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The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

Webb H, Carnall N, Vanhamme L, Rolin S, Van Den Abbeele J, Welburn S, Pays E, Carrington M - J. Cell Biol. (1997)

Bottom Line: 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 mutant trypanosome has been generated by targeted gene deletion.Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation.This phenotype is partially alleviated when the mutant is modified to express low levels of GPI-PLC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cambridge University, United Kingdom.

ABSTRACT
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 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 mutant is modified to express low levels of GPI-PLC.

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Related in: MedlinePlus

Presence of GPI-PLC activity in PLC+ trypanosomes.  Western analysis of bloodstream trypanosomes from four cell  lines; AnTat 1.1, proG Anvers, PLC− and PLC+. Lysates were  prepared by SDS or Triton lysis, Western blotted, and probed  with anti-CRD. All samples are from the same blot and were  probed simultaneously.
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Figure 10: Presence of GPI-PLC activity in PLC+ trypanosomes. Western analysis of bloodstream trypanosomes from four cell lines; AnTat 1.1, proG Anvers, PLC− and PLC+. Lysates were prepared by SDS or Triton lysis, Western blotted, and probed with anti-CRD. All samples are from the same blot and were probed simultaneously.

Mentions: Phleomycin-resistant clones were transmitted through tsetse flies, and the expression of GPI-PLC characterized by Western blotting and assaying mfVSG to sVSG conversion on hypotonic lysis. The PLC+ trypanosomes were found to contain GPI-PLC protein but, by comparison with a twofold dilution series of AnTat 1.1 cells (Fig. 9), the level of GPI-PLC in 2 × 106 PLC+ trypanosomes was roughly equivalent to that in only 1.25 × 105 AnTat1.1 cells. Thus the PLC+ trypanosomes contain approximately one sixteenth of the wild-type, bloodsteam-form GPI-PLC level. The presence of GPI-PLC activity was tested in two assays. The first was detergent lysis of PLC+ trypanosomes; in this assay the endogenous VSG became anti-CRD positive (Fig. 10). In the second assay, lysates of PLC+ and wild-type trypanosomes were assayed in vitro for release of tritium from [3H]-myristyl VSG. The PLC+ trypanosome lysates contained an activity that released 15.5 pmol/min/mg protein or 31% of wild-type activity. There is a discrepency between the estimates of the amount of GPI-PLC protein (5–10% of wild-type) and the activity (30% of wild-type). At this stage it is not clear whether this is an artefact of the enzyme assay or due to some endogenous regulatory phenomenon.


The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

Webb H, Carnall N, Vanhamme L, Rolin S, Van Den Abbeele J, Welburn S, Pays E, Carrington M - J. Cell Biol. (1997)

Presence of GPI-PLC activity in PLC+ trypanosomes.  Western analysis of bloodstream trypanosomes from four cell  lines; AnTat 1.1, proG Anvers, PLC− and PLC+. Lysates were  prepared by SDS or Triton lysis, Western blotted, and probed  with anti-CRD. All samples are from the same blot and were  probed simultaneously.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139819&req=5

Figure 10: Presence of GPI-PLC activity in PLC+ trypanosomes. Western analysis of bloodstream trypanosomes from four cell lines; AnTat 1.1, proG Anvers, PLC− and PLC+. Lysates were prepared by SDS or Triton lysis, Western blotted, and probed with anti-CRD. All samples are from the same blot and were probed simultaneously.
Mentions: Phleomycin-resistant clones were transmitted through tsetse flies, and the expression of GPI-PLC characterized by Western blotting and assaying mfVSG to sVSG conversion on hypotonic lysis. The PLC+ trypanosomes were found to contain GPI-PLC protein but, by comparison with a twofold dilution series of AnTat 1.1 cells (Fig. 9), the level of GPI-PLC in 2 × 106 PLC+ trypanosomes was roughly equivalent to that in only 1.25 × 105 AnTat1.1 cells. Thus the PLC+ trypanosomes contain approximately one sixteenth of the wild-type, bloodsteam-form GPI-PLC level. The presence of GPI-PLC activity was tested in two assays. The first was detergent lysis of PLC+ trypanosomes; in this assay the endogenous VSG became anti-CRD positive (Fig. 10). In the second assay, lysates of PLC+ and wild-type trypanosomes were assayed in vitro for release of tritium from [3H]-myristyl VSG. The PLC+ trypanosome lysates contained an activity that released 15.5 pmol/min/mg protein or 31% of wild-type activity. There is a discrepency between the estimates of the amount of GPI-PLC protein (5–10% of wild-type) and the activity (30% of wild-type). At this stage it is not clear whether this is an artefact of the enzyme assay or due to some endogenous regulatory phenomenon.

Bottom Line: 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 mutant trypanosome has been generated by targeted gene deletion.Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation.This phenotype is partially alleviated when the mutant is modified to express low levels of GPI-PLC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cambridge University, United Kingdom.

ABSTRACT
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 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 mutant is modified to express low levels of GPI-PLC.

Show MeSH
Related in: MedlinePlus