Mitochondrial ROS prime the hyperglycemic shift from apoptosis to necroptosis.

We have previously identified a shift from TNF-α-induced apoptosis to necroptosis that occurs under hyperglycemic conditions. This shift involves the downregulation or silencing of caspases and concurrent upregulation of necroptotic proteins leading to activation of the necrosome. In addition, under hyperglycemic conditions in vivo, this shift in cell death mechanisms exacerbates neonatal hypoxia-ischemia (HI) brain injury. Here, we identify two major factors that drive the hyperglycemic shift to necroptosis: (1) reactive oxygen species (ROS) and (2) receptor-interacting protein kinase 1 (RIP1). ROS, including mitochondrial superoxide, led to the oxidation of RIP1, as well as formation and activation of the necrosome. Concurrently, ROS mediate a decrease in the levels and activation of executioner caspases-3, -6, and -7. Importantly, hyperglycemia and mitochondrial ROS result in the oxidation of RIP1 and loss of executioner caspases prior to death receptor engagement by TNF-α. Moreover, RIP1 partially controlled levels of mitochondrial ROS in the context of hyperglycemia. As a result of its regulation of ROS, RIP1 also regulated necrosome activation and caspase loss. Mitochondrial ROS exacerbated neonatal HI-brain injury in hyperglycemic mice, as a result of the shift from apoptosis to necroptosis

Environmental pH modulates inerolysin activity via post-binding blockade

Abstract The cholesterol dependent cytolysins (CDCs) are a family of pore-forming toxins produced by a wide range of bacteria. Some CDCs are important virulence factors for their cognate organisms, but their activity must be tightly regulated to ensure they operate at appropriate times and within the appropriate subcellular compartments. pH-dependent activity has been described for several CDCs, but the mechanism of such regulation has been studied in depth only for listeriolysin O (LLO), which senses environmental pH through a triad of acidic residues that mediate protein unfolding. Here we present data supporting a distinct mechanism for pH-dependence for inerolysin (INY), the CDC produced by Lactobacillus iners. Inerolysin (INY) has an acidic pH optimum with loss of activity at neutral pH. INY pH-dependence is characterized by reversible loss of pore formation with preservation of membrane binding. Fluorescent membrane probe assays indicated that INY insertion into host cell membranes, but not oligomerization, was defective at neutral pH. These data support the existence of a newly appreciated form of CDC pH-dependence functioning at a late stage of pore formation

Proving Lipid Rafts Exist: Membrane Domains in the Prokaryote <i>Borrelia burgdorferi</i> Have the Same Properties as Eukaryotic Lipid Rafts

<div><p>Lipid rafts in eukaryotic cells are sphingolipid and cholesterol-rich, ordered membrane regions that have been postulated to play roles in many membrane functions, including infection. We previously demonstrated the existence of cholesterol-lipid-rich domains in membranes of the prokaryote, <i>B. burgdorferi</i>, the causative agent of Lyme disease [LaRocca <i>et al.</i> (2010) Cell Host & Microbe 8, 331–342]. Here, we show that these prokaryote membrane domains have the hallmarks of eukaryotic lipid rafts, despite lacking sphingolipids. Substitution experiments replacing cholesterol lipids with a set of sterols, ranging from strongly raft-promoting to raft-inhibiting when mixed with eukaryotic sphingolipids, showed that sterols that can support ordered domain formation are both <i>necessary</i> and <i>sufficient</i> for formation of <i>B. burgdorferi</i> membrane domains that can be detected by transmission electron microscopy or in <i>living</i> organisms by Förster resonance energy transfer (FRET). Raft-supporting sterols were also necessary and sufficient for formation of high amounts of detergent resistant membranes from <i>B. burgdorferi</i>. Furthermore, having saturated acyl chains was required for a biotinylated lipid to associate with the cholesterol-lipid-rich domains in <i>B. burgdorferi</i>, another characteristic identical to that of eukaryotic lipid rafts. Sterols supporting ordered domain formation were also necessary and sufficient to maintain <i>B. burgdorferi</i> membrane integrity, and thus critical to the life of the organism. These findings provide compelling evidence for the existence of lipid rafts and show that the same principles of lipid raft formation apply to prokaryotes and eukaryotes despite marked differences in their lipid compositions.</p></div

<i>B. burgdorferi</i> transfer ³H-cholesterol to HeLa cells.

