<i>Bacillus anthracis</i>Phospholipases C Facilitate Macrophage-Associated Growth and Contribute to Virulence in a Murine Model of Inhalation Anthrax

Heffernan, Brian J.; Thomason, Brendan; Herring-Palmer, Amy; Shaughnessy, Lee; McDonald, Rod A.; Fisher, Nathan; Huffnagle, Gary B.; Hanna, Philip C.

doi:10.1128/iai.00307-06

Cited by 47 publications

(46 citation statements)

References 46 publications

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“…In B. anthracis, PI-PLC is one of three phospholipase C enzymes that contribute to macrophage-associated growth of the bacteria (37). Along with aiding bacterial association with the macrophages, the three enzymes may have overlapping roles in pathogenesis by aiding in release from the phagocyte (38).…”

Section: Resultsmentioning

confidence: 99%

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

Fang

Redfield

et al. 2009

Journal of Biological Chemistry

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The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (X PC ), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31 P NMR methods to estimate internal correlation times ( c ) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 X PC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased c . PI-PLC activity and phospholipid c are constant between 0.1 and 0.5 X PC . Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC c , a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high X PC , kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.Bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) 2 enzymes aid in organism infectivity (1), whereas the structurally homologous mammalian PLC␦1 and related enzymes are required for phosphoinositide metabolism and are important for intracellular signaling (2, 3). These peripheral membrane proteins often have distinct binding modes for substrates and for other lipids that either anchor the protein to the surface or adjust its conformation to enhance catalysis (4). However, using traditional methods, it has been difficult to determine how lipid composition affects phospholipase binding and, conversely, how protein binding alters the lipid environment, particularly in multicomponent vesicles. Current methods for monitoring peripheral membrane protein binding to mixed component lipid vesicles, including centrifugation, gel filtration, and NMR, can provide information on bulk protein partitioning but do not usually provide insight into how the properties of the individual phospholipids change when protein is bound. Most of these methods also require protein concentrations well above the amounts used in enzyme kinetics. Therefore, even when binding to multicomponent vesicles can be measured, it has been difficult to separate how substrates (or substrate analogues)...

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Section: Resultsmentioning

confidence: 99%

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

Fang

Redfield

et al. 2009

Journal of Biological Chemistry

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“…Of these 19 ivi genes, five genes involved in regulation or signalling were identified. Except Va-iv-3, the other four homology genes have been demonstrated to be involved directly or indirectly in bacterial pathogenesis (Heffernan et al 2006;McArthur et al 2006;Ha 2007). For example, Va-iv-4 encoded a glycine cleavage operon activator GcvA, which belongs to LysR family protein and activates GcvTHP and GcvB in response to glycine.…”

Section: Discussionmentioning

confidence: 99%

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Zou

Hao

et al. 2010

Letters in Applied Microbiology

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Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo‐induced antigen technology (IVIAT). Methods and Results: The convalescent‐phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo‐induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion‐mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo‐expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

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“…PbPL-null sporozoites are impaired in their ability to pass through cell monolayers in vitro and to migrate through mammalian tissues to reach the liver, whereas PbLP-deficient liver forms have delayed release from liver cells, presumably due to a defect in PV membrane disruption. More broadly, several intracellular bacteria also secrete phospholipases to break down host membranes and establish successful infections in mammalian hosts (53,54).…”

Section: Discussionmentioning

confidence: 99%

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

Pszenny

Ehrenman

Romano

et al. 2016

Journal of Biological Chemistry

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The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases "AHSLG" and the catalytic triad consisting of serine, aspartate, and histidine (SDH) from LCAT enzymes. TgL-CAT is secreted by the parasite, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals, and exhibit delays in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the developmental stage of the parasite.The phospholipase A 2 (PLA 2 ) 2 family of serine lipases comprises more than 30 enzymes in mammals. The PLA 2 members hydrolyze the sn-2 ester of phospholipids, yielding lysophospholipid and releasing a fatty acid (1). Approximately one-third of the PLA 2 family members are secreted from cells and display various localizations post-secretion, reflecting their specialized biological functions (2). Among the secretory PLA 2 , lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) is characterized by dual activity, PLA 2 and acyltransferase. In mammals, this enzyme catalyzes the transacylation of the sn-2 fatty acid liberated from various phospholipids (e.g. phosphatidylcholine or phosphatidylethanolamine) to the 3-␤-hydroxyl group on the A-ring of cholesterol, thereby forming cholesteryl esters (3-5). The primary sequence of LCAT is well conserved between mammalian species (6). A structural model for LCAT predicts the conformation of a catalytic triad formed by SerAsp-His residues involved in the phospholipase reaction (7). Mammalian LCATs are primarily expressed in the liver and secreted to the plasma where they circulate in association with HDL (8). These enzymes are components of the reverse cholesterol transport pathway by which cholesterol from peripheral cells is delivered to the liver f...

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Bacillus anthracisPhospholipases C Facilitate Macrophage-Associated Growth and Contribute to Virulence in a Murine Model of Inhalation Anthrax

Cited by 47 publications

References 46 publications

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

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