Organelle Membrane Proteomics Reveals Differential Influence of Mycobacterial Lipoglycans on Macrophage Phagosome Maturation and Autophagosome Accumulation

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors ofMycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the twomycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid

Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis

Isotopomer-assisted metabolite analysis was used to investigate the central metabolism of Mycobacterium smegmatis and its transition from normal growth to a non-replicating state under a hypoxic environment. Tween 80 significantly promoted aerobic growth by improving O2 transfer, while only small amount was degraded and metabolized via the TCA cycle for biomass synthesis. As the bacillus encountered hypoxic stress, isotopomer analysis suggested: (1) isocitrate lyase activity increased, which further induced glyoxylate pathway and glycine dehydrogenase for replenishing NAD+; (2) the relative amount of acetyl-CoA entering the TCA cycle was doubled, whereas little entered the glycolytic and pentose phosphate pathways

Membrane proteomics of phagosomes suggests a connection to autophagy

Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance on induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages

Comprehensive evaluation of untargeted metabolomics data processing software in feature detection, quantification and discriminating marker selection

Data analysis represents a key challenge for untargeted metabolomics studies and it commonly requires extensive processing of more than thousands of metabolite peaks included in raw high-resolution MS data. Although a number of software packages have been developed to facilitate untargeted data processing, they have not been comprehensively scrutinized in the capability of feature detection, quantification and marker selection using a well-defined benchmark sample set. In this study, we acquired a benchmark dataset from standard mixtures consisting of 1100 compounds with specified concentration ratios including 130 compounds with significant variation of concentrations. Five software evaluated here (MS-Dial, MZmine 2, XCMS, MarkerView, and Compound Discoverer) showed similar performance in detection of true features derived from compounds in the mixtures. However, significant differences between untargeted metabolomics software were observed in relative quantification of true features in the benchmark dataset. MZmine 2 outperformed the other software in terms of quantification accuracy and it reported the most true discriminating markers together with the fewest false markers. Furthermore, we assessed selection of discriminating markers by different software using both the benchmark dataset and a real-case metabolomics dataset to propose combined usage of two software for increasing confidence of biomarker identification. Our findings from comprehensive evaluation of untargeted metabolomics software would help guide future improvements of these widely used bioinformatics tools and enable users to properly interpret their metabolomics results

Classification: Biological Sciences Title: Membrane Proteomics of Phagosomes Suggests a Connection to Autophagy

Abstract Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance upon induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages

Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis.

Isotopomer-assisted metabolite analysis was used to investigate the central metabolism of Mycobacterium smegmatis and its transition from normal growth to a non-replicating state under a hypoxic environment. Tween 80 significantly promoted aerobic growth by improving O(2) transfer, while only small amount was degraded and metabolized via the TCA cycle for biomass synthesis. As the bacillus encountered hypoxic stress, isotopomer analysis suggested: (1) isocitrate lyase activity increased, which further induced glyoxylate pathway and glycine dehydrogenase for replenishing NAD(+); (2) the relative amount of acetyl-CoA entering the TCA cycle was doubled, whereas little entered the glycolytic and pentose phosphate pathways