Effects of phenothiazine neuroleptic drugs on the microtubular–membrane complex in bloodstream forms of Trypanosoma brucei

Parasitology ◽  
1995 ◽  
Vol 111 (4) ◽  
pp. 493-504 ◽  
Author(s):  
A. M. Page ◽  
J. R. Lagnado
Keyword(s):  
Trypanosoma Brucei ◽  
Structural Integrity ◽  
Membrane Skeleton ◽  
Host Immune System ◽  
Surface Coat ◽  
Neuroleptic Drugs ◽  
African Trypanosomes ◽  
Glycosyl Phosphatidylinositol ◽  
Membrane Complex

SUMMARYAfrican trypanosomes are parasitic protozoa causing sleeping sickness in humans and related diseases in domestic animals against which no entirely satisfactory forms of chemotherapy are yet available. It was previously shown that related species of trypanosomes, as well as procyclic (insect) forms of Trypanosoma brucei are extremely sensitive to the action of phenothiazine neuroleptic drugs in vitro. In this work, we have carried out a more detailed investigation of the effects of thioridazine, one of the most potent neuroleptic phenothiazine drugs known, on the morphology of the infective bloodstream forms of T. brucei, with particular reference to the parasite's prominent pellicular membrane complex. Our data show that this drug induces rapid changes in cell shape that appear to involve some reorganization of the microtubular membrane skeleton, but does not affect the structural integrity of the microtubular complex. Another early consequence of drug action involved damage to nuclear and cytoplasmic membranes and the appearance of tubular arrays of coated membrane within the flagellar pocket. It was also revealed that the drug induces a rapid release of the variant-specific glycoprotein (VSG) which makes up the surface coat protecting bloodstream forms of the parasite against the host immune system. Our evidence suggests that this release of VSG involves cleavage of the protein's glycosyl-phosphatidylinositol (GPI) membrane anchor by endogenous GPI-specific phospholipase C, probably as a consequence of minor damage to the parasite plasma membrane induced by the drug.

scholarly journals In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

2006 ◽  
Vol 5 (8) ◽  
pp. 1276-1286 ◽  
Author(s):  
Sara D. Faulkner ◽  
Monika W. Oli ◽  
Rudo Kieft ◽  
Laura Cotlin ◽  
Justin Widener ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Trypanosoma Brucei ◽  
Density Lipoprotein ◽  
High Density ◽  
Surface Glycoprotein ◽  
Trypanosoma Brucei Brucei ◽  
African Trypanosomes ◽  
Expression Site ◽  
Human High Density Lipoprotein

ABSTRACT The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

scholarly journals Escaping the immune system by DNA repair and recombination in African trypanosomes

Open Biology ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 190182 ◽  
Author(s):  
Núria Sima ◽  
Emilia Jane McLaughlin ◽  
Sebastian Hutchinson ◽  
Lucy Glover
Keyword(s):  
Immune Response ◽  
Dna Repair ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Surface Coat ◽  
African Trypanosomes ◽  
Active Expression ◽  
Expression Site

African trypanosomes escape the mammalian immune response by antigenic variation—the periodic exchange of one surface coat protein, in Trypanosoma brucei the variant surface glycoprotein (VSG), for an immunologically distinct one. VSG transcription is monoallelic, with only one VSG being expressed at a time from a specialized locus, known as an expression site. VSG switching is a predominantly recombination-driven process that allows VSG sequences to be recombined into the active expression site either replacing the currently active VSG or generating a ‘new’ VSG by segmental gene conversion. In this review, we describe what is known about the factors that influence this process, focusing specifically on DNA repair and recombination.

scholarly journals Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N -Acetyltransferase from Trypanosoma brucei

2011 ◽  
Vol 10 (7) ◽  
pp. 985-997 ◽  
Author(s):  
Karina Mariño ◽  
M. Lucia Sampaio Güther ◽  
Amy K. Wernimont ◽  
Wei Qiu ◽  
Raymond Hui ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Trypanosoma Brucei ◽  
Protein Glycosylation ◽  
Surface Glycoprotein ◽  
Surface Coat ◽  
Content Type ◽  
Phosphate Analysis ◽  
Novel Drug

ABSTRACT A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N -acetyltransferase ( TbGNA1 ; EC 2.3.1.4) was cloned and expressed in Escherichia coli . The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly- N -acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.

