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Functional Properties of Helicobacter pylori VacA Toxin m1 and m2 Variants.
Caston RR, Sierra JC, Foegeding NJ, Truelock MD, Campbell AM, Frick-Cheng AE, Bimczok D, Wilson KT, McClain MS, Cover TL
(2020) Infect Immun 88:
MeSH Terms: Amino Acid Sequence, Bacterial Proteins, Bacterial Toxins, Gene Expression Regulation, Bacterial, Genetic Variation, Helicobacter Infections, Helicobacter pylori, Humans, Protein Domains, Protein Multimerization, Protein Transport, Vacuoles
Show Abstract · Added April 15, 2020
colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).
Copyright © 2020 American Society for Microbiology.
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12 MeSH Terms
The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation.
Lopez CA, Beavers WN, Weiss A, Knippel RJ, Zackular JP, Chazin W, Skaar EP
(2019) mBio 10:
MeSH Terms: Clostridioides difficile, Clostridium Infections, Energy Metabolism, Fermentation, Gene Expression Regulation, Bacterial, Leukocyte L1 Antigen Complex, Proline, Zinc
Show Abstract · Added March 11, 2020
The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on colonization is unknown. To define responses to Zn limitation, we performed RNA sequencing on exposed to CP. In medium containing CP, upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select transcripts in a mouse CDI model relative to expression Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts proline fermentation. Overall, these results highlight how colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response. infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of genes necessary for proline fermentation. Paradoxically, this leads to reduced proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce fitness in the intestines. These results emphasize the complexities of how nutrient availability influences colonization and provide insight into critical metabolic processes that drive the pathogen's growth.
Copyright © 2019 Lopez et al.
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8 MeSH Terms
Effect of environmental salt concentration on the Helicobacter pylori exoproteome.
Caston RR, Loh JT, Voss BJ, McDonald WH, Scholz MB, McClain MS, Cover TL
(2019) J Proteomics 202: 103374
MeSH Terms: Bacterial Proteins, Dose-Response Relationship, Drug, Gene Expression Regulation, Bacterial, Helicobacter pylori, Proteome, Proteomics, Sodium Chloride, Dietary
Show Abstract · Added July 14, 2019
Helicobacter pylori infection and a high salt diet are each risk factors for gastric cancer. In this study, we tested the hypothesis that environmental salt concentration influences the composition of the H. pylori exoproteome. H. pylori was cultured in media containing varying concentrations of sodium chloride, and aliquots were fractionated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified proteins that were selectively released into the extracellular space, and we identified selectively released proteins that were differentially abundant in culture supernatants, depending on the environmental salt concentration. We also used RNA-seq analysis to identify genes that were differentially expressed in response to environmental salt concentration. The salt-responsive proteins identified by proteomic analysis and salt-responsive genes identified by RNA-seq analysis were mostly non-concordant, but the secreted toxin VacA was salt-responsive in both analyses. Western blot analysis confirmed that VacA levels in the culture supernatant were increased in response to high salt conditions, and quantitative RT-qPCR experiments confirmed that vacA transcription was upregulated in response to high salt conditions. These results indicate that environmental salt concentration influences the composition of the H. pylori exoproteome, which could contribute to the increased risk of gastric cancer associated with a high salt diet. SIGNIFICANCE: Helicobacter pylori-induced alterations in the gastric mucosa have been attributed, at least in part, to the actions of secreted H. pylori proteins. In this study, we show that H. pylori growth in high salt concentrations leads to increased levels of a secreted VacA toxin. Salt-induced alterations in the composition of the H. pylori exoproteome is relevant to the increased risk of gastric cancer associated with consumption of a high salt diet.
Published by Elsevier B.V.
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7 MeSH Terms
Manganese Detoxification by MntE Is Critical for Resistance to Oxidative Stress and Virulence of .
