using Yop beta-lactamase hybrids and fluorescent staining of live cells from plague-infected animals, that Y. pestis selected immune cells for injection. In vivo, dendritic cells, macrophages, and neutrophils were injected most frequently, whereas B and T lymphocytes were rarely selected. Thus, it appears that Y. pestis disables these cell populations to annihilate host immune responses during plague.     

The intracellular fate of Salmonella depends on the recruitment of kinesin

Salmonella enterica causes a variety of diseases, including gastroenteritis and typhoid fever. The success of this pathogen depends on its capacity to proliferate within host cells in a membrane-bound compartment. We found that the Salmonella-containing vacuole recruited the plus-end-directed motor kinesin. Bacterial effector proteins translocated into the host cell by a type III secretion system antagonistically regulated this event. Among these effectors, SifA targeted SKIP, a host protein that down-regulated the recruitment of kinesin on the bacterial vacuole and, in turn, controlled vacuolar membrane dynamics.     

小麦叶锈菌效应蛋白Pt18906激发TcLr27+31的双层防御反应

【目的】由小麦叶锈菌（Puccinia triticina）引起的小麦叶锈病是影响小麦生产的主要病害之一,在小麦与叶锈菌互作的过程中病菌向寄主细胞分泌效应蛋白,以调控寄主防御反应、发挥毒性功能。开展对小麦叶锈菌效应蛋白的研究,探索小麦叶锈菌的致病机制,为病害的持续防控提供依据。【方法】以小麦叶锈菌13-5-72与感病品种Thatcher互作的cDNA为模板扩增效应蛋白Pt18906,通过SignalP 4.1、TargetP 1.1、TMHMM 2.0和EffectorP 2.0软件对Pt18906进行序列特征分析,利用在线软件Swiss-Model预测Pt18906的三级结构,利用在线软件SOPMA预测Pt18906的二级结构。采用实时荧光定量PCR对Pt18906的表达模式进行分析,借助于烟草的异源表达系统对Pt18906进行抑制Bax和INF1诱导的细胞程序性死亡（programmed cell death, PCD）能力验证,利用酵母系统验证Pt18906的信号肽是否具有分泌功能,采用氨基酸逐步缺失的方法缺失突变Pt18906,从而确定其功能毒性motif;通过在烟草中瞬时表达Pt18906-GFP融合蛋白,结合质壁分离技术分析Pt18906的亚细胞定位,得出效应蛋白的作用位点;利用瞬时表达技术在以Thatcher为背景的不含抗病基因和含有不同抗病基因的全套近等基因系上开展Pt18906无毒性功能分析;采用细菌三型分泌系统（Type Ⅲ secretion system）介导的瞬时转化分析Pt18906对寄主防御反应的调控。【结果】从小麦叶锈菌13-5-72与感病品种Thatcher互作6 d的转录组文库中获得一个在接种24 h后显著高表达的、基因全长序列672 bp、编码223个氨基酸的候选效应蛋白Pt18906,该效应蛋白缺乏已知的功能结构域和保守基序,工作环境偏碱性,在烟草细胞中瞬时表达Pt18906,Pt18906能够抑制Bax和INF1诱导的细胞程序性死亡,表明该效应蛋白具有毒性功能,并且通过构建缺失突变体明确其28—47位氨基酸对其毒性功能具有重要作用,该效应蛋白定位于细胞核和细胞质,表明其作用于细胞内。Pt18906在单基因系抗病品种TcLr27+31和TcLr42上能够引起过敏性坏死反应,表明该效应蛋白的无毒性,Pt18906能够引起TcLr27+31中胼胝质的积累和活性氧的迸发,胼胝质随注射时间的增加而逐渐积累,活性氧在注射后的10 min达到最高。【结论】位于28—47位的氨基酸决定Pt18906的毒性主要功能,Pt18906能激发小麦TcLr27+31双层防御反应。     

