耻垢分枝杆菌MSMEG_6281 过表达对菌株生长和形态的影响

【目的】耻垢分枝杆菌(Mycobacterium smegmatis mc2155,mc2155)MSMEG_6281为结核分枝杆菌自溶素Rv3717的同源蛋白,通过建立过表达MSMEG_6281的耻垢分枝杆菌菌株,推测该蛋白对耻垢分枝杆菌肽聚糖代谢的影响。【方法】利用RT-PCR方法检测乙胺丁醇(Ethambutol,EMB)作用后MSMEG_6281基因的表达变化;以耻垢分枝杆菌基因组DNA为模板,采用PCR技术克隆MSMEG_6281基因,构建分枝杆菌表达质粒p VV16-MSMEG_6281,进一步建立MSMEG_6281过表达的耻垢分枝杆菌菌株;利用生长曲线检测MSMEG_6281过表达对耻垢分枝杆菌生长的影响;利用扫描电子显微镜分析MSMEG_6281过表达引起的耻垢分枝杆菌形态变化。【结果】EMB处理引起MSMEG_6281基因表达上调;构建了过表达MSMGE_6281的耻垢分枝杆菌菌株(mc2155/p VV16-MSMEG_6281);过表达MSMGE_6281的耻垢分枝杆菌生长缓慢,菌体形态由短杆状转变为长杆状。【结论】MSMGE_6281的过表达可改变耻垢分枝杆菌形态。MSMGE_6281的功能与细胞壁肽聚糖水解相关,在mc2155细胞壁形态维持方面发挥重要作用。     

以蛋白激酶G为靶点的抗结核药物筛选模型的建立和初步应用

结核分枝杆菌可以产生11种丝氨酸/苏氨酸蛋白激酶,其中蛋白激酶G(PknG)对于结核分枝杆菌在巨噬细胞内以"持留"状态长期存活有着重要作用。本研究以结核分枝杆菌基因组DNA为模板,在大肠杆菌中克隆表达了MTBPknG蛋白,并分离纯化得到PknG纯酶。本研究还采用三步级联反应方法测定了PknG酶活性,建立和优化了PknG抑制剂高通量筛选模型。利用此模型共筛选发酵液样品2120个,化合物样品2300个,筛选得到阳性化合物1个,阳性发酵液13个,阳性率0.32%。     

分枝杆菌丝氨酸/苏氨酸蛋白激酶PknK的功能研究

【目的】丝氨酸/苏氨酸蛋白激酶K(Serine/Threonine protein kinases K)是分枝杆菌类似真核样的蛋白激酶,预测在分枝杆菌的生长和新陈代谢等生理过程中起着重要的作用,解析PknK的生物功能及作用机制,将为结核病的防治提供一定的理论基础。【方法】通过基因敲除等遗传方法获得结核分枝杆菌疫苗株BCG的pknK敲除菌株△pknK、回补菌株pMV361-pknK/△pknK和过表达菌株pMV261-pknK/BCG;对获得的菌株进行生长曲线测定和抗药性分析;通过pulldown-MS方法及生物信息学方法鉴定了PknK相互作用蛋白。【结果】监测各种分枝杆菌△pknK、pMV361-pknK/△pknK和pMV261-pknK/BCG生长,确定PknK负调控BCG生长;抗药性分析显示PknK降低BCG的耐药性;pulldown-MS方法显示PknK与丝氨酸/苏氨酸蛋白激酶PknA和双组分系统中的反应调节因子MtrA、TrcR、MoxR等蛋白相互作用。【结论】研究发现PknK调控分枝杆菌的生长和耐药性,我们的研究为深入研究PknK在结核分枝杆菌中的功能奠定了基础。     

丝氨酸/苏氨酸蛋白激酶在结核分枝杆菌致病机制中的作用

丝氨酸/苏氨酸蛋白激酶(STPK)是一类真核细胞样的蛋白激酶,是分枝杆菌生长和代谢的重要调节因子,参与其多种细胞活动(如细胞和菌落形态、葡萄糖和谷氨酰胺转运、吞噬体-溶酶体融合、转录因子活性等)的调节.结核分枝杆菌编码产生11种STPK( PknA、PknB、PknD～PknL),可分为5群(ABL群、HED群、FIJ...     

