scholarly journals Enterococcal PrgU mitigates PrgB overexpression toxicity by binding to intergenic RNA downstream of the PQ promoter

2020 ◽  
Author(s):  
Lena Lassinantti ◽  
Martha I Camacho ◽  
Rebecca J B Erickson ◽  
Julia L E Willett ◽  
Nicholas R. De Lay ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Rna Structure ◽  
Rna Binding ◽  
Fine Tuning ◽  
Conjugative Plasmid ◽  
Regulatory Control ◽  
Sequence Specificity ◽  
Secretion Systems

AbstractEfficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the sex pheromone cCF10, which induces the expression of the Type 4 Secretion System (T4SS) genes controlled by the PQ promoter. The pheromone responsive PQ promoter is strictly regulated to prevent overproduction of the prgQ operon, which contains the T4SS, and to limit the cell toxicity caused by overproduction of PrgB, a T4SS adhesin involved in cellular aggregation. PrgU plays an important role in regulating this toxicity by decreasing PrgB production. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. With a combination of lacZ reporter fusion, northern blot, and RNAseq analyses, we provide evidence that PrgU binds a specific RNA sequence within the intergenic region (IGR), ca 400 bp downstream of the PQ promoter. PrgU-IGR binding reduces levels of downstream transcripts, with the strongest decrease seen for prgB messages. Consistent with these findings, we determined that pCF10-carrying cells expressing prgU decreased transcript levels more rapidly than isogenic cells deleted of prgU. Finally, purified PrgU bound RNA in vitro, but without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors to bind its RNA target in vivo. Together, our results support a working model where PrgU binding to the IGR serves to recruit RNase(s) for targeted degradation of downstream transcripts.ImportanceBacteria utilize Type 4 Secretion Systems (T4SS) to efficiently transfer DNA from donor to recipient cells, thereby spreading genes encoding for antibiotic resistance as well as various virulence factors. The conjugative plasmid pCF10 from Enterococcus faecalis, originally isolated from clinical isolates, serves as a model system for these processes in Gram-positive bacteria. It is very important to strictly regulate the expression of the T4SS proteins for the bacteria, as some of these proteins are highly toxic to the cell. Here, we identify the mechanism by which PrgU performs its delicate fine tuning of the expression levels. As prgU genes are present in various conjugative plasmids and transposons, this provides an important new insight into the bacterial repertoire of regulation mechanisms of these clinically important systems.

scholarly journals Antibodies to a Surface-Exposed, N-terminal Domain of Aggregation Substance Are Not Protective in the Rabbit Model of Enterococcus faecalis Infective Endocarditis

2001 ◽  
Vol 69 (5) ◽  
pp. 3305-3314 ◽  
Author(s):  
John K. McCormick ◽  
Helmut Hirt ◽  
Christopher M. Waters ◽  
Timothy J. Tripp ◽  
Gary M. Dunny ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Enterococcus Faecalis ◽  
Rabbit Model ◽  
Surface Protein ◽  
Conjugative Plasmid ◽  
Aggregation Substance ◽  
Exposed Region ◽  
Correct Sequence

ABSTRACT The aggregation substance (AS) surface protein fromEnterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS44–331) was cloned with a C-terminal histidine tag translational fusion and expressed fromEscherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS44–331 reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS44–331 bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS44–331were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS44–331 were challenged intravenously withE. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due toE. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

scholarly journals Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly

2017 ◽  
Author(s):  
Kimberly A. Stanek ◽  
Cameron Mura
Keyword(s):  
Molecular Weight ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Data Bank ◽  
Sequence Specificity ◽  
Sm Proteins ◽  
Vapor Diffusion

AbstractHfq is a bacterial RNA-binding protein that plays key roles in the post–transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA⋯RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways—including stress response, quorum sensing and biofilm formation— has motivated genetic and ‘RNAomic’ studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq⋯RNA interactions (in vitro). Indeed, crystallographic and bio-physical studies first established Hfq as a member of the phylogenetically-conserved Sm superfamily. Crystallography and other biophysical methodologies enable the RNA-binding properties of Hfq to be elucidated in atomic detail, but such approaches have stringent sample requirements, viz.: reconstituting and characterizing an Hfq•RNA complex requires ample quantities of well-behaved (sufficient purity, homogeneity) specimens of Hfq and RNA (sRNA, mRNA fragments, short oligoribonucleotides, or even single nucleotides). The production of such materials is covered in this Chapter, with a particular focus on recombinant Hfq proteins for crystallization experiments.Abbreviations3Dthree-dimensionalAUasymmetric unitCVcolumn volumeDEPCdiethyl pyrocarbonateHDVhepatitis δ virusHDVDhanging-drop vapor diffusionIMACimmobilized metal affinity chromatographyMWmolecular weightMWCOmolecular weight cut-offntnucleotidePDBProtein Data BankRNPribonucleoproteinRTroom temperatureSDVDsitting-drop vapor diffusionJournal formatMethods in Molecular Biology (Springer Protocols series); this volume is entitled “Bacterial Regulatory RNA: Methods and Protocols”; an author guide is linked at http://www.springer.com/series/7651

