Identification of Novel Surface Proteins of Anaplasma phagocytophilum by Affinity Purification and Proteomics

ABSTRACT Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.

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Surface-Exposed Proteins of Ehrlichia chaffeensis

Infection and Immunity ◽

10.1128/iai.00188-07 ◽

2007 ◽

Vol 75 (8) ◽

pp. 3833-3841 ◽

Cited By ~ 45

Author(s):

Yan Ge ◽

Yasuko Rikihisa

Keyword(s):

Vaccine Development ◽

Affinity Purification ◽

Hypothetical Protein ◽

Capillary Liquid Chromatography ◽

Surface Proteins ◽

Mass Spectrometry Analysis ◽

Ehrlichia Chaffeensis ◽

Monocytic Leukemia ◽

Spectrometry Analysis ◽

Host Interactions

ABSTRACT The surface proteins of Ehrlichia chaffeensis provide an important interface for pathogen-host interactions. To investigate the surface proteins of E. chaffeensis, membrane-impermeable, cleavable Sulfo-NHS-SS-Biotin was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin-agarose affinity purification. The affinity-captured proteins were separated by electrophoresis, and five relatively abundant protein bands containing immunoreactive proteins were subjected to capillary-liquid chromatography-nanospray tandem mass spectrometry analysis. Nineteen out of 22 OMP-1/P28 family proteins, including P28 (which previously was shown to be surface exposed), were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. For the first time, with the exception of P28 and P28-1, 17 OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. The surface exposure of OMP-1A and OMP-1N was verified by immunofluorescence microscopy. OMP-1B was undetectable either by surface biotinylation or by Western blotting of the whole bacterial lysate, suggesting that it is not expressed by E. chaffeensis cultured in THP-1 cells. Additional E. chaffeensis surface proteins detected were OMP85, hypothetical protein ECH_0525 (here named Esp73), immunodominant surface protein gp47, and 11 other proteins. The identification of E. chaffeensis surface-exposed proteins provides novel insights into the E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interactions and vaccine development.

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Formation of Strecker Aldehydes in Casein Digestion

Current Developments in Nutrition ◽

10.1093/cdn/nzaa045_133 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 500-500

Author(s):

Yuyin Zhou ◽

Chi Chen

Keyword(s):

Degradation Products ◽

In Vitro Digestion ◽

The United States ◽

Mass Spectrometry Analysis ◽

United States Department ◽

Gastric Digestion ◽

Spectrometry Analysis ◽

Strecker Aldehydes ◽

Intestinal Digestion

Abstract Objectives This study investigated the occurrence of Strecker degradation during in vitro digestion. Methods Casein was first incubated with artificial gastric fluid containing porcine pepsin and HCl (pH = 2) for 60 min. After adjusting pH to 7 with sodium hydroxide, casein gastric digesta was then incubated with artificial intestinal fluid containing porcine pancreatin (pH = 7) for 120 min. Digesta samples were collected at 0, 10, 20, 30, and 60 min of gastric digestion, and then 10, 20, 30, 60, and 120 min of intestinal digestion. Free amino acids and aldehydes in digesta samples were analyzed by the liquid chromatography-mass spectrometry analysis. Results Multiple aldehydes were detected in gastric digestion samples, and their concentrations were further increased by intestinal digestion. Among them, isovaleraldehyde, isobutyraldehyde, phenylacetaldehyde, and acetaldehyde, are the Strecker degradation products of leucine, valine, phenylalanine, and alanine, respectively. Without digestive enzymes, casein incubation did not produce Strecker aldehydes. Conclusions In vitro digestion of proteins can produce Strecker aldehydes. Funding Sources This research was partially supported by the Agricultural Experiment Station project MIN-18-125 (C. C.) from the United States Department of Agriculture (USDA).

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Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation

International Journal of Molecular Sciences ◽

10.3390/ijms22137011 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7011

Author(s):

Barbora Mikolaskova ◽

Matus Jurcik ◽

Ingrid Cipakova ◽

Tomas Selicky ◽

Jan Jurcik ◽

...

