scholarly journals Identification of microsporidia host-exposed proteins reveals a repertoire of large paralogous gene families and rapidly evolving proteins

2016 ◽  
Author(s):  
Aaron W. Reinke ◽  
Keir M. Balla ◽  
Eric J. Bennett ◽  
Emily R. Troemel
Keyword(s):  
Gene Families ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Specific Gene ◽  
Large Species ◽  
Spectrometry Analysis ◽  
C Elegans ◽  
Pathogen Effectors ◽  
Potential Pathogen ◽  
Species Specific

AbstractPathogens use a variety of secreted and surface proteins to interact with and manipulate their hosts, but a systematic approach for identifying such proteins has been lacking. To identify these ‘host-exposed’ proteins, we used spatially restricted enzymatic tagging followed by mass spectrometry analysis of C. elegans infected with two species of Nematocida microsporidia. We identified 82 microsporidia proteins inside of intestinal cells, including several pathogen proteins in the nucleus. These microsporidia proteins are enriched in targeting signals, are rapidly evolving, and belong to large, Nematocida-specific gene families. We also find that large, species-specific families are common throughout microsporidia species. Our data suggest that the use of a large number of rapidly evolving species-specific proteins represents a common strategy for these intracellular pathogens to interact with their hosts. The unbiased method described here for identifying potential pathogen effectors represents a powerful approach for the study of a broad range of pathogens.

Chemical and biological characterization of sclerosin, an antifungal lipopeptide

2012 ◽  
Vol 58 (8) ◽  
pp. 1027-1034 ◽  
Author(s):  
Chrystal L. Berry ◽  
Ann Karen C. Brassinga ◽  
Lynda J. Donald ◽  
W.G. Dilantha Fernando ◽  
Peter C. Loewen ◽  
...  
Keyword(s):  
Ring Opening ◽  
Chemical Structure ◽  
Cell Types ◽  
Mass Spectrometry Analysis ◽  
Bacillus Species ◽  
Biological Characterization ◽  
Spectrometry Analysis ◽  
C Elegans ◽  
Oomycete Pathogen

Pseudomonas sp. strain DF41 produces a lipopeptide, called sclerosin that inhibits the fungal pathogen Sclerotinia sclerotiorum . The aim of the current study was to deduce the chemical structure of this lipopeptide and further characterize its bioactivity. Mass spectrometry analysis determined the structure of sclerosin to be CH3-(CH2)6-CH(OH)-CH2-CO-Dhb-Pro-Ala-Leu/Ile-Ala-Val-Val-Dhb-Thr-Val-Leu/Ile-Dhp-Ala-Ala-Ala-Val-Dhb-Dhb-Ala-Dab-Ser-Val-OH, similar to corpeptins A and B of the tolaasin group, differing by only 3 amino acids in the peptide chain. Subjecting sclerosin to various ring opening procedures revealed no new ions, suggesting that this molecule is linear. As such, sclerosin represents a new member of the tolaasin lipopeptide group. Incubation of S. sclerotinia ascospores and sclerotia in the presence of sclerosin inhibited the germination of both cell types. Sclerosin also exhibited antimicrobial activity against Bacillus species. Conversely, this lipopeptide demonstrated no zoosporicidal activity against the oomycete pathogen Phytophthora infestans . Next, we assessed the effect of DF41 and a lipopeptide-deficient mutant on the growth and development of Caenorhabditis elegans larvae. We discovered that sclerosin did not protect DF41 from ingestion by and degradation in the C. elegans digestive tract. However, another metabolite produced by this bacterium appeared to shorten the life-span of the nematode compared to C. elegans growing on Escherichia coli OP50.

scholarly journals The chromosome-scale reference genome of black pepper provides insight into piperine biosynthesis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lisong Hu ◽  
Zhongping Xu ◽  
Maojun Wang ◽  
Rui Fan ◽  
Daojun Yuan ◽  
...  
Keyword(s):  
Reference Genome ◽  
Gene Families ◽  
Black Pepper ◽  
Piper Nigrum ◽  
Comparative Genomic ◽  
Specific Gene ◽  
Phylogenomic Analysis ◽  
Genomic Analyses ◽  
Species Specific ◽  
Insight Into

