Walls Of Gram-positive Bacteria Research Articles

Staphylococcus aureus Lipoteichoic Acid has a Dual Effect on Inflammatory Responses in Airway Epithelial Cells via the Transcription Factor NFκB

Staphylococcus aureus is a major human pathogen that causes a wide array of infections ranging in severity from mild skin infections to life-threatening diseases such as sepsis. S. aureus can cause severe inflammation whilst, at the same time, modulating specific innate and humoral immune defense mechanisms. Lipoteichoic acid (LTA) is a component of the cell wall of gram-positive bacteria that is abundantly released by S. aureus. We investigated the dose-dependent effects of LTA on inflammation in airway epithelial cells. Purified S. aureus LTA was applied to airway epithelial (NuLi-1) cells in a range of concentrations, followed by assessment of expression of NFκB p50 and p65/RelA via qPCR, immunofluorescence and western blot. Multiplex analysis of cell culture supernatants was carried out to evaluate cytokine levels. NFκB p50 mRNA varied according to the LTA dose (p = 0.013) whereas NFκB p65/RelA did not (p = 0.90). A dose-dependent increase in NFκB p50 mRNA expression was seen up to 10 μg/ml LTA treatment, before falling at the 100μg/ml treatment. Western blot analysis showed nuclear and cytoplasmic NFκB p50 and cytoplasmic NFκB p65 protein reduced with increasing LTA concentrations. Multiplex analysis demonstrated that cytokine levels increased with LTA dose, but then decreased at the highest LTA dose (100 μg/ml). This study identifies LTA as having a dual dose-dependent effect on immune activation at low dosages and ablation at high dosages, associated with altered expression levels of NFκB. It adds LTA to the extensive repertoire of possible immune modulators produced by S. aureus.

In silico Predicted Glucose‐1‐phosphate Uridylyltransferase (GalU) Inhibitors Block a Key Pathway Required for Listeria Virulence

Peptidoglycan walls of gram positive bacteria are functionalized by glycopolymers called wall teichoic acid (WTA). In Listeria monocytogenes, multiple enzymes including the glucose-1-phosphate uridylyltransferase (GalU) were identified as mandatory for WTA galactosylation, so that the inhibition of GalU is associated with a significant attenuation of Listeria virulence. Herein, we report on a series of in silico predicted GalU inhibitors identified using structure-based virtual screening and experimentally validated to be effective in blocking the WTA galactosylation pathway in vitro. Several hits such as C04, a pyrimidinyl benzamide, afforded promising experimental potencies. This proof-of-concept study opens new perspectives for the development of potent and selective GalU inhibitors of high interest to attenuate Listeria virulence. It also underscores the high relevance of using molecular modeling for facilitating the identification of bacterial virulence attenuators and more generally antibacterials.

Using Sand Particles for the Disruption of Cell Walls of Gram-Positive Bacteria and Mycobacteria

Using Sand Particles for the Disruption of Cell Walls of Gram-Positive Bacteria and Mycobacteria

Synthesis of cationized nanofibrillated cellulose and its antimicrobial properties

Two types of cationized nanofibrillated cellulose (NFC) were prepared by redox initiated graft copolymerization and etherification with quaternary ammonium compounds (QAC). The QAC content and charge density of the products were measured. The NFC derivatives were tested for antimicrobial activity against Gram positive and negative bacteria, and yeast. Both NFC types exhibited broad spectrum antimicrobial activity. Etherification resulted in a higher degree of substitution and charge density, and the product also showed higher antimicrobial activity than the copolymerization product. Etherified NFC was more efficient against Gram negative than positive bacteria, whereas the polymer grafted NFC was equally active against both. This was attributed to the ability of the polymeric grafts to penetrate the thick cell wall of Gram positive bacteria, followed by the destabilization of the cellular membrane. Neither cationized NFC type showed cytotoxicity against human cells, providing means to manufacture safe, insoluble, and permanently antimicrobial materials via aqueous synthesis.

