scholarly journals The pentaglycine bridges ofStaphylococcus aureuspeptidoglycan are essential for cell integrity

2018 ◽  
Author(s):  
João M. Monteiro ◽  
Daniela Münch ◽  
Sérgio R. Filipe ◽  
Tanja Schneider ◽  
Hans-Georg Sahl ◽  
...  
Keyword(s):  
Sharp Decrease ◽  
Bacterial Cells ◽  
Cell Integrity ◽  
Protein Activity ◽  
Membrane Rupture ◽  
Cross Bridge ◽  
Cross Bridges ◽  
Main Component

AbstractBacterial cells are surrounded by cell wall, whose main component is peptidoglycan (PG), a macromolecule that withstands the internal turgor of the cell. PG composition can vary considerably between species. The Gram-positive pathogenStaphylococcus aureuspossesses highly crosslinked PG due to the presence of cross bridges containing five glycines, which are synthesised by the FemXAB protein family. FemX adds the first glycine of the cross bridge, while FemA and FemB add the second and the third, and the fourth and the fifth glycines, respectively. Of these, FemX was reported to be essential. To investigate the essentiality of FemAB, we constructed a conditionalS. aureusmutant of thefemABoperon. Depletion offemABwas lethal, with cells appearing as pseudomulticellular forms that eventually lyse due to extensive membrane rupture. This deleterious effect was mitigated by drastically increasing the osmolarity of the medium, indicating that pentaglycine crosslinks are required forS. aureuscells to withstand internal turgor. Despite the absence of canonical membrane targeting domains, FemA has been shown to localise at the membrane. To study its mechanism of localisation, we constructed mutants in key residues present in the putative transferase pocket and the α6 helix of FemA, possibly involved in tRNA binding. Mutations in the α6 helix led to a sharp decrease in protein activityin vivoandin vitrobut did not impair correct membrane localisation, indicating that FemA activity is not required for localisation. Our data indicates that, contrarily to what was previously thought,S. aureuscells do not survive in the absence of a pentaglycine cross bridge.

scholarly journals Reassociation of microvillar core proteins: making a microvillar core in vitro.

1989 ◽  
Vol 108 (2) ◽  
pp. 495-502 ◽  
Author(s):  
L M Coluccio ◽  
A Bretscher
Keyword(s):  
Actin Filaments ◽  
Direct Proof ◽  
Intestinal Epithelia ◽  
The Core ◽  
Cross Bridge ◽  
Core Proteins ◽  
The Cross ◽  
Cross Bridges

Intestinal epithelia have a brush border membrane of numerous microvilli each comprised of a cross-linked core bundle of 15-20 actin filaments attached to the surrounding membrane by lateral cross-bridges; the cross-bridges are tilted with respect to the core bundle. Isolated microvillar cores contain actin (42 kD) and three other major proteins: fimbrin (68 kD), villin (95 kD), and the 110K-calmodulin complex. The addition of ATP to detergent-treated isolated microvillar cores has previously been shown to result in loss of the lateral cross-bridges and a corresponding decrease in the amount of the 110-kD polypeptide and calmodulin associated with the core bundle. This provided the first evidence to suggest that these lateral cross-bridges to the membrane are comprised at least in part by a 110-kD polypeptide complexed with calmodulin. We now demonstrate that purified 110K-calmodulin complex can be readded to ATP-treated, stripped microvillar cores. The resulting bundles display the same helical and periodic arrangement of lateral bridges as is found in vivo. In reconstitution experiments, actin filaments incubated in EGTA with purified fimbrin and villin form smooth-sided bundles containing an apparently random number of filaments. Upon addition of 110K-calmodulin complex, the bundles, as viewed by electron microscopy of negatively stained images, display along their entire length helically arranged projections with the same 33-nm repeat of the lateral cross-bridges found on microvilli in vivo; these bridges likewise tilt relative to the bundle. Thus, reconstitution of actin filaments with fimbrin, villin, and the 110K-calmodulin complex results in structures remarkably similar to native microvillar cores. These data provide direct proof that the 110K-calmodulin is the cross-bridge protein and indicate that actin filaments bundled by fimbrin and villin are of uniform polarity and lie in register. The arrangement of the cross-bridge arms on the bundle is determined by the structure of the core filaments as fixed by fimbrin and villin; a contribution from the membrane is not required.

scholarly journals Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2157-2169 ◽  
Author(s):  
Sudarson Sundarrajan ◽  
Junjappa Raghupatil ◽  
Aradhana Vipra ◽  
Nagalakshmi Narasimhaswamy ◽  
Sanjeev Saravanan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mechanism Of Action ◽  
Catalytic Domain ◽  
Antibiotic Sensitivity ◽  
High Sensitivity ◽  
Common Mechanism ◽  
Cross Bridge ◽  
Sensitivity Pattern

