Publications by authors named "Joshua N Adkins"

146 Publications

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics.

mBio 2021 06 29;12(3):e0053021. Epub 2021 Jun 29.

Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.

Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in and , but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in or , responsible for succinyl-CoA biosynthesis, reversed mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. -dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, , increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance.
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http://dx.doi.org/10.1128/mBio.00530-21DOI Listing
June 2021

efam: an expanded, metaproteome-supported HMM profile database of viral protein families.

Bioinformatics 2021 Jun 16. Epub 2021 Jun 16.

Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA.

Motivation: Viruses infect, reprogram, and kill microbes, leading to profound ecosystem consequences, from elemental cycling in oceans and soils to microbiome-modulated diseases in plants and animals. Although metagenomic datasets are increasingly available, identifying viruses in them is challenging due to poor representation and annotation of viral sequences in databases.

Results: Here we establish efam, an expanded collection of Hidden Markov Model (HMM) profiles that represent viral protein families conservatively identified from the Global Ocean Virome 2.0 dataset. This resulted in 240,311 HMM profiles, each with at least 2 protein sequences, making efam >7-fold larger than the next largest, pan-ecosystem viral HMM profile database. Adjusting the criteria for viral contig confidence from "conservative" to "eXtremely Conservative" resulted in 37,841 HMM profiles in our efam-XC database. To assess the value of this resource, we integrated efam-XC into VirSorter viral discovery software to discover viruses from less-studied, ecologically distinct oxygen minimum zone (OMZ) marine habitats. This expanded database led to an increase in viruses recovered from every tested OMZ virome by ∼24% on average (up to ∼42%) and especially improved the recovery of often-missed shorter contigs (<5 kb). Additionally, to help elucidate lesser-known viral protein functions, we annotated the profiles using multiple databases from the DRAM pipeline and virion-associated metaproteomic data, which doubled the number of annotations obtainable by standard, single-database annotation approaches. Together, these marine resources (efam and efam-XC) are provided as searchable, compressed HMM databases that will be updated bi-annually to help maximize viral sequence discovery and study from any ecosystem.

Availability: The resources are available on the iVirus platform at (doi.org/10.25739/9vze-4143).

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab451DOI Listing
June 2021

Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.

ACS Omega 2021 May 6;6(19):12660-12666. Epub 2021 May 6.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.

Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC-MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as "Mixing QC." This Mixing QC enables the detection of TMT labeling issues by LC-MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.
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http://dx.doi.org/10.1021/acsomega.1c00776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154127PMC
May 2021

The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry.

Nat Commun 2021 02 26;12(1):1339. Epub 2021 Feb 26.

School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA.

Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.
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http://dx.doi.org/10.1038/s41467-021-21579-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910587PMC
February 2021

Amylases in the Human Vagina.

mSphere 2020 12 9;5(6). Epub 2020 Dec 9.

Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, Idaho, USA

Dominance of species in vaginal communities is a hallmark of healthy conditions in the female genital tract. Key nutrients for lactobacilli include sugars produced when glycogen is degraded by α-amylase in the vagina. While α-amylase activity has been demonstrated in vaginal fluids, it is unclear whether α-amylases are produced solely by the host, bacteria in the vagina, or both. We screened cervicovaginal mucus from 23 reproductive-age women, characterized the species composition of vaginal communities, measured vaginal pH, and determined levels of amylase activity, glycogen, and lactic acid. Based on differences in these measured variables, one sample from each of four individual donors was selected for metagenomic and proteomic analyses. Of eight putative bacterial amylases identified in the assembled bacterial metagenomes, we detected four in vaginal fluids. These amylases were produced by various bacteria in different vaginal communities. Moreover, no two communities were the same in terms of which bacteria were producing amylases. Although we detected bacterial amylases in vaginal fluids, there was no clear association between the bacterial species that was dominant in a community and the level of amylase activity. This association was likely masked by the presence of human α-amylase, which was also detected in vaginal fluids. Finally, the levels of amylase activity and glycogen were only weakly associated. Our findings show, for the first time, that multiple amylases from both bacterial and human origins can be present simultaneously in the vagina. This work also suggests that the link between glycogen, amylase, and in the vagina is complex. In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as "common goods" that sustain bacterial populations in vaginal communities. Given the temporal changes that are observed in the human vaginal microbiome, we expect the kinds of bacterial amylases produced will also vary over time. These differences influence the pool of resources that are broadly shared and shape the species composition of the vaginal bacterial community.
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http://dx.doi.org/10.1128/mSphere.00943-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729256PMC
December 2020

Characterization of Local and Systemic Impact of Whitefly () Feeding and Whitefly-Transmitted Tomato Mottle Virus Infection on Tomato Leaves by Comprehensive Proteomics.