青枯雷尔氏菌致病机制及其相关基因的研究进展

阐述青枯雷尔氏菌致病基因以及它们之间的相互调节。由于青枯雷尔氏菌的复杂性,进而发展了许多青枯雷尔氏菌分子鉴定技术,并且对青枯雷尔氏菌的鉴定逐渐走向快速、便捷和灵敏高的趋势。青枯雷尔氏菌基因组约5.8Mb,具有高(G+C)含量和约5 120个可能的编码基因;它是由3.7Mb的染色体和2.1Mb的大质粒所组成,主要的致病因子有Ⅲ型hrp分泌系统产物、胞外多糖、细胞壁降解酶(包括果胶质酶以及纤维素酶等),其涉及的基因主要包括hrp基因簇、avr基因、毒性基因;青枯雷尔氏菌通过Ⅲ型分泌系统(T3SS)、II型分泌系统(T2SS)等分泌系统将多种毒性因子输送到胞外使寄主植物致病。同时,T3SS和T2SS之间也是相互影响的。上述致病因子的协调作用是由一个复杂的网络调节系统控制的,并以PhcA调节基因的启动和转录为核心,自动而精密地调节有关致病基因的表达及关闭,从而控制细菌的生长状态。     

MAPKK-independent activation of p38alpha mediated by TAB1-dependent autophosphorylation of p38alpha

Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.     

Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates

We have developed a proteomic approach for identifying phosphopeptide binding domains that modulate kinase-dependent signaling pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular protein kinase phosphorylation motif is used to isolate phospho-binding domains that bind to proteins phosphorylated by that kinase. Applying this approach to cyclin-dependent kinases (Cdks), we identified the polo-box domain (PBD) of the mitotic kinase polo-like kinase 1 (Plk1) as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif. This motif is present in known Plk1 substrates such as Cdc25, and an optimal phosphopeptide containing the motif disrupted PBD-substrate binding and localization of the PBD to centrosomes. This finding reveals how Plk1 can localize to specific sites within cells in response to Cdk phosphorylation at those sites and provides a structural mechanism for targeting the Plk1 kinase domain to its substrates.     

The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.     

MAPKs在植物逆境信号转导中的作用

蛋白质的可逆磷酸化是细胞信号识别与转导的重要环节。促分裂原活化蛋白激酶（mitogen-activatedprotein kinases,MAPKs）是一类丝氨酸/苏氨酸（Ser/Thr）蛋白激酶,主要催化蛋白质的磷酸化过程。植物中存在大量的MAPKs,它们在多种信号传递过程中起重要作用。笔者着重介绍MAPKs在植物逆境信号识别与转导中的作用。     

福建省青枯雷尔氏菌脂肪酸多态性研究

 【目的】利用气相色谱技术检测福建省的40株青枯雷尔氏菌(Ralstonia solanacearum)菌株细胞内的脂肪酸，分析其脂肪酸分布的多态性；研究青枯雷尔氏菌脂肪酸多态性与青枯雷尔氏菌现有种下分化方法之间的关系。【方法】对40株青枯雷尔氏菌脂肪酸进行气相色谱分析，比较同一寄主分离的青枯雷尔氏菌和不同寄主分离的青枯雷尔氏菌脂肪酸的分布；对40株青枯雷尔氏菌脂肪酸进行聚类分析，分析聚成的各类青枯雷尔氏菌脂肪酸的特点以及脂肪酸多态性与其生理小种、生化型和致病性之间的关系。【结果】同一寄主分离的青枯雷尔氏菌和不同寄主分离的青枯雷尔氏菌，其脂肪酸都存在着明显的多态性；对40株青枯雷尔氏菌的脂肪酸进行聚类分析，可以聚成3类，即group Ⅰ、group Ⅱ和group Ⅲ；青枯雷尔氏菌生理小种1存在着不同的脂肪酸类群，青枯雷尔氏菌脂肪酸多态性与其生化型之间不存在相关性，但是脂肪酸和致病性之间存在一定的相关性：group Ⅰ为无致病性菌株，group Ⅱ为过渡性菌株，group Ⅲ为强致病性菌株。【结论】福建省青枯雷尔氏菌脂肪酸分布存在着明显的多态性；青枯雷尔氏菌脂肪酸多态性与致病性之间存在一定的相关性，脂肪酸有望成为青枯雷尔氏菌小种鉴定的新指标。     

Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation

The canonical Wnt-beta-catenin signaling pathway is initiated by inducing phosphorylation of one of the Wnt receptors, low-density lipoprotein receptor-related protein 6 (LRP6), at threonine residue 1479 (Thr1479) and serine residue 1490 (Ser1490). By screening a human kinase small interfering RNA library, we identified phosphatidylinositol 4-kinase type II alpha and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) as required for Wnt3a-induced LRP6 phosphorylation at Ser1490 in mammalian cells and confirmed that these kinases are important for Wnt signaling in Xenopus embryos. Wnt3a stimulates the formation of phosphatidylinositol 4,5-bisphosphates [PtdIns (4,5)P2] through frizzled and dishevelled, the latter of which directly interacted with and activated PIP5KI. In turn, PtdIns (4,5)P2 regulated phosphorylation of LRP6 at Thr1479 and Ser1490. Therefore, our study reveals a signaling mechanism for Wnt to regulate LRP6 phosphorylation.     

A sorting platform determines the order of protein secretion in bacterial type III systems

Bacterial type III protein secretion systems deliver effector proteins into eukaryotic cells in order to modulate cellular processes. Central to the function of these protein-delivery machines is their ability to recognize and secrete substrates in a defined order. Here, we describe a mechanism by which a type III secretion system from the bacterial enteropathogen Salmonella enterica serovar Typhimurium can sort its substrates before secretion. This mechanism involves a cytoplasmic sorting platform that is sequentially loaded with the appropriate secreted proteins. The sequential loading of this platform, facilitated by customized chaperones, ensures the hierarchy in type III protein secretion. Given the presence of these machines in many important pathogens, these findings can serve as the bases for the development of novel antimicrobial strategies.     

Comparative analysis of the genome of the field isolate V86010 of the rice blast fungus Magnaporthe oryzae from Philippines

Genome dynamics of pathogenic organisms are driven by plant host and pathogenic organism co-evolution,in which pathogen genomes are used to overcome stresses imposed by hosts with various genetic backgrounds through generation of a range of field isolates. This model also applies to the rice host and its fungal pathogen Magnaporthe oryzae. To better understand genetic variation of M. oryzae in nature,the field isolate V86010 from the Philippines was sequenced and analyzed. Genome annotation found that the assembled V86010 genome was composed of 1 931 scaffolds with a combined length of 38.9 Mb. The average GC ratio is 51.3% and repetitive elements constitute 5.1% of the genome. A total of 11 857 genes including 616 effector protein genes were predicted using a combined analysis pipeline. All predicted genes and effector protein genes of isolate V86010 distribute on the eight chromosomes when aligned with the assembled genome of isolate 70-15. Effector protein genes are located disproportionately at several chromosomal ends. The Pot2 elements are abundant in V86010. Seven V86010-specific effector proteins were found to suppress programmed cell death induced by BAX in tobacco leaves using an Agrobacterium-mediated transient assay. Our results may provide useful information for further study of the molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions,and for characterizing novel effectors and AVR genes in the rice blast pathogen.     

A manganese(IV)/iron(III) cofactor in Chlamydia trachomatis ribonucleotide reductase

In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y*). The Y* reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C*), which abstracts the 3'-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y*, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C* in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.     

A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae

Type III secreted "effector" proteins of bacterial pathogens play central roles in virulence, yet are notoriously difficult to identify. We used an in vivo genetic screen to identify 13 effectors secreted by the type III apparatus (called Hrp, for "hypersensitive response and pathogenicity") of the plant pathogen Pseudomonas syringae. Although sharing little overall homology, the amino-terminal regions of these effectors had strikingly similar amino acid compositions. This feature facilitated the bioinformatic prediction of 38 P. syringae effectors, including 15 previously unknown proteins. The secretion of two of these putative effectors was shown to be type III--dependent. Effectors showed high interstrain variation, supporting a role for some effectors in adaptation to different hosts.