Abstract Black pepper (Piper nigrum), dubbed the ‘King of Spices’ and ‘Black Gold’, is one of the most widely used spices. Here, we present its reference genome assembly by integrating PacBio, 10x Chromium, BioNano DLS optical mapping, and Hi-C mapping technologies. The 761.2 Mb sequences (45 scaffolds with an N50 of 29.8 Mb) are assembled into 26 pseudochromosomes. A phylogenomic analysis of representative plant genomes places magnoliids as sister to the monocots-eudicots clade and indicates that black pepper has diverged from the shared Laurales-Magnoliales lineage approximately 180 million years ago. Comparative genomic analyses reveal specific gene expansions in the glycosyltransferase, cytochrome P450, shikimate hydroxycinnamoyl transferase, lysine decarboxylase, and acyltransferase gene families. Comparative transcriptomic analyses disclose berry-specific upregulated expression in representative genes in each of these gene families. These data provide an evolutionary perspective and shed light on the metabolic processes relevant to the molecular basis of species-specific piperine biosynthesis.

scholarly journals Targeting the endothelial progenitor cell surface proteome to identify novel mechanisms that mediate angiogenic efficacy in a rodent model of vascular disease

2013 ◽  
Vol 45 (21) ◽  
pp. 999-1011 ◽  
Author(s):  
Catherine C. Kaczorowski ◽  
Timothy J. Stodola ◽  
Brian R. Hoffmann ◽  
Anthony R. Prisco ◽  
Pengyuan Y. Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Vascular Disease ◽  
Rodent Model ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Major Advance ◽  
Endothelial Progenitor ◽  
Angiogenic Potential ◽  
Spectrometry Analysis

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials suggest autologous EPC-based therapy may be effective in treatment of vascular diseases. Albeit promising, variability in the efficacy of EPCs associated with underlying disease states has hindered the realization of EPC-based therapy. Here we first identify and characterize EPC dysfunction in a rodent model of vascular disease (SS/Mcwi rat) that exhibits impaired angiogenesis. To identify molecular candidates that mediate the angiogenic potential of these cells, we performed a broad analysis of cell surface protein expression using chemical labeling combined with mass spectrometry. Analysis revealed EPCs derived from SS/Mcwi rats express significantly more type 2 low-affinity immunoglobulin Fc-gamma (FCGR2) and natural killer 2B4 (CD244) receptors compared with controls. Genome-wide sequencing (RNA-seq) and qt-PCR confirmed isoforms of CD244 and FCGR2a transcripts were increased in SS/Mcwi EPCs. EPCs with elevated expression of FCGR2a and CD244 receptors are predicted to increase the probability of SS/Mcwi EPCs being targeted for death, providing a mechanistic explanation for their reduced angiogenic efficacy in vivo. Pathway analysis supported this contention, as “key” molecules annotated to cell death paths were differentially expressed in the SS/Mcwi EPCs. We speculate that screening and neutralization of cell surface proteins that “tag” and impair EPC function may provide an alternative approach to utilizing incompetent EPCs in greater numbers, as circulating EPCs are depleted in patients with vascular disease. Overall, novel methods to identify putative targets for repair of EPCs using discovery-based technologies will likely provide a major advance in the field of regenerative medicine.

scholarly journals Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

2002 ◽  
Vol 70 (1) ◽  
pp. 368-379 ◽  
Author(s):  
Silvia Montigiani ◽  
Fabiana Falugi ◽  
Maria Scarselli ◽  
Oretta Finco ◽  
Roberto Petracca ◽  
...  
Keyword(s):  
Chlamydia Pneumoniae ◽  
Genetic Manipulation ◽  
Respiratory Infections ◽  
Surface Protein ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Western Blots ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Definition Of

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

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

2007 ◽  
Vol 189 (21) ◽  
pp. 7819-7828 ◽  
Author(s):  
Yan Ge ◽  
Yasuko Rikihisa
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Affinity Purification ◽  
The United States ◽  
Capillary Liquid Chromatography ◽  
Etiologic Agent ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Major Surface

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.

scholarly journals Comparative Genome Sequence Analysis Reveals the Extent of Diversity and Conservation for Glycan-Associated Proteins inBurkholderiaspp.