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

scholarly journals In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite and Struvite Urolithiasis

Kidney360 ◽  
2020 ◽  
pp. 10.34067/KID.0006942020
Author(s):  
Jessica J. Saw ◽  
Mayandi Sivaguru ◽  
Elena M. Wilson ◽  
Yiran Dong ◽  
Robert A. Sanford ◽  
...  
Keyword(s):  
Calcium Oxalate ◽  
Stone Formation ◽  
Human Kidney ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Stone Formers ◽  
Bacteria And Fungi ◽  
Struvite Stones

Background: Human kidney stones form via repeated events of mineral precipitation, partial dissolution and reprecipitation, which are directly analogous to similar processes in other natural and man-made environments where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods: Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, while one patient each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from 7 of these 20 patients (5 CaOx, 1 brushite and 1 struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, superresolution autofluorescence (SRAF) and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1-V3, V3-V5) and internal transcribed spacer (ITS1, ITS2) regions. Results: Bulk entombed DNA was sequenced from stone fragments in 11 of the 18 CaOx patients, as well as the brushite and struvite patients. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells ~1  µm in diameter were also optically observed to be entombed and well-preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions: These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

scholarly journals IN VITRO ANTIOXIDANT AND IN VIVO ANTIDIABETIC ACTIVITY OF TWO POTENTIAL PROBIOTIC ENTEROCOCCUS SPP. ON ALLOXAN-INDUCED DIABETIC RATS

2021 ◽  
pp. 94-98
Author(s):  
KAMNI RAJPUT ◽  
RAMESH CHANDRA DUBEY
Keyword(s):  
Diabetic Rats ◽  
Antidiabetic Activity ◽  
Control Group ◽  
Albino Rats ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Animal Group ◽  
Enterococcus Spp

Objective: In vitro antioxidant activity, in vivo antidiabetic property and intestinal attachment by two potential probiotic bacterial strains, namely, Enterococcus faecium and Enterococcus hirae were studied using albino rats. Methods: Antioxidant the activity was assessed using 2,2-Diphenyl-1-picrylhydrazyl radicals scavenging assay. Alloxan was administered intraperitoneally to induce diabetic conditions in experimental rats. Animals were treated with oral administration of Enterococcus spp., such as E. faecium, and E. hirae isolated from goat and sheep milk. The control animal group received normal saline for the same days. Glibenclamide drug was used as a positive control against probiotic bacterial cells. Results: However, administration of probiotic bacterial strains E. faecium and E. hirae, in albino rats significantly (p<0.05) at varying doses lowered blood glucose levels in diabetic rats as compared to the diabetic control group. Both the species of Enterococcus increased the bodyweight of experimental rats. However, E. faecium was the best antidiabetic strain having the antioxidant activities also in comparison to E. hirae. The attachment of probiotic bacterial cells E. faecium on the rat’s intestine wall against pathogens was examined. Furthermore, E. faecium showed its aggregation with pathogens by attachment of the intestines of albino rats. This showed that both the bacterial strains exhibited in vivo antidiabetic effect. Conclusion: The results of this study showed that probiotic bacteria possess antioxidant, antidiabetic activities, and attachment of intestine.

scholarly journals Assessment of Minocycline and Polymyxin B Combination against Acinetobacter baumannii

2015 ◽  
Vol 59 (5) ◽  
pp. 2720-2725 ◽  
Author(s):  
Dana R. Bowers ◽  
Henry Cao ◽  
Jian Zhou ◽  
Kimberly R. Ledesma ◽  
Dongxu Sun ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Utility ◽  
Efflux Pump ◽  
Polymyxin B ◽  
Intracellular Concentration ◽  
Bacterial Cells ◽  
Efflux Pump Inhibitor ◽  
Content Type

ABSTRACTAntimicrobial resistance amongAcinetobacter baumanniiis increasing worldwide, often necessitating combination therapy. The clinical utility of using minocycline with polymyxin B is not well established. In this study, we investigated the activity of minocycline and polymyxin B against 1 laboratory isolate and 3 clinical isolates ofA. baumannii. Minocycline susceptibility testing was performed with and without an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN). The intracellular minocycline concentration was determined with and without polymyxin B (0.5 μg/ml). Time-kill studies were performed over 24 h using approximately 106CFU/ml of each strain with clinically relevant minocycline concentrations (2 μg/ml and 8 μg/ml), with and without polymyxin B (0.5 μg/ml). Thein vivoefficacy of the combination was assessed in a neutropenic murine pneumonia model. Infected animals were administered minocycline (50 mg/kg), polymyxin B (10 mg/kg), or both to achieve clinically equivalent exposures in humans. A reduction in the minocycline MIC (≥4×) was observed in the presence of PAβN. The intracellular concentration andin vitrobactericidal effect of minocycline were both enhanced by polymyxin B. With 2 minocycline-susceptible strains, the bacterial burden in lung tissue at 24 h was considerably reduced by the combination compared to monotherapy with minocycline or polymyxin B. In addition, the combination prolonged survival of animals infected with a minocycline-susceptible strain. Polymyxin B increased the intracellular concentration of minocycline in bacterial cells and enhanced the bactericidal activity of minocycline, presumably due to efflux pump disruption. The clinical utility of this combination should be further investigated.

