In addition to AUK2, we identified 11 other cell lines where depletion of the protein kinase led to a loss of fitness in vivo, but not in either the kinome- (Jones et al., 2014) or the genome- wide (Alsford et al., 2011) in vitro RNAi studies (Table 3-5 and Figure 3-13). In order to validate the observation in vitro, independent growth curves were performed in culture for each of the individual RNAi cell lines used in the inoculum pool. Where a growth defect was not
detected after 48 h of RNAi induction in vitro, parasitemia was assessed in mice after infection with the selected clone for 96 h (Figure 3-14). Tb927.10.14770 (AKB1) was not examined as has been shown to be an active kinase involved in regulation of cytokinesis and cell division in bloodstream and procyclic forms upon knock down from 48 h onwards (Inoue et al., 2015). The reason why it was not detected by Jones et al. is that the original cell line used in Replicate 1 did not contain an insert (Table 3-4). Another protein kinase gene, Tb927.7.3880 (DYRK), had a clear and unexpected growth defect after 48 h of RNAi induction
in vitro (Figure 3-14A), and so this gene was not considered further. For the
other protein kinase genes, where no growth defect was detected after 48 h of RNAi induction in vitro, parasitemia was assessed for 72-96 h in mice, with or without induction of RNAi, after inoculation of each individual selected cell line (Figure 3-14B).
Figure 3-14. Validation of RITseq in vivo-mainly loss of fitness phenotypes.
For each RNAi cell line, cumulative in vitro growth curves are shown on the left hand side; and in vivo parasitemia on the right hand side. Growth curves were built in triplicate. In vitro cultures started at 104 cells x ml-1. Mice were culled if parasitemia reached 108 cells x ml-1. A. Tb927.7.3880 (DYRK). B. 9 other cell lines with a loss of fitness phenotype in vivo that is more pronounced than the one observed in vitro. * p < 0.05 and ** p < 0.01 using T-test.
BLAST alignment with protein sequences in other eukaryotes allowed a tentative proposal of the putative function of each protein kinase based on gene ontology (Table 3-6). Tb927.11.9290 (Pseudo-unique), Tb927.2.1820 (CAMKL),
Tb927.11.850 (aPK/Bud32) and Tb927.11.9190 (Other/VPS15) displayed some evidence for growth retardation after RNAi in vitro (Figure 3-14B) but, in each case, the extent of growth impairment or death was more severe in vivo and arose more quickly. For each of the remaining six cell lines there was no evidence of RNAi-induced growth retardation in vitro. For two of the genes, Tb927.3.3920 (AUK2) and Tb927.10.10350 (STE11/Bck1p), RNAi caused growth retardation in vivo from 48 h, indicating a loss of fitness. For three other genes, Tb927.2.2430 (Other/CK2A2), Tb927.7.960 (CMGC/SRPK1) and Tb927.10.14300 (STE11/MRK1), the loss of fitness after in vivo RNAi was even more severe, since a decrease in parasitemia or clearance was seen before 96 h (Figure 3-14B). Taken as a whole, these growth curves validate the kinome-wide RITseq both in
vivo and in vitro for nine of eleven predicted protein kinases (Table 3-3).
Table 3-6. Predicted function of in vivo only protein kinases based on gene ontology with other eukaryotes.
pBLAST Organism Identity E value Protein Putative function
Tb927.2.
2430 (C
K
2A
2)
T. brucei 100% 0 casein kinase II, chain (CK2A2) · Proven kinase activity.
· Mainly nucleolar but ubiquitous (Jensen
et al., 2007), controlled by distribution and
abundance (Pinna, 2003): stress-induced mobilization (Gerber et al., 2000; Davis et
al., 2002).
· Gene expression/protein synthesis: survival, differentiation and proliferation (>300 substrates) (Meggio and Pinna, 2003).
· Downregulation causes autophagy through PI3K/AKT/TOR pathway (Olsen, Svenstrup and Guerra, 2012; Hales, Taub and Matherly, 2014; Sanchez-Casalongue et
al., 2015).
· Intersects with mitogen and stress- activated protein kinases (Shi et al., 2009; Jacks and Koch, 2010).