Grunenwald CM, Choby JE, Juttukonda LJ, Beavers WN, Weiss A, Torres VJ, Skaar EP
(2019) mBio 10:
MeSH Terms: Animals, Cation Transport Proteins, Disease Models, Animal, Gene Expression Regulation, Bacterial, Homeostasis, Iron, Manganese, Mice, Inbred BALB C, Microbial Viability, Oxidative Stress, Staphylococcal Infections, Staphylococcus aureus, Transcription Factors, Transcription, Genetic, Virulence
Show Abstract · Added April 2, 2019
Manganese (Mn) is an essential micronutrient critical for the pathogenesis of , a significant cause of human morbidity and mortality. Paradoxically, excess Mn is toxic; therefore, maintenance of intracellular Mn homeostasis is required for survival. Here we describe a Mn exporter in , MntE, which is a member of the cation diffusion facilitator (CDF) protein family and conserved among Gram-positive pathogens. Upregulation of transcription in response to excess Mn is dependent on the presence of MntR, a transcriptional repressor of the Mn uptake system. Inactivation of or leads to reduced growth in media supplemented with Mn, demonstrating MntE is required for detoxification of excess Mn. Inactivation of results in elevated levels of intracellular Mn, but reduced intracellular iron (Fe) levels, supporting the hypothesis that MntE functions as a Mn efflux pump and Mn efflux influences Fe homeostasis. Strains inactivated for are more sensitive to the oxidants NaOCl and paraquat, indicating Mn homeostasis is critical for resisting oxidative stress. Furthermore, and are required for full virulence of during infection, suggesting experiences Mn toxicity Combined, these data support a model in which MntR controls Mn homeostasis by balancing transcriptional repression of and induction of , both of which are critical for pathogenesis. Thus, Mn efflux contributes to bacterial survival and virulence during infection, establishing MntE as a potential antimicrobial target and expanding our understanding of Mn homeostasis. Manganese (Mn) is generally viewed as a critical nutrient that is beneficial to pathogenic bacteria due to its function as an enzymatic cofactor and its capability of acting as an antioxidant; yet paradoxically, high concentrations of this transition metal can be toxic. In this work, we demonstrate utilizes the cation diffusion facilitator (CDF) family protein MntE to alleviate Mn toxicity through efflux of excess Mn. Inactivation of leads to a significant reduction in resistance to oxidative stress and mediated mortality within a mouse model of systemic infection. These results highlight the importance of MntE-mediated Mn detoxification in intracellular Mn homeostasis, resistance to oxidative stress, and virulence. Therefore, this establishes MntE as a potential target for development of anti- therapeutics.
Copyright © 2019 Grunenwald et al.
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15 MeSH Terms
OxyR Regulates the Transcriptional Response to Hydrogen Peroxide.
Juttukonda LJ, Green ER, Lonergan ZR, Heffern MC, Chang CJ, Skaar EP
(2019) Infect Immun 87:
MeSH Terms: Acinetobacter Infections, Acinetobacter baumannii, Animals, Anti-Infective Agents, Local, Gene Expression Regulation, Bacterial, Hydrogen Peroxide, Mice, Oxidants, Repressor Proteins, Stress, Physiological
Show Abstract · Added April 7, 2019
is a Gram-negative opportunistic pathogen that causes diverse infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and acquired antimicrobial-resistance mechanisms, isolates are commonly multidrug resistant, and infections are notoriously difficult to treat. The World Health Organization recently highlighted carbapenem-resistant as a "critical priority" for the development of new antimicrobials because of the risk to human health posed by this organism. Therefore, it is important to discover the mechanisms used by to survive stresses encountered during infection in order to identify new drug targets. In this study, by use of imaging, we identified hydrogen peroxide (HO) as a stressor produced in the lung during infection and defined OxyR as a transcriptional regulator of the HO stress response. Upon exposure to HO, differentially transcribes several hundred genes. However, the transcriptional upregulation of genes predicted to detoxify hydrogen peroxide is abolished in an strain in which the transcriptional regulator is genetically inactivated. Moreover, inactivation of in both antimicrobial-susceptible and multidrug-resistant strains impairs growth in the presence of HO OxyR is a direct regulator of and , which encode the major HO-degrading enzymes in , as confirmed through measurement of promoter binding by recombinant OxyR in electromobility shift assays. Finally, an mutant is less fit than wild-type during infection of the murine lung. This work reveals a mechanism used by this important human pathogen to survive HO stress encountered during infection.