定量蛋白质组学分析链霉素耐药和敏感结核分枝杆菌临床分离株

[目的]发现结核分枝杆菌(Mycobacterium tuberculosis)链霉素耐药相关的潜在菌体蛋白.[方法]以结核分枝杆菌临床分离链霉素敏感株01105和结核分枝杆菌H37Rv为对照,采用iTRAQ技术和生物信息学鉴定并相对定量结核分枝杆菌临床分离链霉素耐药株01108菌体蛋白,并通过WEGO功能注释聚类分析01108菌株差异表达蛋白的细胞组分、分子功能和生物进程.[结果]01108菌株分别与01105菌株和H37Rv菌株比较差异表达蛋白为194个和146个,01108菌株与01105菌株和H37Rv比较均差异表达蛋白121个(共同差异表达蛋白).差异表达蛋白理论相对分子量和等电点分布广泛,其生物进程主要参与中间代谢、呼吸作用和脂质代谢,分子功能主要为催化活性功能和结合功能.共同差异表达蛋白:7个核糖体蛋白(Rv2785c,Rv0056,Rv0641,Rv0652,Rv0701,Rv1630和Rv2442c)在01108菌株中表达下调;7个蛋白在01108菌株中显著差异表达(上调大于1.20倍或下调小于0.55倍),分别为巯基过氧化物酶(Rv1932)、酰基载体蛋白脱氢酶(Rv0824c)、30S核糖体蛋白S15 (Rv2785c)、丙酮酸脱氢酶E2部分(Rv2215)、双组份转录调控蛋白(Rv3133c)以及假定未知蛋白(Rv2466c和Rv2626c).[结论]iTRAQ发现了链霉素耐药结核分枝杆菌相对于链霉素敏感结核分枝杆菌和H37Rv共同差异表达蛋白,为进一步探讨结核分枝杆菌链霉素耐药机制奠定了基础.     

分枝杆菌脂聚糖的分离、纯化及其结构和功能分析

【目的】建立分离、纯化分枝杆菌脂聚糖的方法,初步比较分析不同菌株来源的脂阿拉伯甘露聚糖(Lipoarabinomannan,LAM)和脂甘露聚糖(Lipomannan,LM)的结构差异及研究脂聚糖刺激对巨噬细胞环氧合酶-2(Cyclooxygenase-2,COX-2)蛋白表达的影响。【方法】应用Triton X-114液相法提取脂聚糖,电洗脱法分离纯化,基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF/TOF-MS)进行分子量鉴定;基于特异性识别非还原性末端α-D-甘露糖基的刀豆球蛋白(Concanavalin A,Con A)分析新诺分枝杆菌JDM601、结核分枝杆菌H37Rv标准株和耻垢分枝杆菌mc2155脂聚糖的结构差异;进一步用Western blot检测脂聚糖刺激的RAW 264.7巨噬细胞COX-2蛋白的表达。【结果】通过电洗脱法成功纯化出3种菌株脂聚糖;MALDI-TOF/TOF-MS鉴定发现,分子量从小到大依次为新诺分枝杆菌JDM601、耻垢分枝杆菌mc2155和结核分枝杆菌H37Rv来源的脂聚糖。Western blot显示,Con A能与结核分枝杆菌H37Rv标准株来源的LAM相互作用,而不能与新诺分枝杆菌JDM601和耻垢分枝杆菌来源的LAM相互作用;并且发现Con A与新诺分枝杆菌JDM601来源的LM有很强的反应,然而与其余两种来源的LM反应很弱。3种菌株来源的脂聚糖均能刺激RAW 264.7巨噬细胞COX-2蛋白的表达。【结论】首次成功对来源于中国临床分枝杆菌分离株的脂聚糖进行了分离纯化,初步探讨了不同菌株来源分枝杆菌脂聚糖的结构差异,并表明LAM和LM均能刺激巨噬细胞诱导COX-2蛋白的表达,为进一步研究其对宿主的毒力和免疫机制奠定了基础。     