Int J Mol Sci 2020 Sep 30;21(19). Epub 2020 Sep 30.

Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA.

Tomato mottle virus (ToMoV) is a single-stranded DNA (ssDNA) begomovirus transmitted to solanaceous crops by the whitefly species complex (), causing stunted growth, leaf mottling, and reduced yield. Using a genetic repertoire of seven genes, ToMoV pathogenesis includes the manipulation of multiple plant biological processes to circumvent antiviral defenses. To further understand the effects of whitefly feeding and whitefly-transmitted ToMoV infection on tomato plants ( 'Florida Lanai'), we generated comprehensive protein profiles of leaves subjected to feeding by either viruliferous whiteflies harboring ToMoV, or non-viruliferous whiteflies, or a no-feeding control. The effects of whitefly feeding and ToMoV infection were measured both locally and systemically by sampling either a mature leaf directly from the site of clip-cage confined whitefly feeding, or from a newly formed leaf 10 days post feeding (dpf). At 3 dpf, tomato's response to ToMoV included proteins associated with translation initiation and elongation as well as plasmodesmata dynamics. In contrast, systemic impacts of ToMoV on younger leaves 10 dpf were more pronounced and included a virus-specific change in plant proteins associated with mRNA maturation and export, RNA-dependent DNA methylation, and other antiviral plant processes. Our analysis supports previous findings and provides novel insight into tomato's local and systemic response to whitefly feeding and ToMoV infection.
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http://dx.doi.org/10.3390/ijms21197241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583044PMC
September 2020

Lignin induced iron reduction by novel sp., Tolumonas lignolytic BRL6-1.

PLoS One 2020 17;15(9):e0233823. Epub 2020 Sep 17.

Department of Microbiology, University of Massachusetts-Amherst, Amherst, MA, United States of America.

Lignin is the second most abundant carbon polymer on earth and despite having more fuel value than cellulose, it currently is considered a waste byproduct in many industrial lignocellulose applications. Valorization of lignin relies on effective and green methods of de-lignification, with a growing interest in the use of microbes. Here we investigate the physiology and molecular response of the novel facultative anaerobic bacterium, Tolumonas lignolytica BRL6-1, to lignin under anoxic conditions. Physiological and biochemical changes were compared between cells grown anaerobically in either lignin-amended or unamended conditions. In the presence of lignin, BRL6-1 accumulates higher biomass and has a shorter lag phase compared to unamended conditions, and 14% of the proteins determined to be significantly higher in abundance by log2 fold-change of 2 or greater were related to Fe(II) transport in late logarithmic phase. Ferrozine assays of the supernatant confirmed that Fe(III) was bound to lignin and reduced to Fe(II) only in the presence of BRL6-1, suggesting redox activity by the cells. LC-MS/MS analysis of the secretome showed an extra band at 20 kDa in lignin-amended conditions. Protein sequencing of this band identified a protein of unknown function with homology to enzymes in the radical SAM superfamily. Expression of this protein in lignin-amended conditions suggests its role in radical formation. From our findings, we suggest that BRL6-1 is using a protein in the radical SAM superfamily to interact with the Fe(III) bound to lignin and reducing it to Fe(II) for cellular use, increasing BRL6-1 yield under lignin-amended conditions. This interaction potentially generates organic free radicals and causes a radical cascade which could modify and depolymerize lignin. Further research should clarify the extent to which this mechanism is similar to previously described aerobic chelator-mediated Fenton chemistry or radical producing lignolytic enzymes, such as lignin peroxidases, but under anoxic conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233823PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497984PMC
October 2020

A porcine ligated loop model reveals new insight into the host immune response against .

Gut Microbes 2020 11;12(1):1-25

School of Molecular Biosciences, College of Veterinary Medicine, Washington State University , Pullman, WA, USA.

The symptoms of infectious diarrheal disease are mediated by a combination of a pathogen's virulence factors and the host immune system. is the leading bacterial cause of diarrhea worldwide due to its near-ubiquitous zoonotic association with poultry. One of the outstanding questions is to what extent the bacteria are responsible for the diarrheal symptoms via intestinal cell necrosis versus immune cell initiated tissue damage. To determine the stepwise process of inflammation that leads to diarrhea, we used a piglet ligated intestinal loop model to study the intestinal response to . Pigs were chosen due to the anatomical similarity between the porcine and the human intestine. We found that the abundance of neutrophil related proteins increased in the intestinal lumen during infection, including proteins related to neutrophil migration (neutrophil elastase and MMP9), actin reorganization (Arp2/3), and antimicrobial proteins (lipocalin-2, myeloperoxidase, S100A8, and S100A9). The appearance of neutrophil proteins also corresponded with increases of the inflammatory cytokines IL-8 and TNF-α. Compared to infection with the wild-type strain, infection with the noninvasive mutant resulted in a blunted inflammatory response, with less inflammatory cytokines and neutrophil markers. These findings indicate that intestinal inflammation is driven by virulence and that neutrophils are the predominant cell type responding to infection. We propose that this model can be used as a platform to study the early immune events during infection with intestinal pathogens.
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http://dx.doi.org/10.1080/19490976.2020.1814121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524355PMC
November 2020

Molecular Transducers of Physical Activity Consortium (MoTrPAC): Mapping the Dynamic Responses to Exercise.