Copyright © 2018 American Society for Microbiology.
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10 MeSH Terms
HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis.
Choby JE, Grunenwald CM, Celis AI, Gerdes SY, DuBois JL, Skaar EP
(2018) mBio 9:
MeSH Terms: Aldehyde Oxidoreductases, Bacterial Proteins, Gene Deletion, Gene Expression, Gene Expression Regulation, Bacterial, Heme, Methyltransferases, Staphylococcus aureus
Show Abstract · Added March 15, 2018
is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme homeostasis. In this work, we describe a posttranscriptional regulatory strategy in heme biosynthesis. The first committed enzyme in the heme biosynthetic pathway, glutamyl-tRNA reductase (GtrR), is regulated by heme abundance and the integral membrane protein HemX. GtrR abundance increases dramatically in response to heme deficiency, suggesting a mechanism by which responds to the need to increase heme synthesis. Additionally, HemX is required to maintain low levels of GtrR in heme-proficient cells, and inactivation of leads to increased heme synthesis. Excess heme synthesis in a Δ mutant activates the staphylococcal heme stress response, suggesting that regulation of heme synthesis is critical to reduce self-imposed heme toxicity. Analysis of diverse organisms indicates that HemX is widely conserved among heme-synthesizing bacteria, suggesting that HemX is a common factor involved in the regulation of GtrR abundance. Together, this work demonstrates that regulates heme synthesis by modulating GtrR abundance in response to heme deficiency and through the activity of the broadly conserved HemX. is a leading cause of skin and soft tissue infections, endocarditis, bacteremia, and osteomyelitis, making it a critical health care concern. Development of new antimicrobials against requires knowledge of the physiology that supports this organism's pathogenesis. One component of staphylococcal physiology that contributes to growth and virulence is heme. Heme is a widely utilized cofactor that enables diverse chemical reactions across many enzyme families. relies on many critical heme-dependent proteins and is sensitive to excess heme toxicity, suggesting must maintain proper intracellular heme homeostasis. Because provides heme for heme-dependent enzymes via synthesis from common precursors, we hypothesized that regulation of heme synthesis is one mechanism to maintain heme homeostasis. In this study, we identify that posttranscriptionally regulates heme synthesis by restraining abundance of the first heme biosynthetic enzyme, GtrR, via heme and the broadly conserved membrane protein HemX.
Copyright © 2018 Choby et al.
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8 MeSH Terms
Fur regulation of Staphylococcus aureus heme oxygenases is required for heme homeostasis.
Lojek LJ, Farrand AJ, Weiss A, Skaar EP
(2018) Int J Med Microbiol 308: 582-589
MeSH Terms: Aerobiosis, Bacterial Proteins, Gene Expression Regulation, Bacterial, Heme, Heme Oxygenase (Decyclizing), Homeostasis, Iron, Mixed Function Oxygenases, Oxygenases, Repressor Proteins, Staphylococcus aureus
Show Abstract · Added March 15, 2018
Heme is a cofactor that is essential for cellular respiration and for the function of many enzymes. If heme levels become too low within the cell, S. aureus switches from producing energy via respiration to producing energy by fermentation. S. aureus encodes two heme oxygenases, IsdI and IsdG, which cleave the porphyrin heme ring releasing iron for use as a nutrient source. Both isdI and isdG are only expressed under low iron conditions and are regulated by the canonical Ferric Uptake Regulator (Fur). Here we demonstrate that unregulated expression of isdI and isdG within S. aureus leads to reduced growth under low iron conditions. Additionally, the constitutive expression of these enzymes leads to decreased heme abundance in S. aureus, an increase in the fermentation product lactate, and increased resistance to gentamicin. This work demonstrates that S. aureus has developed tuning mechanisms, such as Fur regulation, to ensure that the cell has sufficient quantities of heme for efficient ATP production through aerobic respiration.
Copyright © 2018 Elsevier GmbH. All rights reserved.
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11 MeSH Terms
High-Salt Conditions Alter Transcription of Helicobacter pylori Genes Encoding Outer Membrane Proteins.