<p><b>A.. </b><i>B. burgdorferi</i> utilized ³H-cholesterol as a substrate for cholesterol glycolipid synthesis; DPM's derived from scraped silica from HPTLC plate. <i>B. burgdorferi</i> incubated with ³H-cholesterol (black bars) utilized ³H-cholesterol as a substrate for cholesterol-glycolipid synthesis and incorporated the radiolabeled sterol into their lipid fraction when compared to unlabeled <i>B. burgdorferi</i> (gray bars). <b>B.</b> Exposure of the HPTLC plate to Kodak BioMax MR film without scraping demonstrates that the ³H-cholesterol was incorporated into the cholesterol-glycolipids. Radioactive cholesterol was not observed in <i>B. burgdorferi</i> incubated with unlabeled cholesterol (middle lane). This data supports the results from HPTLC exposed to UV light from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003109#ppat-1003109-g001" target="_blank">Figure 1E</a>. <b>C.</b> Spirochetes labeled with 10.0 µCi ³H-cholesterol transfer membrane lipids to HeLa cells. Spirochetes were incubated at an MOI of 1∶1, 10∶1, 20∶1, and 40∶1 with HeLa cells for 2 hours at 37°C in BSK-II. HeLa cells were analyzed for transfer by liquid scintillation counting. <b>D.. </b><i>B. burgdorferi</i> transfer ³H-cholesterol labeled glycolipids to HeLa cells. Radiolabeled <i>B. burgdorferi</i> were incubated with HeLa cells. The bacteria were removed and the HeLa cell lipid extracts were analyzed by liquid scintillation. Experiments <b>A, C,</b> and <b>D</b> represent the mean ± standard error of the mean from three separate experiments.</p

<i>B. burgdorferi</i> exchange BODIPY-cholesterol to HeLa cells.

<p><i>B. burgdorferi</i> labeled with BODIPY-cholesterol were incubated with HeLa cells for 15 min, 30 min, 1 hr, and 2 hr. <b>A–D:</b> HeLa cells exposed to <i>B. burgdorferi</i> labeled with BODIPY-cholesterol. <b>E–H:</b> HeLa cells exposed to conditioned medium from <i>B. burgdorferi</i> labeled with BODIPY-cholesterol. <b>I–J:</b> HeLa cells exposed to cell free wash supernatant from <i>B. burgdorferi</i> labeled with BODIPY-cholesterol for 2 hrs. <b>I.</b> Photograph of negative control representative of all temperatures; <b>J.</b> Phase contrast. Scale Bar = 20 µm. <b>K.</b> The mean relative fluorescence intensity (RFI) +/− standard error of the mean of HeLa cells from 10 microscope fields were calculated from the different experimental conditions. ANOVA ***p<0.001, ###p<0.001(negative control is significantly less than associated condition). <b>L.</b> Mean geometric fluorescence +/− standard error of the mean from three separate flow cytometry analysis of HeLa cells incubated following experimental conditions. ANOVA *p<0.05, *p<0.01 ***p<0.001, ##p<0.01, ###p<0.001(negative control is significantly less than associated experimental condition). <b>M–P.</b> Single confocal microscopy optical section illustrates that during the 2 hr incubation the <i>B. burgdorferi</i> (yellow) adhered to HeLa cells (green). Scale bar = 20 µm. <b>Q–T. </b><i>B. burgdorferi</i> derived cholesterol are processed by the HeLa cell and localize to the Golgi complex. Confocal microscopy demonstrated that the cis-Golgi marker, GM130 (red), co-localizes (yellow) with the BODIPY-cholesterol fluorescence (green). In the control image (<b>T</b>) where no <i>Borrelia</i> were added, the perinuclear localization (DAPI nuclear stain, blue) of GM130 is consistent with the other images. Scale bar = 20 µm.</p

<i>B. burgdorferi</i> attach to HeLa cells and acquire cholesterol from the epithelial cell membranes.