Cell 2020 06;181(7):1464-1474

Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Exercise provides a robust physiological stimulus that evokes cross-talk among multiple tissues that when repeated regularly (i.e., training) improves physiological capacity, benefits numerous organ systems, and decreases the risk for premature mortality. However, a gap remains in identifying the detailed molecular signals induced by exercise that benefits health and prevents disease. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to address this gap and generate a molecular map of exercise. Preclinical and clinical studies will examine the systemic effects of endurance and resistance exercise across a range of ages and fitness levels by molecular probing of multiple tissues before and after acute and chronic exercise. From this multi-omic and bioinformatic analysis, a molecular map of exercise will be established. Altogether, MoTrPAC will provide a public database that is expected to enhance our understanding of the health benefits of exercise and to provide insight into how physical activity mitigates disease.
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http://dx.doi.org/10.1016/j.cell.2020.06.004DOI Listing
June 2020

Phloem Exudate Protein Profiles during Drought and Recovery Reveal Abiotic Stress Responses in Tomato Vasculature.

Int J Mol Sci 2020 Jun 23;21(12). Epub 2020 Jun 23.

Plant Biology Graduate Program, The Pennsylvania State University, University Park, PA 16802, USA.

Drought is the leading cause of agricultural yield loss among all abiotic stresses, and the link between water deficit and phloem protein contents is relatively unexplored. Here we collected phloem exudates from leaves during periods of drought stress and recovery. Our analysis identified 2558 proteins, the most abundant of which were previously localized to the phloem. Independent of drought, enrichment analysis of the total phloem exudate protein profiles from all samples suggests that the protein content of phloem sap is complex, and includes proteins that function in chaperone systems, branched-chain amino acid synthesis, trehalose metabolism, and RNA silencing. We observed 169 proteins whose abundance changed significantly within the phloem sap, either during drought or recovery. Proteins that became significantly more abundant during drought include members of lipid metabolism, chaperone-mediated protein folding, carboxylic acid metabolism, abscisic acid signaling, cytokinin biosynthesis, and amino acid metabolism. Conversely, proteins involved in lipid signaling, sphingolipid metabolism, cell wall organization, carbohydrate metabolism, and a mitogen-activated protein kinase are decreased during drought. Our experiment has achieved an in-depth profiling of phloem sap protein contents during drought stress and recovery that supports previous findings and provides new evidence that multiple biological processes are involved in drought adaptation.
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http://dx.doi.org/10.3390/ijms21124461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352395PMC
June 2020

Phage-specific metabolic reprogramming of virocells.

ISME J 2020 04 2;14(4):881-895. Epub 2020 Jan 2.

Department of Ecology and Evolutionary Biology, University of Michigan, 1105 North University Ave, Ann Arbor, MI, 48109, USA.

Ocean viruses are abundant and infect 20-40% of surface microbes. Infected cells, termed virocells, are thus a predominant microbial state. Yet, virocells and their ecosystem impacts are understudied, thus precluding their incorporation into ecosystem models. Here we investigated how unrelated bacterial viruses (phages) reprogram one host into contrasting virocells with different potential ecosystem footprints. We independently infected the marine Pseudoalteromonas bacterium with siphovirus PSA-HS2 and podovirus PSA-HP1. Time-resolved multi-omics unveiled drastically different metabolic reprogramming and resource requirements by each virocell, which were related to phage-host genomic complementarity and viral fitness. Namely, HS2 was more complementary to the host in nucleotides and amino acids, and fitter during infection than HP1. Functionally, HS2 virocells hardly differed from uninfected cells, with minimal host metabolism impacts. HS2 virocells repressed energy-consuming metabolisms, including motility and translation. Contrastingly, HP1 virocells substantially differed from uninfected cells. They repressed host transcription, responded to infection continuously, and drastically reprogrammed resource acquisition, central carbon and energy metabolisms. Ecologically, this work suggests that one cell, infected versus uninfected, can have immensely different metabolisms that affect the ecosystem differently. Finally, we relate phage-host genome complementarity, virocell metabolic reprogramming, and viral fitness in a conceptual model to guide incorporating viruses into ecosystem models.
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http://dx.doi.org/10.1038/s41396-019-0580-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082346PMC
April 2020

Assessment of the Utility of the Oral Fluid and Plasma Proteomes for Hydrocodone Exposure.

