Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei

Abstract

The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub-Saharan Africa. In mammalian blood, two developmental forms of the parasite exist: proliferative 'slender' forms and arrested 'stumpy' forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. This response is triggered by an elusive 'stumpy induction factor' (SIF) whose intracellular signalling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell-permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNA interference library screen to identify the signalling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) or 8-pCPT-2'-O-methyl-5'-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide Ion Torrent based RNAi target sequencing identified cohorts of genes implicated in each step of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo. The putative RNA-binding protein, RBP7, was required for normal quorum sensing and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that quorum sensing signalling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for quorum-sensing interference-based therapeutics.

Figure 1. Identification of trypanosome QS regulators

a. Selection for genes whose RNAi silencing renders trypanosomes resistant to pCPTcAMP or 8-pCPT-2′-O-Me-5′-AMP, identifying molecules that promote stumpy formation. b. RNAi libaries were exposed to pCPTcAMP or 8-pCPT-2′-O-Me-5′-AMP, RNAi being induced (1 μg/ml tetracycline), or not. The curves for uninduced samples are combined for clarity (mean ±s.e.m, n=5). c. Ion Torrent™ read-density from the selected parasites aligned to the trypanosome genome. Since amplicons were fragmented prior to sequencing, reads with and without the flanking primers are shown.

Figure 2. RNAi to the identified genes prevents growth control and morphological transformation

a. In vivo growth of pleomorphic RNAi lines targeting distinct genes identified from the genome-wide screen for QS-signal resistance. RNAi was induced by provision of doxycyline to the infected animals (n=3, red lines), with parallel infections remaining uninduced (n=3, blue lines). Infections were terminated when the ascending parasitaemias were predicted to become lethal within 12 hours (crosses). b.Morphology of PP1 RNAi cells and parental T. brucei AnTat1.1 90:13 cells (each grown in mice ±doxycycline) at 6 days post-infection. The induced PP1 cells remained predominently slender in morphology. Cells with slender (SL), intermediate (INT) or stumpy (ST) morphology are labelled. Bar=15μm.

Figure 3. Silencing the identified genes reduces G1 arrest and differentiation competence

a. Cell-cycle status of pleomorphic RNAi lines. n=6; mean ±s.e.m. percentage 1 kinetoplast (K), 1 nucleus (N) (G0/G1, plus S-phase cells; left Y-axis), 2K1N (G2-phase cells) or 2K2N (post-mitotic cells) (both right Y-axis) are shown. Test genes showed a signficant difference (GLMM, p<0.001) in comparison to AnTat1.1 90:13 +doxycycline on at least one day of infection. b. PAD1 expression on day 6 post-infection (n=3/group, mean ±s.e.m.). PP1 RNAi cells show reduced PAD1 expression (GLM, F1,4=22.35, p=0.009). c. PP1-depleted cells show signficantly reduced Procyclin expression during differentiation (GLM, F1,4=10.87, p=0.030) Bars represents mean ±s.e.m; n=3.

Figure 4. RBP7 drives cell cycle arrest and differentiation competence

a. Inducible overexpression of RBP7B mRNA on day 3 post-infection. Stumpy RNA is also shown; Ethidium bromide stained rRNA indicates loading. b. AnTat1.1 90:13 induced with doxycycline (red lines) to overexpress RBP7B show reduced parasitaemia in mice (n=3 per group). GLM, F_1,4=55.25, p=0.002 and F_1,4=233.1, p<0.001 on day 2 and 3, respectively. c. Cells accumulate in G1 upon RBP7B ectopic expression. Values shown are mean ±s.e.m at day 3 post-infection; n=3 per group. GLM, F_1,4=15.1, p=0.018 (1K1N); F_1,4=8.9, p=0.041 (2K1N); F_1,4=5.17, p=0.085 (2K2N). d. Parasites isolated on day 3 post-infection were exposed to 6mM cis aconitate (CCA) and EP-procyclin expression monitored by flow cytometry. At 0h enhanced cold induction of EP procyclin expression is seen in the induced population. n=3; GLM; F_1,4=8.54, p=0.043 (0h); F_1,4=9.99, p=0.034 (4h); F_1,4=10.36, p=0.032 (6h) F_1,4=6.84 , p=0.059 (8h). e. Schematic of the proposed SIF-signaling pathway in T. brucei. Major identified components (Supp. Table 2) are shown; those in bold italics are experimentally confirmed. The order and potential branching in the pathway is unknown as is the position of pathway inhibitors, TbTOR4 (Tb927.1.1930) ZFK (Tb927.11.9270) and MAPK5 (Tb927.6.4220).