结核病疫苗和耻垢分枝杆菌引起树突状细胞差异免疫反应的蛋白质组学分析

研究结核分枝杆菌和免疫细胞的相互作用对发展新的结核病防治策略至关重要.由于结核分枝杆菌生长缓慢,且需要在高等级生物安全实验室中进行操作,快速生长非致病的耻垢分枝杆菌(Mycobacterium smegmatis)经常用来作为结核分枝杆菌的模式菌开展相关研究.为了探讨耻垢分枝杆菌和结核分枝杆菌对免疫系统产生的不同影响,本课题组利用缓慢生长的结核菌的减毒活疫苗卡介苗(BCG)和耻垢分枝杆菌mc2155分别感染小鼠(Mus musculus)树突状细胞DC2.4细胞,通过蛋白质组学分析,阐述宿主细胞对BCG和耻垢分枝杆菌不同的免疫反应.结果表明,BCG在DC2.4中生存时间比耻垢分枝杆菌长.定量蛋白质组学发现耻垢分枝杆菌激活了Ⅰ型干扰素信号通路,上调了AIM2,IFI204,IFIT1和ISG15蛋白的表达.相反,BCG的侵染对宿主细胞的蛋白组影响不大.分泌组学的结果显示,耻垢分枝杆菌的侵染诱导树突状细胞分泌更多的细胞趋化因子以及ISG15,TNF和IL-6等细胞因子,说明耻垢分枝杆菌的侵染促进了宿主细胞的细胞因子和趋化因子的释放,从而迅速地清除侵染的细菌.与此一致的是,耻垢分枝杆菌侵染的小鼠原代免疫细胞比BCG侵染产生更多的IFN-γ.进一步的研究发现,ISG15过表达阻止分枝杆菌进入宿主细胞.综上所述,本研究证明与缓慢生长的BCG相比,耻垢分枝杆菌引起宿主细胞更强烈的免疫反应,致使耻垢分枝杆菌在宿主细胞中被迅速清除.同时,本研究组发现耻垢分枝杆菌侵染引起的ISG15上调阻止了分枝杆菌进入宿主细胞.     

发菜磷酸葡糖胺变位酶(glmM)对干旱胁迫的响应及其基因克隆

glmM编码的磷酸葡糖胺变位酶是肽聚糖合成前体的关键酶。为探究发菜glmM响应干旱胁迫的表达调控机制及明确其分子信息,本研究对干旱胁迫条件下发菜glmM在转录水平的差异表达进行了分析,并对glmM的表达水平、磷酸化修饰、乙酰化修饰和琥珀酰化修饰水平进行了检测,克隆了发菜glmM,进行了序列分析和原核表达。结果表明,干旱胁迫条件下,发菜glmM在转录水平上的表达量先增加后减少,glmM上调表达,glmM的磷酸化修饰水平逐渐增加,乙酰化修饰水平相对稳定,琥珀酰化修饰水平有明显变化。设计特异性引物克隆glmM基因,获得全长1416 bp发菜glmM基因,与肺衣(5183)glmM的核苷酸序列同源性为95%,氨基酸同源性为97%。将glmM在大肠杆菌中表达,获得一个51.45 kD的外源蛋白,MALDI-TOF-TOF/MS分析证明该蛋白为磷酸葡糖胺变位酶。研究结果为深入研究发菜glmM的分子信息、生物学功能及其响应干旱胁迫的分子机制提供帮助。     

