Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth

Toxoplasma gondii is an intracellular protozoan parasite capable of causing devastating infections in immunocompromised and immunologically immature individuals. In this report, we demonstrate the relative independence of T. gondii from its host cell for aminoglycerophospholipid synthesis. The parasite can acquire the lipid precursors serine, ethanolamine, and choline from its environment and use them for the synthesis of its major lipids, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), respectively. Dimethylethanolamine (Etn(Me)(2)), a choline analog, dramatically interfered with the PtdCho metabolism of T. gondii and caused a marked inhibition of its growth within human foreskin fibroblasts. In tissue culture medium supplemented with 2 mm Etn(Me)(2), the parasite-induced lysis of the host cells was dramatically attenuated, and the production of parasites was inhibited by more than 99%. The disruption of parasite growth was paralleled by structural abnormalities in its membranes. In contrast, no negative effect on host cell growth and morphology was observed. The data also reveal that the Etn(Me)(2)-supplemented parasite had a time-dependent decrease in its PtdCho content and an equivalent increase in phosphatidyldimethylethanolamine, whereas other major lipids, PtdSer, PtdEtn, and PtdIns, remained largely unchanged. Relative to host cells, the parasites incorporated more than 7 times as much Etn(Me)(2) into their phospholipid. These findings reveal that Etn(Me)(2) selectively alters parasite lipid metabolism and demonstrate how selective inhibition of PtdCho synthesis is a powerful approach to arresting parasite growth.     

BCL-xL-dependent light scattering by apoptotic cells

We measured the intensity ratio of wide-to-narrow angle scatter, optical scatter image ratio (OSIR), in single cells during apoptosis and after overexpression of the mitochondria-bound antiapoptotic protein BCL-xL. OSIR is sensitive to particle size/shape for objects with wavelength-scale dimensions, and was used as a morphometric measure of cellular response. Three cell variants were treated with staurosporine (STS): nontransfected parental CSM14.1, CSM14.1 stably expressing yellow fluorescent protein (YFP) with diffuse YFP fluorescence, and apoptosis-resistant CSM14.1 stably expressing the fusion protein construct YFP-BCL-xL with YFP fluorescence localized on the mitochondria. After treatment with 1 or 2 muM STS, the measured OSIR decreased monotonically by approximately 25% in the nontransfected and YFP variants, and reached a steady-state value 40-60 min after STS treatment. The decrease in OSIR at the onset of apoptosis preceded phosphatidyl serine exposure by 5 h. In the YFP-BCL-xL cell variant, the initial OSIR was already approximately 24% lower than the initial OSIR in YFP and nontransfected cells, and only decreased by <10% after STS treatment. Alterations in light scattering by cells overexpressing BCL-xL even before apoptosis induction raise interesting questions as to the role of BCL-xL in conferring apoptosis resistance by preconditioning the cells and possibly altering mitochondrial morphology.     

Transmembrane domain modulates sorting of membrane proteins in Toxoplasma gondii

Overlapping mechanisms that function simultaneously in the intracellular sorting of mammalian membrane proteins often confound delineation of individual sorting pathways. By analyzing sorting in the evolutionarily simpler organism Toxoplasma gondii, we demonstrate a role for transmembrane domain (TMD) length in modulating the signal-dependent segregation of membrane proteins to distinct intracellular organelles. The dense granule localization of the single pass transmembrane protein GRA4 could be completely rerouted to the Golgi and cell surface simply by replacement of its TMD with that from either vesicular stomatitis virus G or the low density lipoprotein (LDL) receptor. Mutational and biochemical analyses suggested that this effect was not caused by any specific sequence motif or strength of membrane association of the GRA4 TMD. Instead, a property imparted by the vesicular stomatitis virus G or LDL receptor TMDs, both of which are longer than the GRA4 TMD, appeared to be a decisive factor. Indeed, shortening the LDL receptor TMD to a length similar to that of GRA4 resulted in dense granule localization, whereas lengthening the GRA4 TMD resulted in rerouting to the Golgi. From these data, we conclude that although the TMD may not necessarily be a sole determinant in membrane protein sorting, its properties can markedly modulate the utilization of more conventional signal-mediated sorting pathways.     