Theoretical evaluation of wall teichoic acids in the cavitation-mediated pores formation in Gram-positive bacteria subjected to an electric field

BackgroundElectroporation is a method of choice to transform living cells. The ability of electroporation to transfer small or large chemicals across the lipid bilayer membrane of eukaryotic cells or Gram-negative bacteria relies on the formation of transient pores across the membrane. To exist, these pores rely on an insulator (the bilayer membrane) and the presence of a potential difference on either side of the membrane mediated by an external electric field. In Gram-positive bacteria, however, the wall is not an insulator but pores can still form when an electric field is applied. Past works have shown that the electrostatic charge of teichoic acids, a major wall component; sensitizes the wall to pore formation when an external electric field is applied. These results suggest that teichoic acids mediate the formation of defects in the wall of Gram-positive bacteria. MethodsWe model the electrostatic repulsion between teichoic acids embedded in the bacterial wall composed of peptidoglycan when an electric field is applied. The repulsion between teichoic acids gives rise to a stress pressure that is able to rupture the wall when a threshold value has been reached. The size of such small defects can diverge leading to the formation of pores. ResultsIt is demonstrated herein that for a bonding energy of about ~1−10kBT between peptidoglycan monomers an intra-wall pressure of about ~5−120kBT/nm3 generates spherical defects of radius ~0.1−1nm diverging in size to create pores. ConclusionThe electrostatic cavitation of the bacterial wall theory has the potential to highlight the role of teichoic acids in the formation pores, providing a new step in the understanding of electroporation in Gram-positive bacteria without requiring the use of an insulator.

Revised model of calcium and magnesium binding to the bacterial cell wall.

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membraneas lipoteichoic acid or covalently bound to the cell wallas wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, whichcontradicts previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, whichmeans that the binding affinity decreases as more ions become bound to the sample. For Ca(2+), Region I has a KA=(1.0±0.2)×10(6)M(-1) and Region II has a KA=(0.075±0.058)×10(6)M(-1). For Mg(2+), KA1=(1.5±0.1)×10(6) and KA2=(0.17±0.10)×10(6). A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70±0.04 and 0.67±0.03µmol/mg for Ca(2+) and Mg(2+) respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.

Peptidoglycan Hydrolases of Local Lactic Acid Bacteria from Kazakh Traditional Food.

IntroductionPeptidoglycan (PG) is a major component of the cell wall of Gram-positive bacteria and is essential for maintaining the integrity of the bacterial cell and its shape. The bacteria synthesize PG hydrolases, which are capable of cleaving the covalent bonds of PG. They also play an important role in modeling PG, which is required for bacterial growth and division. In an era of increasing antibiotic-resistant pathogens, PG hydrolases that destroy these important structures of the cell wall act as a potential source of new antimicrobials. The aim of this study is to identify the main PG hydrolases of local lactic acid bacteria isolated from traditional foods that enhance probiotic activity of a biological preparation.MethodsLactococcus lactis 17A and Lactococcus garvieae 19A were isolated from the traditional sausage-like meat product called kazy. They were isolated according to standards methods of microbiology. Genetic identification of the isolates were tested by determining the nucleotide sequences of 16S rDNA. The Republican collection of microorganisms took strains of Lactobacillus casei subsp. Rhamnosus 13-P, L. delbrueckii subsp. lactis CG-1 B-RKM 0044 from cheese, Lactobacillus casei subsp. casei B-RKM 0202 from homemade butter. They used the standard technique of renaturating polyacrylamide gel electrophoresis to detect PG hydrolases activity.ResultsAccording to the profiles of PG hydrolase activity on zymograms, the enzymes of Lactococci 17A and 19A in kazy are similar in electrophoretic mobility to major autolysin AcmA, while the lactobacilli of industrial and home-made dairy products have enzymes similar to extracellular proteins p40 and p75, which have probiotic activity.ConclusionsUse of peptidoglycan hydrolases seems to be an interesting approach in the fight against multi-drug resistant strains of bacteria and could be a valuable tool for the treatment of diseases caused by these microorganisms in Kazakhstan.