L. major 60% 4x10-148 putative casein kinase II, chain
T. cruzi 73% 0 casein kinase II, chain
S. cerevisiae 48% 4x10-95 Cka2p
C. elegans 48% 1x10-87 Casein kinase II subunit
Human 51% 10-109 Chain A, Crystal Structure Of Ck2
Tb927.10 .143 00 (S TE1 1/ M R K 1)
T. brucei 100% 0 MEKK-related kinase 1, putative (MRK1)
· Member of the HOG pathway (Zhi et al., 2013)
· Response to stress signalling, mainly hyperosmotic shock (through actin recovery pathway (Zhi et al., 2013) · Essential for promastigote survival, in Leishmania (Agron, Reed and Engel, 2005)
L. major 37% 6x10-174 MEKK-related kinase 1, putative
(MRK1)
cruzi 50% 0 MEKK-related kinase 1, putative (MRK1)
S. cerevisiae 36% 1x10-43 Ssk2p
C. elegans 34% 7x10-37 Germinal Center Kinase family
Human 36% 1x10-46 MAPKKK19 isoform 3
Tb927.10 .103 50 (S TE1 1/ B ck1p)
T. brucei 100% 0 protein kinase, putative · Contains 8 amino terminal “MORN motif” sequences: subcellular localization, membrane fusion, fission and mobility in other organisms (Lee, Han and Hur, 2010). · Cell integrity via MAPK pathway in response to growth factors and stress (hyperosmotic, oxidative and sheer fluid
L. major 60% 0 protein kinase, putative
T. cruzi 73% 0 protein kinase, putative
S. cerevisiae 41% 8x10-55 Bck1p
Human 45% 4x10-65 MEK kinase 3
shocks) (Widmann et al., 1999) · Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways (Ellinger-Ziegelbauer et al., 1997)
Tb927.11 .850 (aP K /B ud32)
T. brucei 100% 0 protein kinase, putative · KEOPS/EKC complex: translational regulation throughout tRNA modification (Srinivasan et al., 2011; Rojas-Benítez, Ibar and Glavic, 2013).
· Transducer for TOR activation that, in turn, has also been related with autophagy and endocytosis under stress conditions (Ibar et al., 2013; Rojas-Benítez, Ibar and Glavic, 2013).
L. major 48% 1x10-70 protein kinase, putative
T. cruzi 63% 2x10-103 protein kinase, putative
S. cerevisiae 32% 4x10-24 Bud32p
C.elegans 35% 2x10-40 Uncharacterized protein
CELE_F52C12.6
Human 40% 2x10-44 TP53 regulating kinase
Tb927.11 .919 0 (aP K /V P S15)
T. brucei 100% 0 protein kinase, putative
· PI3K complex mamber: autophagosome formation/regulation of protein and vesicular trafficking and sorting (Abraham, 2004; Araki et al., 2013; Liu et al., 2014; Anding and Baehrecke, 2015).
L. major 33% 2x10-64 protein kinase, putative
T. cruzi 50% 0 protein kinase, putative
S. cerevisiae 23% 6x10-32 Vps15p
C.elegans 33% 7x10-23 Vacuolar Protein Sorting factor
Human 24% 6x10-38 PI3K regulatory subunit 4
Tb927.7. 960 (C M G C /S R P K )
T. brucei 100% 0 protein kinase, putative
· Nucleus/cytoplasm localization · Phosphorylation of splicing factors containing serine/arginine-rich domains · Regulation of constitutive/alternative splicing (Zhou et al., 2012).
Stress-response mechanism in eukaryotes (Zhong et al., 2009).
L. major 53% 8x10-173 protein kinase, putative
T. cruzi 51% 0 protein kinase, putative
S. cerevisiae 40% 5x10-26 Sky1p
C.elegans 39% 6x10-24 SR Protein Kinase
Human 41% 2x10-27 SFRS protein kinase 1, isoform
CRA_c Tb927.11 .929 0 (P se ud o - O rphan/ FA Z20)
T. brucei 100% 0 protein tyrosine kinase, putative
· Parasite-specific
· Localizes to the tip of the flagellum attachment zone in both the old and the new cell Generated during cytokinesis (Zhou, Hu and Li, 2016)
· None predicted kinase activity (Figure 3-18) and (Taylor et al., 2013)
L. major 32% 2x10-35 protein kinase, putative
T. cruzi 34% 2x10-110 protein kinase, putative
S. cerevisiae 26% 8x10-7 Gin4p
C.elegans 25% 3x10-4 unc-82
Human 25% 4x10-4 NUAK family SNF1-like kinase 2
Tb927.3. 3920 ( A U K 2)
T. brucei 100% 0 protein kinase, putative
· Associates to the spindle poles · Regulates entry in mitosis and it is often overexpressed in human cancers (D’Assoro, Haddad and Galanis, 2015)
· DNA repair candidate (Stortz, 2016)
L. major 49% 9x10-89 protein kinase, putative
T. cruzi 60% 0 protein kinase, putative
S. cerevisiae 31% 1x10-33 Kin82p
C.elegans 34% 4x10-50 Aurora/IPL1-related protein
kinase 2
Human 37% 7x10-54 Chain A, Structure Of Aurora-2
(AUKA) Tb927.2. 1820 (C A M K
L) T. brucei 100% 0 protein kinase, putative
· Energy balance maintenance (Salminen A, Kauppinen A and Kaarniranta K, 2016) · Phosphorylation of metabolic enzymes and transcription factors
· Epigenetic regulation
· Calcium binding (contains EF hands)
L. major 57% 0 protein kinase, putative
T. cruzi 63% 0 protein kinase, putative
S. cerevisiae 40% 8x10-57 Ampk Homolog Snf1
C.elegans 61% 7x10-59 Ampk subunit alpha-2
Human 38% 1x10-55 Ampk subunit alpha-2
Results of BLASTp analysis aligning against a non-redundant protein sequence database. E = Expect value.