J Med Toxicol 2020 01 1;16(1):49-60. Epub 2019 Nov 1.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.

Introduction: Non-medical use and abuse of prescription opioids is a growing problem in both the civilian and military communities, with minimal technologies for detecting hydrocodone use. This study explored the proteomic changes that occur in the oral fluid and blood plasma following controlled hydrocodone administration in 20 subjects.

Methods: The global proteomic profile was determined for samples taken at four time points per subject: pre-exposure and 4, 6, or 168 hours post-exposure. The oral fluid samples analyzed herein provided greater differentiation between baseline and response time points than was observed with blood plasma, at least partially due to significant person-to-person relative variability in the plasma proteome.

Results: A total of 399 proteins were identified from oral fluid samples, and the abundance of 118 of those proteins was determined to be significantly different upon metabolism of hydrocodone (4 and 6 hour time points) as compared to baseline levels in the oral fluid (pre-dose and 168 hours).

Conclusions: We present an assessment of the oral fluid and plasma proteome following hydrocodone administration, which demonstrates the potential of oral fluid as a noninvasive sample that may reveal features of hydrocodone in opioid use, and with additional study, may be useful for other opioids and in settings of misuse.
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http://dx.doi.org/10.1007/s13181-019-00731-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942069PMC
January 2020

Nipah Virus-Like Particle Egress Is Modulated by Cytoskeletal and Vesicular Trafficking Pathways: a Validated Particle Proteomics Analysis.

mSystems 2019 Sep 24;4(5). Epub 2019 Sep 24.

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA

Classified as a biosafety level 4 (BSL4) select agent, Nipah virus (NiV) is a deadly henipavirus in the family, with a nearly 75% mortality rate in humans, underscoring its global and animal health importance. Elucidating the process of viral particle production in host cells is imperative both for targeted drug design and viral particle-based vaccine development. However, little is understood concerning the functions of cellular machinery in paramyxoviral and henipaviral assembly and budding. Recent studies showed evidence for the involvement of multiple NiV proteins in viral particle formation, in contrast to the mechanisms understood for several paramyxoviruses as being reliant on the matrix (M) protein alone. Further, the levels and purposes of cellular factor incorporation into viral particles are largely unexplored for the paramyxoviruses. To better understand the involvement of cellular machinery and the major structural viral fusion (F), attachment (G), and matrix (M) proteins, we performed proteomics analyses on virus-like particles (VLPs) produced from several combinations of these NiV proteins. Our findings indicate that NiV VLPs incorporate vesicular trafficking and actin cytoskeletal factors. The involvement of these biological processes was validated by experiments indicating that the perturbation of key factors in these cellular processes substantially modulated viral particle formation. These effects were most impacted for NiV-F-modulated viral particle formation either autonomously or in combination with other NiV proteins, indicating that NiV-F budding relies heavily on these cellular processes. These findings indicate a significant involvement of the NiV fusion protein, vesicular trafficking, and actin cytoskeletal processes in efficient viral particle formation. Nipah virus is a zoonotic biosafety level 4 agent with high mortality rates in humans. The genus to which Nipah virus belongs, , includes five officially recognized pathogens; however, over 20 species have been identified in multiple continents within the last several years. As there are still no vaccines or treatments for NiV infection, elucidating its process of viral particle production is imperative both for targeted drug design as well as for particle-based vaccine development. Developments in high-throughput technologies make proteomic analysis of isolated viral particles a highly insightful approach to understanding the life cycle of pathogens such as Nipah virus.
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http://dx.doi.org/10.1128/mSystems.00194-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759566PMC
September 2019

Unified feature association networks through integration of transcriptomic and proteomic data.

PLoS Comput Biol 2019 09 17;15(9):e1007241. Epub 2019 Sep 17.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA, United States of America.

High-throughput multi-omics studies and corresponding network analyses of multi-omic data have rapidly expanded their impact over the last 10 years. As biological features of different types (e.g. transcripts, proteins, metabolites) interact within cellular systems, the greatest amount of knowledge can be gained from networks that incorporate multiple types of -omic data. However, biological and technical sources of variation diminish the ability to detect cross-type associations, yielding networks dominated by communities comprised of nodes of the same type. We describe here network building methods that can maximize edges between nodes of different data types leading to integrated networks, networks that have a large number of edges that link nodes of different-omic types (transcripts, proteins, lipids etc). We systematically rank several network inference methods and demonstrate that, in many cases, using a random forest method, GENIE3, produces the most integrated networks. This increase in integration does not come at the cost of accuracy as GENIE3 produces networks of approximately the same quality as the other network inference methods tested here. Using GENIE3, we also infer networks representing antibody-mediated Dengue virus cell invasion and receptor-mediated Dengue virus invasion. A number of functional pathways showed centrality differences between the two networks including genes responding to both GM-CSF and IL-4, which had a higher centrality value in an antibody-mediated vs. receptor-mediated Dengue network. Because a biological system involves the interplay of many different types of molecules, incorporating multiple data types into networks will improve their use as models of biological systems. The methods explored here are some of the first to specifically highlight and address the challenges associated with how such multi-omic networks can be assembled and how the greatest number of interactions can be inferred from different data types. The resulting networks can lead to the discovery of new host response patterns and interactions during viral infection, generate new hypotheses of pathogenic mechanisms and confirm mechanisms of disease.
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http://dx.doi.org/10.1371/journal.pcbi.1007241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748406PMC
September 2019