scholarly journals OmpA-Mediated Biofilm Formation Is Essential for the Commensal Bacterium Sodalis glossinidius To Colonize the Tsetse Fly Gut

2012 ◽  
Vol 78 (21) ◽  
pp. 7760-7768 ◽  
Author(s):  
Michele A. Maltz ◽  
Brian L. Weiss ◽  
Michelle O'Neill ◽  
Yineng Wu ◽  
Serap Aksoy
Keyword(s):  
Immune System ◽  
Pathogenic Bacteria ◽  
Tsetse Fly ◽  
Symbiotic Bacteria ◽  
Tsetse Flies ◽  
Host Immune System ◽  
Content Type ◽  
African Trypanosomes ◽  
Sodalis Glossinidius

ABSTRACTMany bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relationship between biofilms and host colonization by symbiotic bacteria is less well understood. Tsetse flies (Glossinaspp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined thatSodalisforms biofilms in the tsetse gut and that this process is influenced by theSodalisouter membrane protein A (OmpA). MutantSodalisstrains that do not produce OmpA (SodalisΔOmpA mutants) fail to form biofilmsin vitroand are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms,SodalisΔOmpA mutant cells are exposed to and eliminated by tsetse's innate immune system, suggesting that biofilms helpSodalisevade the host immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly gut. Acquiring a better understanding of the dynamics that promoteSodaliscolonization of the tsetse gut may enhance the development of novel disease control strategies.

scholarly journals GPI-anchored Proteins and Free GPI Glycolipids of Procyclic Form Trypanosoma brucei Are Nonessential for Growth, Are Required for Colonization of the Tsetse Fly, and Are Not the Only Components of the Surface Coat

2006 ◽  
Vol 17 (12) ◽  
pp. 5265-5274 ◽  
Author(s):  
Maria Lucia Sampaio Güther ◽  
Sylvia Lee ◽  
Laurence Tetley ◽  
Alvaro Acosta-Serrano ◽  
Michael A.J. Ferguson
Keyword(s):  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Current Model ◽  
Apparent Molecular Weight ◽  
Tsetse Fly ◽  
Surface Coat ◽  
Procyclic Form ◽  
Cell Surface Biotinylation ◽  
A Cell

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine–containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine–containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI–anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.

scholarly journals Activation of Endocytosis as an Adaptation to the Mammalian Host by Trypanosomes

2007 ◽  
Vol 6 (11) ◽  
pp. 2029-2037 ◽  
Author(s):  
Senthil Kumar A. Natesan ◽  
Lori Peacock ◽  
Keith Matthews ◽  
Wendy Gibson ◽  
Mark C. Field
Keyword(s):  
Antigenic Variation ◽  
Subsequent Development ◽  
Tsetse Fly ◽  
Mammalian Host ◽  
Surface Coat ◽  
Down Regulation ◽  
African Trypanosomes ◽  
Endocytic Activity

ABSTRACT Immune evasion in African trypanosomes is principally mediated by antigenic variation, but rapid internalization of surface-bound immune factors may contribute to survival. Endocytosis is upregulated approximately 10-fold in bloodstream compared to procyclic forms, and surface coat remodeling accompanies transition between these life stages. Here we examined expression of endocytosis markers in tsetse fly stages in vivo and monitored modulation during transition from bloodstream to procyclic forms in vitro. Among bloodstream stages nonproliferative stumpy forms have endocytic activity similar to that seen with rapidly dividing slender forms, while differentiation of stumpy forms to procyclic forms is accompanied by rapid down-regulation of Rab11 and clathrin, suggesting that modulation of endocytic and recycling systems accompanies this differentiation event. Significantly, rapid down-regulation of endocytic markers occurs upon entering the insect midgut and expression of Rab11 and clathrin remains low throughout subsequent development, which suggests that high endocytic activity is not required for remodeling the parasite surface or for survival within the fly. However, salivary gland metacyclic forms dramatically increase expression of clathrin and Rab11, indicating that emergence of mammalian infective forms is coupled to reacquisition of a high-activity endocytic-recycling system. These data suggest that high-level endocytosis in Trypanosoma brucei is an adaptation required for viability in the mammalian host.

scholarly journals Identification and characterization of two repetitive non-variable antigens from African trypanosomes which are recognized early during infection

