Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells

Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml-1 LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo®) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker® Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells. © 2016 John Wiley & Sons A/S

Inhibition of LtxA Toxicity by Blocking Cholesterol Binding with Peptides

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of 334LEEYSKR340, in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC336 motif of LtxA (CRAC336WT). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controllinginfections of repeats-in-toxin-producing organisms. © 2016 John Wiley & Sons A/S

Membrane Association and Destabilization by Aggregatibacter Actinomycetemcomitans Leukotoxin Requires Changes in Secondary Structures

Summary: Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin\u27s pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell\u27s plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Aggregatibacter Actinomycetemcomitans Leukotoxin is Post-Translationally Modified by Addition of Either Saturated or Hydroxylated Fatty Acyl Chains

Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27-29%) and palmitolyl (C16:1 cis Δ9, 43-44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12-14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys562 and Lys687 are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coliα-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys562 and Lys687 with Arg blocks acylation, resulting in a \u3e75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these post-translational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity. © 2011 John Wiley & Sons A/S

Aggregatibacter Actinomycetemcomitans Leukotoxin is Post-Translationally Modified by Addition of Either Saturated or Hydroxylated Fatty Acyl Chains

Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27–29%) and palmitolyl (C16:1 cis Δ9, 43–44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12–14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys562 and Lys687 are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coli α-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys562 and Lys687 with Arg blocks acylation, resulting in a \u3e75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these posttranslational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity

Membrane Localization of the Repeats-in-Toxin (RTX) Leukotoxin (LtxA) Produced by Aggregatibacter Actinomycetemcomitans

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane. © 2018 Brown et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

GRB 180418A:A Possibly Short Gamma-Ray Burst with a Wide-angle Outflow in a Faint Host Galaxy

We present X-ray and multiband optical observations of the afterglow and host galaxy of GRB 180418A, discovered by Swift/BAT and Fermi/GBM. We present a reanalysis of the GBM and BAT data deriving durations of the prompt emission of T 90 ≈ 2.56 and 1.90 s, respectively. Modeling the Fermi/GBM catalog of 1405 bursts (2008-2014) in the hardness-T 90 plane, we obtain a probability of ≈60% that GRB 180418A is a short-hard burst. From a combination of Swift/XRT and Chandra observations, the X-ray afterglow is detected to ≈38.5 days after the burst and exhibits a single power-law decline with F X ∝ t -0.98. Late-time Gemini observations reveal a faint r ≈ 25.69 mag host galaxy at an angular offset of ≈0.″16. At the likely redshift range of z ≈ 1-2.25, we find that the X-ray afterglow luminosity of GRB 180418A is intermediate between short and long gamma-ray bursts (GRBs) at all epochs during which there are contemporaneous data and that GRB 180418A lies closer to the E γ,peak-E γ,iso correlation for short GRBs. Modeling the multiwavelength afterglow with the standard synchrotron model, we derive the burst explosion properties and find a jet opening angle of θ j 9°-14°. If GRB 180418A is a short GRB that originated from a neutron star merger, it has one of the brightest and longest-lived afterglows along with an extremely faint host galaxy. If, instead, the event is a long GRB that originated from a massive star collapse, it has among the lowest-luminosity afterglows and lies in a peculiar space in terms of the hardness-T 90 and E γ,peak-E γ,iso planes. </p

GRB 180418A:A Possibly Short Gamma-Ray Burst with a Wide-angle Outflow in a Faint Host Galaxy

We present X-ray and multiband optical observations of the afterglow and host galaxy of GRB 180418A, discovered by Swift/BAT and Fermi/GBM. We present a reanalysis of the GBM and BAT data deriving durations of the prompt emission of T 90 ≈ 2.56 and 1.90 s, respectively. Modeling the Fermi/GBM catalog of 1405 bursts (2008-2014) in the hardness-T 90 plane, we obtain a probability of ≈60% that GRB 180418A is a short-hard burst. From a combination of Swift/XRT and Chandra observations, the X-ray afterglow is detected to ≈38.5 days after the burst and exhibits a single power-law decline with F X ∝ t -0.98. Late-time Gemini observations reveal a faint r ≈ 25.69 mag host galaxy at an angular offset of ≈0.″16. At the likely redshift range of z ≈ 1-2.25, we find that the X-ray afterglow luminosity of GRB 180418A is intermediate between short and long gamma-ray bursts (GRBs) at all epochs during which there are contemporaneous data and that GRB 180418A lies closer to the E γ,peak-E γ,iso correlation for short GRBs. Modeling the multiwavelength afterglow with the standard synchrotron model, we derive the burst explosion properties and find a jet opening angle of θ j 9°-14°. If GRB 180418A is a short GRB that originated from a neutron star merger, it has one of the brightest and longest-lived afterglows along with an extremely faint host galaxy. If, instead, the event is a long GRB that originated from a massive star collapse, it has among the lowest-luminosity afterglows and lies in a peculiar space in terms of the hardness-T 90 and E γ,peak-E γ,iso planes. © 2021. The American Astronomical Society. All rights reserved..Immediate accessThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The SOS Pilot Study: a RCT of routine oxygen supplementation early after acute stroke—effect on recovery of neurological function at one week

Mild hypoxia is common after stroke and associated with poor long-term outcome. Oxygen supplementation could prevent hypoxia and improve recovery. A previous study of routine oxygen supplementation showed no significant benefit at 7 and 12 months. This pilot study reports the effects of routine oxygen supplementation for 72 hours on oxygen saturation and neurological outcomes at 1 week after a stroke