Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

The virulence-attenuated Leptospira interrogans serovar Lai strain IPAV was derived by prolonged laboratory passage from a highly virulent ancestral strain isolated in China. We studied the genetic variations of IPAV that render it avirulent via comparative analysis against the pathogenic L. interrogans serovar Lai strain 56601. The complete genome sequence of the IPAV strain was determined and used to compare with, and then rectify and reannotate the genome sequence of strain 56601. Aside from their highly similar genomic structure and gene order, a total of 33 insertions, 53 deletions and 301 single-nucleotide variations (SNVs) were detected throughout the genome of IPAV directly affecting 101 genes, either in their 5' upstream region or within their coding region. Among them, the majority of the 44 functional genes are involved in signal transduction, stress response, transmembrane transport and nitrogen metabolism. Comparative proteomic analysis based on quantitative liquid chromatography (LC)-MS/MS data revealed that among 1 627 selected pairs of orthologs, 174 genes in the IPAV strain were upregulated, with enrichment mainly in classes of energy production and lipid metabolism. In contrast, 228 genes in strain 56601 were upregulated, with the majority enriched in the categories of protein translation and DNA replication/repair. The combination of genomic and proteomic approaches illustrated that altered expression or mutations in critical genes, such as those encoding a Ser/Thr kinase, carbon-starvation protein CstA, glutamine synthetase, GTP-binding protein BipA, ribonucleotide-diphosphate reductase and phosphate transporter, and alterations in the translational profile of lipoproteins or outer membrane proteins are likely to account for the virulence attenuation in strain IPAV.     

Proteomic alterations of <Emphasis Type="Italic">Brassica napus</Emphasis> root in response to boron deficiency

Boron (B) deficiency is a worldwide problem, and Brassica napus is one of the most sensitive crops to B deficiency. To better understand the B starvation response of Brassica napus, we conducted a comparative proteomic analysis of seedling stage Brassica napus root between B-sufficient and B-limited conditions: 45 differentially expressed proteins were successfully identified by 2-DE coupled with MALDI-TOF/TOF-MS and LTQ-ESI-MS/MS analysis. Among these proteins, 10 were down-regulated and 35 were up-regulated under B-limited condition. Combining GO and KEGG analyses with data from previous reports, proteins were categorized into several functional groups, including antioxidant and detoxification, defense-related proteins, signaling and regulation, carbohydrate and energy metabolism, amino acid and fatty acid metabolism, protein translation and degradation, cell wall structure, and transporter. The genes of selected proteins were analyzed by quantitative RT-PCR. Our results provide novel information for better understanding the physiological and biochemical responses to B deficiency in plants.     

N-terminal processing of Lhca3 Is a key step in remodeling of the photosystem I-light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii

Iron deficiency induces a remodeling of the photosynthetic apparatus in Chlamydomonas reinhardtii. In this study we showed that a key mechanistic event in the remodeling process of photosystem I (PSI) and its associated light-harvesting proteins (LHCI) is the N-terminal processing of Lhca3. N-terminal processing of Lhca3 is documented independently by two-dimensional gel electrophoresis and tandem mass spectrometric (MS/MS) analysis as well as by quantitative comparative MS/MS peptide profiling using isotopic labeling of proteins. Dynamic remodeling of the LHCI complex under iron deficiency is further exemplified by depletion of Lhca5 and up-regulation of Lhca4 and Lhca9 polypeptides in respect to photosystem I. Most importantly, the induction of N-terminal processing of Lhca3 by progression of iron deficiency correlates with the functional drop in excitation energy transfer efficiency between LHCI and PSI as assessed by low temperature fluorescence emission spectroscopy. Using an RNA interference (RNAi) strategy, we showed that the truncated form of Lhca3 is essential for the structural stability of LHCI. Depletion of Lhca3 by RNAi strongly impacted the efficiency of excitation energy transfer between PSI and LHCI, as is the case for iron deficiency. However, in contrast to iron deficiency, comparative MS/MS peptide profiling using isotopic labeling of proteins demonstrated that RNAi depletion of Lhca3 caused strong reduction of almost all Lhca proteins in isolated PSI particles.     

Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis

Arh1p is an essential mitochondrial protein of yeast with reductase activity. Here we show that this protein is involved in iron metabolism. A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter. Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer. Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron. Iron trafficking within the cell was also disturbed. Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control. Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered. Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron. Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins. Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins.     

应用蛋白质组学的方法分析缺铁培养对化脓性链球菌MGAS5005的影响

【目的】研究铁缺失对化脓性链球菌的影响,并寻找摄铁系统中的关键蛋白。【方法】以化脓链球菌为模型,利用含Fe和不含Fe的培养基对细菌进行培养,收集全细胞蛋白进行双向电泳,定量软件分析电泳图谱,质谱鉴定差异蛋白,进而通过生物信息学分析蛋白上下游关系,从中找到关键蛋白。【结果】鉴定出20个差异蛋白,并用Cytoscape软件对差异蛋白相互关系网络进行节点分析找到其中5个瓶颈分子。【结论】在培养基中的Fe3+缺乏时,细菌的生物合成和含氮化合物、生物大分子等重要代谢受到很大影响,这为进一步阐明细菌铁代谢机制奠定了基础。     

