Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

小菜蛾溴氰菊酯敏感和抗性品系蛋白质表达谱的比较分析

杀虫剂抗性是害虫防治中的一个主要挑战。为有效治理抗性, 我们必须了解杀虫剂诱导的害虫生理生化变化。目前害虫抗药性的一些机理已经清楚, 但更多的相关机制还有待探究。本研究通过蛋白质组学方法检测了小菜蛾Plutella xylostella溴氰菊酯敏感和抗性品系间蛋白质组的表达差异。结果显示： SDS-PAGE胶上有大约300个蛋白差异点, 其中23个蛋白点具2.5倍以上的表达差异, 通过MALDI-TOF-MS, 我们成功鉴定出8个蛋白, 其中包括化感蛋白CSP2、 铜锌超氧化物歧化酶和peroxiredoxin样蛋白。通过实时定量PCR（real-time quantitative PCR, qPCR）分析了其中5个蛋白的mRNA 表达水平, 结果表明mRNA 表达水平不能真实反映蛋白的表达水平。免疫印迹验证了双向电泳中SOD1的表达差异。本研究有力地证明溴氰菊酯诱导小菜蛾成虫蛋白质组表达变化, 这为进一步筛选抗性靶标提供很大的帮助。     

Proteome analysis of Desulfovibrio desulfuricans G20 mutants using the accurate mass and time (AMT) tag approach

Abundance values obtained from direct LC-MS analyses were used to compare the proteomes of six transposon-insertion mutants of Desulfovibrio desulfuricans G20, the lab strain (G20lab) and a sediment-adapted strain (G20sediment). Three mutations were in signal transduction histidine kinases, and three mutations were in other regulatory proteins. The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to analyze the proteomes. A total of 1318 proteins was identified with high confidence, approximately 35% of all predicted proteins in the D. desulfuricans G20 genome. Proteins from all functional categories were identified. Significant differences in the abundance of 30 proteins were detected between the G20lab strain and the G20sediment strain. Abundances of proteins for energy metabolism, ribosomal synthesis, membrane biosynthesis, transport, and flagellar synthesis were affected in the mutants. Specific examples of proteins down-regulated in mutants include a putative tungstate transport system substrate-binding protein and several proteins related to energy production, for example, 2-oxoacid:acceptor oxidoreductase, cytochrome c-553, and formate acetyltransferase. In addition, several signal transduction mechanism proteins were regulated in one mutant, and the abundances of ferritin and hybrid cluster protein were reduced in another mutant. However, the similar abundance of universal stress proteins, heat shock proteins, and chemotaxis proteins in the mutants revealed that regulation of chemotactic behavior and stress regulation might not be observed under our growth conditions. This study provides the first proteomic overview of several sediment fitness mutants of G20, and evidence for the difference between lab strains and sediment-adapted strains at the protein level.     

Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins

Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects. In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pl's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span. By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins. More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level.     

In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.     

A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.