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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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FACS® analysis showing the loss of VSG and the appearance of procyclin during the in vitro differentiation of PLC−  trypanosomes to procyclic forms.
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Figure 5: FACS® analysis showing the loss of VSG and the appearance of procyclin during the in vitro differentiation of PLC− trypanosomes to procyclic forms.

Mentions: FACS® analysis (Roditi et al., 1989; Ziegelbauer et al., 1990) was used to follow the kinetics of appearance of cell surface procyclin and loss of VSG in the PLC− trypanosomes. In preliminary experiments, the VSG expressed by the nonclonal PLC− trypanosomes was investigated by immunofluorescence. Of 12 antibodies tried (anti-AnTat 1.1 to anti-AnTat 1.12), 4 (AnTat 1.1, 1.2, 1.3, and 1.6) were found to react weakly with some cells in the population. The strength of the fluorescence indicated that the VSG(s) expressed by the PLC− trypanosomes was slightly cross-reactive with these antibodies as opposed to actually being one of these VSGs. A mixture of three of the antisera (AnTat 1.1, 1.2, and 1.3) was sufficient to produce a signal for the VSG that was detectable on FACS® analysis. Fig. 5 shows that surface VSG has been completely replaced by procyclin between 7 and 9 h after initiation of differentiation. Once again, this is consistent with previous studies on this cell line (Rolin et al., 1993). Furthermore, the transient activation of adenylate cyclase, which follows VSG release during differentiation, occurs normally in the PLC− trypanosomes (Rolin et al., 1996). Finally, when bloodstream forms were fed to tsetse flies, PLC− trypanosomes established a typical procyclic infection in the midgut (data not shown). This indicates that the essentially normal differentiation observed in vitro also occurs in vivo.


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)

FACS® analysis showing the loss of VSG and the appearance of procyclin during the in vitro differentiation of PLC−  trypanosomes to procyclic forms.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: FACS® analysis showing the loss of VSG and the appearance of procyclin during the in vitro differentiation of PLC− trypanosomes to procyclic forms.
Mentions: FACS® analysis (Roditi et al., 1989; Ziegelbauer et al., 1990) was used to follow the kinetics of appearance of cell surface procyclin and loss of VSG in the PLC− trypanosomes. In preliminary experiments, the VSG expressed by the nonclonal PLC− trypanosomes was investigated by immunofluorescence. Of 12 antibodies tried (anti-AnTat 1.1 to anti-AnTat 1.12), 4 (AnTat 1.1, 1.2, 1.3, and 1.6) were found to react weakly with some cells in the population. The strength of the fluorescence indicated that the VSG(s) expressed by the PLC− trypanosomes was slightly cross-reactive with these antibodies as opposed to actually being one of these VSGs. A mixture of three of the antisera (AnTat 1.1, 1.2, and 1.3) was sufficient to produce a signal for the VSG that was detectable on FACS® analysis. Fig. 5 shows that surface VSG has been completely replaced by procyclin between 7 and 9 h after initiation of differentiation. Once again, this is consistent with previous studies on this cell line (Rolin et al., 1993). Furthermore, the transient activation of adenylate cyclase, which follows VSG release during differentiation, occurs normally in the PLC− trypanosomes (Rolin et al., 1996). Finally, when bloodstream forms were fed to tsetse flies, PLC− trypanosomes established a typical procyclic infection in the midgut (data not shown). This indicates that the essentially normal differentiation observed in vitro also occurs in vivo.

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