Limits...
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.

Show MeSH

Related in: MedlinePlus

Analysis of the VSG in PLC− and control bloodstream  trypanosomes. (a) Cell extracts were prepared by SDS lysis, hypotonic lysis, and with addition of exogenous B. cereus PI-PLC to  the hypotonic lysate. Control cell extracts were loaded in lanes 1–3,  and the equivalent PLC− trypanosome extracts in lanes 4–6. The  top panel shows a Coomassie blue-stained gel (2 × 106 cells/ track). Beneath is an equivalent gel (2 × 105 cells/track) that has  been Western blotted and probed with an anti-CRD polyclonal  antibody. (b) Trypanosome extracts were prepared by triton lysis  and incubated at 37°C in the absence or presence of GPI-PLC.  The hydrolysis of the VSG by the GPI-PLC was monitored by  Western blotting the whole reaction using anti-CRD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139819&req=5

Figure 3: Analysis of the VSG in PLC− and control bloodstream trypanosomes. (a) Cell extracts were prepared by SDS lysis, hypotonic lysis, and with addition of exogenous B. cereus PI-PLC to the hypotonic lysate. Control cell extracts were loaded in lanes 1–3, and the equivalent PLC− trypanosome extracts in lanes 4–6. The top panel shows a Coomassie blue-stained gel (2 × 106 cells/ track). Beneath is an equivalent gel (2 × 105 cells/track) that has been Western blotted and probed with an anti-CRD polyclonal antibody. (b) Trypanosome extracts were prepared by triton lysis and incubated at 37°C in the absence or presence of GPI-PLC. The hydrolysis of the VSG by the GPI-PLC was monitored by Western blotting the whole reaction using anti-CRD.

Mentions: Anti–GPI-PLC was prepared by immunizing a rabbit with a λcro-β-galactosidase–GPI-PLC fusion protein. This was prepared by cloning the end-filled DraI–EcoRI fragment from pBS1, containing a GPI-PLC cDNA (Carrington et al., 1989), into the SmaI site of pEX3 (Genofit, Grand-Lancy, France). The fusion protein was insoluble and was separated from the other insoluble proteins by preparative SDS-PAGE. The antibodies were affinity purified on a glutathione-S-transferase–GPI-PLC fusion protein (gst-plc) attached to Sepharose beads. The gst-plc was made by cloning the entire GPI-PLC coding sequence into the EcoRI and BamHI sites of pGEX2T (Pharmacia Fine Chemicals, Piscataway, NJ), the restriction sites being added to the cDNA using PCR. The gst-plc was insoluble and was purified by preparative SDS-PAGE. The anti-CRD was a kind gift of Dr. Paul Englund (Johns Hopkins Medical School, Baltimore, MD; see Fig. 3 a) and Dr. Linda Allen (Cambridge University, Cambridge, UK; see other figures).


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)

Analysis of the VSG in PLC− and control bloodstream  trypanosomes. (a) Cell extracts were prepared by SDS lysis, hypotonic lysis, and with addition of exogenous B. cereus PI-PLC to  the hypotonic lysate. Control cell extracts were loaded in lanes 1–3,  and the equivalent PLC− trypanosome extracts in lanes 4–6. The  top panel shows a Coomassie blue-stained gel (2 × 106 cells/ track). Beneath is an equivalent gel (2 × 105 cells/track) that has  been Western blotted and probed with an anti-CRD polyclonal  antibody. (b) Trypanosome extracts were prepared by triton lysis  and incubated at 37°C in the absence or presence of GPI-PLC.  The hydrolysis of the VSG by the GPI-PLC was monitored by  Western blotting the whole reaction using anti-CRD.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Analysis of the VSG in PLC− and control bloodstream trypanosomes. (a) Cell extracts were prepared by SDS lysis, hypotonic lysis, and with addition of exogenous B. cereus PI-PLC to the hypotonic lysate. Control cell extracts were loaded in lanes 1–3, and the equivalent PLC− trypanosome extracts in lanes 4–6. The top panel shows a Coomassie blue-stained gel (2 × 106 cells/ track). Beneath is an equivalent gel (2 × 105 cells/track) that has been Western blotted and probed with an anti-CRD polyclonal antibody. (b) Trypanosome extracts were prepared by triton lysis and incubated at 37°C in the absence or presence of GPI-PLC. The hydrolysis of the VSG by the GPI-PLC was monitored by Western blotting the whole reaction using anti-CRD.
Mentions: Anti–GPI-PLC was prepared by immunizing a rabbit with a λcro-β-galactosidase–GPI-PLC fusion protein. This was prepared by cloning the end-filled DraI–EcoRI fragment from pBS1, containing a GPI-PLC cDNA (Carrington et al., 1989), into the SmaI site of pEX3 (Genofit, Grand-Lancy, France). The fusion protein was insoluble and was separated from the other insoluble proteins by preparative SDS-PAGE. The antibodies were affinity purified on a glutathione-S-transferase–GPI-PLC fusion protein (gst-plc) attached to Sepharose beads. The gst-plc was made by cloning the entire GPI-PLC coding sequence into the EcoRI and BamHI sites of pGEX2T (Pharmacia Fine Chemicals, Piscataway, NJ), the restriction sites being added to the cDNA using PCR. The gst-plc was insoluble and was purified by preparative SDS-PAGE. The anti-CRD was a kind gift of Dr. Paul Englund (Johns Hopkins Medical School, Baltimore, MD; see Fig. 3 a) and Dr. Linda Allen (Cambridge University, Cambridge, UK; see other figures).

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