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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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The course of infection in mice infected with control  and PLC− trypanosomes for comparison of parasitemias. Results  are shown as geometric mean ± 2 standard errors, n = 6. Arrowheads indicate the survival time of individual mice.
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Figure 8: The course of infection in mice infected with control and PLC− trypanosomes for comparison of parasitemias. Results are shown as geometric mean ± 2 standard errors, n = 6. Arrowheads indicate the survival time of individual mice.

Mentions: The course of chronic infection of mice with control trypanosomes was monitored in parallel with the PLC− infections described above. Passage through a tsetse fly can greatly alter the growth characteristics of a trypanosome cell line (Turner, 1990). Therefore, the most comparable trypanosome population available was used as a control in infections. This population had been derived from the same procyclic cell line as the PLC− mutant and was transmitted through tsetse flies in parallel, but had a chloramphenicol acetyl transferase-hygromycinR gene construct integrated into the tubulin gene cluster (Berberof et al., 1995). Since isolation from tsetse flies, both of the trypanosome stocks had been amplified twice in immunosuppressed NMRI mice and stabilates prepared from the second mouse. These stabilates were used to infect immunosuppressed CFLP mice, and blood from these mice was used to establish persistent infections in matched sets of six CFLP mice. The innoculum for control and PLC− trypanosomes was matched as closely as possible; both were collected while the parasitemia was rising and contained a preponderence of long slender and intermediate forms as opposed to stumpy forms. The differences between the two parasitemic profiles are illustrated in Fig. 8, which shows log10 geometric mean parasite density through time. Although each infection within a group gave the same basic profile, slight variation in the timing of peaks and troughs in individual mice means that the second and subsequent troughs are evident only by large standard error bars.


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)

The course of infection in mice infected with control  and PLC− trypanosomes for comparison of parasitemias. Results  are shown as geometric mean ± 2 standard errors, n = 6. Arrowheads indicate the survival time of individual mice.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: The course of infection in mice infected with control and PLC− trypanosomes for comparison of parasitemias. Results are shown as geometric mean ± 2 standard errors, n = 6. Arrowheads indicate the survival time of individual mice.
Mentions: The course of chronic infection of mice with control trypanosomes was monitored in parallel with the PLC− infections described above. Passage through a tsetse fly can greatly alter the growth characteristics of a trypanosome cell line (Turner, 1990). Therefore, the most comparable trypanosome population available was used as a control in infections. This population had been derived from the same procyclic cell line as the PLC− mutant and was transmitted through tsetse flies in parallel, but had a chloramphenicol acetyl transferase-hygromycinR gene construct integrated into the tubulin gene cluster (Berberof et al., 1995). Since isolation from tsetse flies, both of the trypanosome stocks had been amplified twice in immunosuppressed NMRI mice and stabilates prepared from the second mouse. These stabilates were used to infect immunosuppressed CFLP mice, and blood from these mice was used to establish persistent infections in matched sets of six CFLP mice. The innoculum for control and PLC− trypanosomes was matched as closely as possible; both were collected while the parasitemia was rising and contained a preponderence of long slender and intermediate forms as opposed to stumpy forms. The differences between the two parasitemic profiles are illustrated in Fig. 8, which shows log10 geometric mean parasite density through time. Although each infection within a group gave the same basic profile, slight variation in the timing of peaks and troughs in individual mice means that the second and subsequent troughs are evident only by large standard error bars.

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