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform.

Anal Chem 2019 10 25;91(20):13119-13127. Epub 2019 Sep 25.

Environmental Molecular Sciences Laboratory , Pacific Northwest National Laboratory , Richland , Washington 99354 , United States.

Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ∼1 600 proteins with a median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2 300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.
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http://dx.doi.org/10.1021/acs.analchem.9b03349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192326PMC
October 2019

Prediction of bacterial E3 ubiquitin ligase effectors using reduced amino acid peptide fingerprinting.

PeerJ 2019 7;7:e7055. Epub 2019 Jun 7.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, United States of America.

Background: Although pathogenic Gram-negative bacteria lack their own ubiquitination machinery, they have evolved or acquired virulence effectors that can manipulate the host ubiquitination process through structural and/or functional mimicry of host machinery. Many such effectors have been identified in a wide variety of bacterial pathogens that share little sequence similarity amongst themselves or with eukaryotic ubiquitin E3 ligases.

Methods: To allow identification of novel bacterial E3 ubiquitin ligase effectors from protein sequences we have developed a machine learning approach, the SVM-based Identification and Evaluation of Virulence Effector Ubiquitin ligases (SIEVE-Ub). We extend the string kernel approach used previously to sequence classification by introducing reduced amino acid (RED) alphabet encoding for protein sequences.

Results: We found that 14mer peptides with amino acids represented as simply either hydrophobic or hydrophilic provided the best models for discrimination of E3 ligases from other effector proteins with a receiver-operator characteristic area under the curve (AUC) of 0.90. When considering a subset of E3 ubiquitin ligase effectors that do not fall into known sequence based families we found that the AUC was 0.82, demonstrating the effectiveness of our method at identifying novel functional family members. Feature selection was used to identify a parsimonious set of 10 RED peptides that provided good discrimination, and these peptides were found to be located in functionally important regions of the proteins involved in E2 and host target protein binding. Our general approach enables construction of models based on other effector functions. We used SIEVE-Ub to predict nine potential novel E3 ligases from a large set of bacterial genomes. SIEVE-Ub is available for download at https://doi.org/10.6084/m9.figshare.7766984.v1 or https://github.com/biodataganache/SIEVE-Ub for the most current version.
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http://dx.doi.org/10.7717/peerj.7055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557245PMC
June 2019

Demonstrates Conserved Proteomic and Transcriptomic Responses When Co-cultured With Human INT 407 and Caco-2 Epithelial Cells.

Front Microbiol 2019 11;10:755. Epub 2019 Apr 11.

School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, United States.

Major foodborne bacterial pathogens, such as , have devised complex strategies to establish and foster intestinal infections. For more than two decades, researchers have used immortalized cell lines derived from human intestinal tissue to dissect -host cell interactions. Known from these studies is that virulence is multifactorial, requiring a coordinated response to produce virulence factors that facilitate host cell interactions. This study was initiated to identify proteins that contribute to adaptation to the host cell environment and cellular invasion. We demonstrated that responds to INT 407 and Caco-2 cells in a similar fashion at the cellular and molecular levels. Active protein synthesis was found to be required for to maximally invade these host cells. Proteomic and transcriptomic approaches were then used to define the protein and gene expression profiles of co-cultured with cells. By focusing on those genes showing increased expression by when co-cultured with epithelial cells, we discovered that quickly adapts to co-culture with epithelial cells by synthesizing gene products that enable it to acquire specific amino acids for growth, scavenge for inorganic molecules including iron, resist reactive oxygen/nitrogen species, and promote host cell interactions. Based on these findings, we selected a subset of the genes involved in chemotaxis and the regulation of flagellar assembly and generated deletion mutants for phenotypic analysis. Binding and internalization assays revealed significant differences in the interaction of chemotaxis and flagellar regulatory mutants. The identification of genes involved in adaptation to culture with host cells provides new insights into the infection process.
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http://dx.doi.org/10.3389/fmicb.2019.00755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470190PMC
April 2019

Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle.

Nat Microbiol 2019 05 11;4(5):864-875. Epub 2019 Mar 11.

