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. 2015 Aug 14;9(8):e0003933.
doi: 10.1371/journal.pntd.0003933. eCollection 2015.

ApiAP2 Factors as Candidate Regulators of Stochastic Commitment to Merozoite Production in Theileria annulata

Marta Pieszko  1 , William Weir  1 , Ian Goodhead  2 , Jane Kinnaird  1 , Brian Shiels  1
Affiliations

ApiAP2 Factors as Candidate Regulators of Stochastic Commitment to Merozoite Production in Theileria annulata

Marta Pieszko et al. PLoS Negl Trop Dis. .

Abstract

Background: Differentiation of one life-cycle stage to the next is critical for survival and transmission of apicomplexan parasites. A number of studies have shown that stage differentiation is a stochastic process and is associated with a point that commits the cell to a change over in the pattern of gene expression. Studies on differentiation to merozoite production (merogony) in T. annulata postulated that commitment involves a concentration threshold of DNA binding proteins and an auto-regulatory loop.

Principal findings: In this study ApiAP2 DNA binding proteins that show changes in expression level during merogony of T. annulata have been identified. DNA motifs bound by orthologous domains in Plasmodium were found to be enriched in upstream regions of stage-regulated T. annulata genes and validated as targets for the T. annulata AP2 domains by electrophoretic mobility shift assay (EMSA). Two findings were of particular note: the gene in T. annulata encoding the orthologue of the ApiAP2 domain in the AP2-G factor that commits Plasmodium to gametocyte production, has an expression profile indicating involvement in transmission of T. annulata to the tick vector; genes encoding related domains that bind, or are predicted to bind, sequence motifs of the type 5'-(A)CACAC(A) are implicated in differential regulation of gene expression, with one gene (TA11145) likely to be preferentially up-regulated via auto-regulation as the cell progresses to merogony.

