Molecular Modeling of Structures and Interaction of Short Peptides and Sortase Family Protein of Enterococcus Faecalis: Basis for Developing Peptide-Based Therapeutics Against Multidrug Resistant Strains

2020 ◽  
Author(s):  
Muthusaravanan S ◽  
Ram Kothandan ◽  
Kumaravel Kandaswamy ◽  
Cashlin Anna Suveetha Gnana Rajan ◽  
Janamitra Arjun ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Enterococcus Faecalis ◽  
Bacterial Colonization ◽  
Van Der Waals ◽  
Surface Proteins ◽  
Binding Modes ◽  
Flexible Docking ◽  
Docking Simulations ◽  
Initial Adhesion

<p>The <i>Enterococcus faecalis</i> (<i>E. faecalis</i>) infection starts with initial adhesion to a host cell or abiotic surface by multiple adhesions on its cell membrane. The pathogenicity is due to virulence factors SrtA, SrtC, EbpA, EbpB, EbpC, and Aggregation Substance. <i>E. faecalis</i> developed resistance to the majority of standard therapies. Additionally, a notable key feature of <i>E. faecalis</i> is its ability to form biofilm <i>in vivo</i>. <i>E. faecalis</i> strains show resistance to aminoglycosides and β-lactam antibiotics with different degree of susceptibility. Sortases (SrtA and SrtC) are enzymes spatially localized at the septal region in majority of gram-positive bacteria during the cell cycle, which in-turn plays an important role in proper assembling of adhesive surface proteins and pilus on cell membrane. The studies have also proved that the both SrtA and SrtC were focally localized in <i>E. faecalis</i> and essential for efficient bacterial colonization and biofilm formation on the host tissue surfaces Using homology modeling and protein-peptide flexible docking methods, we report here the detailed interaction between peptides and <i>Ef</i>Srt (Q836L7) enzyme. Plausible binding modes between <i>Ef</i>Srt and the selected short biofilm active peptides were revealed from protein-peptide flexible docking. The simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the peptides and <i>Ef</i>Srt. The flexible docking simulations showed that the peptide-<i>Ef</i>Srt binding was achieved through hydrogen bonding, hydrophobic, and van der Waals interaction. The strength of interactions between peptide-<i>Ef</i>Srt complexes were calculated using standard energy calculations involving non-bonded interactions like electrostatic, van der Waals, and hydrogen bonds.</p>

Molecular Modeling of Structures and Interaction of Short Peptides and Sortase Family Protein of Enterococcus Faecalis: Basis for Developing Peptide-Based Therapeutics Against Multidrug Resistant Strains

2020 ◽  
Author(s):  
Muthusaravanan S ◽  
Ram Kothandan ◽  
Kumaravel Kandaswamy ◽  
Cashlin Anna Suveetha Gnana Rajan ◽  
Janamitra Arjun ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Enterococcus Faecalis ◽  
Bacterial Colonization ◽  
Van Der Waals ◽  
Surface Proteins ◽  
Binding Modes ◽  
Flexible Docking ◽  
Docking Simulations ◽  
Initial Adhesion

<p>The <i>Enterococcus faecalis</i> (<i>E. faecalis</i>) infection starts with initial adhesion to a host cell or abiotic surface by multiple adhesions on its cell membrane. The pathogenicity is due to virulence factors SrtA, SrtC, EbpA, EbpB, EbpC, and Aggregation Substance. <i>E. faecalis</i> developed resistance to the majority of standard therapies. Additionally, a notable key feature of <i>E. faecalis</i> is its ability to form biofilm <i>in vivo</i>. <i>E. faecalis</i> strains show resistance to aminoglycosides and β-lactam antibiotics with different degree of susceptibility. Sortases (SrtA and SrtC) are enzymes spatially localized at the septal region in majority of gram-positive bacteria during the cell cycle, which in-turn plays an important role in proper assembling of adhesive surface proteins and pilus on cell membrane. The studies have also proved that the both SrtA and SrtC were focally localized in <i>E. faecalis</i> and essential for efficient bacterial colonization and biofilm formation on the host tissue surfaces Using homology modeling and protein-peptide flexible docking methods, we report here the detailed interaction between peptides and <i>Ef</i>Srt (Q836L7) enzyme. Plausible binding modes between <i>Ef</i>Srt and the selected short biofilm active peptides were revealed from protein-peptide flexible docking. The simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the peptides and <i>Ef</i>Srt. The flexible docking simulations showed that the peptide-<i>Ef</i>Srt binding was achieved through hydrogen bonding, hydrophobic, and van der Waals interaction. The strength of interactions between peptide-<i>Ef</i>Srt complexes were calculated using standard energy calculations involving non-bonded interactions like electrostatic, van der Waals, and hydrogen bonds.</p>

