16 research outputs found
AGEs Secreted by Bacteria Are Involved in the Inflammatory Response
Advanced Glycated End Products (AGEs) are formed by non-enzymatic protein glycation and are implicated in several physiological aspects including cell aging and diseases. Recent data indicate that bacteria – although short lived – produce, metabolize and accumulate AGEs. Here we show that Escherichia coli cells secret AGEs by the energy-dependent efflux pump systems. Moreover, we show that in the presence of these AGEs there is an upshift of pro-inflammatory cytokins by mammalian cells. Thus, we propose that secretion of AGEs by bacteria is a novel avenue of bacterial-induced inflammation which is potentially important in the pathophysiology of bacterial infections. Moreover, the sensing of AGEs by the host cells may constitute a warning system for the presence of bacteria
Metabolism of AGEs--bacterial AGEs are degraded by metallo-proteases.
Advanced Glycation End Products (AGEs) are the final products of non-enzymatic protein glycation that results in loss of protein structure and function. We have previously shown that in E. coli AGEs are continually formed as high-molecular weight protein complexes. Moreover, we showed that AGEs are removed from the cells by an active, ATP-dependent secretion and that these secreted molecules have low molecular weight. Taken together, these results indicate that E. coli contains a fraction of low molecular weight AGEs, in addition to the high-molecular weight AGEs. Here we show that the low-molecular weight AGEs originate from high-molecular weight AGEs by proteolytic degradation. Results of in-vitro and in vivo experiments indicated that this degradation is carried out not by the major ATP-dependent proteases that are responsible for the main part of bacterial protein quality control but by an alternative metal-dependent proteolysis. This proteolytic reaction is essential for the further secretion of AGEs from the cells. As the biochemical reactions involving AGEs are not yet understood, the implication of a metalloprotease in breakdown of high molecular weight AGEs and their secretion constitutes an important step in the understanding of AGEs metabolism
Effect of protein synthesis arrest on AGEs secretion.
<p>Bacteria were grown in MOPS minimal medium and samples were collected for AGEs determination as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017974#s4" target="_blank">materials and methods</a>. AGEs-specific fluorescence (Ex. 370, Em. 440) was determined and normalized to cells density. A) Intracellular and extracellular AGEs levels following exposure to chloramphenicol. B) Effect of chloramphenicol (open circles) and Arsenate (triangles) on AGEs secretion kinetics. The data represent three independent experiments.</p
Effect of metallo-protease inhibition on AGEs accumulation and secretion.
<p>Bacterial growth and sample collection were described in materials and methods. AGEs-specific fluorescence was determined and normalized to cells density. A) Extracellular fraction was further separated into proteins and low molecular weight compounds fractions. B) Effect of 100 µM and 1 mM of 1,10 phenantholine (Sigma) on intracellular AGEs accumulation. C) Effect of 100 µM of 1,10 phenantholine (Sigma) on AGEs secretion after 2 hours in a non-growing cultures.</p
Effect of extracellular AGEs on secretion of TNF-alpha by THP-1 cells.
<p>The levels of TNF-alpha secreted by THP-1 cells were measured using sandwich-ELISA (see methods). A) TNF-alpha levels following exposure to elevated concentration of AGEs-containing fraction. B) Relative TNF-alpha levels secreted from THP-1 cells following exposure to the extracellular fractions (1/80 stock dilutions in distilled water) secreted from wild type cells, peprazine-treated cells and Δ<i>tolc</i> mutant cells. The data represent three independent experiments.</p
Size profile of AGEs following metallo-protease inhibition <i>in-vivo</i>.
<p>Effect of 100 µM 1,10 phenantholine (Sigma) on AGEs degradation. Bacteria were grown as described in materials and methods. Lysate was extracted from treated and untreated cells and further separated by size using HiLoad 16/600 Superdex 75 pg 120 ml filtration column. After 30 ml of elution, 4 ml fractions were collected and AGEs specific fluorescence was determined. </p
Effect of arsenate and chloramphenicol on AGEs size profile.
<p>Lysates were extracted from exponentially growing cultures at time intervals after addition of arsenate and chloramphenicol. Samples were separated into proteins and low molecular weight compounds fractions, as described in Materials and Methods. AGEs-specific fluorescence was monitored and normalized to cell density. AGEs level in the high molecular weight proteins fraction (full circles) and low-molecular weight fractions (empty circles). </p
AGEs degradation <i>in-vitro</i>.
<p>Lysates were extracted from exponentially growing cultures and separated into high and low-molecular-mass fractions, as described in Materials and Methods. The high molecular weight fraction was incubated at 37°C for 3 hours with various protease inhibitors and then separated again into high and low-molecular-mass fractions. AGE-specific fluorescence was determined. The graph represents the percentage of low-molecular-mass fraction from the total AGEs in an untreated sample. The treatments were with 3M urea, protease inhibitor cocktail (Sigma), 25 mM MPSF (serine-protease inhibitor), 10 mM EDTA (metallo-protease inhibitor), 0.3 mM E-64 (cystein-protease inhibitor), 0.3 mM Pepstatin A (aspartate -protease inhibitor) and 2 mM Bestatin (amino-protease inhibitor).</p
AGEs size exultation filtration <i>in-vitro</i>.
<p>Lysate was extracted from exponentially growing cultures and further separated into high and low-molecular-mass fractions, as described in Materials and Methods. The high molecular weight fraction was treated and incubated at 37°C for 3 hours and then separated by size using HiLoad 16/600 Superdex 75 pg 120 ml filtration column. After 30 ml of elution, 4 ml fractions were collected and AGEs specific fluorescence was determined. The graph represents the size distribution of AGEs in an untreated sample, lystae treated with 3M urea and lystae treated with protease inhibitor cocktail (Sigma). </p
Effect of an efflux pumps inhibitor and <i>tolC</i> deletion mutant on AGEs secretion.
<p>Effect of A) 50 ng/ml and 100 ng/ml of piperazine or B) <i>tolC</i> deletion on the kinetics of AGEs secretion. The data represent three independent experiments.</p