古菌蛋白激酶的研究进展

磷酸化是蛋白质翻译后修饰(post-translational modification)的主要方式,可由蛋白激酶、磷酸转移酶、磷酸化酶等多种方式催化进行。其中,由蛋白激酶(protein kinases)/磷酸酶(protein phosphatases)介导的可逆的蛋白磷酸化是细胞中信号转导的重要机制,在DNA复制、转录、蛋白质翻译、DNA损伤修复等生命过程中起广泛的调节作用。目前,古菌中蛋白激酶的研究尚属于初期阶段。虽然磷酸化蛋白质组学研究表明,古菌中存在大量的磷酸化蛋白质,但是我们对其具体催化作用的酶及调控机制尚不清楚。本文总结了古菌中已报道的蛋白激酶所参与的生命过程,包括古菌的DNA代谢、细胞代谢、细胞周期和运动机制等四个方面,并对今后的研究提出展望。     

定量蛋白质组分析蚯蚓血红蛋白样蛋白MSMEG_3312介导的分枝杆菌耐红霉素机制（英文）

【目的】细菌耐药机制是个复杂的机制,系统生物学是系统性揭示耐药机制的有力研究手段。我们课题组前期研究结果显示,蚯蚓血红蛋白样蛋白msmeg_3312基因敲除后能够增加耻垢分枝杆菌对红霉素的耐药性,本文系统研究MSMEG_3312参与红霉素耐药性形成的机制。【方法】首先纯化MSMEG_3312蛋白,利用光谱及圆二色谱描述MSMEG-3312蛋白。利用定量蛋白质组学的方法比较分析敲除菌株Δmsmeg_3312与野生型菌株mc~2155蛋白表达的差异,并通过qRT-PCR进行验证。利用红霉素ELASA试剂盒测定Δmsmeg_3312与mc~2155的胞内药物浓度。【结果】光谱及圆二色谱分析确定MSMEG_3312是蚯蚓血红蛋白样蛋白。定量蛋白质组学分析发现,红霉素未处理的条件下,相比于野生型菌株mc~2155,敲除菌株Δmsmeg_3312有包括3种转运蛋白在内的8种蛋白表达水平上调,14种蛋白表达下调;而红霉素处理后,Δmsmeg_3312中有448种蛋白差异表达,其中有11种转运蛋白表达上调,26种蛋白与氨基酸合成通路相关。胞内药物浓度检测显示敲除菌株Δmsmeg_3312的胞内红霉素浓度显著低于野生型菌株。【结论】蚯蚓血红蛋白样蛋白MSMEG_3312调控改变了细菌对红霉素药物处理的反应网络,其介导的红霉素耐药是一种集合抗生素耐受机制。     

Analysis of SecA2‐dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector

The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2‐dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2‐dependent substrates. Immunoblotting procedures confirmed SecA2‐dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG‐overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2‐dependent membrane localization of PknG is an important determinant for M. marinum virulence.     

Novel mycobacteria antigen 85 complex binding motif on fibronectin

The members of the antigen 85 protein family (Ag85), consisting of members Ag85A, Ag85B, and Ag85C, are the predominantly secreted proteins of mycobacteria and possess the ability to specifically interact with fibronectin (Fn). Because Fn-binding proteins are likely to be important virulence factors of Mycobacterium spp., Ag85 may contribute to the adherence, invasion, and dissemination of organisms in host tissue. In this study, we reported the Fn binding affinity of Ag85A, Ag85B, and Ag85C from Mycobacterium avium subsp. paratuberculosis (MAP) (K(D) values were determined from 33.6 to 68.4 nm) and mapped the Ag85-binding motifs of Fn. Fn14, a type III module located on the heparin-binding domain II (Hep-2) of Fn, was discovered to interact with Ag85 from MAP. The peptide inhibition assay subsequently demonstrated that a peptide consisting of residues 17-26 from Fn14 ((17)SLLVSWQPPR(26), termed P17-26) could interfere with Ag85B binding to Fn (73.3% reduction). In addition, single alanine substitutions along the sequence of P17-26 revealed that the key residues involved in Ag85-Fn binding likely contribute through hydrophobic and charge interactions. Moreover, binding of Ag85 on Fn siRNA-transfected Caco2 cells was dramatically reduced (44.6%), implying the physiological significance of the Ag85-Fn interaction between mycobacteria and host cells during infection. Our results indicate that Ag85 binds to Fn at a novel motif and plays a critical role in mycobacteria adherence to host cells by initiating infection. Ag85 might serve as an important colonization factor potentially contributing to mycobacterial virulence.     