Keyword(s):

Gene Expression ◽

Rna Processing ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Terminal Region ◽

Mass Spectrometry Analysis ◽

Post Translational Modification ◽

Vitro Assay ◽

Spectrometry Analysis

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.

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Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses

Circulation Research ◽

10.1161/circresaha.121.320029 ◽

2021 ◽

Author(s):

Andreas W Heumüller ◽

Alisha Nicole Jones ◽

André Mourão ◽

Marius Klangwart ◽

Chenyue Shi ◽

...

Keyword(s):

Endothelial Cells ◽

Laminar Flow ◽

Affinity Purification ◽

Mass Spectrometry Analysis ◽

Circular Rnas ◽

Cell Orientation ◽

Spectrometry Analysis ◽

Functional Conservation

Background : Circular RNAs (circRNAs) are generated by back-splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. Methods: Mechanistically, we identified the protein syndesmos (SDOS) to specifically interact with cZNF292 in endothelial cells by RNA affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganisation thereby recapitulating cZfp292 phenotypes. Results: Combining published endothelial RNA sequencing datasets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localisation and focal adhesion organisation. Conclusion: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

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Phytochemicals of Minthostachys diffusa Epling and Their Health-Promoting Bioactivities

Foods ◽

10.3390/foods9020144 ◽

2020 ◽

Vol 9 (2) ◽

pp. 144

Author(s):

Immacolata Faraone ◽

Daniela Russo ◽

Lucia Chiummiento ◽

Eloy Fernandez ◽

Alka Choudhary ◽

...

Keyword(s):

Ethyl Acetate ◽

Radical Scavenging ◽

Ethyl Acetate Fraction ◽

Mass Spectrometry Analysis ◽

Antioxidant Power ◽

Spectrometry Analysis ◽

In Silico Docking ◽

Aerial Parts ◽

Health Promoting

The genus Minthostachys belonging to the Lamiaceae family, and is an important South American mint genus used commonly in folk medicine as an aroma in cooking. The phytochemical-rich samples of the aerial parts of Minthostachys diffusa Epling. were tested for pharmacological and health-promoting bioactivities using in vitro chemical and enzymatic assays. A range of radical scavenging activities of the samples against biological radicals such as nitric oxide and superoxide anion and against synthetic 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, the ferric reducing antioxidant power and the lipid peroxidation inhibition were determined and ranked using the ‘relative antioxidant capacity index’ (RACI). The ethyl acetate fraction showed the highest RACI of +1.12. Analysis of the various fractions’ inhibitory ability against enzymes involved in diabetes (α-amylase and α-glucosidase), and against enzymes associated with Parkinson’s or Alzheimer’s diseases (acetylcholinesterase and butyrylcholinesterase) also suggested that the ethyl acetate fraction was the most active. Liquid chromatography–tandem mass spectrometry analysis of the ethyl acetate fraction showed more than 30 polyphenolic compounds, including triterpenes. The inhibitory cholinesterase effects of the triterpenes identified from M. diffusa were further analysed by in silico docking of these compounds into 3D-structures of acetylcholinesterase and butyrylcholinesterase. This is the first study on pharmacological activities and phytochemical profiling of the aerial parts of M. diffusa, showing that this plant, normally used as food in South America, is also rich in health-promoting phytochemicals.

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Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

International Journal of Molecular Sciences ◽

10.3390/ijms22137099 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7099

Author(s):

Pradeep Kumar Kopparapu ◽

Meghshree Deshmukh ◽

Zhicheng Hu ◽

Majd Mohammad ◽

Marco Maugeri ◽

...