Ecology Department, Earth and Environmental Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet.
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http://dx.doi.org/10.1038/s41564-019-0384-yDOI Listing
May 2019

Rare Earth Elements Alter Redox Balance in 20Z.

Front Microbiol 2018 27;9:2735. Epub 2018 Nov 27.

Biology Department, Viral Information Institute, San Diego State University, San Diego, CA, United States.

Rare Earth Elements (REEs) control methanol utilization in both methane- and methanol-utilizing microbes. It has been established that the addition of REEs leads to the transcriptional repression of MxaFI-MeDH [a two-subunit methanol dehydrogenase (MeDH), calcium-dependent] and the activation of XoxF-MeDH (a one-subunit MeDH, lanthanum-dependent). Both enzymes are pyrroquinoline quinone-dependent alcohol dehydrogenases and show significant homology; however, they display different kinetic properties and substrate specificities. This study investigates the impact of the MxaFI to XoxF switch on the behavior of metabolic networks at a global scale. In this study we investigated the steady-state growth of 20Z in media containing calcium (Ca) or lanthanum (La, a REE element). We found that cells supplemented with La show a higher growth rate compared to Ca-cultures; however, the efficiency of carbon conversion, estimated as biomass yield, is higher in cells grown with Ca. Three complementary global-omics approaches-RNA-seq transcriptomics, proteomics, and metabolomics-were applied to investigate the mechanisms of improved growth vs. carbon conversion. Cells grown with La showed the transcriptional activation of the gene, a homolog of the formaldehyde-activating enzyme (), a putative transporter, genes for hemin-transport proteins, and nitrate reductase. In contrast, genes for and associated cytochrome () expression were downregulated. Proteomic profiling suggested additional adjustments of the metabolic network at the protein level, including carbon assimilation pathways, electron transport systems, and the tricarboxylic acid (TCA) cycle. Discord between gene expression and protein abundance changes points toward the possibility of post-transcriptional control of the related systems including key enzymes of the TCA cycle and a set of electron-transport carriers. Metabolomic data followed proteomics and showed the reduction of the ribulose-monophosphate (RuMP) pathway intermediates and the increase of the TCA cycle metabolites. Cells exposed to REEs display higher rates of growth but have lower carbon conversion efficiency compared to cells supplemented with Ca. The most plausible explanation for these physiological changes is an increased conversion of methanol into formate by XoxF-MeDH, which further stimulates methane oxidation but limits both the supply of reducing power and flux of formaldehyde into the RuMP pathway.
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http://dx.doi.org/10.3389/fmicb.2018.02735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277846PMC
November 2018

New mass spectrometry technologies contributing towards comprehensive and high throughput omics analyses of single cells.

Analyst 2019 Jan;144(3):794-807

Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.

Mass-spectrometry based omics technologies - namely proteomics, metabolomics and lipidomics - have enabled the molecular level systems biology investigation of organisms in unprecedented detail. There has been increasing interest for gaining a thorough, functional understanding of the biological consequences associated with cellular heterogeneity in a wide variety of research areas such as developmental biology, precision medicine, cancer research and microbiome science. Recent advances in mass spectrometry (MS) instrumentation and sample handling strategies are quickly making comprehensive omics analyses of single cells feasible, but key breakthroughs are still required to push through remaining bottlenecks. In this review, we discuss the challenges faced by single cell MS-based omics analyses and highlight recent technological advances that collectively can contribute to comprehensive and high throughput omics analyses in single cells. We provide a vision of the potential of integrating pioneering technologies such as Structures for Lossless Ion Manipulations (SLIM) for improved sensitivity and resolution, novel peptide identification tactics and standards free metabolomics approaches for future applications in single cell analysis.
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http://dx.doi.org/10.1039/c8an01574kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349538PMC
January 2019

Individual Variability of Protein Expression in Human Tissues.

J Proteome Res 2018 11 18;17(11):3914-3922. Epub 2018 Oct 18.

Biological Sciences Division , Pacific Northwest National Laboratory , Richland , Washington 99336 , United States.

Human tissues are known to exhibit interindividual variability, but a deeper understanding of the different factors affecting protein expression is necessary to further apply this knowledge. Our goal was to explore the proteomic variability between individuals as well as between healthy and diseased samples, and to test the efficacy of machine learning classifiers. In order to investigate whether disparate proteomics data sets may be combined, we performed a retrospective analysis of proteomics data from 9 different human tissues. These data sets represent several different sample prep methods, mass spectrometry instruments, and tissue health. Using these data, we examined interindividual and intertissue variability in peptide expression, and analyzed the methods required to build accurate tissue classifiers. We also evaluated the limits of tissue classification by downsampling the peptide data to simulate situations where less data is available, such as clinical biopsies, laser capture microdissection or potentially single-cell proteomics. Our findings reveal the strong potential for utilizing proteomics data to build robust tissue classifiers, which has many prospective clinical applications for evaluating the applicability of model clinical systems.
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http://dx.doi.org/10.1021/acs.jproteome.8b00580DOI Listing
November 2018

Multiple mechanisms drive phage infection efficiency in nearly identical hosts.