Antimicrobial-peptide coating that ruptures the wall of Gram positive bacteria

Antimicrobial-peptide coating that ruptures the wall of Gram positive bacteria

Circulating microRNA signatures in mice exposed to lipoteichoic acid

BackgroundPreviously, we had identified a specific whole blood–derived microRNAs (miRNAs) signature in mice following in vivo injection of lipopolysaccharide (LPS) originated from Gram-negative bacteria. This study was designed to profile the circulating miRNAs expression in mice exposed to lipoteichoic acid (LTA) which is a major component of the wall of Gram-positive bacteria.ResultsC57BL/6 mice received intraperitoneal injections of 100 μg of LTA originated from Bacillus subtilis, Streptococcus faecalis, and Staphylococcus aureus were killed 6 h and the whole blood samples were obtained for miRNA expression analysis using a miRNA array (Phalanx miRNA OneArray® 1.0). Up-regulated expression of miRNA targets in the whole blood, serum and white blood cells (WBCs) of C57BL/6 and Tlr2−/− mice upon LTA treatment in 10, 100, or 1000 ug concentrations was quantified at indicated time (2, 6, 24, and 72 h) using real-time RT-PCR and compared with that in the serum of C57BL/6 mice injected with 100 ug of LPS. A significant increase of 4 miRNAs (miR-451, miR-668, miR-1902, and miR-1904) was observed in the whole blood and the serum in a dose- and time-dependent fashion following LTA injection. Induction of miRNA occurred in the serum after 2 h and persisted for at least 6 h. No increased expression of these 4 miRNAs was found in the WBCs. Higher but not significant expression level of these 4 miRNAs were observed following LTA treatment in the serum of Tlr2−/−against that of C57BL6 mice. In contrast, LPS exposure induced moderate expression of miR-451 but not of the other 3 miRNA targets.ConclusionsWe identified a specific circulating miRNA signature in mice exposed to LTA. That expression profile is different from those of mice exposed to LPS. Those circulating miRNAs induced by LTA or LPS treatment may serve as promising biomarkers for the differentiation between exposures to Gram-positive or Gram-negative bacteria.

Efficient N‐Terminal Labeling of Proteins by Use of Sortase

Efficient N‐Terminal Labeling of Proteins by Use of Sortase

48 Anti-inflammatory and bactericidal action of LED medolight device at treatment of dental diseases

48 Anti-inflammatory and bactericidal action of LED medolight device at treatment of dental diseases

The effect of nebivolol on the production of nitric oxide induced by bacterial lipopolysaccharide and peptidoglycan in mice

Nitric oxide (NO) plays a pivotal role in main- taining balance of physiological events in many systems including the autonomic, cardiovas- cular, hematological, and pulmonary systems. Lipopolysaccharide (LPS) and peptidoglycan (PGN), components of the outer cell membranes of Gram-negative bacteria and cell walls of Gram-positive bacteria respectively, are in- criminated in NO-induced septic shock. Ne- bivolol is a third generation β1- adrenoceptor blocker with a vasodilatory property attributed to enhanced availability of nitric oxide and re- duction of cellular oxidative stress through an unknown mechanism. The current study ex- plored the hypothesis that if nebivolol enhances the availability of NO, pretreatment with ne- bivolol may enhance production of NO in re- sponse to subsequent treatment with LPS and PGN, an observation that may have relevance in clinical septic shock. Groups of female BALB/c mice each containing 12 mice (6-8 weeks old) were injected intraperitoneally with LPS (30 µg/mouse), PGN (100 µg/mouse), nebivolol (0.25 µg/g, 0.35 µg/g, 0.7 µg/g), LPS and nebivolol (0.25 µg/g), LPS and nebivolol (0.35 µg/g), LPS and nebivolol (0.7 µg/g), PGN and nebivolol (0.25 µg/g), PGN and nebivolol (0.35µg/g). One group of mice was injected with saline and an- other served as control. Three mice from each group were bled 1, 3, 6 and 9 hours post-injec- tion, the blood was pooled and the nitrite serum levels, reflecting NO concentration, were de- termined using Greiss reagent. The following results were obtained: 1) Treatment with saline did not induce NO production; 2) LPS induced NO production to a maximal limit of 545% at 9 hours as compared to treatment with saline; 3) PGN did not induce NO production; 4) nebivolol at most doses and periods (7 out of 10 deter- minations) increased NO production over a range of 18-110% as compared to treatment with saline; 5) Nebivolol enhanced LPS-induced production of NO by 58% at a dose of 0.7 µg/gm at 9 hours. It is concluded that nebivolol in- duces NO production. At low doses nebivolol initially appeared to have a suppressive or no effect on NO production induced by LPS. In- crease in the dose of nebivolol resulted in augmentation of LPS-induced production of NO. PGN, in the dose tested, did not have an effect on NO production.

