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Search Results: 1 - 10 of 201537 matches for " Joshua N Adkins "
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Diverse Secreted Effectors Are Required for Salmonella Persistence in a Mouse Infection Model
Afshan S. Kidwai, Ivy Mushamiri, George S. Niemann, Roslyn N. Brown, Joshua N. Adkins, Fred Heffron
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0070753
Abstract: Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.
Controlling the Response: Predictive Modeling of a Highly Central, Pathogen-Targeted Core Response Module in Macrophage Activation
Jason E. McDermott,Michelle Archuleta,Brian D. Thrall,Joshua N. Adkins,Katrina M. Waters
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0014673
Abstract: We have investigated macrophage activation using computational analyses of a compendium of transcriptomic data covering responses to agonists of the TLR pathway, Salmonella infection, and manufactured amorphous silica nanoparticle exposure. We inferred regulatory relationship networks using this compendium and discovered that genes with high betweenness centrality, so-called bottlenecks, code for proteins targeted by pathogens. Furthermore, combining a novel set of bioinformatics tools, topological analysis with analysis of differentially expressed genes under the different stimuli, we identified a conserved core response module that is differentially expressed in response to all studied conditions. This module occupies a highly central position in the inferred network and is also enriched in genes preferentially targeted by pathogens. The module includes cytokines, interferon induced genes such as Ifit1 and 2, effectors of inflammation, Cox1 and Oas1 and Oasl2, and transcription factors including AP1, Egr1 and 2 and Mafb. Predictive modeling using a reverse-engineering approach reveals dynamic differences between the responses to each stimulus and predicts the regulatory influences directing this module. We speculate that this module may be an early checkpoint for progression to apoptosis and/or inflammation during macrophage activation.
Decon2LS: An open-source software package for automated processing and visualization of high resolution mass spectrometry data
Navdeep Jaitly, Anoop Mayampurath, Kyle Littlefield, Joshua N Adkins, Gordon A Anderson, Richard D Smith
BMC Bioinformatics , 2009, DOI: 10.1186/1471-2105-10-87
Abstract: With a variety of options that include peak processing, deisotoping, isotope composition, etc, Decon2LS supports processing of multiple raw data formats. Deisotoping can be performed on an individual scan, an individual dataset, or on multiple datasets using batch processing. Other processing options include creating a two dimensional view of mass and liquid chromatography (LC) elution time features, generating spectrum files for tandem MS data, creating total intensity chromatograms, and visualizing theoretical peptide profiles. Application of Decon2LS to deisotope different datasets obtained across different instruments yielded a high number of features that can be used to identify and quantify peptides in the biological sample.Decon2LS is an efficient software package for discovering and visualizing features in proteomics studies that require automated interpretation of mass spectra. Besides being easy to use, fast, and reliable, Decon2LS is also open-source, which allows developers in the proteomics and bioinformatics communities to reuse and refine the algorithms to meet individual needs.Decon2LS source code, installer, and tutorials may be downloaded free of charge at http://http:/ncrr.pnl.gov/software/ webcite.High resolution mass spectrometry (MS) is used extensively in proteomics and metabolomics studies to identify and quantify proteins and metabolites [1]. This information is inferred from peak patterns observed in either mass spectra of intact proteins, digested proteins (i.e., peptides) or metabolites, or tandem mass spectra (MS/MS) of proteins, peptides, or metabolites fragmented as a result of collision-induced dissociation within the instrument. While hundreds of individual species can be resolved from a single mass spectrum, even relatively simple proteomics and metabolomics samples can result in thousands of overlapping isotopic patterns. As these patterns may not be readily separable by the instrument or discernible by downstream processing algori
Systems analysis of multiple regulator perturbations allows discovery of virulence factors in Salmonella
Hyunjin Yoon, Charles Ansong, Jason E McDermott, Marina Gritsenko, Richard D Smith, Fred Heffron, Joshua N Adkins
BMC Systems Biology , 2011, DOI: 10.1186/1752-0509-5-100
Abstract: In this study we present a systems biology approach in which sample-matched multi-omic measurements of fourteen virulence-essential regulator mutants were coupled with computational network analysis to efficiently identify Salmonella virulence factors. Immunoblot experiments verified network-predicted virulence factors and a subset was determined to be secreted into the host cytoplasm, suggesting that they are virulence factors directly interacting with host cellular components. Two of these, SrfN and PagK2, were required for full mouse virulence and were shown to be translocated independent of either of the type III secretion systems in Salmonella or the type III injectisome-related flagellar mechanism.Integrating multi-omic datasets from Salmonella mutants lacking virulence regulators not only identified novel virulence factors but also defined a new class of translocated effectors involved in pathogenesis. The success of this strategy at discovery of known and novel virulence factors suggests