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.

scholarly journals Trypanosoma brucei Interaction with Host: Mechanism of VSG Release as Target for Drug Discovery for African Trypanosomiasis

2019 ◽  
Vol 20 (6) ◽  
pp. 1484 ◽  
Author(s):  
Cláudia Moreno ◽  
Adriana Temporão ◽  
Taffarel Torres ◽  
Marcelo Sousa Silva
Keyword(s):  
Drug Discovery ◽  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
African Trypanosomiasis ◽  
Variable Surface Glycoprotein ◽  
Variable Surface ◽  
Major Surface

The protozoan Trypanosoma brucei, responsible for animal and human trypanosomiasis, has a family of major surface proteases (MSPs) and phospholipase-C (PLC), both involved in some mechanisms of virulence during mammalian infections. During parasitism in the mammalian host, this protozoan is exclusively extracellular and presents a robust mechanism of antigenic variation that allows the persistence of infection. There has been incredible progress in our understanding of how variable surface glycoproteins (VSGs) are organised and expressed, and how expression is switched, particularly through recombination. The objective of this manuscript is to create a reflection about the mechanisms of antigenic variation in T. brucei, more specifically, in the process of variable surface glycoprotein (VSG) release. We firstly explore the mechanism of VSG release as a potential pathway and target for the development of anti-T. brucei drugs.

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 Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

1989 ◽  
Vol 108 (2) ◽  
pp. 737-746 ◽  
Author(s):  
I Roditi ◽  
H Schwarz ◽  
T W Pearson ◽  
R P Beecroft ◽  
M K Liu ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Tsetse Fly ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Mammalian Host ◽  
Cylindrical Shape ◽  
Surface Coat ◽  
Insect Vector ◽  
Molecular Arrangement

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.

scholarly journals Black-necked spitting cobra (Naja nigricollis) phospholipases A2 cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket

2021 ◽  
Author(s):  
Andrea Martos-Esteban ◽  
Olivia J. S. Macleod ◽  
Isabella Maudlin ◽  
Konstantinos Kalogeropoulos ◽  
Jonas A. Jurgensen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Adaptive Immune Response ◽  
Surface Glycoprotein ◽  
Covalent Attachment ◽  
Mammalian Host ◽  
Flagellar Pocket ◽  
African Trypanosomes ◽  
External Face

African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to a glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components including proteases and phospholipases. Here, we investigated the effect of Naja nigricollis on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VGS hydrolysis however N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) a cell cycle arrest prior to cytokinesis. A single protein family, the phospholipases A2s present in N. nigricollis venom, was necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.

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 Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro

2021 ◽  
Author(s):  
Federico Rojas ◽  
Mathieu Cayla ◽  
Keith Matthews
Keyword(s):  
Quorum Sensing ◽  
Basement Membrane ◽  
Trypanosoma Brucei ◽  
Life Cycle Stage ◽  
Mammalian Host ◽  
Connective Tissues ◽  
Basement Membrane Extract ◽  
Differentiation Marker

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process.  For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo , we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms . BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro , procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

scholarly journals Kinetics and In Vivo Induction of Genetic Variation of vlsE in Borrelia burgdorferi

1998 ◽  
Vol 66 (8) ◽  
pp. 3689-3697 ◽  
Author(s):  
Jing-Ren Zhang ◽  
Steven J. Norris
Keyword(s):  
Borrelia Burgdorferi ◽  
Sequence Variation ◽  
Antigenic Variation ◽  
Pcr Amplification ◽  
Scid Mice ◽  
Mammalian Host ◽  
Expression Site ◽  
Disease Agent

ABSTRACT The Lyme disease agent, Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host.vlsE, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in B. burgdorferi B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice. In this study, we examined the kinetics of vlsEsequence variation in C3H/HeN mice at 4, 7, 14, 21, and 28 days and at 7 and 12 months postinfection. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection. The sequence changes were consistent with sequential recombination of segments from multiple silent vls cassette sites into thevlsE expression site. No vlsE sequence changes were detected in organisms cultured in vitro for up to 84 days. These results indicate that vlsE recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation. The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.

scholarly journals A major surface glycoprotein of Trypanosoma brucei is expressed transiently during development and can be regulated post-transcriptionally by glycerol or hypoxia

2000 ◽  
Vol 14 (5) ◽  
pp. 615-626
Author(s):  
Erik Vassella ◽  
Jan Van Den Abbeele ◽  
Peter Bütikofer ◽  
Christina Kunz Renggli ◽  
André Furger ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Glycoprotein ◽  
Tsetse Flies ◽  
Extracellular Signals ◽  
Major Surface Glycoprotein ◽  
Host Parasite Interactions ◽  
Major Surface ◽  
Host Parasite

Differentiation is a means by which unicellular parasites adapt to different environments. In some cases, the developmental program may be modulated by interactions with the host, but the mechanisms are largely unknown. Trypanosoma brucei is transmitted between mammals by tsetse flies. The development of the procyclic form in the tsetse midgut is marked by the synthesis of a new glycoprotein coat, composed of EP and GPEET procyclins, that is important for survival. Here we demonstrate that the composition of the coat changes in response to extracellular signals in vitro and during development in vivo. EP and GPEET are coinduced when differentiation is initiated. Subsequently, EP expression is maintained, whereas GPEET is repressed after 7–9 days. The timepoint at which GPEET is repressed coincides with the appearance of parasites in a new compartment of the fly midgut. In culture, down-regulation of GPEET can be prevented by exogenous glycerol or accelerated by hypoxia. Regulation is post-transcriptional, and is conferred by the GPEET 3′ untranslated region. The same sequence also regulates expression of a reporter gene in the fly. The finding that GPEET is expressed during a defined window during the establishment of infection suggests that it has a specific function in host-parasite interactions rather than a generalized role in shielding underlying membrane molecules.

Antigenic variation in clones of animal-infective Trypanosoma brucei derived and maintained in vitro

Parasitology ◽  
1980 ◽  
Vol 80 (2) ◽  
pp. 359-369 ◽  
Author(s):  
J. J. Doyle ◽  
H. Hirumi ◽  
K. Hirumi ◽  
E. N. Lupton ◽  
G. A. M. Cross
Keyword(s):  
Immune Response ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Antigenic Composition ◽  
New Variant ◽  
First Relapse ◽  
Variable Antigen ◽  
Immunofluorescent Analysis

SummaryEighteen clones of variable antigen type 052 of Trypanosoma brucei stock S. 427 were derived and maintained as animal-infective bloodstream forms in vitro for up to 60 days of cultivation. The antigenic composition of such clones was monitored weekly by immunofluorescent analysis of viable trypanosomes, using antisera raised to isolated variant-specific surface glycoproteins of both 052 and a variable antigen type (221) which consistently appeared in the first relapse population of type 052 in vitro. The appearance of new variants was detected in 9 of the 18 clones 18–46 days following initiation of the clone and variable antigen type 221 was found in all 9 clones. On one or more occasions in 8 of such clones, viable trypanosomes were found which did not react with either antiserum but were mouse-infective on the 4 occasions tested and probably represent other variable antigen types. The process of antigen, variation in vitro appears to resemble the process in vivo except that new variant types are detected earlier in vivo. This possibly results from different growth rates of the trypanosomes in vivo and in vitro, together with the fact that elimination of the initial variant population by the host's immune response facilitates the detection of newly arising variable antigen types in vivo.

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