Probing Teichoic Acid Genetics with Bioactive Molecules Reveals New Interactions among Diverse Processes in Bacterial Cell Wall Biogenesis

The bacterial cell wall has been a celebrated target for antibiotics and holds real promise for the discovery of new antibacterial chemical matter. In addition to peptidoglycan, the walls of Gram-positive bacteria contain large amounts of the polymer teichoic acid, covalently attached to peptidoglycan. Recently, wall teichoic acid was shown to be essential to the proper morphology of Bacillus subtilis and an important virulence factor for Staphylococcus aureus. Additionally, recent studies have shown that the dispensability of genes encoding teichoic acid biosynthetic enzymes is paradoxical and complex. Here, we report on the discovery of a promoter (P(ywaC)), which is sensitive to lesions in teichoic acid synthesis. Exploiting this promoter through a chemical-genetic approach, we revealed surprising interactions among undecaprenol, peptidoglycan, and teichoic acid biosynthesis that help explain the complexity of teichoic acid gene dispensability. Furthermore, the new reporter assay represents an exciting avenue for the discovery of antibacterial molecules.

Butanol-extracted lipoteichoic acid induces in vivo leukocyte adhesion

Lipoteichoic acid (LTA), an immunostimulatory component of the cell walls of Gram positive bacteria, has pro-inflammatory effects in vitro and in vivo. However, one in vivo study concluded that LTA had no noticeable effects on leukocyte recruitment. In this study we investigated the effects of highly purified LTA, prepared by butanol extraction (Bu-LTA) at room temperature, on in vivo leukocyte adhesion. Using intravital microscopy we measured adhesion of leukocytes in mesenteric post-capillary venules of rats and mice. Topical superfusion of Bu-LTA (1 μg/ml) in rats significantly ( p < 0.05) increased adhesion within 30 min. By contrast, hot phenol-extracted LTA did not increase adhesion. Alkaline hydrolysis of Bu-LTA removed alanine residues and prevented adhesion. Also, pre-administration of anti-rat β 2-integrin antibody abolished Bu-LTA-induced adhesion. Finally, intraperitoneal injection of Bu-LTA (100 μg/ml) into mice also significantly ( p < 0.01) increased leukocyte adhesion measured at 60 min. In conclusion, Bu-LTA with intact alanine residues promotes β 2-integrin-dependent leukocyte adhesion in vivo.

Salivary lysozyme levels in patients with primary immunodeficiencies

Background Lysozyme is a muramidase that acts on the peptideoglycan wall of Gram positive bacteria, causing cell death. It plays part in innate immunity and is present in blood, external fluid, as well in Iysossomal granules of the phagocytes. Primary Immunodeficiencies are a diverse group of iIInesses that, as a result of abnormalities of the immune system, increase susceptibility to infection. Among the examples of impaired natural immunity are defects in phagocytes and in the complement system. Innate immunity could be important in protecting mucosas against infections in patients with different forms of primary immunodeficiencies. The aim of this study was to investigate Iysozyme concentrations in saliva from patients with primary immunodeficiencies. Methods Lysozyme levels in saliva samples from 34 patients with primary immunodeficiency (30 children and adolescents between the age of 3-13 years and 4 adults between the age of 20-33) and 60 agematched healthy controls (49 children and adolescents between the ages of 3-15 and 11 adults between the ages of 22-42) were determined by the Iysoplate method. Results There was no statistically significant difference between the Iysozyme concentrations in the saliva of the immunodeficient subjects and those of the healthy controls. Conclusion The results in the present work clearly show that salivary Iysozyme levels in primary immunodeficient patients are equivalent to those found in healthy controls, suggesting that this enzyme still represents a remaining (but not a compensatory mechanism), contributing to the protection of there patients against infections.