that the approach may have applicability for other bacterial pathogens.The interactions between intracellular pathogen and host can be complex involving sophisticated offensive and defensive strategies by both organisms. Developing a systems level understanding of the virulence program of a pathogen, both in terms of the regulatory pathways and the virulence-related proteins that execute this program is important to effectively combat persistent and adapting pathogens [1-3]. Combining high-throughput characterization of proteins and gene transcripts under multiple different conditions relevant to virulence provides a wealth of information that can be mined to provide useful leads for further investigation or used as the basis of predictive models.Salmonella enterica serovar Typhimurium (STM) is a facultative intracellular bacterial pathogen with a broad host range capable of infecting birds, reptiles, mice, humans and other mammals. In humans, it is a leading causative agent
Comparative Bacterial Proteomics: Analysis of the Core Genome Concept
Stephen J. Callister, Lee Ann McCue, Joshua E. Turse, Matthew E. Monroe, Kenneth J. Auberry, Richard D. Smith, Joshua N. Adkins, Mary S. Lipton
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0001542
Abstract: While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits.
An experimentally-supported genome-scale metabolic network reconstruction for Yersinia pestis CO92
Pep Charusanti, Sadhana Chauhan, Kathleen McAteer, Joshua A Lerman, Daniel R Hyduke, Vladimir L Motin, Charles Ansong, Joshua N Adkins, Bernhard O Palsson
BMC Systems Biology , 2011, DOI: 10.1186/1752-0509-5-163
Abstract: Here we report a genome-scale reconstruction and mathematical model of metabolism for Y. pestis CO92 and supporting experimental growth and metabolite measurements. The model contains 815 genes, 678 proteins, 963 unique metabolites and 1678 reactions, accurately simulates growth on a range of carbon sources both qualitatively and quantitatively, and identifies gaps in several key biosynthetic pathways and suggests how those gaps might be filled. Furthermore, our model presents hypotheses to explain certain known nutritional requirements characteristic of this strain.Y. pestis continues to be a dangerous threat to human health during modern times. The Y. pestis genome-scale metabolic reconstruction presented here, which has been benchmarked against experimental data and correctly reproduces known phenotypes, provides an in silico platform with which to investigate the metabolism of this important human pathogen.Yersinia pestis is a gram-negative bacterium within the family Enterobacteriaceae that, along with Yersinia pseudotuberculosis and Yersinia enterocolitica, is one of three members of its genus that can cause disease in humans. Y. pestis diverged from Y. pseudotuberculosis only 1,500 - 20,000 years ago, but Y. pestis and Y. pseudotuberculosis diverged from Y. enterocolitica in the more distant past [1]. Despite their close evolutionary relationship, the diseases they cause differ markedly. Whereas Y. pseudotuberculosis and Y. enterocolitica are primarily gastrointestinal pathogens in humans, Y. pestis infections lead to a systemic disease known as plague that can become fatal rapidly. In the last 2000 years, there have been three distinct outbreaks of plague that have led to a large number of fatalities: the Justinian plague between the 5th and 7th centuries, the Black Death in Europe between the 13th and 15th centuries, and the modern plague from the latter half of the 1800s to the present. These outbreaks of fatal infections that continue to occur [2,3], the
A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions
Roslyn N. Brown,James A. Sanford,Jea H. Park,Brooke L. Deatherage,Boyd L. Champion,Richard D. Smith,Fred Heffron,Joshua N. Adkins
International Journal of Proteomics , 2012, DOI: 10.1155/2012/123076
Abstract: Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB and PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein locations for Salmonella and a framework for further investigations using computational modeling. 1. Introduction The pursuit of a systems-level understanding of bacterial physiology requires not only knowledge about the identity, function, and relative abundance of proteins, but also insight into the subcellular localization of these proteins. Subcellular protein localization is linked to protein function, potential protein-protein interactions, and to interactions between a cell and its exterior environment. The observation of proteins in unexpected cellular compartments gives clues about the presence of possible alternate functions. Hence, there is a growing appreciation for the presence of bacterial “moonlighting proteins,” that is, those proteins that have a secondary function depending on subcellular location [1–3]. Experimentally verified localization also provides a foundation for describing proteins that are “hypothetical,” uncharacterized, or that contain domains of unknown function. Furthermore, with the increasing use of systems biology approaches, including genome-scale models of metabolism [4] and regulation to study microbial functions, experimentally founded protein localization on a global scale is necessary to produce more accurate model constraints. Subcellular proteomics has emerged as
Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation
Charles Ansong, Hyunjin Yoon, Steffen Porwollik, Heather Mottaz-Brewer, Brianne O. Petritis, Navdeep Jaitly, Joshua N. Adkins, Michael McClelland, Fred Heffron, Richard D. Smith
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004809
Abstract: Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella.