Keyword(s):

Extracellular Vesicles ◽

Inflammatory Responses ◽

Surface Proteins ◽

Host Cells ◽

Immune Stimulation ◽

Domains Of Life ◽

Major Surface ◽

Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

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A Comparative Study of the Antioxidative Effects of Helichrysum italicum and Helichrysum arenarium Infusions

Antioxidants ◽

10.3390/antiox10030380 ◽

2021 ◽

Vol 10 (3) ◽

pp. 380

Author(s):

Katja Kramberger ◽

Zala Jenko Pražnikar ◽

Alenka Baruca Arbeiter ◽

Ana Petelin ◽

Dunja Bandelj ◽

...

Keyword(s):

Oxidative Stress ◽

High Performance ◽

Radical Scavenging Activity ◽

Radical Scavenging ◽

Morphological Type ◽

Mass Spectrometry Analysis ◽

In Vitro Assays ◽

Spectrometry Analysis ◽

Stress Related Genes

Helichrysum arenarium (L.) Moench (abbrev. as HA) has a long tradition in European ethnomedicine and its inflorescences are approved as a herbal medicinal product. In the Mediterranean part of Europe, Helichrysum italicum (Roth) G. Don (abbrev. as HI) is more common. Since infusions from both plants are traditionally used, we aimed to compare their antioxidative potential using in vitro assays. Two morphologically distinct HI plants, HIa and HIb, were compared to a commercially available HA product. Genetic analysis using microsatellites confirmed a clear differentiation between HI and HA and suggested that HIb was a hybrid resulting from spontaneous hybridization from unknown HI subspecies. High-performance liquid chromatography–mass spectrometry analysis showed the highest amounts of hydroxycinnamic acids and total arzanol derivatives in HIa, whereas HIb was richest in monohydroxybenzoic acids, caffeic acids, and coumarins, and HA contained the highest amounts of flavonoids, especially flavanones. HIa exhibited the highest radical scavenging activity; it was more efficient in protecting different cell lines from induced oxidative stress and in inducing oxidative stress-related genes superoxide dismutase 1, catalase, and glutathione reductase 1. The antioxidative potential of HI was not only dependent on the morphological type of the plant but also on the harvest date, revealing important information for obtaining the best possible product. Considering the superior properties of HI compared to HA, the evaluation of HI as a medicinal plant could be recommended.

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Phytotoxicity of Essential Oils on Selected Weeds: Potential Hazard on Food Crops

Plants ◽

10.3390/plants7040079 ◽

2018 ◽

Vol 7 (4) ◽

pp. 79 ◽

Cited By ~ 12

Author(s):

María Ibáñez ◽

María Blázquez

Keyword(s):

Seed Germination ◽

Essential Oil ◽

Essential Oils ◽

Mass Spectrometry Analysis ◽

Gas Chromatography Mass Spectrometry ◽

Food Crops ◽

Potential Hazard ◽

Spectrometry Analysis

The chemical composition of winter savory, peppermint, and anise essential oils, and in vitro and in vivo phytotoxic activity against weeds (Portulaca oleracea, Lolium multiflorum, and Echinochloa crus-galli) and food crops (maize, rice, and tomato), have been studied. Sixty-four compounds accounting for between 97.67–99.66% of the total essential oils were identified by Gas Chromatography-Mass Spectrometry analysis. Winter savory with carvacrol (43.34%) and thymol (23.20%) as the main compounds produced a total inhibitory effect against the seed germination of tested weed. Menthol (48.23%), menthone (23.33%), and iso-menthone (16.33%) from peppermint only showed total seed germination inhibition on L. multiflorum, whereas no significant effects were observed with trans-anethole (99.46%) from anise at all concentrations (0.125–1 µL/mL). Low doses of peppermint essential oil could be used as a sustainable alternative to synthetic agrochemicals to control L. multiflorum. The results corroborate that in vivo assays with a commercial emulsifiable concentrate need higher doses of the essential oils to reproduce previous in vitro trials. The higher in vivo phytotoxicity of winter savory essential oil constitutes an eco-friendly and less pernicious alternative to weed control. It is possible to achieve a greater in vivo phytotoxicity if less active essential oil like peppermint is included with other active excipients.

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