Low-dose hyper-radiosensitivity: a consequence of ineffective cell cycle arrest of radiation-damaged G2-phase cells

This review highlights the phenomenon of low-dose hyper- radiosensitivity (HRS), an effect in which cells die from excessive sensitivity to small single doses of ionizing radiation but become more resistant (per unit dose) to larger single doses. Established and new data pertaining to HRS are discussed with respect to its possible underlying molecular mechanisms. To explain HRS, a three-component model is proposed that consists of damage recognition, signal transduction and damage repair. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell cycle checkpoint that is specific to cells irradiated in the G2phase. This checkpoint exhibits a dose expression profile that is identical to the cell survival pattern that characterizes HRS and is probably the key control element of low-dose radiosensitivity. This premise is strengthened by the recent observation coupling low- dose radiosensitivity of G2-phase cells directly to HRS. The putative role of known damage response factors such as ATM, PARP, H2AX, 53BP1 and HDAC4 is also included within the framework of the HRS model.     

Renal damage in the mouse: the effect of d(4)-Be neutrons

A further study on the response of the mouse kidney to d(4)-Be neutrons (EN = 2.3 MeV) is described. The results confirm and augment the work published previously by Stewart et al. [Br. J. Radiol. 57, 1009-1021 (1984)]; the present paper includes the data from a "top-up" design of experiment which extends the measurements of neutron RBE (relative to 240 kVp X rays) down to X-ray doses of 0.75 Gy per fraction. The mean RBE for these neutrons increases from 5.8 to 7.3 as X-ray dose per fraction decreases from 3.0 to 1.5 Gy in the kidney. This agrees with the predictions from the linear quadratic (LQ) model, based on the renal response to X-ray doses above 4 Gy per fraction. The mean RBE estimate from a single dose group at 0.75 Gy per fraction of X rays is, however, 3.9. This is below the LQ prediction and may indicate increasing X-ray sensitivity at low doses. Data from this study and from those published previously have been used to determine more accurately the shape of the underlying response to d(4)-Be neutrons; an alpha/beta ratio of 20.5 +/- 3.7 Gy was found. The best value of alpha/beta for X rays determined from these experiments was 3.04 +/- 0.35 Gy, in agreement with previous values.     

C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella montevideo

We studied the population of LPS molecules on Salmonella montevideo that bind C3 during alternative pathway activation in serum. LPS molecules of Salmonella are composed of lipid A:core oligosaccharide (one copy per molecule), substituted by an O-polysaccharide (O-PS) side chain, which is a linear polymer of 0 to greater than 60 O-antigen repeat units containing mannose. A mutant of S. montevideo called SL5222 that inserts galactose only into core oligosaccharide and mannose only into O-antigen subunits was grown with [3H]mannose and [14C]galactose, so that LPS molecules bearing large numbers of O-antigen subunits have high 3H to 14C ratios, whereas molecules with few O-antigen subunits have lower 3H to 14C ratios. Double-labeled SL5222 was incubated in C8-deficient (C8D) serum or C8D serum with 2 mM Mg++Cl2 and 10 mM ethylene glycoltetraacetic acid (MgEGTA C8D). LPS molecules with covalently attached C3 were identified by binding to anti-C3. LPS molecules that bound C3 under both incubation conditions had O chains seven to eight times longer than the average LPS molecule. SL5222 was then grown in suboptimal concentrations of mannose in order to decrease the number of LPS molecules with long O-PS side chains. C3 attached to progressively shorter chain molecules of LPS as the mannose input was lowered, but still chose the longest available molecules. This finding and recently published observations indicate that C3 can bind to LPS molecules with short O-PS side chains. We postulate that preferential attachment of C3 to long-chain LPS in SL5222 results because long-chain LPS molecules sterically hinder shorter chain LPS molecules from macromolecules. This study provides direct proof that the O-PS of LPS sterically hinders access of large molecules to the outer membrane and indicates that the LPS coat of these bacteria functions as a barrier against large protein molecules.     

Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases

Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.