scholarly journals Complete Structure of the Enterococcal Polysaccharide Antigen (EPA) of Vancomycin-Resistant Enterococcus faecalis V583 Reveals that EPA Decorations Are Teichoic Acids Covalently Linked to a Rhamnopolysaccharide Backbone

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Yann Guerardel ◽  
Irina Sadovskaya ◽  
Emmanuel Maes ◽  
Sylviane Furlan ◽  
Marie-Pierre Chapot-Chartier ◽  
...  
Keyword(s):  
Biological Activity ◽  
Cell Growth ◽  
Enterococcus Faecalis ◽  
Virulence Factor ◽  
Critical Role ◽  
Complete Structure ◽  
Host Colonization ◽  
Content Type ◽  
Teichoic Acids ◽  
Surface Polysaccharide

ABSTRACT All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the “decorations” of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex Enterococcus faecalis V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-N-acetylglucosamine (β-GlcNAc). The so-called “EPA decorations” consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor. IMPORTANCE Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (enterococcal polysaccharide antigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called “EPA decorations” essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.

scholarly journals EfaR Is a Major Regulator of Enterococcus faecalis Manganese Transporters and Influences Processes Involved in Host Colonization and Infection

2013 ◽  
Vol 81 (3) ◽  
pp. 935-944 ◽  
Author(s):  
M. C. Abrantes ◽  
J. Kok ◽  
M. de F. Lopes
Keyword(s):  
Oxidative Stress ◽  
Metal Ions ◽  
Enterococcus Faecalis ◽  
Bacterial Endocarditis ◽  
Nosocomial Pathogen ◽  
Host Colonization ◽  
Content Type ◽  
Bacterial Pathogenicity

ABSTRACTMetal ions, in particular manganese, are important modulators of bacterial pathogenicity. However, little is known about the role of manganese-dependent proteins in the nosocomial pathogenEnterococcus faecalis, a major cause of bacterial endocarditis. The present study demonstrates that the DtxR/MntR family metalloregulator EfaR ofE. faecaliscontrols the expression of several of its regulon members in a manganese-dependent way. We also show thatefaRinactivation impairs the ability ofE. faecalisto form biofilms, to survive inside macrophages, and to tolerate oxidative stress. Our results reveal that EfaR is an important modulator ofE. faecalisvirulence and link manganese homeostasis to enterococcal pathogenicity.

Modulation of meningioma cell growth by sex steroid hormones in vitro

1985 ◽  
Vol 62 (5) ◽  
pp. 757-762 ◽  
Author(s):  
Jeffrey R. Jay ◽  
David T. MacLaughlin ◽  
Kathleen R. Riley ◽  
Robert L. Martuza
Keyword(s):  
Biological Activity ◽  
Cell Growth ◽  
Tumor Tissue ◽  
Growth Stimulation ◽  
Tissue Cell ◽  
Hormone Binding ◽  
Content Type ◽  
Human Meningiomas ◽  
Meningioma Cell

✓ Meningiomas were removed from four patients and estradiol binding was measured in the tumor tissue. Cell cultures were established and an in vitro system was developed to test the biological activity of physiologically relevant concentrations (10−7 M and 10−9 M) of estradiol-17pβ, progesterone, and the antiestrogen, tamoxifen, on the growth of meningioma cells in early culture (passages 3 to 5). Assays of the frozen surgical specimens demonstrated cytosolic estradiol binding, with levels of 0.3 to 26.7 femtomoles (fM)/mg protein, in all four tumors. Nuclear estradiol binding was detected in three tumors, with levels of 16.8 to 39.5 fM/mg protein. In cell culture, estradiol at either 10−7 M or 10−9 M consistently stimulated cell growth in all four cultures. When tested alone, progesterone stimulated the growth of all four tumors and tamoxifen stimulated the growth of three of the four tumors, but the levels of stimulation produced by either of these compounds were less pronounced than the level produced by estradiol. When tested in combination with estradiol, progesterone significantly inhibited the growth stimulation produced by estradiol in all four meningioma cultures and tamoxifen significantly inhibited estradiol-induced growth stimulation in three of four cultures. It is not known if these effects are mediated by a hormone receptor or by a hormone binder different from a true receptor, or if they are caused by alterations in cellular metabolism that are independent of specific hormone binding. However, the authors conclude that this in vitro technique can be used to further study the biological activity of hormones on human meningiomas in order to answer these questions.