scholarly journals RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein

2021 ◽  
Vol 22 (16) ◽  
pp. 9103
Author(s):  
Julita Gumna ◽  
Angelika Andrzejewska-Romanowska ◽  
David J. Garfinkel ◽  
Katarzyna Pachulska-Wieczorek
Keyword(s):  
Rna Structure ◽  
Rna Binding ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Mobility Shift ◽  
Gag Protein ◽  
Rna Dimerization ◽  
Electrophoretic Mobility Shift Assays

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

scholarly journals A Deep Learning Approach for Learning Intrinsic Protein-RNA Binding Preferences

2018 ◽  
Author(s):  
Ilan Ben-Bassat ◽  
Benny Chor ◽  
Yaron Orenstein
Keyword(s):  
Neural Network ◽  
Rna Structure ◽  
Rna Binding ◽  
Gene Expression Regulation ◽  
Supplementary Information ◽  
Joint Analysis ◽  
Binding Prediction ◽  
Binding Models

AbstractMotivationThe complexes formed by binding of proteins to RNAs play key roles in many biological processes, such as splicing, gene expression regulation, translation, and viral replication. Understanding protein-RNA binding may thus provide important insights to the functionality and dynamics of many cellular processes. This has sparked substantial interest in exploring protein-RNA binding experimentally, and predicting it computationally. The key computational challenge is to efficiently and accurately infer RNA-binding models that will enable prediction of novel protein-RNA interactions to additional transcripts of interest.ResultsWe developed DLPRB, a new deep neural network (DNN) approach for learning protein-RNA binding preferences and predicting novel interactions. We present two different network architectures: a convolutional neural network (CNN), and a recurrent neural network (RNN). The novelty of our network hinges upon two key aspects: (i) the joint analysis of both RNA sequence and structure, which is represented as a probability vector of different RNA structural contexts; (ii) novel features in the architecture of the networks, such as the application of RNNs to RNA-binding prediction, and the combination of hundreds of variable-length filters in the CNN. Our results in inferring accurate RNA-binding models from high-throughput in vitro data exhibit substantial improvements, compared to all previous approaches for protein-RNA binding prediction (both DNN and non-DNN based). A highly significant improvement is achieved for in vitro binding prediction, and a more modest, yet statistically significant,improvement for in vivo binding prediction. When incorporating experimentally-measured RNA structure compared to predicted one, the improvement on in vivo data increases. By visualizing the binding specificities, we can gain novel biological insights underlying the mechanism of protein RNA-binding.AvailabilityThe source code is publicly available at https://github.com/ilanbb/[email protected] informationSupplementary data are available at Bioinformatics online.

scholarly journals Enterococcus faecalis Sex Pheromone cCF10 Enhances Conjugative Plasmid Transfer In Vivo

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Helmut Hirt ◽  
Kerryl E. Greenwood-Quaintance ◽  
Melissa J. Karau ◽  
Lisa M. Till ◽  
Purna C. Kashyap ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
High Frequency ◽  
Intestinal Tract ◽  
Bacteriocin Production ◽  
Plasmid Transfer ◽  
Conjugative Plasmid ◽  
Conjugative Plasmid Transfer ◽  
Pheromone Signaling

ABSTRACT Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis, an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency plasmid transfer of E. faecalis plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.

scholarly journals An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

2014 ◽  
Vol 14 (2) ◽  
pp. 149-157 ◽  
Author(s):  
Natalie M. McAdams ◽  
Michelle L. Ammerman ◽  
Julee Nanduri ◽  
Kaylen Lott ◽  
John C. Fisk ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Mitochondrial Rna ◽  
Insect Vector ◽  
Sequence Specificity ◽  
Content Type

ABSTRACT In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo , inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei . TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

scholarly journals Direct Evidence for Control of the Pheromone-Inducible prgQ Operon of Enterococcus faecalis Plasmid pCF10 by a Countertranscript-Driven Attenuation Mechanism

2010 ◽  
Vol 192 (6) ◽  
pp. 1634-1642 ◽  
Author(s):  
Christopher M. Johnson ◽  
Dawn A. Manias ◽  
Heather A. H. Haemig ◽  
Sonia Shokeen ◽  
Keith E. Weaver ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Direct Evidence ◽  
Conjugative Plasmid ◽  
Receptor Protein ◽  
Genetic Studies ◽  
Regulatory Circuits ◽  
Attenuation Mechanism ◽  
Q Function

