Proteomics to study macrophage response to viral infection

Viral infections are a major burden to human and animal health. Immune response against viruses consists of innate and adaptive immunity which are both critical for the eradication of the viral infection. The innate immune system is the first line of defense against viral infections. Proper innate immune response is required for the activation of adaptive, humoral and cell-mediated immunity. Macrophages are innate immune cells which have a central role in detecting viral infections including influenza A and human immunodeficiency viruses. Macrophages and other host cells respond to viral infection by modulating their protein expression levels, proteins' posttranslational modifications, as well as proteins' intracellular localization and secretion. Therefore the detailed characterization how viruses dynamically manipulate host proteome is needed for understanding the molecular mechanisms of viral infection. It is critical to identify cellular host factors which are exploited by different viruses, and which are less prone for mutations and could serve as potential targets for novel antiviral compounds. Here, we review how proteomics studies have enhanced our understanding of macrophage response to viral infection with special focus on Influenza A and Human immunodeficiency viruses, and virus infections of swine. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

From Inflammasome to Exosome-Does Extracellular Vesicle Secretion Constitute an Inflammasome-Dependent Immune Response?

Inflammasomes are intracellular protein complexes of pattern recognition receptors and caspase-1, with essential functions in regulating inflammatory responses of macrophages and dendritic cells. The primary role of inflammasomes is to catalyze processing and secretion of pro-inflammatory cytokines IL-1 beta and IL-18. Recently, intracellular non-canonical inflammasome activation by caspases-4/5, which are also regulators of pyroptosis via processing gasdermin D, has been elucidated. Caspase-1, the effector protease of inflammasome complex, is also known to modulate secretion of large number of other proteins. Thereby, besides its known role in processing pro-inflammatory cytokines, the inflammasome turns into a universal regulator of protein secretion, which allows the danger-exposed cells to release various proteins in order to alert and guide neighboring cells. Majority of these proteins are not secreted through the conventional ER-Golgi secretory pathway. Instead, they are segregated in membrane-enclosed compartment and secreted in nanosized extracellular vesicles, which protect their cargo and guide it for delivery. Growing evidence indicates that inflammasome activity correlates with enhanced secretion of extracellular vesicles and modulation of their protein cargo. This inflammasome-driven unconventional, vesicle-mediated secretion of multitude of immunoregulatory proteins may constitute a novel paradigm in inflammatory responses. In this mini review we discuss the current knowledge and highlight unsolved questions about metabolic processes, signals, and mechanisms linking inflammasome activity with regulated extracellular vesicle secretion of proteins. Further investigations on this relationship may in the future help understanding the significance of extracellular vesicle secretion in inflammatory diseases such as atherosclerosis, gouty arthritis, asthma, Alzheimer's and many others.Peer reviewe

Mass spectrometry-based proteomic exploration of the human immune system : focus on the inflammasome, global protein secretion, and T cells

Introduction: The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses.Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses.Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.Peer reviewe

PhosFox : a bioinformatics tool for peptide-level processing of LC-MS/MS-based phosphoproteomic data

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Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus

Structural and Functional Dynamics of Staphylococcus aureus Biofilms and Biofilm Matrix Proteins on Different Clinical Materials

Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials—borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene—at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-β-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points—from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points

Food-Like Growth Conditions Support Production of Active Vitamin B12 by Propionibacterium freudenreichii 2067 without DMBI, the Lower Ligand Base,or Cobalt Supplementation

Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein andmetabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.Peer reviewe

Growth Mode and Carbon Source Impact the Surfaceome Dynamics of Lactobacillus rhamnosus GG

Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.Peer reviewe

Pripper: prediction of caspase cleavage sites from whole proteomes

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PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker Diabetic Fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.Peer reviewe