ISME J 2018 06 22;12(6):1605-1618. Epub 2018 Mar 22.

Department of Microbiology, The Ohio State University, Columbus, OH, USA.

Phage-host interactions are critical to ecology, evolution, and biotechnology. Central to those is infection efficiency, which remains poorly understood, particularly in nature. Here we apply genome-wide transcriptomics and proteomics to investigate infection efficiency in nature's own experiment: two nearly identical (genetically and physiologically) Bacteroidetes bacterial strains (host18 and host38) that are genetically intractable, but environmentally important, where phage infection efficiency varies. On host18, specialist phage phi18:3 infects efficiently, whereas generalist phi38:1 infects inefficiently. On host38, only phi38:1 infects, and efficiently. Overall, phi18:3 globally repressed host18's transcriptome and proteome, expressed genes that likely evaded host restriction/modification (R/M) defenses and controlled its metabolism, and synchronized phage transcription with translation. In contrast, phi38:1 failed to repress host18's transcriptome and proteome, did not evade host R/M defenses or express genes for metabolism control, did not synchronize transcripts with proteins and its protein abundances were likely targeted by host proteases. However, on host38, phi38:1 globally repressed host transcriptome and proteome, synchronized phage transcription with translation, and infected host38 efficiently. Together these findings reveal multiple infection inefficiencies. While this contrasts the single mechanisms often revealed in laboratory mutant studies, it likely better reflects the phage-host interaction dynamics that occur in nature.
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http://dx.doi.org/10.1038/s41396-018-0099-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955906PMC
June 2018

Salmonella-Mediated Inflammation Eliminates Competitors for Fructose-Asparagine in the Gut.

Infect Immun 2018 05 23;86(5). Epub 2018 Apr 23.

Department of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio, USA

elicits intestinal inflammation to gain access to nutrients. One of these nutrients is fructose-asparagine (F-Asn). The availability of F-Asn to during infection is dependent upon pathogenicity islands 1 and 2, which in turn are required to provoke inflammation. Here, we determined that F-Asn is present in mouse chow at approximately 400 pmol/mg (dry weight). F-Asn is also present in the intestinal tract of germfree mice at 2,700 pmol/mg (dry weight) and in the intestinal tract of conventional mice at 9 to 28 pmol/mg. These findings suggest that the mouse intestinal microbiota consumes F-Asn. We utilized heavy-labeled precursors of F-Asn to monitor its formation in the intestine, in the presence or absence of inflammation, and none was observed. Finally, we determined that some members of the class encode F-Asn utilization pathways and that they are eliminated from highly inflamed -infected mice. Collectively, our studies identify the source of F-Asn as the diet and that -mediated inflammation is required to eliminate competitors and allow the pathogen nearly exclusive access to this nutrient.
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http://dx.doi.org/10.1128/IAI.00945-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913863PMC
May 2018

Comprehensive computational design of ordered peptide macrocycles.

Science 2017 12;358(6369):1461-1466

Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.

Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with l-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of l- and d-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.
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http://dx.doi.org/10.1126/science.aap7577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860875PMC
December 2017

The food-borne pathogen Campylobacter jejuni responds to the bile salt deoxycholate with countermeasures to reactive oxygen species.

Sci Rep 2017 11 13;7(1):15455. Epub 2017 Nov 13.

School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, 99164-7520, USA.

Bile plays an important role in digestion, absorption of fats, and the excretion of waste products, while concurrently providing a critical barrier against colonization by harmful bacteria. Previous studies have demonstrated that gut pathogens react to bile by adapting their protein synthesis. The ability of pathogens to respond to bile is remarkably complex and still incompletely understood. Here we show that Campylobacter jejuni, a leading bacterial cause of human diarrheal illness worldwide, responds to deoxycholate, a component of bile, by altering global gene transcription in a manner consistent with a strategy to mitigate exposure to reactive oxygen stress. More specifically, continuous growth of C. jejuni in deoxycholate was found to: 1) induce the production of reactive oxygen species (ROS); 2) decrease succinate dehydrogenase activity (complex II of the electron transport chain); 3) increase catalase activity that is involved in HO breakdown; and 4) result in DNA strand breaks. Congruently, the addition of 4-hydroxy-TEMPO (TEMPOL), a superoxide dismutase mimic that reacts with superoxide, rescued the growth of C. jejuni cultured in the presence of deoxycholate. We postulate that continuous exposure of a number of enteric pathogens to deoxycholate stimulates a conserved survival response to this stressor.
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http://dx.doi.org/10.1038/s41598-017-15379-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684402PMC
November 2017

Protein abundances can distinguish between naturally-occurring and laboratory strains of Yersinia pestis, the causative agent of plague.

