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ABSTRACT: The Helicobacter pylori virulence factor CagA targets a variety of host proteins to alter different cellular responses, including the induction of pro-inflammatory cytokines. We have previously shown that CagA-facilitated lysine 63-linked ubiquitination of TAK1 is essential for the H. pylori-induced NF-κB activation and the expression of proinflammatory cytokines. However, the molecular mechanism for TAK1 ubiquitination and activation in H. pylori-mediated NF-κB activation remains elusive. Here, we identify lysine 158 of TAK1 as the key residue undergoing lysine 63-linked ubiquitination in response to H. pylori infection. Mutation of lysine 158 to arginine prevents the ubiquitination of TAK1 and impairs H. pylori-induced TAK1 and NF-κB activation. Moreover, we demonstrate that E2 ubiquitin conjugating enzyme Ubc13 is involved in H. pylori-mediated TAK1 ubiquitination. Suppressing the activity of Ubc13 by a dominant-negative mutant or siRNA abolishes CagA-facilitated and H. pylori-induced TAK1 and NF-κB activation. These findings further underscore the importance of lysine 63-linked ubiquitination of TAK1 in H. pylori-induced NF-κB activation and NF-κB-mediated inflammatory response. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
Journal of Cellular Biochemistry 04/2013; · 2.87 Impact Factor
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Michael C Brothers,
Mengfei Ho,
Ram Maharjan,
Nathan C Clemons,
Yuka Bannai,
Mark A Waites,
Melinda J Faulkner,
Theresa B Kuhlenschmidt,
Mark S Kuhlenschmidt, Steven R Blanke,
Chad M Rienstra,
Brenda A Wilson
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ABSTRACT: Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.
FEBS Journal 09/2011; 278(23):4633-48. · 3.79 Impact Factor
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ABSTRACT: A number of pathogenic bacteria target mitochondria to modulate the host's apoptotic machinery. Studies here revealed that infection with the human gastric pathogen Helicobacter pylori disrupts the morphological dynamics of mitochondria as a mechanism to induce host cell death. The vacuolating cytotoxin A (VacA) is both essential and sufficient for inducing mitochondrial network fragmentation through the mitochondrial recruitment and activation of dynamin-related protein 1 (Drp1), which is a critical regulator of mitochondrial fission within cells. Inhibition of Drp1-induced mitochondrial fission within VacA-intoxicated cells inhibited the activation of the proapoptotic Bcl-2-associated X (Bax) protein, permeabilization of the mitochondrial outer membrane, and cell death. Our data reveal a heretofore unrecognized strategy by which a pathogenic microbe engages the host's apoptotic machinery.
Proceedings of the National Academy of Sciences 09/2011; 108(38):16032-7. · 9.68 Impact Factor
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ABSTRACT: Aerobic respiration in bacteria, Archaea, and mitochondria is performed by oxygen reductase members of the heme-copper oxidoreductase superfamily. These enzymes are redox-driven proton pumps which conserve part of the free energy released from oxygen reduction to generate a proton motive force. The oxygen reductases can be divided into three main families based on evolutionary and structural analyses (A-, B- and C-families), with the B- and C-families evolving after the A-family. The A-family utilizes two proton input channels to transfer protons for pumping and chemistry, whereas the B- and C-families require only one. Generally, the B- and C-families also have higher apparent oxygen affinities than the A-family. Here we use whole cell proton pumping measurements to demonstrate differential proton pumping efficiencies between representatives of the A-, B-, and C-oxygen reductase families. The A-family has a coupling stoichiometry of 1 H(+)/e(-), whereas the B- and C-families have coupling stoichiometries of 0.5 H(+)/e(-). The differential proton pumping stoichiometries, along with differences in the structures of the proton-conducting channels, place critical constraints on models of the mechanism of proton pumping. Most significantly, it is proposed that the adaptation of aerobic respiration to low oxygen environments resulted in a concomitant reduction in energy conservation efficiency, with important physiological and ecological consequences.
