Cryopreservation of Pathogenic African Trypanosomes In Situ: Metacyclic and Bloodstream Forms

SYNOPSIS. Of several methods developed for cryopreservation of Trypanosoma vivax, Trypanosoma congolense , and Trypanosoma brucei metacyclic forms in tsetse fly organs, as well as bloodstream forms in host blood, one proved the most satisfactory. In this method, infected fly proboscises and salivary glands were placed in glass capillary tubes containing fetal calf serum with 8% (v/v) glycerol as the cryoprotectant. The method for bloodstream forms involved the addition of glycerol directly to infected blood, which was then dispensed into capillary tubes. Next the tubes were placed in paper containers inside a glass test tube with a 5 mm thick plasticine jacket. The insulated assembly was suspended in the liquid nitrogen vapor phase in an LR-35 (Union Carbide) refrigerator for 45 min. Under these conditions, the cooling rate was 2 C/min. The frozen samples were transferred to permanent storage. The viability and infectivity of the preserved organisms were found to be satisfactory upon testing, and no antigenic changes were observed. Laboratory and field applications of the method are discussed.     

A New Organelle of Bloodstream Salivarian Trypanosomes

SYNOPSIS. Electron micrographs of the bloodstream forms of Trypanosoma brucei, T. congolense and T. vivax revealed a group of 4 subpellicular microtubules lying near the point of attachment of the flagellum and associated with a vesicular membrane which, in T. brucei at least, is continuous with the outer nuclear membrane. Since its function is unknown, the whole structure is provisionally termed the "subpellicular organelle."     

The Surface of the African Trypanosomes1,2

The African trypanosomes bear on the outside of their cell membrane a single 10–15 nm thick coat of a glycoprotein. This glycoprotein may differ in structure in the predominant populations of parasitemic waves found in relapsing infections. Variant Specific Glycoprotein (VSG) range in MW between 53,000–63,000 d and may have variable amounts of carbohydrate attached at one, two, or several loci. Such differences in carbohydrate content may account in part for their range in molecular size. Approximately 30 C-terminal residues demonstrate isotypy; i.e. these regions fall into classes having similar amino acid sequence. Modest homology has been demonstrated in two VSGs of T. congolense arising in relapsing infections although comparison of many VSG show little or no obvious homology. More recently, lipid-associated forms of VSG have been described and it is believed that these forms may be transmembrane proteins. Different VSGs appear to have different amounts of the primary sequence which have alpha-helix-forming potential. In some VSG, in excess of 80% of the structure is helical as judged by both Chou-Fasman calculations and by circular dichroism. This raises the possibility that different VSG may have different folding patterns. The arrangement of VSG on the trypanosome surface probably places the basic amino acid-rich carbohydrate-bearing C-terminus of the polypeptide chain close to the membrane. There is some protein-protein association between VSGs for which (in T. evansi) the C-terminal tail is not required. The importance of VSG structure lies not only in the fact that the molecule mediates the phenomenon of antigenic variation but also in the recent observation that VSG may act on the cellular immune system to suppress the humoral immune responses of the host.     

Purification and Properties of Purified Hexokinase from the African Trypanosomes and Trypanosoma equiperdum

SYNOPSIS. Hexokinase was in both soluble and particulate fractions of extracts of Trypanosoma brucei, T. gambiense, T. rhodesiense and T. equiperdum. These enzymes have been purified some 350-fold from crude extracts by sonication, (NH₄)₂SO₄ fractionation, and DEAE Sephadex column chromatography. Purified hexokinases had: A) temperature optimum 45-50 C; B) pH optimum 6.5-7.0; C) Mg⁺⁺ and ATP requirements; D) inhibition by ADP, p -hydroxymercuribenzoate, glucose-6-phosphate, and competitive inhibition by mannose, glucosamine, N -acetyl-D-glucosamine and xylose; E) ability to phosphorylate glucose, fructose, mannose, 2-deoxy-D-glucose and glucosamine; F) a glucose requirement for stabilization. T. equiperdum hexokinase was inhibited 33% by ADP as compared to 17-20% with brucei group hexokinase, and showed 22% activity when ITP was substituted for ATP in contrast to 35-40% with brucei group hexokinase.     

