Authors

Discussion

In organisms such as yeast, mammals and plants, ‘mitochondrial retrograde signalling’ pathways, including the ‘unfolded protein response (UPR^mt), convey information on the functional status of this organelle to the nucleus and modulate expression of nuclear genes accordingly^1,2,3. Although successful completion of the life cycle of Trypanosoma brucei depends on stringent regulation of mitochondrial activity, it is not known if similar signalling pathways exist in these parasites. As T. brucei differentiates from the slender bloodstream form via the transmission competent stumpy form to the procyclic insect form it undergoes dramatic remodelling of its morphology and metabolism, including mitochondrial activity. For example, respiratory complexes III (cIII, cytochrome bc₁ complex) and IV (cIV, cytochrome c oxidase), composed of both mitochondrially and nuclearly-encoded subunits, are repressed in slender forms but fully active in procyclic forms. As a first step towards exploring potential retrograde signalling pathways in T. brucei we have compared the transcriptome of strain EATRO 1125 (AnTat1.1 90:13⁴) with a genetically engineered derivate devoid of mitochondrial DNA (akinetoplastic, or AK cells), before and after differentiation from slender to stumpy forms. Our main findings are:

(i) In control (‘WT’) and AK parasites, 242 and 433 genes, respectively, showed significant upregulation in stumpy forms (we considered a difference of ≥ 2-fold with a p-value ≤ 0.05 as significant). 102 upregulated genes were shared, including genes previously reported to be upregulated in stumpy cells such as PIP39 (Tb927.9.6090), dihydrolipoyl dehydrogenase (Tb927.11.16730) and EP1 procyclin (Tb927.10.10260). The upregulation of other procyclins was much less pronounced in AK stumpy cells compared to WT stumpy cells and data from the literature. This is perhaps a reflection of the reduced life span of AK stumpy cells⁵. On the other hand, 10 hypothetical proteins showed more than 4-fold upregulation exclusively in stumpy AK cells.

(ii) As reported before^6,7, genes involved in the glycolytic pathway were generally downregulated in stumpy cells. We also observed robust downregulation in stumpy cells of numerous histones and of two genes involved in kDNA maintenance, mitochondrial DNA ligase LIG k alpha (Tb927.7.610) and cysteine peptidase PNT1 (Tb927.11.6550).

(iii) When we compared slender AK vs. WT cells we observed only 27 robust changes (either up or down), which mostly concerned genes annotated as ‘pseudogene’, or ‘hypothetical unlikely’, including a putative UDP-Gal or UDP-GlcNAc-dependent glycosyltransferase pseudogene that was ~5-fold increased in AK slender cells. The same mRNA was ~3-fold increased in AK stumpy cells compared to WT stumpy cells. Other changes in AK stumpy cells concerned a hypothetical protein (~3-fold upregulated) and a putative adenylosuccinate lyase (~3-fold downregulated), but overall we observed only a limited number of robust changes (13 up, 9 down), including the decreased levels of procyclin mRNAs mentioned above.

In summary, our transcriptomic studies suggest that absence of the mitochondrial genome has a surprisingly limited effect on levels of nuclearly encoded mRNAs in bloodstream stage T. brucei.

References

¹Qureshi MA et al. (2017). J Biol Chem 292(33):13500-13506.

²Knorre DA et al. (2016). Microb Cell 3(11):532-539.

³Kleine T, Leister D (2016). Biochim Biophys Acta 1857(8):1313-1325.

⁴Engstler M, Boshart M (2004). Genes Dev 18(22):2798-811.

⁵Dewar CE et al. (submitted).

⁶Capewell P et al. (2013). PLoS One 8(6):e67069. doi: 10.1371/journal.pone.0067069.

⁷Nilsson et al. (2010). PLoS Pathog 6(8):e1001037. doi: 10.1371/journal.ppat.1001037.