Control of cell wall assembly by a histone-like protein in Mycobacteria

Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.     

Insights from the molecular docking of withanolide derivatives to the target protein PknG from Mycobacterium tuberculosis

A crucial virulence factor for intracellular Mycobacterium tuberculosis survival is Protein kinase G (PknG), a eukaryotic-like serinethreonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG results in mycobacterial transfer to lysosomes. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show various pharmacological activities. We describe the docking of 26 withanferin and 14 withanolides from Withania somnifera into the three dimensional structure of PknG of M. tuberculosis using GLIDE. The inhibitor binding positions and affinity were evaluated using scoring functions- Glidescore. The withanolide E, F and D and Withaferin - diacetate 2 phenoxy ethyl carbonate were identified as potential inhibitors of PknG. The available drug molecules and the ligand AX20017 showed hydrogen bond interaction with the aminoacid residues Glu233 and Val235. In addition to Val235 the other amino acids, Gly237, Gln238 and Ser239 are important for withanolide inhibitor recognition via hydrogen bonding mechanisms.     

Survival of Pathogenic Mycobacteria in Macrophages Is Mediated through Autophosphorylation of Protein Kinase G

Pathogenic mycobacteria survive within macrophages through the inhibition of phagosome-lysosome fusion. A crucial factor for avoiding lysosomal degradation is the mycobacterial serine/threonine protein kinase G (PknG). PknG is released into the macrophage cytosol upon mycobacterial infection, suggesting that PknG might exert its activity by interfering with host signaling cascades, but the mode of action of PknG remains unknown. Here, we show that PknG undergoes autophosphorylation on threonine residues located at the N terminus. In contrast to all other mycobacterial kinases investigated thus far, autophosphorylation of PknG was not involved in the regulation of its kinase activity. However, autophosphorylation was crucial for the capacity of PknG to promote mycobacterial survival within macrophages. These results will contribute to a better understanding of the virulence mechanisms of pathogenic mycobacteria and may help to design improved inhibitors of PknG to be developed as antimycobacterial compounds.The genome of Mycobacterium tuberculosis comprises 11 serine/threonine protein kinases which are considered key players in signaling cascades by transferring phosphate groups from γ-ATP to the side chain hydroxyl group of serine and threonine residues in their substrates (1, 25). One of these kinases, protein kinase G (PknG), is secreted into the cytosol of infected macrophages and prevents the intracellular destruction of mycobacteria by blocking phagosome-lysosome fusion (30). Its essential role in mycobacterial virulence makes PknG an attractive drug target; the precise mode of action of PknG remains unclear, however (24, 30).Kinases in general can be classified into so-called RD and non-RD kinases based on the presence of a conserved arginine (R) and aspartate (D) sequence in the catalytic loop of the kinase domain. The RD arginine interacts with the phosphorylated activation loop and controls its fold, finally resulting in the activation of kinase activity (13, 18). Interestingly, PknG is unique among mycobacterial kinases in that the critical arginine residue in the kinase domain is lacking. The fact that PknG is classified as a non-RD kinase therefore suggests the absence of autophosphorylation in the activation loop (24).Although PknG is known to undergo autophosphorylation (14, 30), neither the localization of the autophosphorylation sites of PknG nor the importance of autophosphorylation for PknG activity is known. Here, we identify the autophosphorylation sites of PknG and provide evidence that autophosphorylation is essential for the capacity of PknG to modulate the survival of intracellularly residing mycobacteria.     