The Plasma Membrane of Bloodstream-form African Trypanosomes Confers Susceptibility and Specificity to Killing by Hydrophobic Peptides

Trypanosoma brucei is the causative agent of both a veterinary wasting disease and human African trypanosomiasis, or sleeping sickness. The cell membrane of the developmental stage found within the mammalian host, the bloodstream form (BSF), is highly dynamic, exhibiting rapid rates of endocytosis and lateral flow of glycosylphosphatidylinositol-anchored proteins. Here, we show that the cell membrane of these organisms is a target for killing by small hydrophobic peptides that increase the rigidity of lipid bilayers. Specifically, we have derived trypanocidal peptides that are based upon the hydrophobic N-terminal signal sequences of human apolipoproteins. These peptides selectively partitioned into the plasma membrane of BSF trypanosomes, resulting in an increase in the rigidity of the bilayer, dramatic changes in cell motility, and subsequent cell death. No killing of the developmental stage found within the insect midgut, the procyclic form, was observed. Additionally, the peptides exhibited no toxicity toward mammalian cell lines and did not induce hemolysis. Studies with model liposomes indicated that bilayer fluidity dictates the susceptibility of membranes to manipulation by hydrophobic peptides. We suggest that the composition of the BSF trypanosome cell membrane confers a high degree of fluidity and unique susceptibility to killing by hydrophobic peptides and is therefore a target for the development of trypanocidal drugs.     

Modulation of Iron-Mediated Oxidant Damage in Erythrocytes by Cellular Energy Levels

In this work we have investigated the effects of iron-induced oxidative stress on erythrocytes and their membranes, the importance of haemoglobin oxidation and of the maintenance of the metabolic properties of the cells. The results show that by maintaining the energy requirements of the erythrocyte, methaemog-lobin production is minimised under conditions of iron-stress. However, in this situation, the membranes of the erythrocytes become more susceptible to the oxidative damage and increased lipid peroxidation ensues.     

Antigens and Antigenic Variability of the African Trypanosomes*†

SYNOPSIS. The recent literature on the physical, chemical and biological characterization of antigens from the African trypanosomes is reviewed. The antigens are divided into three major groups: a) variant-specific antigens, b) common antigens, and c) host-like antigens. The variant-specific antigen(s) are relatively small molecular weight proteins located on the surface of the trypanosome, and are involved in protection and agglutination. It is suggested that there is more than a single variant-specific antigen on the cell surface. In contrast, the common antigens are internal or somatic antigens which are believed to have structural or enzymatic functions but are not involved in either protection or agglutination. In addition, evidence is also presented which suggests that there are host-like antigens within the surface coat and/or membranes of the trypanosomes. The importance of these three groups of antigens, and the mechanis(s) of antigenic variation are discussed in relationship to the immune response of the host to the African trypanosomes. Several possible approaches for future investigations are described.     

Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes.     

Purification of African Trypanosomes Can Cause Biochemical Changes in the Parasites1

Bloodstream forms of African trypanosomes are routinely purified from blood components by a combination of centrifugation and chromatography on DEAE cellulose at pH 8.0, Here we report that the nonphysiological conditions used for DEAE chromatography of the parasites result in changes in the ATP levels of the trypanosomes and an enhanced release from the parasites of proteins such as variable surface glycoprotein, peptidase, and phospholipase. Some of these changes can be reduced by the addition of nucleosides to the elution buffer and, after the elution of the parasites, by immediate readjustment of the external pH to the normal physiological level of blood (pH 7.4).     

Polymyxin E Induces Rapid Paenibacillus polymyxa Death by Damaging Cell Membrane while Ca2+ Can Protect Cells from Damage

Polymyxin E, produced by Paenibacillus polymyxa, is an important antibiotic normally against Gram-negative pathogens. In this study, we found that polymyxin E can kill its producer P. polymyxa, a Gram-positive bacterium, by disrupting its cell membrane. Membrane damage was clearly revealed by detecting the leakage of intracellular molecules. The observation using scanning electron microscopy also supported that polymyxin E can destroy the cell membrane and cause an extensive cell surface alteration. On the other hand, divalent cations can give protection against polymyxin E. Compared with Mg²⁺, Ca²⁺ can more effectively alleviate polymyxin E-induced damage to the cell membrane, thus remarkably increasing the P. polymyxa survival. Our findings would shed light on a not yet described bactericidal mechanism of polymyxin E against Gram-positive bacteria and more importantly the nature of limited fermentation output of polymyxin E from P. polymyxa.     