Abstract 319: Dalcetrapib-Mediated Modulation of Cholesteryl Ester Transfer Protein Activity Increases HDL-Cholesterol, Apolipoprotein A-I Levels and Cholesterol Efflux Capacity in Chow-Fed Rabbits

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Mathieu R Brodeur ◽  
David Rhainds ◽  
Daniel Charpentier ◽  
Téodora Mihalache-Avram ◽  
Cyrille Maugeais ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Cholesteryl Ester Transfer Protein ◽  
Cholesterol Efflux ◽  
Hdl Cholesterol ◽  
Lipid Transfer ◽  
Protein Activity ◽  
Cholesterol Efflux Capacity ◽  
Transfer Protein

Introduction: A potential approach to reduce CV risk is to increase HDL-C levels. This could be achieved by reducing cholesteryl ester transfer protein (CETP) activity. Dalcetrapib, which modulates CETP activity by changing its conformation and raises HDL-C without inhibiting CETP-induced pre-β-HDL formation in humans, was shown to decrease progression of atherosclerosis in rabbits. Hypothesis: Investigate the modifications of HDL particle size distribution and cholesterol efflux capacity of serum produced by dalcetrapib in normocholesterolemic rabbits. Methods: New Zealand white rabbits were treated with dalcetrapib (300 mg/kg as food admix) or placebo for 14 days. We evaluated CETP conformation and mass by ELISAs (including antibodies sensitive to conformational change), CETP activity by fluorescent lipid transfer, lipid profile and apoA-I distribution in HDL subclasses by 2D-non denaturing gradient gels (2D-NDGGE). Cholesterol efflux capacity of rabbit sera was determined after loading cells with 3 H-free cholesterol, using HepG2 hepatocytes to measure SR-BI-dependent efflux and by inducing ABCA1 or ABCG1 expression in BHK cells. Results: Dalcetrapib modified the conformation of rabbit CETP in vitro and in vivo and, after 14 days, this was associated with increased CETP mass (+50%, p<0.001) and reduced CETP activity (-86%, p<0.001). Total cholesterol was increased with dalcetrapib (+178%, p<0.001), due to a higher HDL-C level. In contrast, dalcetrapib reduced LDL-C and triglycerides by 41% (p<0.01) and 48% (p<0.001). Serum analysis by 2D-NDGGE showed that total rabbit apoA-I was increased 1.7- fold in animals treated with dalcetrapib. This was associated with an increase in large HDL but also in small α-migrating HDL with pre-β-HDL size. Cholesterol efflux assays showed that ABCA1-, ABCG1- and SR-BI-dependent efflux were all increased in dalcetrapib-treated rabbits (+24%, p=0.038; +21%, p=0.021; +44%, p<0.001). Conclusion: Modulation of CETP activity and conformation by dalcetrapib increases HDL-C and apoA-I levels and affects apoA-I distribution in HDL subclasses. These changes are associated with increased cholesterol efflux capacity, suggesting that HDL functionality is preserved in dalcetrapib-treated chow-fed rabbits.

scholarly journals Non-Lytic Antibacterial Peptides That Translocate Through Bacterial Membranes to Act on Intracellular Targets

2019 ◽  
Vol 20 (19) ◽  
pp. 4877 ◽  
Author(s):  
Marlon H. Cardoso ◽  
Beatriz T. Meneguetti ◽  
Bruna O. Costa ◽  
Danieli F. Buccini ◽  
Karen G. N. Oshiro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Biological Properties ◽  
Antibacterial Peptides ◽  
Bacterial Cells ◽  
Bacterial Membranes ◽  
Intracellular Targets