Loh JT, Beckett AC, Scholz MB, Cover TL
(2018) Infect Immun 86:
MeSH Terms: Bacterial Outer Membrane Proteins, Gene Expression Regulation, Bacterial, Helicobacter Infections, Helicobacter pylori, Humans, Operon, Sodium Chloride, Transcription, Genetic, Up-Regulation
Show Abstract · Added July 29, 2018
infection and high dietary salt intake are risk factors for the development of gastric adenocarcinoma. One possible mechanism by which a high-salt diet could influence gastric cancer risk is by modulating gene expression. In this study, we utilized transcriptome sequencing (RNA-seq) methodology to compare the transcriptional profiles of grown in media containing different concentrations of sodium chloride. We identified 118 differentially expressed genes (65 upregulated and 53 downregulated in response to high-salt conditions), including multiple members of 14 operons. Twenty-nine of the differentially expressed genes encode proteins previously shown to undergo salt-responsive changes in abundance, based on proteomic analyses. Real-time reverse transcription (RT)-PCR analyses validated differential expression of multiple genes encoding outer membrane proteins, including adhesins (SabA and HopQ) and proteins involved in iron acquisition (FecA2 and FecA3). Transcript levels of , , and are increased under high-salt conditions, whereas transcript levels of and are decreased under high-salt conditions. Transcription of , , , and is derepressed in an mutant strain, but salt-responsive transcription of these genes is not mediated by the ArsRS two-component system, and the CrdRS and FlgRS two-component systems do not have any detectable effects on transcription of these genes. In summary, these data provide a comprehensive view of transcriptional alterations that occur in response to high-salt environmental conditions.
Copyright © 2018 American Society for Microbiology.
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9 MeSH Terms
Hydrogen Sulfide and Reactive Sulfur Species Impact Proteome S-Sulfhydration and Global Virulence Regulation in Staphylococcus aureus.
Peng H, Zhang Y, Palmer LD, Kehl-Fie TE, Skaar EP, Trinidad JC, Giedroc DP
(2017) ACS Infect Dis 3: 744-755
MeSH Terms: Gene Expression Regulation, Bacterial, Hydrogen Sulfide, Proteome, Staphylococcus aureus, Sulfur, Virulence
Show Abstract · Added September 23, 2017
Hydrogen sulfide (HS) is thought to protect bacteria from oxidative stress, but a comprehensive understanding of its function in bacteria is largely unexplored. In this study, we show that the human pathogen Staphylococcus aureus (S. aureus) harbors significant effector molecules of HS signaling, reactive sulfur species (RSS), as low molecular weight persulfides of bacillithiol, coenzyme A, and cysteine, and significant inorganic polysulfide species. We find that proteome S-sulfhydration, a post-translational modification (PTM) in HS signaling, is widespread in S. aureus. RSS levels modulate the expression of secreted virulence factors and the cytotoxicity of the secretome, consistent with an S-sulfhydration-dependent inhibition of DNA binding by MgrA, a global virulence regulator. Two previously uncharacterized thioredoxin-like proteins, denoted TrxP and TrxQ, are S-sulfhydrated in sulfide-stressed cells and are capable of reducing protein hydrodisulfides, suggesting that this PTM is potentially regulatory in S. aureus. In conclusion, our results reveal that S. aureus harbors a pool of proteome- and metabolite-derived RSS capable of impacting protein activities and gene regulation and that HS signaling can be sensed by global regulators to affect the expression of virulence factors.
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6 MeSH Terms
Structure of a DNA glycosylase that unhooks interstrand cross-links.
Mullins EA, Warren GM, Bradley NP, Eichman BF
(2017) Proc Natl Acad Sci U S A 114: 4400-4405
MeSH Terms: Anti-Bacterial Agents, Bacterial Proteins, DNA Glycosylases, DNA, Bacterial, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Intercellular Signaling Peptides and Proteins, Models, Molecular, Mutation, Naphthalenes, Peptides, Protein Binding, Protein Conformation, Protein Folding, Streptomyces
Show Abstract · Added August 26, 2019
DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.
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15 MeSH Terms