Proceedings of the National Academy of Sciences 08/2011; 108(34):14109-14. · 9.68 Impact Factor
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ABSTRACT: The lantibiotic nisin inhibits growth of vegetative Gram-positive bacteria by binding to lipid II, which disrupts cell wall biosynthesis and facilitates pore formation. Nisin also inhibits the outgrowth of bacterial spores, including spores of Bacillus anthracis, whose structural and biochemical properties are fundamentally different from those of vegetative bacteria. The molecular basis of nisin inhibition of spore outgrowth had not been identified, as previous studies suggested that inhibition of spore outgrowth involved either covalent binding to a spore target or loss of membrane integrity; disruption of cell wall biosynthesis via binding to lipid II had not been investigated. To provide insights into the latter possibility, the effects of nisin were compared with those of vancomycin, another lipid II binding antibiotic that inhibits cell wall biosynthesis but does not form pores. Nisin and vancomycin both inhibited the replication of vegetative cells, but only nisin inhibited the transition from a germinated spore to a vegetative cell. Moreover, vancomycin prevented nisin's activity in competition studies, suggesting that the nisin-lipid II interaction is important for inhibition of spore outgrowth. In experiments with fluorescently labeled nisin, no evidence was found for a covalent mechanism for inhibition of spore outgrowth. Interestingly, mutants in the hinge region (N20P/M21P and M21P/K22P) that still bind lipid II but cannot form pores had potent antimicrobial activity against vegetative B. anthracis cells but did not inhibit spore outgrowth. Therefore, pore formation is essential for the latter activity but not the former. Collectively, these studies suggest that nisin utilizes lipid II as the germinated spore target during outgrowth inhibition and that nisin-mediated membrane disruption is essential to inhibit spore development into vegetative cells.
ACS Chemical Biology 05/2011; 6(7):744-52. · 6.45 Impact Factor
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ABSTRACT: Enzyme inhibition via allostery, in which the ligand binds remotely from the active site, is a poorly understood phenomenon and represents a significant challenge to structure-based drug design. Dipicolinic acid (DPA), a major component of Bacillus spores, is shown to inhibit glutamate racemase from Bacillus anthracis , a monosubstrate/monoproduct enzyme, in a novel allosteric fashion. Glutamate racemase has long been considered an important drug target for its integral role in bacterial cell wall synthesis. The DPA binding mode was predicted via multiple docking studies and validated via site-directed mutagenesis at the binding locus, while the mechanism of inhibition was elucidated with a combination of Blue Native polyacrylamide gel electrophoresis, molecular dynamics simulations, and free energy and pK(a) calculations. Inhibition by DPA not only reveals a novel cryptic binding site but also represents a form of allosteric regulation that exploits the interplay between enzyme conformational changes, fluctuations in the pK(a) values of buried residues and catalysis. The potential for future drug development is discussed.
The Journal of Physical Chemistry B 03/2011; 115(13):3416-24. · 3.70 Impact Factor
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ABSTRACT: During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.
Using an in vitro model of infection, we evaluated the influence of the germination state of B. anthracis spores, as controlled by defined culture conditions, on the outcome of infection. Spores prepared from B. anthracis Sterne 7702 germinated in a variety of common cell culture media supplemented with fetal bovine serum (FBS) while, in the absence of FBS, germination was strictly dependent on medium composition. RAW264.7 macrophage-like cells internalized spores to the same extent in either germinating or non-germinating media. However, significantly more viable, intracellular B. anthracis were recovered from cells infected under non-germinating conditions compared to germinating conditions. At the same time, RAW264.7 cells demonstrated a significant loss in viability when infected under non-germinating conditions.
These results suggest that the outcome of host cell infection is sensitive to the germination state of spores at the time of uptake. Moreover, this study demonstrates the efficacy of studying B. anthracis spore infection of host cells within a defined, non-germinating, in vitro environment.
BMC Microbiology 02/2011; 11:46. · 3.04 Impact Factor
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ABSTRACT: Plasma membrane sphingomyelin (SM) binds the Helicobacter pylori vacuolating toxin (VacA) to the surface of epithelial cells. To evaluate the importance of SM for VacA cellular entry, we characterized toxin uptake and trafficking within cells enriched with synthetic variants of SM, whose intracellular trafficking properties are strictly dependent on the acyl chain lengths of their sphingolipid backbones. While toxin binding to the surface of cells was independent of acyl chain length, cells enriched with 12- or 18-carbon acyl chain variants of SM (e.g. C12-SM or C18-SM) were more sensitive to VacA, as indicated by toxin-induced cellular vacuolation, than those enriched with shorter 2- or 6-carbon variants (e.g. C2-SM or C6-SM). In C18-SM-enriched cells, VacA was taken into cells by a previously described Cdc42-dependent pinocytic mechanism, localized initially to GPI-enriched vesicles, and ultimately trafficked to Rab7/Lamp1 compartments. In contrast, within C2-SM-enriched cells, VacA was taken up at a slower rate by a Cdc42-independent mechanism and trafficked to Rab11 compartments. VacA-associated predominantly with detergent-resistant membranes (DRMs) in cells enriched with C18-SM, but predominantly with non-DRMs in C2-SM-enriched cells. These results suggest that SM is required for targeting VacA to membrane rafts important for subsequent Cdc42-dependent pinocytic cellular entry.
