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

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

Figure 1

Structure and restriction enzyme maps of the PLC and tubulin loci, the constructs with which they were targeted, and the desired homologous recombination products. None of the constructs contains promoters but rely on polycistronic transcription for selectable marker expression. (a) Deletion of the PLC gene; structure of genome shows the sites of polyadenylation (pA) for the PLC gene and the main sites of mini-exon addition (ME) for both the PLC gene and the downstream gene, gene 3. These were mapped by RNase protection analysis (data not shown). Boxes represent coding sequences. K* (KpnI) and X* (XbaI) mark the positions of restriction sites introduced into genomic clones by site-directed mutagenesis. Constructs shows the PLC elimination constructs in which the PLC gene has been replaced by a chimeric expression cassette. The two constructs differ in the presence of either the long or short version of an RFLP in the intergenic region. The BglI (Bg) sites were used to linearize constructs before electroporation. Desired recombination product shows the genome after targeting. (b) PLC replacement. Structure of genome shows a representative section of the T. brucei tubulin gene cluster. Construct shows the PLC replacement construct, based on a PstI to PstI fragment that includes two complete α tubulin genes separated by one entire β tubulin gene. BamHI sites were used in Southern analysis of transformed cell lines. The other restriction sites were involved in construction: BstEII (Bs), ClaI (C), HindIII (H), MluI (M), NcoI (N), StuI (St).

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.

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.

Figure 2

Southern analysis demonstrating elimination and subsequent replacement of the PLC gene in EATRO 1125 proG procyclic T. brucei. About 4 μg of genomic DNA from (a) parent EATRO 1125 proG cells, (b) a G418 resistant line cloned after a single PLC knock-out, (c) the PLC null mutant (PLC ⁻) clone and (d) a PLC ⁺ null clone were digested with BamHI and probed with probe B or PLC-coding sequence as indicated. Blots were washed in 0.1 × SSC, 0.1% SDS for 2 h at 60°C. Sizes on the left correspond to the HindIII fragments of λ DNA.

Figure 4

Time course of trypanosome growth on differentiation of control and PLC ⁻ trypanosomes from bloodstream to procyclic forms in vitro.

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.

Figure 6

The course of infection with PLC ⁻ trypanosomes in six individual mice. Parasite densities are expressed as log₁₀ number of trypanosomes/ml of blood.

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.

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.

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.

Figure 9

Expression of GPI-PLC protein in PLC ⁺ trypanosomes. Lysates of bloodstream trypanosomes from three lines; AnTat 1.1, PLC ⁻, and PLC ⁺ trypanosomes were Western blotted and probed with anti–GPI-PLC. All samples are from the same blot and were probed simultaneously.

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.