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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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Antigenic variation in PLC− trypanosomes. (a) Immunofluorescence of trypanosomes from the first and a successive peak of  parasitemia using antiserum from a mouse that had just cleared the first peak. (b) A Western blot, probed with anti-CRD, showing the  effect of GPI-PLC treatment of total cell lysates from PLC− trypanosomes from successive peaks of parasitemia. The enlarged panel  shows the CRD-positive VSGs from the two peaks; the densitometric scan of this region is shown below. Bar, 50 μm.
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Figure 7: Antigenic variation in PLC− trypanosomes. (a) Immunofluorescence of trypanosomes from the first and a successive peak of parasitemia using antiserum from a mouse that had just cleared the first peak. (b) A Western blot, probed with anti-CRD, showing the effect of GPI-PLC treatment of total cell lysates from PLC− trypanosomes from successive peaks of parasitemia. The enlarged panel shows the CRD-positive VSGs from the two peaks; the densitometric scan of this region is shown below. Bar, 50 μm.

Mentions: Although it is barely credible that such an infection could persist without antigenic variation, two experiments were performed to show that VSG switching occurs in the PLC− trypanosomes. A clonal line of PLC− trypanosomes was first established by infecting immunosuppressed mice with single trypanosomes, and stabilates were made 10–14 d later. Three mice were then infected using a single stabilate. The first mouse was used to prepare trypanosomes for immunofluorescence during the first peak of parasitemia. The second mouse was used to prepare serum just after the first peak of parasitemia had declined, and the third mouse was used to prepare trypanosomes for immunofluorescence during the second peak of parasitemia. The results are shown in Fig. 7 a; the serum clearly recognizes trypanosomes in the first peak, but not in the second.


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)

Antigenic variation in PLC− trypanosomes. (a) Immunofluorescence of trypanosomes from the first and a successive peak of  parasitemia using antiserum from a mouse that had just cleared the first peak. (b) A Western blot, probed with anti-CRD, showing the  effect of GPI-PLC treatment of total cell lysates from PLC− trypanosomes from successive peaks of parasitemia. The enlarged panel  shows the CRD-positive VSGs from the two peaks; the densitometric scan of this region is shown below. Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Antigenic variation in PLC− trypanosomes. (a) Immunofluorescence of trypanosomes from the first and a successive peak of parasitemia using antiserum from a mouse that had just cleared the first peak. (b) A Western blot, probed with anti-CRD, showing the effect of GPI-PLC treatment of total cell lysates from PLC− trypanosomes from successive peaks of parasitemia. The enlarged panel shows the CRD-positive VSGs from the two peaks; the densitometric scan of this region is shown below. Bar, 50 μm.
Mentions: Although it is barely credible that such an infection could persist without antigenic variation, two experiments were performed to show that VSG switching occurs in the PLC− trypanosomes. A clonal line of PLC− trypanosomes was first established by infecting immunosuppressed mice with single trypanosomes, and stabilates were made 10–14 d later. Three mice were then infected using a single stabilate. The first mouse was used to prepare trypanosomes for immunofluorescence during the first peak of parasitemia. The second mouse was used to prepare serum just after the first peak of parasitemia had declined, and the third mouse was used to prepare trypanosomes for immunofluorescence during the second peak of parasitemia. The results are shown in Fig. 7 a; the serum clearly recognizes trypanosomes in the first peak, but not in the second.

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