Conclusions: We postulate that the Theileria factor possessing the AP2 domain orthologous to that of Plasmodium AP2-G may regulate gametocytogenesis in a similar manner to AP2-G. In addition, paralogous ApiAP2 factors that recognise 5'-(A)CACAC(A) type motifs could operate in a competitive manner to promote reversible progression towards the point that commits the cell to undergo merogony. Factors possessing AP2 domains that bind (or are predicted to bind) this motif are present in the vector-borne genera Theileria, Babesia and Plasmodium, and other Apicomplexa; leading to the proposal that the mechanisms that control stage differentiation will show a degree of conservation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Life cycle stage and differentiation time course associated gene expression profiles in T. annulata identified by microarray analysis.
Heat-map and hierarchical clustering of T. annulata gene expression profiles of 3,792 predicted protein-encoding genes generated by microarray analysis of RNA derived from a stage-differentiation time-course: Day 0 (macroschizont), Day 4, Day 7 and Day 9 (merozoite production (merogony) occurred at Day 7 and Day 9 time-points); piroplasms (PI) isolated from erythrocytes and tick-derived sporozoites (SP). Each horizontal line represents an individual gene, replicate samples are labelled a and b. Green bands represent genes expressed at low levels, while black and red bands represent intermediate and highly expressed genes respectively.
Fig 2
Fig 2. Temporal expression profiles of selected ApiAP2 genes in T. annulata.
A. Microarray log2 expression values (Y-axis) of up-regulated ApiAP2 genes generated from RNA derived from an in vitro stage-differentiation time-course from Day 0 (macroschizont) to Day 9 (merozoite), and piroplasm stage (x-axis). B. Microarray log2 expression values (Y-axis) of down-regulated APiAP2 genes generated from RNA derived from an in vitro stage-differentiation time-course from Day 0 (macroschizont) to Day 9 (merozoite), and piroplasm stage (x-axis). C. QRT-PCR analysis of ApiAP2 gene, TA11145, expression, plotted as fold change in expression relative to the Day 0 (macroschizont) at Day 4, Day 7 and Day 9 (merozoite), and piroplasm stage. D. QRT-PCR analysis of ApiAP2 gene, TA13515, expression, plotted as fold change in expression relative to the Day 0 (macroschizont) at Day 4, Day 7 and Day 9 (merozoite), and piroplasm stage. Significant difference (P value ≤ 0.05) *, relative to Day 0 and +, relative to the preceding time-point/stage.
Fig 3
Fig 3. Alignment of T. annulata ApiAP2 domains, encoded by genes identified as up-regulated following merogony, with orthologous domains from related species and genera.
A. Alignment of ApiAP2 domain encoded by TA13515 with orthologous domains identified by BLAST analysis from T. parva, T. orientalis, B. bovis, and P. falciparum or C. parvum: strong conservation of the domain across the related species is indicated (up to 100% identity) and genera (92% identity, 96% similarity with B. bovis; 80% identity, 94% similarity with P. falciparum). B. Alignment of ApiAP2 domain encoded by TA11145: strong conservation of the domain across the related species (100% identity) and genera (96% identity, 98% similarity with B. bovis; 72% identity, 82% similarity with P. falciparum) is apparent. C. Alignment of ApiAP2 domain encoded by TA16485: strong conservation of the domain across the related species (100% identity) and genera (96% identity, 100% similarity with B. bovis; 87% identity, 90% similarity with P. falciparum) is evident. D. Alignment of ApiAP2 domain encoded by TA12015: strong conservation of the domain across related species (91% identity with T. parva; 85% identity with T. orientalis), with strong to good conservation across genera (70% identity, 81% similarity with B. bovis; 52% identity, 68% similarity with C. parvum. Regions of predicted secondary structure are indicated above the alignment and were predicted by Phyre2 using three independent secondary structure prediction programs: Psi-Pred [58], SSPro [59] and JNet [60]. * identity,:. similarity.
Fig 4
Fig 4. TA13515D AP2 domain (TaAP2.g) fusion protein domain binds to the motif identified for the orthologous AP2G domain of Plasmodium.
EMSA performed with 0.7 μg of purified GST-TA13515D and 20 fmol of biotin-labelled double-stranded oligo probe containing the GTGTACAC motif recognised by the AP2 domain of Plasmodium AP2-G: lane 1, probe only; lane 2, probe + GST-TA13515D; lane 3, probe + GST-TA13515D + cold competitor (4 pmol); lane 4, probe + GST-TA13515D + competitor (6 pmol); lane 5, probe + GST-TA13515D + competitor (8 pmol); lane 6, mutated ATATAAAA probe (G/C in motif replaced with A) + GST-TA13515D; arrow indicates probe-specific shift.
Fig 5
Fig 5. TA11145D AP2 domain (TaAP2.me1) fusion protein and PNE factor(s) bind to an (A)CACAC(A) type motif upstream of the TA11145 gene.
A. EMSA performed with 0.7μg of purified GST-TA11145D and 20 fmol of biotin-labelled double stranded oligo probe containing a double (A)CACAC(A) motif: lane 1, probe alone; lane 2, probe + GST-TA11145D; lane 3, mutant probe with both motifs mutated * denotes position of specific shift. B. EMSA performed with PNE and the double (A)CACAC(A) motif probe: lane 1, probe alone; lane 2, probe + PNE Day 0; lane 3, probe + PNE Day 9. Letters denote shifts detected, shift B was only present in Day 9 PNE. C. EMSA performed with PNE, and above probe: lane 1, probe only; lane 2, probe + PNE Day 9; lane 3, mutated probe + PNE Day 9, the infection associated shift detected in Day 9 PNE was not obtained with the mutant probe.
Fig 6
Fig 6. Phylogenetic tree of AP2 domains from P. falciparum and Theileria orthologues predicted to bind to (A)CACAC(A) type motifs.
A maximum likelihood phylogenetic tree was constructed using the amino acid sequence of AP2 domains of related genes in T. annulata, T. parva, T. orientalis and P. falciparum. Three clusters of orthologous domains with a representative from each species can be observed for TA11145, TA02615 and TA07100. No clear Plasmodium orthologue was detected for the TA19920 domain. Percentage bootstrap values are shown at each node on the tree.
Fig 7
Fig 7. Expression of TA11145 (TaAP2.me1) is significantly higher in the D7 cell line compared to a cell line that has lost the ability to differentiate to the merozoite.
A. QRT-PCR data plotted as fold change in elevated expression (log2) for ApiAP2 domain encoding genes TA11145 and TA07100 in the differentiation competent D7 cell line versus the attenuated D7B12 cell line. Fold change in expression between cell lines was computed at Day 0 (macroschizont) and Day 4 and Day 7 points of a time-course of differentiation to the merozoite; * denotes significant (P value ≤ 0.05) fold change elevated expression in D7 vs D7B12.
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