金属蛋白质组学研究锌离子匮乏对肺炎链球菌的影响

【目的】研究锌离子缺乏对肺炎链球菌的影响,找到其适应性生长机制。【方法】以肺炎链球菌为模型,利用加锌和不加锌的培养基对细菌进行培养,收集细胞蛋白,采用双向凝胶电泳,结合金属亲和层析和质谱技术鉴定差异表达蛋白,进而通过生物信息学分析蛋白质相互关系,从中找到细菌适应锌离子匮乏条件的关键代谢通路和蛋白。【结果】测定了在限制培养条件下肺炎链球菌的最适生长浓度,建立了锌离子调控蛋白双向凝胶电泳图谱,鉴定到了96个差异表达蛋白斑点,共67个差异蛋白,其中32个表达下调,35个表达上调,锌离子调控蛋白的作用可能主要体现在糖代谢、核酸代谢、氧化还原作用、辅助蛋白质翻译、合成及折叠等方面。建立了锌结合蛋白的差异表达图谱,鉴定到了10个差异表达蛋白斑点,共7个差异蛋白,其中1个表达下调,6个表达上调。锌离子结合蛋白的作用可能主要体现在应对压力、蛋白质折叠和转运、氨基酸代谢等方面。【结论】肺炎链球菌主要通过调控碳水化合物代谢和核酸代谢等多个代谢通路来应对宿主锌金属离子匮乏的环境,从而使自身能够存活并对宿主形成感染。本研究为揭示细菌在宿主环境,特别是金属离子匮乏条件下的适应性生长机制提供理论基础。     

Proteomic profiling of CHO cells with enhanced rhBMP-2 productivity following co-expression of PACEsol

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant protein biopharmaceuticals. The purpose of this study was to investigate differences in the proteome of CHO DUKX cells expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) (G5 cells) compared to cells also expressing soluble exogenous paired basic amino acid cleaving enzyme soluble paired basic amino acid cleaving enzyme (PACEsol) (3C9 cells), which has been previously found to improve the post-translational processing of the mature rhBMP-2 dimer. PACEsol co-expression was also associated with a significant increase (almost four-fold) in cellular productivity of rhBMP-2 protein. Differential proteomic expression profiling using 2-D DIGE and MALDI-TOF MS was performed to compare 3C9 and G5 cells, and revealed a list of 60 proteins that showed differential expression (up/downregulated), with a variety of different cellular functions. A substantial number of these altered proteins were found to have chaperone activity, involved with protein folding, assembly and secretion, as well as a number of proteins involved in protein translation. These results support the use of proteomic profiling as a valuable tool towards understanding the biology of bioprocess cultures.     

Differential analysis of protein expression of Bifidobacterium grown on different carbohydrates

We observed recently that colonic fermentation of lactose might be a major factor in the pathophysiology of lactose intolerance. Proteomic techniques could be helpful in interpreting the metabolic pathways of lactose fermentation. The objective of this study was to explore proteomic methodologies for studying bacterial lactose metabolism that can be used to detect and identify proteins associated with the onset of intolerance symptoms. Differential expression of cytoplasmic proteins of Bifidobacterium animalis, Bifidobacterium breve and Bifidobacterium longum grown on different carbohydrates (lactose, glucose, galactose) was analyzed with surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After fractionation by SDS-PAGE, differentially-expressed proteins were identified with LC-MS/MS. The three strains grown on the same carbohydrate or the same strain grown on glucose or lactose showed differences in SELDI-TOF MS protein profiles. Differences in protein expression were observed in B. breve grown on glucose, galactose or lactose as analyzed with SDS-PAGE. With LC-MS/MS, proteins from Bifidobacterium were identified, which included enzymes for metabolism of lactose, glucose and galactose. In conclusion, the applied techniques can discern differences in protein expression of bacteria metabolizing different carbohydrates. These techniques are promising in studying metabolism of lactose and other substrates in a complex bacterial ecosystem such as the colonic microbiota.     

Proteomic analysis of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) pollen

This paper presents an analysis of Holm oak pollen proteome, together with an evaluation of the potentiality that a proteomic approach may have in the provenance variability assessment. Proteins were extracted from pollen of four Holm oak provenances, and they were analyzed by gel-based (1- and 2-DE in combination with MALDI-TOF/TOF) and gel-free (nLC-LTQ Orbitrap MS) approaches. A comparison of 1- and 2-DE protein profiles of the four provenances revealed significant differences, both qualitative and quantitative, in abundance (18 bands and 16 spots, respectively). Multivariate statistical analysis carried out on bands and spots clearly showed distinct associations between provenances, which highlight their geographical origins. A total of 100 spots selected from the 402 spots observed on 2-DE gels were identified by MALDI-TOF/TOF. Moreover, a complementary gel-free shotgun approach was performed by nLC-LTQ Orbitrap MS. The identified proteins were classified according to biological processes, and most proteins in both approaches were related to metabolism and defense/stress processes. The nLC-LTQ Orbitrap MS analysis allowed us the identification of proteins belonging to the cell wall and division, transport and translation categories. Besides providing the first reference map of Holm oak pollen, our results confirm previous studies based on morphological observations and acorn proteomic analysis. Moreover, our data support the valuable use of proteomic techniques as phylogenetic tool in plant studies.     

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2‐DE. The differential 2‐DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH‐dependent N10 genes using Western blot and real‐time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH‐dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell‐shape mode of pH‐dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH‐responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic‐adaptive mechanism.