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. 2018 Jul 24;13(7):e0201263.
doi: 10.1371/journal.pone.0201263. eCollection 2018.

The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei

Anna Kelner  1 , Michele Tinti  1 , Maria Lucia S Guther  1 , Bernardo J Foth  2 , Lia Chappell  2 , Matthew Berriman  2 , Victoria Haigh Cowling  3 , Michael A J Ferguson  1
Affiliations

The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei

Anna Kelner et al. PLoS One. .

Abstract

Messenger RNA is modified by the addition of a 5' methylated cap structure, which protects the transcript and recruits protein complexes that mediate RNA processing and/or the initiation of translation. Two genes encoding mRNA cap methyltransferases have been identified in T. brucei: TbCMT1 and TbCGM1. Here we analysed the impact of TbCMT1 gene deletion on bloodstream form T. brucei cells. TbCMT1 was dispensable for parasite proliferation in in vitro culture. However, significantly decreased parasitemia was observed in mice inoculated with TbCMT1 null and conditional null cell lines. Using RNA-Seq, we observed that several cysteine peptidase mRNAs were downregulated in TbCMT1 null cells lines. The cysteine peptidase Cathepsin-L was also shown to be reduced at the protein level in TbCMT1 null cell lines. Our data suggest that TbCMT1 is not essential to bloodstream form T. brucei growth in vitro or in vivo but that it contributes significantly to parasite virulence in vivo.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. TbCMT1 is not required for T. brucei Lister 427 BSF proliferation in cell culture.
(A) Total RNA was purified from TbCMT1 conditional null cells cultured with (+tet) and without tetracycline for 24, 48 and 72 h. TbCMT1 expression was analysed by qRT-PCR normalised to telomerase reverse transcriptase (TERT). The delta Ct values (dCt) for nine measurements for each condition are visualised as a swarm plot to show all observations along with representations of the underlying distributions. (B) Cumulative cell counts of triplicate TbCMT1 conditional null mutant cell cultures grown with (plus) and without (minus) tetracycline. (C) Cumulative cell counts of triplicate wild type (WT) and TbCMT1 null cells cultured in parallel. For the data in panels B and C, the cell counts of three biological replicates are reported after 2, 4, 6, 8 and 10 days. The cultures were counted and diluted to 105 cells/ml every two days in (B) and to 104 cells/ml every two days in (C). The cell counts are reported as the log10 value of the cumulative number of parasites per ml of cell culture allowing for the aforementioned dilution factors.
Fig 2
Fig 2. TbCMT1 is required for T. brucei proliferation in vivo.
Mice were inoculated with wild type (WT) parasites, three independent clones of TbCMT1 null mutants (null-1, null-2 and null-3), and a TbCMT1 conditional-null clone (c-null). Mice receiving the latter were dosed with (+dox) or without (-dox) doxycycline in the drinking water for seven days before and following inoculation. Blood parasitemias were measured in triplicate for each animal three days after infection and a total of 14 (WT), 5 (null-1), 5 (null-2), 10 (null-3), 20 (c-null +dox) and 5 (c-null -dox) animals were used. The parasitemia is reported as the number of parasites per ml of blood.
Fig 3
Fig 3. Differential gene expression analysis in wild type and TbCMT1 null mutant cells.
The figure shows the log10 transformation of the normalized mean for the transcript reads detected in the WT and TbCMT1 null mutant samples computed by DESeq2 (Base Mean, x axis) versus the log2 transformation of their mean fold change (y axis). A positive fold change indicates transcript upregulation and a negative fold change indicates downregulation in the TbCMT1 null samples relative to WT. Highlighted with arrows are the top 5 up-regulated VSG-related transcripts (VSGs) and top 3 down-regulated cysteine peptidases transcripts (Cysteine Peptidases). The plot also highlights the position of the top downregulated VSG transcript (VSG), the position of the downregulated TbCMT1 transcript (TbCMT1) and the position of the unchanged MITat1.2 VSG transcript. (B) The plot shows the quantification of the Cathepsin-L protein determined by quantitative Western blot in wild type (WT) and three TbCMT1 null mutants clones (null-1, null-2 and null-3). The values of the Cathepsin-L protein are normalized to the HSP-70 protein and divided by the value for the WT sample.
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