Identification of secreted and surface proteins from Enterococcus faecalis

2009 ◽  
Vol 55 (8) ◽  
pp. 967-974 ◽  
Author(s):  
Abdellah Benachour ◽  
Thierry Morin ◽  
Laurent Hébert ◽  
Aurélie Budin-Verneuil ◽  
André Le Jeune ◽  
...  
Keyword(s):  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Virulence Factors ◽  
Gel Electrophoresis ◽  
Ion Trap ◽  
Surface Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Proteomic Approach ◽  
Associated Proteins

Secreted and surface proteins of bacteria are key molecules that interface the cell with the environment. Some of them, corresponding to virulence factors, have already been described for Enterococcus faecalis , the predominant species involved in enterococcal nosocomial infections. In a global proteomic approach, the identification of the most abundant secreted and surface-associated proteins of E. faecalis JH2-2 strain was carried out. These proteins were separated by gel electrophoresis or directly subjected to in vivo trypsinolysis and then analyzed by liquid chromatography – electrospray ion trap tandem mass spectrometry. Putative functions were assigned by homology to the translated genomic database of E. faecalis. A total of 44 proteins were identified, eight secreted proteins from the supernatant culture and 38 cell surface proteins from two-dimensional gel electrophoresis and in vivo trypsinolysis among which two are common to the two groups. Their sequences analysis revealed that 35 of the 44 proteins harbour characteristic features (signal peptide or transmembrane domains) consistent with an extracellular localization. This study may be considered as an important step to encourage proteomic-based investigations of E. faecalis cell surface associated proteins that could lead to the discovery of virulence factors and to the development of new therapeutic tools.

scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase Increases the Adhesion of Lactobacillus reuteri to Host Mucin to Enhance Probiotic Effects

2020 ◽  
Vol 21 (24) ◽  
pp. 9756
Author(s):  
Zhaoxi Deng ◽  
Tian Dai ◽  
Wenming Zhang ◽  
Junli Zhu ◽  
Xin M. Luo ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Membrane Permeability ◽  
Bacterial Cell ◽  
Lactobacillus Reuteri ◽  
Surface Proteins ◽  
Cell Membrane Permeability ◽  
In Vivo Studies ◽  
Bacterial Surface

The ability to adhere to the intestinal mucus layer is an important property of probiotic bacteria. Lactobacillus reuteri strains ZJ615 and ZJ617 show low and high adhesion, respectively, to intestinal epithelial cells. In this study, we quantified bacterial cell wall-associated glyceraldehyde-3-phosphate dehydrogenases (cw-GAPDH) and bacterial cell membrane permeability in both strains using immunoblotting and flow cytometry, respectively. Highly adhesive L. reuteri ZJ617 possessed significantly more cw-GAPDH, higher cell membrane permeability, and significantly higher adhesive ability toward mucin compared with low-adhesive L. reuteri ZJ615. In vitro adhesion studies and analysis of interaction kinetics using the Octet, the system revealed significantly decreased interaction between L. reuteri and mucin when mucin was oxidized when bacterial surface proteins were removed when bacteria were heat-inactivated at 80 °C for 30 min, and when the interaction was blocked with an anti-GAPDH antibody. SWISS-MODEL analysis suggested intensive interactions between mucin glycans (GalNAcα1-O-Ser, GalNAcαSer, and Galβ3GalNAc) and GAPDH. Furthermore, in vivo studies revealed significantly higher numbers of bacteria adhering to the jejunum, ileum, and colon of piglets orally inoculated with L. reuteri ZJ617 compared with those inoculated with L. reuteri ZJ615; this led to a significantly decreased rate of diarrhea in piglets inoculated with L. reuteri ZJ617. In conclusion, there are strong correlations among the abundance of cw-GAPDH in L. reuteri, the ability of the bacterium to adhere to the host, and the health benefits of this probiotic.

Molecular Docking Studies on Isocytosine Analogues as Xanthine Oxidase Inhibitors

Drug Research ◽  
2018 ◽  
Vol 68 (07) ◽  
pp. 395-402 ◽  
Author(s):  
Subhajit Roy ◽  
Bawneet Narang ◽  
Manish Gupta ◽  
Vikrant Abbot ◽  
Virender Singh ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Docking Studies ◽  
Binding Modes ◽  
Free Energies ◽  
Flexible Docking ◽  
Docking Simulations ◽  
Xanthine Oxidase Inhibitors ◽  
Best Fit ◽  
Insight Into ◽  
Binding Free Energies

AbstractFlexible docking simulations were carried out on a series of isocytosine analogs as xanthine oxidase (XO) inhibitors. This was done by analysing the interaction of these compounds at the active site of XO. The binding free energies of the analogs were calculated using GoldScore. The binding modes of the best-fit conformation were studied, providing some handy important interactions. The results obtained henceforth provided an insight into the pharmacophoric structural requirements for XO inhibition for this class of molecules.