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Hui San Ong ◽  
Rahmah Mohamed ◽  
Mohd Firdaus-Raih
Keyword(s):  
Comparative Analysis ◽  
Genome Sequence ◽  
Defense Mechanisms ◽  
Surface Proteins ◽  
Ecological Niches ◽  
Specific Gene ◽  
Sequence Comparisons ◽  
Virulence Protein ◽  
Associated Proteins ◽  
Species Specific

Members of theBurkholderiafamily occupy diverse ecological niches. In pathogenic family members, glycan-associated proteins are often linked to functions that include virulence, protein conformation maintenance, surface recognition, cell adhesion, and immune system evasion. Comparative analysis of availableBurkholderiagenomes has revealed a core set of 178 glycan-associated proteins shared by allBurkholderiaof which 68 are homologous to known essential genes. The genome sequence comparisons revealed insights into species-specific gene acquisitions through gene transfers, identified an S-layer protein, and proposed that significantly reactive surface proteins are associated to sugar moieties as a potential means to circumvent host defense mechanisms. The comparative analysis using a curated database of search queries enabled us to gain insights into the extent of conservation and diversity, as well as the possible virulence-associated roles of glycan-associated proteins in members of theBurkholderiaspp. The curated list of glycan-associated proteins used can also be directed to screen other genomes for glycan-associated homologs.

scholarly journals Discovery of a Natural Microsporidian Pathogen with a Broad Tissue Tropism in Caenorhabditis elegans

2016 ◽  
Author(s):  
Robert J. Luallen ◽  
Aaron W. Reinke ◽  
Linda Tong ◽  
Michael R. Botts ◽  
Marie-Anne Félix ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Species ◽  
Microbial Pathogens ◽  
Specific Gene ◽  
Tissue Tropism ◽  
Microsporidian Parasite ◽  
C Elegans ◽  
Host Microbe Interactions ◽  
Species Specific ◽  
Host Tissues

AbstractMicrobial pathogens often establish infection within particular niches of their host for replication. Determining how infection occurs preferentially in specific host tissues is a key aspect of understanding host-microbe interactions. Here, we describe the discovery of a natural microsporidian parasite of the nematode Caenorhabditis elegans that has a unique tissue tropism compared to other parasites of C. elegans. We characterize the life cycle of this new species, Nematocida displodere, including pathogen entry, intracellular replication, and exit. N. displodere can invade multiple host tissues, including the epidermis, muscle, neurons, and intestine of C. elegans. Despite robust invasion of the intestine very little replication occurs there, with the majority of replication occurring in the muscle and epidermis. This feature distinguishes N. displodere from two closely related microsporidian pathogens, N. parisii and N. sp. 1, which exclusively invade and replicate in the intestine. Comparison of the N. displodere genome with N. parisii and N. sp. 1 reveals that N. displodere is the earliest diverging species of the Nematocida genus and devotes over 10% of its genome to a single species-specific gene family that may be mediating host interactions upon infection. Altogether, this system provides a convenient whole-animal model to investigate factors responsible for pathogen growth in different tissue niches.

scholarly journals Determination of the lipid composition of the GPI anchor

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256184
Author(s):  
Auxiliadora Aguilera-Romero ◽  
Susana Sabido-Bozo ◽  
Sergio Lopez ◽  
Alejandro Cortes-Gomez ◽  
Sofia Rodriguez-Gallardo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Composition ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Gpi Anchor ◽  
Spectrometry Analysis ◽  
Lipid Structure ◽  
Lipid Moiety ◽  
Protein Lipidation

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.

scholarly journals Miniaturized Digestion and Extraction of Surface Proteins from Candida albicans following Treatment with Histatin 5 for Mass Spectrometry Analysis

2016 ◽  
Vol 2016 ◽  
pp. 1-12
Author(s):  
Shirley Fan ◽  
Eduardo B. Moffa ◽  
Yizhi Xiao ◽  
Walter L. Siqueira ◽  
Ken K.-C. Yeung
Keyword(s):  
Mass Spectrometry ◽  
Candida Albicans ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Bacterial Cells ◽  
Intact Cells ◽  
Histatin 5 ◽  
Spectrometry Analysis ◽  
Glass Slides ◽  
Glass Surfaces

A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on the surface of intact cells suspended in solution. This paper describes miniaturization of this technique, in which cells are adhered on glass surfaces, and all sample treatments are conducted at μL volumes. Specifically, Candida albicans cells were attached onto HSA-coated glass slides. By depositing the appropriate reagent solutions on the adhered cells, we successfully performed cell washing, treatment with antifugal peptide, Histatin 5, and a proteolysis on intact cells with trypsin. The resulting peptides were subsequently analysed by mass spectrometry. In general, the data obtained was similar to that collected with suspended cells in much larger sample volumes. However, our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents.

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