Quantitative proteome analysis of temporally resolved phagosomes following uptake via key phagocytic receptors. Molecular & cellular proteomics : MCP Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis. 10.1074/mcp.M114.044594

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells. Proceedings of the National Academy of Sciences of the United States of America Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by or human macrophages. In contrast, and accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by or macrophages. Adding extracellular polyphosphate increased survival after phagocytosis by and macrophages. Reducing expression of polyphosphate kinase 1 in reduced extracellular polyphosphate and reduced survival in and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed or cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed and Polyphosphate inhibited phagosome acidification and lysosome activity in and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation. 10.1073/pnas.2012009117

and Escape From the Clearance of Macrophages via Controlling TFEB. Rao Shanshan,Xu Tao,Xia Yu,Zhang Hongfeng Frontiers in microbiology Phagosome- and xenophagosome-lysosome systems play a critical role in the defense of pathogenic bacteria, such as and , in macrophages. A great part of the bacteria escapes from the digestion and can survive through some mechanisms that are still poorly understood and which require further exploration. Here we identified that inhibited the expression and activation of TFEB to blunt the functions of lysosomes and defense of clearance by activating caspase-1. The expression and activation of TFEB were enhanced early under the infection of , which was followed by shrinkage to weaken lysosomal functions due to the delayed activation of ERK, mTOR, and STAT3. Thus, we have identified novel escape mechanisms for and to deepen and strengthen our strategies fighting with pathogens. 10.3389/fmicb.2020.573844

Electron microscopic identification of hydrogen peroxide detected in fixed human polymorphonuclear leukocytes during phagocytosis. Moriguchi Keiichi,Ohno Norikazu Okajimas folia anatomica Japonica Polymorphonuclear leukocytes (PMNs) engaged in phagocytosis produce reactive oxygen species (ROS), such as those that occur in an activated NADPH oxidase reaction, to eliminate ingested microorganisms. The translocation of NADPH oxidase components to produce antimicrobial free radicals from the vesicles to the phagosomes may be important. Hydrogen peroxide (H2O2) derived from O2- has been observed by electron microscopy using a cerium method. However, 2'-7'-dichlorofluorescin diacetate can also detect H2O2 through fluorescence. The main objective of the present study was to measure the H2O2-dependent fluorescence of PMNs after opsonized zymosan A (OPZ) phagocytosis using a microplate reader under different fixation conditions, including 0.5, 1, and 10% glutaraldehyde (GA) individually for 1, 5, 10, or 30 min. An additional objective was to visualize, through the use of electron microscopic cytochemistry, the process of H2O2 generation in OPZ phagocytic fixed PMNs. The fixed PMNs showed that the largest fluorescent value was produced by a concentration of 0.5% GA for all fixation times. This suggested that the fixation of PMNs with a high concentration of GA inhibited phagocytosis and produced ROS. In the fixed PMNs, electron microscopic results showed that after 1 min of mixing, some PMNs attached to particles and exhibited mild deposits in their secretory vesicles. When PMNs engulfed particles, free radical-producing vesicles had enhanced reaction deposits 10 min later and fused to the phagosomal membrane, releasing numerous free radicals into the lumen. Time-dependent H2O2 production was enhanced in the secretory vesicles, some of which were fused exactly to the phagosome membranes.