Parasitology ◽  
1992 ◽  
Vol 104 (1) ◽  
pp. 111-120 ◽  
Author(s):  
N. Müller ◽  
A. Hemphill ◽  
M. Imboden ◽  
G. Duvallet ◽  
R. H. Dwinger ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Skeleton ◽  
Immunogold Electron Microscopy ◽  
Host Immune System ◽  
Amino Acid Repeat ◽  
African Trypanosomes ◽  
Repeat Units ◽  
High Molecular Weight Component ◽  
Repetitive Motif ◽  
Common Antigens

SUMMARYThe present paper describes two repetitive proteins representing common antigens of African trypanosomes which are non-variant and which are recognized early in infection by the host immune system. These antigens were identified by their ability to immunoreact with bovine serum taken during the early phase of a cyclic trypanosomal infection. Screening of a cDNA library fromT. b. gambiensewith such early infection serum identified a protein which contains a repetitive motif consisting of 68 amino acid repeat units (GM6). Immunofluorescence and immunogold electron microscopy revealed that GM6 is located on fibres which connect the microtubules of the membrane skeleton with the flagellum. A second repetitive antigen detected by this serum is MARP1 (microtubule-associated repetitive protein 1), a protein previously characterized in this laboratory as a high-molecular weight component of the membrane skeleton, which consists of more than 50 tandemly repeated, near-identical 38 amino acid repeat units. Beta-galactosidase fusion products of both proteins demonstrated a strong immunoreactivity with sera fromT. b. bruceiandT. congolense-infected cattle. The result from this preliminary immunological evaluation indicates a high immunodiagnostic sensititivy (90%) of the two recombinant antigens which make them interesting candidates for immunodiagnosis of trypanosomiasis in cattle.

scholarly journals A Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei

2010 ◽  
Vol 30 (6) ◽  
pp. 1319-1328 ◽  
Author(s):  
Jane C. Hines ◽  
Dan S. Ray
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Cell Growth ◽  
Trypanosoma Brucei ◽  
Dna Polymerase ◽  
Rnase H ◽  
Kinetoplast Dna ◽  
African Trypanosomes ◽  
Dna Primase

ABSTRACT Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the kinetoplast and is essential for both cell growth and kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the kinetoplast DNA. A 3′-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.

Studies on phospholipid turnover argue against sloughing of tegumental membranes in adult Schistosoma mansoni

Parasitology ◽  
1999 ◽  
Vol 119 (3) ◽  
pp. 287-294 ◽  
Author(s):  
J. F. H. M. BROUWERS ◽  
P. J. SKELLY ◽  
L. M. G. VAN GOLDE ◽  
A. G. M. TIELENS
Keyword(s):  
Fatty Acid ◽  
Immune System ◽  
Schistosoma Mansoni ◽  
Immune Evasion ◽  
Crucial Role ◽  
Host Immune System ◽  
Pulse Labelling ◽  
High Turnover ◽  
Membrane Complex

The tegumental membrane complex of Schistosoma mansoni is the site of interaction between the parasite and the host. The tegument is involved in uptake of many nutrients, but also plays a crucial role in the evasion of the actions of the host immune system. Essential for the success of this evasion is maintaining the integrity of the tegumental membranes. The rate of turnover of phospholipids was investigated by pulse-labelling worms cultured in vitro, followed by additional incubation in the presence of unlabelled substrates. Tegumental membranes were isolated, characterized using antibodies against specific tegumental proteins, and analysed. It was demonstrated that the most prominent fatty acid found in tegumental phospholipids, palmitate, incorporated rapidly into the phospholipid fraction during a 30 min pulse labelling. In a subsequent 20 h chase with unlabelled substrates, the incorporated radioactivity was lost again from the tegumental membrane complex. This high turnover of palmitate was found to be limited to phosphatidylcholine (PC) only. The turnover was due to deacylation/reacylation, and not to the sloughing of membranes as is the case in schistosomula. It is speculated that this rapid turnover of PC in the tegument of adult schistosomes plays a new and important role in the immune evasion by the parasite.

scholarly journals The GPI-Phospholipase C of Trypanosoma brucei Is Nonessential But Influences Parasitemia in Mice

1997 ◽  
Vol 139 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Helena Webb ◽  
Nicola Carnall ◽  
Luc Vanhamme ◽  
Sylvie Rolin ◽  
Jakke Van Den Abbeele ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Covalent Attachment ◽  
Mammalian Host ◽  
Surface Coat ◽  
Null Mutant

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

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