Cellular Microbiology 10/2010; 12(10):1517-33. · 5.46 Impact Factor
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ABSTRACT: Glutamate racemase is an attractive antimicrobial drug target. Virtual screening using a transition-state conformation of the enzyme resulted in the discovery of several μM competitive inhibitors, dissimilar from current amino acid-like inhibitors, providing novel scaffolds for drug discovery. The most effective of these competitive inhibitors possesses a very high ligand efficiency value of -0.6 kcal/mol/heavy atom, and is effective against three distinct glutamate racemases representing two species of Bacillus. The benefits of employing the transition-state conformation of the receptor in virtual screening are discussed.
ACS Medicinal Chemistry Letters 04/2010; 1(1):9-13. · 3.36 Impact Factor
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EMBO Reports 01/2010; 11(1):11-2. · 7.36 Impact Factor
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ABSTRACT: Chronic infection of the human stomach by Helicobacter pylori is an important risk factor for gastric cancer. H. pylori produces a cache of virulence factors that promote colonization and persistence, which, in turn, contributes to a robust inflammatory response at the host-pathogen interface. Recently, we reported that H. pylori activates the abundant nuclear regulator poly(ADP-ribose) polymerase (PARP)-1, resulting in the production of the catabolite poly(ADP-ribose) (PAR). PARP-1 is emerging as a key player in establishing homeostasis at the host-pathogen interface. In this article, we summarize the discovery of H. pylori-dependent PARP-1 activation, and discuss potential roles for PARP-1 in H. pylori-mediated gastric disease. In light of the remarkable successes that have reported for treating inflammatory disorders and cancers with PARP-1 inhibitors, we discuss the prospects of targeting PARP-1 for treatment of H. pylori-associated gastric disease.
Gut Microbes 01/2010; 1(6):373-8.
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ABSTRACT: Modification of eukaryotic proteins is a powerful strategy used by pathogenic bacteria to modulate host cells during infection. Previously, we demonstrated that Helicobacter pylori modify an unidentified protein within mammalian cell lysates in a manner consistent with the action of a bacterial ADP-ribosylating toxin. Here, we identified the modified eukaryotic factor as the abundant nuclear factor poly(ADP-ribose) polymerase-1 (PARP-1), which is important in the pathologies of several disease states typically associated with chronic H. pylori infection. However, rather than being ADP-ribosylated by an H. pylori toxin, the intrinsic poly(ADP-ribosyl) polymerase activity of PARP-1 is activated by a heat- and protease-sensitive H. pylori factor, resulting in automodification of PARP-1 with polymers of poly(ADP-ribose) (PAR). Moreover, during infection of gastric epithelial cells, H. pylori induce intracellular PAR-production by a PARP-1-dependent mechanism. Activation of PARP-1 by a pathogenic bacterium represents a previously unrecognized strategy for modulating host cell signaling during infection.
Proceedings of the National Academy of Sciences 11/2009; 106(47):19998-20003. · 9.68 Impact Factor
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ABSTRACT: Helicobacter pylori-initiated chronic gastritis is characterized by the cag pathogenicity island-dependent upregulation of proinflammatory cytokines, which is largely mediated by the transcription factor nuclear factor (NF)-kappaB. However, the cag pathogenicity island-encoded proteins and cellular signalling molecules that are involved in H. pylori-induced NF-kappaB activation and inflammatory response remain unclear. Here, we show that H. pylori virulence factor CagA and host protein transforming growth factor-beta-activated kinase 1 (TAK1) are essential for H. pylori-induced activation of NF-kappaB. CagA physically associates with TAK1 and enhances its activity and TAK1-induced NF-kappaB activation through the tumour necrosis factor receptor-associated factor 6-mediated, Lys 63-linked ubiquitination of TAK1. These findings show that polyubiquitination of TAK1 regulates the activation of NF-kappaB, which in turn is used by H. pylori CagA for the H. pylori-induced inflammatory response.
EMBO Reports 10/2009; 10(11):1242-9. · 7.36 Impact Factor
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Steven R Blanke
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ABSTRACT: After she stepped through a looking glass into a mirror image of her own world, Lewis Carroll's adventuress Alice soon recognized
that the two worlds were perhaps not so similar after all. She intuits that something about the very make-up of “looking-glass
milk” was fundamentally different from that of the wholesome beverage she typically enjoyed (1). Indeed, nature has stocked our universe with biological substances that exist as two mirror image forms of one another.
Nineteen of the 20 naturally occurring amino acids that make up proteins are “chiral,” meaning that each can be arranged in
two orientations around a central carbon atom. The result is a mixture of “mirror-image compounds” called l- and d-amino acids, which cannot be superimposed (see the figure), much in the way a person's left and right hands are not superimposable.