Control of CydB and GltA1 expression by the SenX3 RegX3 two component regulatory system of Mycobacterium tuberculosis

Two component regulatory systems are used widely by bacteria to coordinate changes in global gene expression profiles in response to environmental signals. The SenX3-RegX3 two component system of Mycobacterium tuberculosis has previously been shown to play a role in virulence and phosphate-responsive control of gene expression. We demonstrate that expression of SenX3-RegX3 is controlled in response to growth conditions, although the absolute changes are small. Global gene expression profiling of a RegX3 deletion strain and wild-type strain in different culture conditions (static, microaerobic, anaerobic), as well as in an over-expressing strain identified a number of genes with changed expression patterns. Among those were genes previously identified as differentially regulated in aerobic culture, including ald (encoding alanine dehydrogenase) cyd,encoding a subunit of the cytochrome D ubiquinol oxidase, and gltA1, encoding a citrate synthase. Promoter activity in the upstream regions of both cydB and gltA1 was altered in the RegX3 deletion strain. DNA-binding assays confirmed that RegX3 binds to the promoter regions of ald, cydB and gltA1 in a phosphorylation-dependent manner. Taken together these data suggest a direct role for the SenX-RegX3 system in modulating expression of aerobic respiration, in addition to its role during phosphate limitation.     

Regulation of glutamate metabolism by protein kinases in mycobacteria

Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit α‐ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD⁺‐specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.     

PknG supports mycobacterial adaptation in acidic environment

Mycobacterium tuberculosis (Mtb), causative agent of human tuberculosis (TB), has the remarkable ability to adapt to the hostile environment inside host cells. Eleven eukaryotic like serine-threonine protein kinases (STPKs) are present in Mtb. Protein kinase G (PknG) has been shown to promote mycobacterial survival inside host cells. A homolog of PknG is also present in Mycobacterium smegmatis (MS), a fast grower, non-pathogenic mycobacterium. In the present study, we have analyzed the role of PknG in mycobacteria during exposure to acidic environment. Expression of pknG in MS was decreased in acidic medium. Recombinant MS ectopically expressing pknG (MS-G) showed higher growth in acidic medium compared to wild type counterpart. MS-G also showed higher resistance upon exposure to 3.0 pH and better adaptability to acidic pH. Western blot analysis showed differential threonine but not serine phosphorylation of cellular proteins in MS at acidic pH which was restored by ectopic expression of pknG in MS. In Mtb H37Ra (Mtb-Ra), expression of pknG was increased at acidic pH. We also observed decreased expression of pknG in MS during infection in macrophages while the expression of pknG in Mtb-Ra was increased in similar conditions. Taken together, our data strongly suggests that pknG regulates growth of mycobacteria in acidic environment and is differentially transcribed in MS and Mtb under these conditions.     

PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5

ESX-5 is one of the five type VII secretion systems found in mycobacteria. These secretion systems are also known as ESAT-6-like secretion systems. Here, we have determined the secretome of ESX-5 by a proteomic approach in two different strains of Mycobacterium marinum . Comparison of the secretion profile of wild-type strains and their ESX-5 mutants showed that a number of PE_PGRS and PPE-MPTR proteins are dependent on ESX-5 for transport. The PE and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum , and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that nearly all PE_PGRS proteins that are recognized by this antibody are missing in the supernatant of ESX-5 mutants. In addition to PE_PGRS and PPE proteins, the ESX-5 secretion system is responsible for the secretion of a ESAT-6-like proteins. Together, these data show that ESX-5 is probably a major secretion pathway for mycobacteria and that this system is responsible for the secretion of recently evolved PE_PGRS and PPE proteins.     

Effects of human and porcine bile on the proteome of Helicobacter hepaticus

Background

Helicobacter hepaticus colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, H. hepaticus must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of H. hepaticus modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry.

Results

The 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of H. hepaticus were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of H. hepaticus in response to human or porcine bile.

Conclusions

Overall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.