<p>HeLa cells incubated with BODIPY-cholesterol (green) and washed with MβCD were incubated with <i>B. burgdorferi</i> (red) for 1 hr at an MOI of 40∶1. Cells were fixed, stained with CB2 (red) and examined by confocal fluorescence microscopy. Four individual optical sections are shown at 2.0 µm, 2.5 µm, 3.0 µm and 3.5 µm. Confocal micrographs show colocalization (yellow, arrows) of BODIPY-cholesterol (green) and OspB (red) on the spirochete at the point of attachment. Scale bars = 10 µm. Confocal micrographs taken at the start of the experiment, 0 min panels (bottom), show that acquisition of BODIPY-cholesterol (green) has not occurred on the spirochetes (red) because there is no colocalization (yellow). The lack of colocalization is highlighted in the individual panels (arrows). Scale bars = 20 µm.</p

<i>B. burgdorferi</i> labeled with BODIPY-cholesterol release the fluorescent cholesterol probe in OMV.

<p><b>A.</b> BODIPY-cholesterol labeled and unlabeled <i>B. burgdorferi</i> release similar amounts of membrane material. DPH was added to the supernatants collected from <i>B. burgdorferi</i> labeled with BODIPY-cholesterol and unlabeled spirochetes to measure released OMV. Using the SpectraMaxM2, the DPH fluorescence was calculated for each sample at the specific time points. Supernatant from labeled <i>B. burgdorferi</i> (black); supernatant from unlabeled <i>B. burgdorferi</i> (gray). <b>B.</b> Transmission electron micrograph showing isolated vesicles from both labeled and unlabeled <i>B. burgdorferi</i> OMV. Immunogold labeling of OspB (18 nm) and <i>B. burgdorferi</i> glycolipids (6 nm) is seen throughout the vesicles. Scale Bar = 100 nM. Experiment <b>A</b> represents the mean ± standard error of the mean from three separate experiments.</p

<i>B. burgdorferi</i> incorporate BODIPY-cholesterol into its outer membrane as a component of the cholesterol-glycolipids.

<p><b>A.</b> Spirochetes were grown for 6 hrs in BSK-II media lacking cholesterol and their fluorescence was measured by spectrophotometry. 4.0 mg/L BODIPY-cholesterol in BSK-II without free cholesterol (green); 0.25% DMSO in BSK-II without free cholesterol (purple); 2.0 mg/L BODIPY-cholesterol BSK-II without free cholesterol (red); 0.175% DMSO in BSK-II without free cholesterol (dark blue); 0.2 mg/L BODIPY-cholesterol in BSK-II without free cholesterol (light blue); 0.0125% DMSO in BSK-II without free cholesterol (black). <b>B... </b><i>B. burgdorferi</i> incubated with and without BODIPY-cholesterol were analyzed by flow cytometry. ANOVA ***p<0.001. <b>C.</b> Unfixed, live spirochetes grown in the presence of 4.0 mg/L BODIPY-cholesterol for 4 hrs were observed by fluorescent microscopy. The spirochetes did not exhibit morphological or motility defects. Scale bar = 20 µm. <b>D.</b> Following incubation of <i>B. burgdorferi</i> with 4.0 mg/L BODIPY-cholesterol for 4 hrs, the spirochetes were grown in BSK-II for up to 96 hrs, and there were no significant differences in growth compared to controls. Untreated control (black diamond); labeled with BODIPY-cholesterol (blue square); Incubated with 0.25% DMSO (red triangle). <b>E... </b><i>B. burgdorferi</i> were treated and incubated with 4.0 mg/L. <i>B. burgdorferi</i> pellets were extracted by Bligh and Dyer lipid extraction and analyzed using a chloroform-methanol (85/15) HPTLC. The HPTLC plate was developed using iodine and exposure to UV light. <b>F... </b><i>B. burgdorferi</i> were grown in BSK-II media lacking free cholesterol with BODIPY-cholesterol or cholesterol as the primary source of sterol in the media to assess long term growth and cytotoxicity in the presence of exogenously added sterol. BSK-II (green); 4.0 mg/L BODIPY-cholesterol in cholesterol free BSK-II without serum (blue); 4.0 mg/L cholesterol in cholesterol free BSK-II without serum (black). Experiments <b>A, B, D,</b> and <b>F</b> represent the mean ± standard error of the mean from three separate experiments.</p