Interactions of bacterial lipopolysaccharide and peptidoglycan with a 70 kDa and an 80 kDa protein on the cell surface of CD14 + and CD14 − cells

Bacterial cell wall components, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) are known to stimulate cells of the immune, inflammatory and vascular systems contributing to septic shock. CD14 has been identified as the main LPS receptor, a process that is accelerated by the serum protein LPS-binding protein (LBP). CD14 has also been found to bind LTA and PGN from the cell wall of gram positive bacteria. Recently, toll-like receptor proteins TLR-2 and TLR-4 have been shown to be required for LPS and LTA-induced intracellular signalling. Although CD14 functions as either a glycosylphosphatidylinositol (GPI)-anchored molecule that does not transverse the cell membrane or as a soluble serum protein, the mechanisms by which the CD14-LPS/LTA complex interacts with the TLRs remains to be elucidated. We have looked directly for cell surface protein(s) that bind LPS or LTA in a CD14-dependent manner. Using biochemical approaches we have identified two proteins of molecular weight 70 kDa (LAP-1) and 80 kDa (LAP-2) that can be precipitated from both CD14 + and CD14 − cells with LPS- or LTA-specific antibodies. Binding of LPS and LTA to LAP-1 and -2 required serum. While soluble CD14 (sCD14) was sufficient to allow precipitation of these two proteins from CD14 − cells, serum could not be replaced by purified sCD14 and/or LBP when mCD14-expressing cells were used.

The possible role of porins in bacterial antibiotic resistance.

Excerpt Antibiotic agents are thought to diffuse freely through the cell wall of gram-positive bacteria. However, in gram-negative bacteria the diffusion of a given antibiotic agent depends on the ...

Hydration of synthetic polydialkylphosphate (PPF) ? a simplified model for natural teichoic acids

The hydration of synthetic poly(P-hydroxy 1,3 propylenephosphate) in hydrogen and mixed H+-Mg+2 forms has been studied by means of the pulsed NMR method. This polymer may be regarded as a simplified model of natural teichoic acids appearing in a cell wall of gram-positive bacteria. The investigations of nuclear magnetic relaxation of water protons in polymer gels prove the existence of two phases of water. One is water strongly bound to phosphate groups which does not freeze at low temperature and the other is weakly bound water freezing at 243–253 K. The distribution of correlation times has been stated for water in the first hydration shell of phosphate groups. For both acid and magnesium salt forms the hydration shell is composed of three water molecules.

Biosynthesis of the unit that links teichoic acid to the bacterial wall: Inhibition by tunicamycin

The walls of Gram-positive bacteria contain acidic polysaccharides and teichoic acids that are covalently linked through phosphodiesters to muramic acid residues in the peptidoglycan. It has been shown recently that in St@yZococcus agrees H [ 1 ] and in a mutant strain that lacks N-acetylglucosaminyl substituents on its wall teichoic acid [2] a ‘linkage unit’ that is a trimer of glycerol phosphate is interposed between the phosphate-terminal end of the teichoic acid chain and a muramic acid residue of the peptidoglycan to which it is attached. Evidence for similar linkage units has been found in Micrococcus sp. 2102 and in Bacillus subtilis W32 [J. Coley and E. Tarelli, unpublished work]. None of the bacteria examined contains a glycerol teichoic acid in its wall, and it has been demonstrated that the glycerol membrane teichoic acids are synthesized from phosphatidylglycerol [3,4]. Nevertheless, we have found [5] that membrane preparations from all of them catalyse the synthesis from CDP-glycerol of material containing a glycerol phosphate oligomer. Maximal synthesis of this product requires the addition of UDP-N-acetylglucosamine and the substrate for the synthesis of the backbone of the wall teichoic acid. The product is amphiphilic and appears to consist of the teichoic acid chain linked through the glycerol phosphate oligomer to a lipid acceptor. Bracha and Glaser [6] found that synthesis of the ribitol teichoic acid covalently linked to wall, in a wall-membrane preparation from S. aure’eus H, also required the addition of CDP-glycerol and UDP-N-acetylglucosamine. If therefore appears that teichoic acid attached to linkage unit is synthesized in

The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.