The GPI-Phospholipase C of Trypanosoma brucei Is Nonessential ...

Table I. Comparison of Parasitaemias Produced on Infection of Mice with Various Trypanosome Lines; Mann-Whitney Tests Were
Used to Compare Experimental Groups with the Control (Ia) or PLC � with PLC � (Ib), and p Values Are Given in Brackets
Trypanosome line
Ia
Median no.
of troughs*
Parasitaemia
at first peak ‡
significantly (P � 0.01) lower than in PLC � trypanosome
infections, where the mean survival time was 81 d, and
three of the six mice were still alive at the end of the experiment
on day 99. Passaging and innoculum size of the
PLC � trypanosomes had no significant effect on the survival
time of the infected mice despite the higher overall
level of parasitemia compared with the unpassaged PLC �
trypanosomes. Survival time remained significantly different
from control infections at both inoculation size (P �
0.05 and 0.01 for 10 6 and 10 7 trypanosomes, respectively).
The Altered Parasitemia in PLC� Trypanosome Infections is Partially Rescued by
Low Level GPI-PLC Expression
The results above clearly showed that mice infected with
PLC� trypanosomes had an altered parasitemic profile
when compared to the best available control. To address
the question as to whether this alteration was due to the
absence of the PLC gene, or some other event, a copy of
the PLC gene was returned to the PLC� trypanosome. It
was decided to place the PLC gene in the tubulin locus (to
produce PLC� trypanosomes) as opposed to returning it
to the endogenous position, as the latter manipulation
could conceivably repair any lesion in a flanking gene. The
targeting construct employed bleR as a selectable marker
and is shown in Fig. 1. Southern analysis of BamHI digested
DNA from one phleomycin resistant clone is shown
in Fig. 2 and shows that the PLC probe hybridises to a 6.6kb
band, as expected after the desired integration into the
tubulin locus.
Phleomycin-resistant clones were transmitted through
tsetse flies, and the expression of GPI-PLC characterized
by Western blotting and assaying mfVSG to sVSG conversion
on hypotonic lysis. The PLC� trypanosomes were
found to contain GPI-PLC protein but, by comparison
with a twofold dilution series of AnTat 1.1 cells (Fig. 9),
the level of GPI-PLC in 2 � 106 PLC� trypanosomes was
roughly equivalent to that in only 1.25 � 105 AnTat1.1
cells. Thus the PLC� trypanosomes contain approximately
one sixteenth of the wild-type, bloodsteam-form GPI-PLC
level. The presence of GPI-PLC activity was tested in two
assays. The first was detergent lysis of PLC� trypanosomes;
in this assay the endogenous VSG became anti-
Webb et al. GPI-PLC Null T. brucei 111
No. of days
pd �1 � 10 8 (%) §
No. of days
pd �1 � 10 7 (%) §
Mean
survival time
Control 1 1.4 � 10 8 3.5 (17.5) 6.5 (32.5) 28.0
PLC � 3 4.1 � 10 7 (P � 0.01) 0 12.8 (64.2) 81.3 (P � 0.01)
PLC � 22p 2 7.4 � 10 7 (P � 0.01) 1.2 (6.0) 11.2 (56) 72.8 (P � 0.05)
PLC � 22p 1 � 10 7 1–2 7.2 � 10 7 (P � 0.11) 1.2 (6.0) 10.2 (50.8) 90.5 (P � 0.01)
Ib
PLC � 3 2.4 � 10 7 0.2 (0.8) 14.7 (73.3) 74.3
PLC � 1–2 9.4 � 10 7 (P � 0.01) 2.2 (10.8) 9.7 (48.3) 56.7 (P � 0.26)
*A trough is defined as a drop in parasitaemia greater than one order of magnitude between adjacent time points (calculated for the first 40 d).
‡ Geometric means of parasite density at the peak of the first wave of parasitaemia (n � 6, except for PLC � 22p, where n � 5).
§ The mean number of days on which parasite density (pd) was �1 � 10 8 cells per ml of blood, or �1 � 10 7 cells per ml of blood. These were determined for the first 20 d after
infection and are expressed as percentages in parentheses.
CRD positive (Fig. 10). In the second assay, lysates of
PLC � and wild-type trypanosomes were assayed in vitro
for release of tritium from [ 3 H]-myristyl VSG. The PLC �
trypanosome lysates contained an activity that released
15.5 pmol/min/mg protein or 31% of wild-type activity.
There is a discrepency between the estimates of the
amount of GPI-PLC protein (5–10% of wild-type) and the
activity (30% of wild-type). At this stage it is not clear
whether this is an artefact of the enzyme assay or due to
some endogenous regulatory phenomenon.
Matched sets of six mice were infected with either PLC �
or PLC � trypanosomes and the parasitemias monitored
over 93 d. The data are summarized in Table I b. The parasitemia
and mouse survival data for the PLC � parasites
in the two experiments were slightly different as might be
expected, but statistical comparison of parasitemias at first
peak and survivorship showed no significant differences
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.
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