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. Author manuscript; available in PMC: 2018 Mar 4.
Published in final edited form as: Nat Microbiol. 2017 Sep 4;2(11):1471–1479. doi: 10.1038/s41564-017-0014-5

Figure 2. A T. congolense orthologue of a T. brucei QS response pathway component, TbHYP2, can restore stumpy formation in a T. brucei TbHYP2 null mutant in murine infections.

Figure 2

a) A T. brucei EATRO 1125 TbHyp2 null mutant was generated by sequential allelic replacement in the parental cell line T. brucei AnTat1.1 90:13. The TbHyp2 null mutant (T. brucei AnTat1.1 90:13 TbHYP2 dKO) was then transfected with a construct for the doxycycline-inducible ectopic expression of TcHYP2 (TcHYP2 OE in KO).

b) Growth of the T. brucei EATRO 1125 TbHyp2 null mutant cell line with doxycycline-inducible TcHYP2 expression (TcHyp2 OE in KO) in mice. Parasite population growth was limited in infections where TcHYP2 ectopic expression was induced (+Dox, red lines, n=3) relative to uninduced controls (-Dox, blue lines, n=3). Parental T. brucei AnTat 1.1 90:13 (black line, n=1) and T. brucei EATRO 1125 TbHYP2 null mutant (navy line, n=1) cell lines were included as controls (further replicates are provided in Supplementary Figure 4). Crosses indicate humane euthanasia when infections were anticipated to be lethal within 12 hours.

c) Expression of the stumpy marker PAD1 was higher when TcHYP2 was ectopically expressed in the T. brucei EATRO 1125 TbHyp2 null mutant than in the uninduced control. TY1-tagged TcHYP2 protein was detected with BB2 antibody in +Dox samples but not in –Dox samples. Glucose-6-phosphate dehydrogenase, loading control.

d) Cells induced to express TcHYP2 (+Dox, n=3) had increased capacity to differentiate to procyclic forms relative to uninduced cells (-Dox, n=3) following treatment with cis-aconitate, as determined by EP procyclin expression at 4h and 24h after treatment (unpaired t test, ★★★★p<0.0001, ★★p<0.01, two-sided). Bars represent mean ± SD). Parental T. brucei AnTat 1.1 90:13 stumpy (green, n=1) and culture-derived slender (orange, n=2) cells, as well as T. brucei EATRO 1125 TbHYP2 null mutant (navy, n=1) cells were included as controls. Culture-derived procyclic cells (purple, n=1) were used as a positive control for procyclin expression.