scholarly journals The Coxiella burnetii QpH1 Plasmid Is a Virulence Factor for Colonizing Bone Marrow-Derived Murine Macrophages

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Shengdong Luo ◽  
Shanshan Lu ◽  
Huahao Fan ◽  
Zeliang Chen ◽  
Zhihui Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Coxiella Burnetii ◽  
Virulence Factor ◽  
Unknown Function ◽  
Shuttle Vector ◽  
Critical Role ◽  
Growth Defect ◽  
Content Type ◽  
Murine Macrophages

ABSTRACT Coxiella burnetii strains carry one of four large, conserved, autonomously replicating plasmids (QpH1, QpRS, QpDV, or QpDG) or a QpRS-like chromosomally integrated sequence of unknown function. Here, we report the characterization of the QpH1 plasmid of C. burnetii Nine Mile phase II by making QpH1-deficient strains. A shuttle vector pQGK containing the CBUA0036 to CBUA0039a region (predicted as being required for QpH1 maintenance) was constructed. The pQGK vector can be stably transformed into Nine Mile II and maintained at a similar low copy number like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of an RSF1010-ori-based vector, the pQGK transformant shows a similar growth curve in both axenic media and Buffalo green monkey kidney cells, a variable growth defect in macrophage-like THP-1 cells depending on the origin of inoculum, and dramatically reduced ability to colonize wild-type bone marrow-derived murine macrophages. Furthermore, we found that CBUA0037 to CBUA0039 open reading frames (ORFs) are essential for plasmid maintenance, and CBUA0037 and CBUA0038 ORFs account for plasmid compatibility. In addition, plasmid-deficient C. burnetii can be isolated by using CBUA0037 or CBUA0038 deletion vectors. Furthermore, QpH1-deficient C. burnetii strains caused a lesser extent of splenomegaly in SCID mice, but, intriguingly, they had significant growth in SCID mouse-sourced macrophages. Taken together, our data suggest that QpH1 encodes a factor(s) essential for colonizing murine, not human, macrophages. This study suggests a critical role of QpH1 for C. burnetii persistence in rodents and expands the toolkit for genetic studies in C. burnetii. IMPORTANCE All C. burnetii isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence. The plasmid is a candidate virulence factor of unknown function. Here, we describe the construction of novel shuttle vectors that allow making plasmid-deficient C. burnetii mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the plasmid plays a critical role for C. burnetii growth in murine macrophages. Our work suggests an essential role of the QpH1 plasmid for the acquisition of colonizing capability in rodents by C. burnetii. This study represents a major step toward unravelling the mystery of the C. burnetii cryptic plasmids.

scholarly journals AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

2013 ◽  
Vol 81 (5) ◽  
pp. 1696-1708 ◽  
Author(s):  
Kristi L. Frank ◽  
Pascale S. Guiton ◽  
Aaron M. T. Barnes ◽  
Dawn A. Manias ◽  
Olivia N. Chuang-Smith ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Bacterial Species ◽  
Rabbit Model ◽  
Intestinal Microbiome ◽  
Genetic Determinants ◽  
Content Type ◽  
Health Care Associated Infections ◽  
Transposon Insertions

ABSTRACTEnterococcus faecalisis part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of manyE. faecalisinfections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible forE. faecalisbiofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci:ahrC,argR,atlA,opuBC,pyrC,recN, andsepF. Only theahrCmutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss ofahrCcaused defects in early attachment and accumulation of biofilm biomass. Characterization ofahrCtranscription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species.

scholarly journals Myxoma Virus M083 Is a Virulence Factor Which Mediates Systemic Dissemination

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
A. M. Wolfe ◽  
K. M. Dunlap ◽  
A. C. Smith ◽  
M. Y. Bartee ◽  
E. Bartee
Keyword(s):  
Virulence Factor ◽  
Binding Protein ◽  
Critical Role ◽  
Physiological Role ◽  
Myxoma Virus ◽  
Target Cells ◽  
Human Pathogens ◽  
Regional Lymph Nodes ◽  
Content Type