The advent of multidrug resistance among pathogenic bacteria has attracted great attention worldwide. As a response to this growing challenge, diverse studies have focused on the development of novel anti-infective therapies, including antimicrobial peptides (AMPs). The biological properties of this class of antimicrobials have been thoroughly investigated, and membranolytic activities are the most reported mechanisms by which AMPs kill bacteria. Nevertheless, an increasing number of works have pointed to a different direction, in which AMPs are seen to be capable of displaying non-lytic modes of action by internalizing bacterial cells. In this context, this review focused on the description of the in vitro and in vivo antibacterial and antibiofilm activities of non-lytic AMPs, including indolicidin, buforin II PR-39, bactenecins, apidaecin, and drosocin, also shedding light on how AMPs interact with and further translocate through bacterial membranes to act on intracellular targets, including DNA, RNA, cell wall and protein synthesis.

scholarly journals ADME/Tox Properties and Biochemical Interactions of Silybin Congeners: In silico Study

2017 ◽  
Vol 12 (2) ◽  
pp. 1934578X1701200 ◽  
Author(s):  
Antonia Diukendjieva ◽  
Merilin Al Sharif ◽  
Petko Alov ◽  
Tania Pencheva ◽  
Ivanka Tsakovska ◽  
...  
Keyword(s):  
In Silico ◽  
Estrogen Receptor Alpha ◽  
Estrogenic Activity ◽  
Qsar Model ◽  
Quantitative Structure Activity Relationship ◽  
Toxic Effects ◽  
Active Constituent ◽  
Main Component

Silymarin, the active constituent of Silybum marianum (milk thistle), and its main component, silybin, are products with well-known hepatoprotective, cytoprotective, antioxidant, and chemopreventative properties. Despite substantial in vitro and in vivo investigations of these flavonolignans, their mechanisms of action and potential toxic effects are not fully defined. In this study we explored important ADME/Tox properties and biochemical interactions of selected flavonolignans using in silico methods. A quantitative structure–activity relationship (QSAR) model based on data from a parallel artificial membrane permeability assay (PAMPA) was used to estimate bioavailability after oral administration. Toxic effects and metabolic transformations were predicted using the knowledge-based expert systems Derek Nexus and Meteor Nexus (Lhasa Ltd). Potential estrogenic activity of the studied silybin congeners was outlined. To address further the stereospecificity of this effect the stereoisomeric forms of silybin were docked into the ligand-binding domain of the human estrogen receptor alpha (ERα) (MOE software, CCG). According to our results both stereoisomers can be accommodated into the ERα active site, but different poses and interactions were observed for silybin A and silybin B.

scholarly journals Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

2020 ◽  
Vol 295 (32) ◽  
pp. 10926-10939 ◽  
Author(s):  
Benoit Darlot ◽  
James R. O. Eaton ◽  
Lucia Geis-Asteggiante ◽  
Gopala K. Yakala ◽  
Kalimuthu Karuppanan ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Titration Calorimetry ◽  
Protein Activity ◽  
Binding Interface ◽  
Chemokine Ligand ◽  
Therapeutic Development ◽  
Hydrogen Deuterium ◽  
Anti Inflammatory

Chemokines mediate leukocyte migration and homeostasis and are key targets in inflammatory diseases including atherosclerosis, cytokine storm, and chronic autoimmune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines to overcome redundancy and are effective in several preclinical disease models. Their clinical development has not progressed because of concerns regarding potential immunogenicity, parenteral delivery, and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen–deuterium exchange MS to map the binding interface of the evasin P672 that physically interacts with C–C motif chemokine ligand (CCL) 8 and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native MS approaches showed that BK1.1 binds CCL8, CCL7, and CCL18 and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2, and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2, and CCL3 chemotactic function in vitro. We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo. Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

scholarly journals TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 192 ◽  
Author(s):  
Feng Wang ◽  
Xinyu Ji ◽  
Qiupeng Li ◽  
Guanling Zhang ◽  
Jiani Peng ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Infection Model ◽  
Bacterial Cells ◽  
Skin Damage ◽  
Gram Positive ◽  
Antibiotic Resistant ◽  
Gram Negative

New strategies against antibiotic-resistant bacterial pathogens are urgently needed but are not within reach. Here, we present in vitro and in vivo antimicrobial activity of TSPphg, a novel phage lysin identified from extremophilic Thermus phage TSP4 by sequencing its whole genome. By breaking down the bacterial cells, TSPphg is able to cause bacteria destruction and has shown bactericidal activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains of Klebsiella pneumoniae, in which the complete elimination and highest reduction in bacterial counts by greater than 6 logs were observed upon 50 μg/mL TSPphg treatment at 37 °C for 1 h. A murine skin infection model further confirmed the in vivo efficacy of TSPphg in removing a highly dangerous and multidrug-resistant Staphylococcus aureus from skin damage and in accelerating wound closure. Together, our findings may offer a therapeutic alternative to help fight bacterial infections in the current age of mounting antibiotic resistance, and to shed light on bacteriophage-based strategies to develop novel anti-infectives.

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