scholarly journals Multiple Functional Domains of Enterococcus faecalis Aggregation Substance Asc10 Contribute to Endocarditis Virulence

2008 ◽  
Vol 77 (1) ◽  
pp. 539-548 ◽  
Author(s):  
Olivia N. Chuang ◽  
Patrick M. Schlievert ◽  
Carol L. Wells ◽  
Dawn A. Manias ◽  
Timothy J. Tripp ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Bacterial Load ◽  
Rabbit Model ◽  
Surface Proteins ◽  
Functional Domains ◽  
Mammalian Host ◽  
Bacterial Cells ◽  
Wild Type ◽  
Aggregation Substance

ABSTRACT Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo.

A Global Role for EKLF in Definitive and Primitive Erythropoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1745-1745
Author(s):  
Andrew C. Perkins ◽  
Elise Coghill ◽  
Tina Maguire ◽  
Belinda Hartmann ◽  
Alasdair McDowall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Membrane ◽  
Target Genes ◽  
Globin Gene ◽  
Surface Proteins ◽  
Erythroid Cell ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Reporter Assays

Abstract Erythroid Kruppel-like factor (EKLF or Klf1) is an erythroid specific C2H2 zinc-finger transcription factor which is essential for definitive erythropoiesis and β-globin gene expression. The absence of EKLF results in fatal anaemia but correction of globin chain imbalance does result in rescue, suggesting the existence of additional EKLF target genes. The aim of this study was to search for such genes by expression profiling. We performed profiling on fetal livers from wild-type versus EKLF null litter mates, and also EKLF null erythroid cell lines containing an inducible EKLF-ERTM fusion construct. Hybridisations were performed on microarray slides printed with a 23K oligo library from Compugen. Target gene validation was performed by real-time RT-PCR, chromatin immuno-precipitation (ChIP) and promoter-reporter assays. A large number of genes were down regulated in the absence of EKLF but few were up regulated, suggesting EKLF acts primarily as a transcriptional activator in vivo. One hundred genes were EKLF dependent in both systems. These include heme synthesis enzymes, red cell surface proteins including Rh and the transferrin receptor, and erythroid transcription factors. Two interesting highly EKLF-dependent genes are α-haemoglobin stabilising protein (AHSP), a key chaperone for free a-globin chains, and dematin (band 4.9) which links the cytoskeleton to the red cell membrane. A search for EKLF binding sites within the dematin and AHSP genes demonstrated a number of phylogenetically conserved CACC sites, and ChIP demonstrated in vivo EKLF occupancy at some but not all of these. Promoter-reporter assays showed EKLF directly activates dematin gene transcription through two promoters containing these sites. Lastly, investigation of EKLF target genes in the yolk sac lead to the discovery of unexpected defects in the embryonic red cell membrane and cytoskeleton. In conclusion, EKLF regulates global erythroid gene expression which is critical for development of primitive as well as definitive red cells.

Approach in improving potency and selectivity of kinase inhibitors: Allosteric kinase inhibitors

2020 ◽  
Vol 21 ◽  
Author(s):  
Shangfei Wei ◽  
Tianming Zhao ◽  
Jie Wang ◽  
Xin Zhai
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Binding Modes ◽  
Allosteric Inhibitors ◽  
Wide Range ◽  
Allosteric Sites ◽  
Protein Functions ◽  
Regulate Protein

: Allostery is an efficient and particular regulatory mechanism to regulate protein functions. Different from conserved orthosteric sites, allosteric sites have distinctive functional mechanism to form the complex regulatory network. In drug discovery, kinase inhibitors targeting the allosteric pockets have received extensive attention for the advantages of high selectivity and low toxicity. The approval of trametinib as the first allosteric inhibitor validated that allosteric inhibitors could be used as effective therapeutic drugs for treatment of diseases. To date, a wide range of allosteric inhibitors have been identified. In this perspective, we outline different binding modes and potential advantages of allosteric inhibitors. In the meantime, the research processes of typical and novel allosteric inhibitors are described briefly in terms of structureactivity relationships, ligand-protein interactions and in vitro and in vivo activity. Additionally, challenges as well as opportunities are presented.

Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

2019 ◽  
Vol 16 (3) ◽  
pp. 175-180
Author(s):  
Fengjin Hao ◽  
Yueqin Feng ◽  
Yifu Guan
Keyword(s):  
Biological Activity ◽  
Botulinum Toxin ◽  
Cell Membrane ◽  
Western Blot ◽  
Biological Function ◽  
C6 Cells ◽  
Green Fluorescence ◽  
Bv2 Cells

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity. Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25. Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

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.

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