PLoS One 2017 30;12(8):e0183478. Epub 2017 Aug 30.

Chemical and Biological Signature Sciences, Pacific Northwest National Laboratory, Richland, Washington, United States of America.

The rapid pace of bacterial evolution enables organisms to adapt to the laboratory environment with repeated passage and thus diverge from naturally-occurring environmental ("wild") strains. Distinguishing wild and laboratory strains is clearly important for biodefense and bioforensics; however, DNA sequence data alone has thus far not provided a clear signature, perhaps due to lack of understanding of how diverse genome changes lead to convergent phenotypes, difficulty in detecting certain types of mutations, or perhaps because some adaptive modifications are epigenetic. Monitoring protein abundance, a molecular measure of phenotype, can overcome some of these difficulties. We have assembled a collection of Yersinia pestis proteomics datasets from our own published and unpublished work, and from a proteomics data archive, and demonstrated that protein abundance data can clearly distinguish laboratory-adapted from wild. We developed a lasso logistic regression classifier that uses binary (presence/absence) or quantitative protein abundance measures to predict whether a sample is laboratory-adapted or wild that proved to be ~98% accurate, as judged by replicated 10-fold cross-validation. Protein features selected by the classifier accord well with our previous study of laboratory adaptation in Y. pestis. The input data was derived from a variety of unrelated experiments and contained significant confounding variables. We show that the classifier is robust with respect to these variables. The methodology is able to discover signatures for laboratory facility and culture medium that are largely independent of the signature of laboratory adaptation. Going beyond our previous laboratory evolution study, this work suggests that proteomic differences between laboratory-adapted and wild Y. pestis are general, potentially pointing to a process that could apply to other species as well. Additionally, we show that proteomics datasets (even archived data collected for different purposes) contain the information necessary to distinguish wild and laboratory samples. This work has clear applications in biomarker detection as well as biodefense.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576697PMC
October 2017

Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi.

Protein Sci 2017 Sep 19;26(9):1738-1748. Epub 2017 Jun 19.

Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue, Chicago, Illinois, 60637.

Salmonella enterica serovar Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded β-barrel transmembrane domain and a C-terminal domain (OmpA ). The OmpA and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpA from two pathogens: S. typhimurium (STOmpA ) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpA ), in closed form. In the open form of STOmpA , an aspartate residue from a long β2-α3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpA and in the structure of BbOmpA , a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpA , suggest a large conformational change that includes an extension of α3 helix by ordering a part of β2-α3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpA suggesting PG-anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpA , or possibly that of full length STOmpA.
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http://dx.doi.org/10.1002/pro.3209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563150PMC
September 2017

Redeploying β-Lactam Antibiotics as a Novel Antivirulence Strategy for the Treatment of Methicillin-Resistant Staphylococcus aureus Infections.

J Infect Dis 2017 Jan 14;215(1):80-87. Epub 2016 Nov 14.

Department of Microbiology, School of Natural Sciences.

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that β-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner.
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http://dx.doi.org/10.1093/infdis/jiw461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225249PMC
January 2017

Identification of Novel Host Interactors of Effectors Secreted by and .

mSystems 2016 Jul-Aug;1(4). Epub 2016 Jul 12.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

Many pathogenic bacteria of the family use type III secretion systems to inject virulence proteins, termed "effectors," into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the intracellular pathogens serovar Typhimurium and . We identified 54 high-confidence host interactors for the effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of and effectors, which will help elucidate their mechanisms of action.
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http://dx.doi.org/10.1128/mSystems.00032-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5069955PMC
July 2016

Persister formation in Staphylococcus aureus is associated with ATP depletion.

Nat Microbiol 2016 Apr 18;1:16051. Epub 2016 Apr 18.

Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.

Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics(1). Persisters are associated with chronic infections and antibiotic treatment failure(1-3). In Escherichia coli, toxin-antitoxin modules have been linked to persister formation(4-6). The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting toxin-antitoxin modules in S. aureus did not affect the level of persisters. Here, we show that S. aureus persisters are produced due to a stochastic entrance into the stationary phase accompanied by a drop in intracellular adenosine triphosphate. Cells expressing stationary-state markers are present throughout the growth phase, and increase in frequency with cell density. Cell sorting revealed that the expression of stationary markers is associated with a 100-1,000-fold increase in the likelihood of survival to antibiotic challenge. The adenosine triphosphate level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.
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http://dx.doi.org/10.1038/nmicrobiol.2016.51DOI Listing
April 2016
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