One of the great mysteries of life has been the emergence of a strictly “left-handed” protein world where great attention
has been paid to the l-amino acid building blocks (2–4), while d-amino acids are largely regarded as red-headed cousins who are easy to ignore. On page 1552 of this issue, Lam et al. challenge this perception, describing a role for d-amino acids in controlling bacterial responses to environmental cues (5).
Science 09/2009; 325(5947):1505-6. · 31.20 Impact Factor
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ABSTRACT: Glutamate racemases (EC 5.1.1.3) catalyze the cofactor-independent stereoinversion of D- and L-glutamate and are important for viability in several gram-negative and -positive bacteria. As the only enzyme involved in the stereoinversion of L- to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents. However, the development of competitive tight-binding inhibitors has been problematic and highly species specific. Despite a number of recent crystal structures of cofactor-independent epimerases and racemases, cocrystallized with substrates or substrate analogues, the source of these enzymes' catalytic power and their ability to acidify the C alpha of amino acids remains unknown. The present integrated computational and experimental study focuses on the glutamate racemase from Bacillus subtilis (RacE). A particular focus is placed on the interaction of the glutamate carbanion intermediate with RacE. Results suggest that the reactive form of the RacE-glutamate carbanion complex, vis-à-vis proton abstraction from C alpha, is significantly different than the RacE-D-glutamate complex on the basis of the crystal structure and possesses dramatically stronger enzyme-ligand interaction energy. In silico and experimental site-directed mutagenesis indicates that the strength of the RacE-glutamate carbanion interaction energy is highly distributed among numerous electrostatic interactions in the active site, rather than being dominated by strong hydrogen bonds. Results from this study are important for laying the groundwork for discovery and design of high-affinity ligands to this class of cofactor-independent racemases.
Journal of the American Chemical Society 05/2009; 131(14):5274-84. · 9.91 Impact Factor
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Tae Kyung Kim,
Susan M Thomas,
Mengfei Ho,
Shobha Sharma,
Claudia I Reich,
Jeremy A Frank,
Kathleen M Yeater,
Diana R Biggs,
Noriko Nakamura,
Rebecca Stumpf,
Steven R Leigh,
Richard I Tapping, Steven R Blanke,
James M Slauch,
H Rex Gaskins,
Jon S Weisbaum,
Gary J Olsen,
Lois L Hoyer,
Brenda A Wilson
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ABSTRACT: Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.
Journal of clinical microbiology 02/2009; 47(4):1181-9. · 4.16 Impact Factor
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Soumitra Barua,
Matthew McKevitt,
Kevin DeGiusti,
Elaine E Hamm,
Jason Larabee,
Salika Shakir,
Katie Bryant,
Theresa M Koehler, Steven R Blanke,
David Dyer,
Allison Gillaspy,
Jimmy D Ballard
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ABSTRACT: In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.
Infection and immunity 11/2008; 77(1):23-31. · 4.21 Impact Factor
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ABSTRACT: The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC(50)) and an IC(90) of 0.57 microM and 0.90 microM, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC(90). These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.
Antimicrobial Agents and Chemotherapy 10/2008; 52(12):4281-8. · 4.84 Impact Factor
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ABSTRACT: Helicobacter pylori VacA is a secreted pore-forming toxin that is comprised of two domains, designated p33 and p55. The p55 domain has an important role in the binding of VacA to eukaryotic cell surfaces. A total of 111 residues at the amino terminus of p55 (residues 312 to 422) are essential for the intracellular activity of VacA, which suggests that this region may constitute a subdomain with an activity distinct from cell binding. To investigate the properties of this subdomain, a small deletion mutation (targeting aspartic acid 346 and glycine 347) was introduced into the H. pylori chromosomal vacA gene. Similar to wild-type VacA, the VacA Delta346-347 mutant protein was proteolytically processed, secreted, and bound to eukaryotic cells. However, VacA Delta346-347 did not cause cell vacuolation or membrane depolarization, and it was impaired in the ability to assemble into large water-soluble oligomeric structures. Interestingly, VacA Delta346-347 was able to physically interact with wild-type VacA to form mixed oligomeric complexes, and VacA Delta346-347 inhibited wild-type vacuolating activity in a dominant-negative manner. These data indicate that the assembly of functional oligomeric VacA complexes is dependent on specific sequences, including amino acids 346 and 347, within the p55 amino-terminal subdomain.
Infection and immunity 08/2008; 76(7):2843-51. · 4.21 Impact Factor
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ABSTRACT: The engulfment of Bacillus anthracis spores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a high-throughput, nonsubjective manner, we labeled B. anthracis Sterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.
Applied and environmental microbiology 07/2008; 74(16):5201-10. · 3.69 Impact Factor