ABSTRACTPoxviruses are large, DNA viruses whose protein capsid is surrounded by one or more lipid envelopes. Embedded into these lipid envelopes are three conserved viral proteins which are thought to mediate binding of virions to target cells. While the function of these proteins has been studiedin vitro, their specific roles during the pathogenesis of poxviral disease remain largely unclear. Here we present data demonstrating that the putative chondroitin binding protein M083 from the leporipoxvirus myxoma virus is a significant virulence factor during infection of susceptibleOryctolagusrabbits. Removal of M083 results in a reduced capacity of virus to spread beyond the regional lymph nodes and completely eliminates infection-mediated mortality.In vitro, removal of M083 results in only minor intracellular replication defects but causes a significant reduction in the ability of myxoma virus to spread from infected epithelial cells onto primary lymphocytes. We hypothesize that the physiological role of M083 is therefore to mediate the spread of myxoma virus onto rabbit lymphocytes, allowing these cells to disseminate virus throughout infected rabbits.IMPORTANCEPoxviruses represent both a class of human pathogens and potential therapeutic agents for the treatment of human malignancy. Understanding the basic biology of these agents is therefore significant to human health in a variety of ways. While the mechanisms mediating poxviral binding have been well studiedin vitro, how these mechanisms impact poxviral pathogenesisin vivoremains unclear. The current study advances our understanding of how poxviral binding impacts viral pathogenesis by demonstrating that the putative chondroitin binding protein M083 plays a critical role during the pathogenesis of myxoma virus in susceptibleOryctolagusrabbits by impacting viral dissemination through changes in the transfer of virions onto primary splenocytes.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

Accessing resources for service innovation – the critical role of network relationships

2014 ◽  
Vol 25 (1) ◽  
pp. 2-29 ◽  
Author(s):  
Helena Rusanen ◽  
Aino Halinen ◽  
Elina Jaakkola
Keyword(s):  
Critical Role ◽  
Service Innovation ◽  
Innovation Process ◽  
Research Strategy ◽  
Future Research ◽  
Theoretical Understanding ◽  
Resource Access ◽  
Content Type ◽  
External Resources ◽  
Different Types

Purpose – This paper aims to explore how companies access resources through network relationships when developing service innovations. The paper identifies the types of resource that companies seek from other actors and examines the nature of relationships and resource access strategies that can be applied to access each type of resource. Design/methodology/approach – A longitudinal, multi-case study is conducted in the field of technical business-to-business (b-to-b) services. An abductive research strategy is applied to create a new theoretical understanding of resource access. Findings – Companies seek a range of resources through different types of network relationships for service innovation. Four types of resource access strategies were identified: absorption, acquisition, sharing, and co-creation. The findings show how easily transferable resources can be accessed through weak relationships and low-intensity collaboration. Access to resources that are difficult to transfer, instead, necessitates strong relationships and high-intensity collaboration. Research limitations/implications – The findings are valid for technical b-to-b services, but should also be tested for other kinds of innovations. Future research should also study how actors integrate the resources gained through networks in the innovation process. Practical implications – Managers should note that key resources for service innovation may be accessible through a variety of actors and relationships ranging from formal arrangements to miscellaneous social contacts. To make use of tacit resources such as knowledge, firms need to engage in intensive collaboration. Originality/value – Despite attention paid to network relationships, innovation collaboration, and external resources, previous research has neither linked these issues nor studied their mutual contingencies. This paper provides a theoretical model that characterizes the service innovation resources accessible through different types of relationships and access strategies.

scholarly journals The regio-selective synthesis of 10-hydroxy camptothecin norcantharidin conjugates and their biological activity evaluation in vitro

2018 ◽  
Vol 5 (6) ◽  
pp. 172317 ◽  
Author(s):  
Chang K. Zhao ◽  
Chan Li ◽  
Xian H. Wang ◽  
Yu J. Bao ◽  
Fu H. Yang ◽  
...  
Keyword(s):  
Biological Activity ◽  
Cell Growth ◽  
Cancer Treatment ◽  
Cancer Cell ◽  
Therapeutic Potential ◽  
Selective Synthesis ◽  
Cancer Cell Growth ◽  
Activity Evaluation ◽  
Moderate Yield

A series of conjugates of 10-hydroxy camptothecin (HCPT) with functionalized norcantharidin derivatives were regio-selectively synthesized in the condition of (3-dimethylaminopropyl) ethyl-carbodiimide monohydrochloride in a moderate yield. The synthesized conjugate HCPT pro-drugs can also suppress cancer cell growth in vitro . These conjugated pro-drug constructs possess therapeutic potential as novel bi-functional conjugate platforms for cancer treatment.

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