ABSTRACT The mating response of Enterococcus faecalis cells carrying the conjugative plasmid pCF10 is controlled by multiple regulatory circuits. Initiation of transcription of the prgQ conjugation operon is controlled by the peptide receptor protein PrgX; binding of the pheromone peptide cCF10 to PrgX abolishes PrgX repression, while binding of the inhibitor peptide iCF10 enhances repression. The results of molecular analysis of prgQ transcripts and genetic studies suggested that the elongation of prgQ transcripts past a putative terminator (IRS1) may be controlled by the interaction of nascent prgQ mRNAs with a small antisense RNA (Anti-Q) encoded within prgQ. Direct evidence for interaction of these RNAs, as well as the resulting effects on readthrough of prgQ transcription, has been limited. Here we report the results of experiments that (i) determine the inherent termination properties of prgQ transcripts in the absence of Anti-Q; (ii) determine the direct effects of the interaction of Anti-Q with nascent prgQ transcripts in the absence of complicating effects of the PrgX protein; and (iii) begin to dissect the structural components involved in these interactions. The results confirm the existence of alternative terminating and antiterminating forms of nascent prgQ transcripts in vivo and demonstrate that the interaction of Anti-Q with these transcripts leads to termination via inhibition of antiterminator formation. In vitro transcription assays support the major results of the in vivo studies. The data support a model for Anti-Q function suggested from recent studies of these RNAs and their interactions in vitro (S. Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication).

scholarly journals Enterococcus faecalis exploits the human fibrinolytic system to drive excess collagenolysis: implications in gut healing and identification of druggable targets

2020 ◽  
Vol 318 (1) ◽  
pp. G1-G9 ◽  
Author(s):  
Richard A. Jacobson ◽  
Kiedo Wienholts ◽  
Ashley J. Williamson ◽  
Sara Gaines ◽  
Sanjiv Hyoju ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Healing Process ◽  
Abdominal Sepsis ◽  
Infectious Complications ◽  
Fibrinolytic System ◽  
Clinical Safety ◽  
High Concentrations ◽  
Clinically Significant

Perforations, anastomotic leak, and subsequent intra-abdominal sepsis are among the most common and feared complications of invasive interventions in the colon and remaining intestinal tract. During physiological healing, tissue protease activity is finely orchestrated to maintain the strength and integrity of the submucosa collagen layer in the wound. We (Shogan, BD et al. Sci Trans Med 7: 286ra68, 2015.) have previously demonstrated in both mice and humans that the commensal microbe Enterococcus faecalis selectively colonizes wounded colonic tissues and disrupts the healing process by amplifying collagenolytic matrix-metalloprotease activity toward excessive degradation. Here, we demonstrate for the first time, to our knowledge, a novel collagenolytic virulence mechanism by which E. faecalis is able to bind and locally activate the human fibrinolytic protease plasminogen (PLG), a protein present in high concentrations in healing colonic tissue. E. faecalis-mediated PLG activation leads to supraphysiological collagen degradation; in this study, we demonstrate this concept both in vitro and in vivo. This pathoadaptive response can be mitigated with the PLG inhibitor tranexamic acid (TXA) in a fashion that prevents clinically significant complications in validated murine models of both E. faecalis- and Pseudomonas aeruginosa-mediated colonic perforation. TXA has a proven clinical safety record and is Food and Drug Administration approved for topical application in invasive procedures, albeit for the prevention of bleeding rather than infection. As such, the novel pharmacological effect described in this study may be translatable to clinical trials for the prevention of infectious complications in colonic healing. NEW & NOTEWORTHY This paper presents a novel mechanism for virulence in a commensal gut microbe that exploits the human fibrinolytic system and its principle protease, plasminogen. This mechanism is targetable by safe and effective nonantibiotic small molecules for the prevention of infectious complications in the healing gut.

scholarly journals 99mTc Labelling Strategies for the Development of Potential Nitroimidazolic Hypoxia Imaging Agents

Inorganics ◽  
2019 ◽  
Vol 7 (11) ◽  
pp. 128 ◽  
Author(s):  
Giglio ◽  
Rey
Keyword(s):  
Rational Design ◽  
Biological Properties ◽  
Fine Tuning ◽  
Oxidation States ◽  
Hypoxia Imaging ◽  
Biological Target ◽  
99Mtc Radiopharmaceuticals ◽  
99Mtc Labelling

Technetium-99m has a rich coordination chemistry that offers many possibilities in terms of oxidation states and donor atom sets. Modifications in the structure of the technetium complexes could be very useful for fine tuning the physicochemical and biological properties of potential 99mTc radiopharmaceuticals. However, systematic study of the influence of the labelling strategy on the “in vitro” and “in vivo” behaviour is necessary for a rational design of radiopharmaceuticals. Herein we present a review of the influence of the Tc complexes’ molecular structure on the biodistribution and the interaction with the biological target of potential nitroimidazolic hypoxia imaging radiopharmaceuticals presented in the literature from 2010 to the present. Comparison with the gold standard [18F]Fluoromisonidazole (FMISO) is also presented.

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

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