A Novel Strategy to Screen Bacillus Calmette-Guérin Protein Antigen Recognized by γδ TCR

BACKGROUND: Phosphoantigen was originally identified as the main γδ TCR-recognized antigen that could activate γδ T cells to promote immune protection against mycobacterial infection. However, new evidence shows that the γδ T cells activated by phosphoantigen can only provide partial immune protection against mycobacterial infection. In contrast, whole lysates of Mycobacterium could activate immune protection more potently, implying that other γδ TCR-recognized antigens that elicit protective immune responses. To date, only a few distinct mycobacterial antigens recognized by the γδ TCR have been characterized. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we established a new approach to screen epitopes or protein antigens recognized by the γδ TCR using Bacillus Calmette-Guérin- (BCG-) specific γ TCR transfected cells as probes to pan a 12-mer random-peptide phage-displayed library. Through binding assays and functional analysis, we identified a peptide (BP3) that not only binds to the BCG-specific γδ TCR but also effectively activates γδ T cells isolated from human subjects inoculated with BCG. Importantly, the γδ T cells activated by peptide BP3 had a cytotoxic effect on THP-1 cells infected with BCG. Moreover, the oxidative stress response regulatory protein (OXYS), a BCG protein that matches perfectly with peptide BP3 according to bioinformatics analysis, was confirmed as a ligand for the γδ TCR and was found to activate γδ T cells from human subjects inoculated with BCG. CONCLUSIONS/SIGNIFICANCE: In conclusion, our study provides a novel strategy to identify epitopes or protein antigens for the γδ TCR, and provides a potential means to screen mycobacterial vaccines or candidates for adjuvant

Advances in the optimization of central carbon metabolism in metabolic engineering

Abstract Central carbon metabolism (CCM), including glycolysis, tricarboxylic acid cycle and the pentose phosphate pathway, is the most fundamental metabolic process in the activities of living organisms that maintains normal cellular growth. CCM has been widely used in microbial metabolic engineering in recent years due to its unique regulatory role in cellular metabolism. Using yeast and Escherichia coli as the representative organisms, we summarized the metabolic engineering strategies on the optimization of CCM in eukaryotic and prokaryotic microbial chassis, such as the introduction of heterologous CCM metabolic pathways and the optimization of key enzymes or regulatory factors, to lay the groundwork for the future use of CCM optimization in metabolic engineering. Furthermore, the bottlenecks in the application of CCM optimization in metabolic engineering and future application prospects are summarized

Stress Responsive Proteins Are Actively Regulated during Rice (<i>Oryza sativa</i>) Embryogenesis as Indicated by Quantitative Proteomics Analysis

<div><p>Embryogenesis is the initial step in a plant’s life, and the molecular changes that occur during embryonic development are largely unknown. To explore the relevant molecular events, we used the isobaric tags for relative and absolute quantification (iTRAQ) coupled with the shotgun proteomics technique (iTRAQ/Shotgun) to study the proteomic changes of rice embryos during embryogenesis. For the first time, a total of 2 165 unique proteins were identified in rice embryos, and the abundances of 867 proteins were actively changed based on the statistical evaluation of the quantitative MS/MS signals. The quantitative data were then confirmed using multiple reactions monitoring (MRM) and were also supported by our previous study based on two-dimensional gel electrophoresis (2 DE). Using the proteome at 6 days after pollination (DAP) as a reference, cluster analysis of these differential proteins throughout rice embryogenesis revealed that 25% were up-regulated and 75% were down-regulated. Gene Ontology (GO) analysis implicated that most of the up-regulated proteins were functionally categorized as stress responsive, mainly including heat shock-, lipid transfer-, and reactive oxygen species-related proteins. The stress-responsive proteins were thus postulated to play an important role during seed maturation.</p></div

Scatter plot of iTRAQ quantified log2 (protein ratio) and MRM quantified log2 (protein ratio).

<p>(A) iTRAQ versus MRM log2(12 DAP/6 DAP). (B) iTRAQ versus MRM log2(18 DAP/6 DAP). (C) iTRAQ versus MRM log2(24 DAP/6 DAP). (D) iTRAQ versus MRM log2(30 DAP/6 DAP).</p

Rice embryogenesis stages and embryo phenotypes for each stage.

<p>(A) Embryogenesis stages and sampling time settings. (B) Pattern of embryo length change during embryogenesis. (C) Pattern of embryo weight change during embryogenesis. (D) Newly germinated bud length of embryos during embryogenesis. For (B) (C) and (D), the error bars indicate the standard derivation.</p

Evaluation of three technical replicates.

<p>(A) Venn chart showing the overlap of the identified proteins from the three replications. (B) Accumulated frequency graph of the coefficient of variation.</p

Cluster result of the significantly regulated proteins.

<p>(A) Cluster heatmap of significantly regulated proteins using the protein abundance information. The five columns from left to right are labeled 6, 12, 18, 24 and 30 DAP at the top of the heatmap. The left braces indicate the two groups classified by cluster analysis. (B) Color diagram of the heatmap.</p