The Genome Organization of Thermotoga maritima Reflects Its Lifestyle
Haythem Latif equal contributor,Joshua A. Lerman equal contributor,Vasiliy A. Portnoy,Yekaterina Tarasova,Harish Nagarajan,Alexandra C. Schrimpe-Rutledge,Richard D. Smith,Joshua N. Adkins,Dae-Hee Lee,Yu Qiu,Karsten Zengler
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003485
Abstract: The generation of genome-scale data is becoming more routine, yet the subsequent analysis of omics data remains a significant challenge. Here, an approach that integrates multiple omics datasets with bioinformatics tools was developed that produces a detailed annotation of several microbial genomic features. This methodology was used to characterize the genome of Thermotoga maritima—a phylogenetically deep-branching, hyperthermophilic bacterium. Experimental data were generated for whole-genome resequencing, transcription start site (TSS) determination, transcriptome profiling, and proteome profiling. These datasets, analyzed in combination with bioinformatics tools, served as a basis for the improvement of gene annotation, the elucidation of transcription units (TUs), the identification of putative non-coding RNAs (ncRNAs), and the determination of promoters and ribosome binding sites. This revealed many distinctive properties of the T. maritima genome organization relative to other bacteria. This genome has a high number of genes per TU (3.3), a paucity of putative ncRNAs (12), and few TUs with multiple TSSs (3.7%). Quantitative analysis of promoters and ribosome binding sites showed increased sequence conservation relative to other bacteria. The 5′UTRs follow an atypical bimodal length distribution comprised of “Short” 5′UTRs (11–17 nt) and “Common” 5′UTRs (26–32 nt). Transcriptional regulation is limited by a lack of intergenic space for the majority of TUs. Lastly, a high fraction of annotated genes are expressed independent of growth state and a linear correlation of mRNA/protein is observed (Pearson r = 0.63, p<2.2×10?16 t-test). These distinctive properties are hypothesized to be a reflection of this organism's hyperthermophilic lifestyle and could yield novel insights into the evolutionary trajectory of microbial life on earth.
Experimental annotation of post-translational features and translated coding regions in the pathogen Salmonella Typhimurium
Charles Ansong, Nikola Toli?, Samuel O Purvine, Steffen Porwollik, Marcus Jones, Hyunjin Yoon, Samuel H Payne, Jessica L Martin, Meagan C Burnet, Matthew E Monroe, Pratap Venepally, Richard D Smith, Scott N Peterson, Fred Heffron, Michael McClelland, Joshua N Adkins
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-433
Abstract: We experimentally annotated the bacterial pathogen Salmonella Typhimurium 14028, using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. The data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in Salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. Additionally, we uncovered 12 non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel ORFs in Salmonella pathogenesis. We also characterized post-translational features in the Salmonella genome, including chemical modifications and proteolytic cleavages. We find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. Our in vivo proteolysis data identified more than 130 signal peptide and N-terminal methionine cleavage events critical for protein function.This work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of Salmonella as a resource for systems analysis.Many aspects of modern biological research are dependent on accurate identification of the protein-coding genes in each genome, as well as the nature of the mature functional protein products, a process commonly referred to as genome annotation. With the exponential increase in the number of sequenced prokaryotic genomes afforded by advances in genome sequencing technologies over the last decade, present day prokaryotic genome annotation is essentially an automated high-throughput process that relies heavily on de novo gene prediction programs [1-3].While de novo gene prediction programs have significantly improved for prokaryotic genomes consider
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