Global and Ocular Hypothermic Preconditioning Protect the Rat Retina from Ischemic Damage

Retinal ischemia could provoke blindness. At present, there is no effective treatment against retinal ischemic damage. Strong evidence supports that glutamate is implicated in retinal ischemic damage. We investigated whether a brief period of global or ocular hypothermia applied 24 h before ischemia (i.e. hypothermic preconditioning, HPC) protects the retina from ischemia/reperfusion damage, and the involvement of glutamate in the retinal protection induced by HPC. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. One day before ischemia, animals were submitted to global or ocular hypothermia (33°C and 32°C for 20 min, respectively) and fourteen days after ischemia, animals were subjected to electroretinography and histological analysis. Global or ocular HPC afforded significant functional (electroretinographic) protection in eyes exposed to ischemia/reperfusion injury. A marked alteration of the retinal structure and a decrease in retinal ganglion cell number were observed in ischemic retinas, whereas global or ocular HPC significantly preserved retinal structure and ganglion cell count. Three days after ischemia, a significant decrease in retinal glutamate uptake and glutamine synthetase activity was observed, whereas ocular HPC prevented the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate induced alterations in retinal function and histology which were significantly prevented by ocular HPC. These results support that global or ocular HPC significantly protected retinal function and histology from ischemia/reperfusion injury, probably through a glutamate-dependent mechanism.     

过氧化物酶和PeroxiBase过氧化物酶数据库

过氧化物酶是存在于生物体中的一大类以过氧化物为电子受体催化底物氧化的酶,在生物体的生命活动中发挥着重要作用.过氧化物酶由多态性丰富的多基因家族所编码,其结构和功能具有多样性.近年来,随着研究和应用的深入,过氧化物酶领域迫切需要建立一个专业的信息交流平台.PeroxiBase数据库收集了大量的过氧化物酶数据,并进行生物信息学加工,为生命科学研究人员免费共享、最大化利用与综合开发过氧化物酶资源搭建了信息平台,在过氧化物酶的研究与应用中发挥着重要作用.     

Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

The variant surface glycoprotein (VSG) of bloodstream form Trypanosoma brucei (Tb) is a critical virulence factor. The VSG glycosylphosphatidylinositol (GPI)-anchor strongly influences passage through the early secretory pathway. Using a dominant-negative mutation of TbSar1, we show that endoplasmic reticulum (ER) exit of secretory cargo in trypanosomes is dependent on the coat protein complex II (COPII) machinery. Trypanosomes have two orthologues each of the Sec23 and Sec24 COPII subunits, which form specific heterodimeric pairs: TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1. RNA interference silencing of each subunit is lethal but has minimal effects on trafficking of soluble and transmembrane proteins. However, silencing of the TbSec23.2/TbSec24.1 pair selectively impairs ER exit of GPI-anchored cargo. All four subunits colocalize to one or two ER exit sites (ERES), in close alignment with the postnuclear flagellar adherence zone (FAZ), and closely juxtaposed to corresponding Golgi clusters. These ERES are nucleated on the FAZ-associated ER. The Golgi matrix protein Tb Golgi reassembly stacking protein defines a region between the ERES and Golgi, suggesting a possible structural role in the ERES:Golgi junction. Our results confirm a selective mechanism for GPI-anchored cargo loading into COPII vesicles and a remarkable degree of streamlining in the early secretory pathway. This unusual architecture probably maximizes efficiency of VSG transport and fidelity in organellar segregation during cytokinesis.     

Disaccharides Protect Antigens from Drying-Induced Damage in Routinely Processed Tissue Sections

Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions.     

Evidence for Recycling of Invariant Surface Transmembrane Domain Proteins in African Trypanosomes

Intracellular trafficking is a vital component of both virulence mechanisms and drug interactions in Trypanosoma brucei, the causative agent of human African trypanosomiasis and n''agana of cattle. Both maintaining the surface proteome composition within a life stage and remodeling the composition when progressing between life stages are important features of immune evasion and development for trypanosomes. Our recent work implicates the abundant transmembrane invariant surface glycoproteins (ISGs) in the uptake of first-line therapeutic suramin, suggesting a potential therapeutic route into the cell. RME-8 is a mediator of recycling pathways in higher eukaryotes and is one of a small cohort of intracellular transport gene products upregulated in mammal-infective trypanosomes, suggesting a role in controlling the copy number of surface proteins in trypanosomes. Here we investigate RME-8 function and its contribution to intracellular trafficking and stability of ISGs. RME-8 is a highly conserved protein and is broadly distributed across multiple endocytic compartments. By knockdown we find that RME-8 is essential and mediates delivery of endocytic probes to late endosomal compartments. Further, we find ISG accumulation within endosomes, but that RME-8 knockdown also increases ISG turnover; combined with previous data, this suggests that it is most probable that ISGs are recycled, and that RME-8 is required to support recycling.