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Assembling the components of the quorum sensing pathway in African trypanosomes.

Mony BM, Matthews KR - Mol. Microbiol. (2015)

Bottom Line: These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress.Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing 'slender retainer' and 'stumpy inducer' arms described.As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Charlotte Auerbach Road, Edinburgh, EH9 3FL, UK.

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In vitro induction of stumpy-like forms using 8pCPT-cAMP/AMP (A) and use of this response in a genome-wide RNAi screen to identify components of the stumpy induction pathway (B).A. Pleomorphic parasites are capable of responding to SIF, giving rise to stumpy forms. However, monomorphs are non-responsive to SIF but, instead, are capable of being induced to stumpy-like forms by cell permeable, hydrolysable, cAMP/AMP analogues.B. A monomorphic RNAi library was exposed to 8pCPT cAMP/AMP. Those parasites that had a gene required in the cAMP/AMP response pathway depleted (red), remained slender and continued proliferating, whereas the others (green) underwent growth arrest. The resistant parasites eventually outgrew and predominated the population. DNA was extracted from the selected cells, the RNAi inserts amplified by PCR and then subjected to ion-torrent deep sequencing to identify the genes (A and B) involved in relaying the 8pCPT-cAMP/AMP signal. The identified genes were then validated through individual RNAi lines in pleomorphs to confirm their role in physiological SIF signalling (adapted from Mony et al., 2013).
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fig02: In vitro induction of stumpy-like forms using 8pCPT-cAMP/AMP (A) and use of this response in a genome-wide RNAi screen to identify components of the stumpy induction pathway (B).A. Pleomorphic parasites are capable of responding to SIF, giving rise to stumpy forms. However, monomorphs are non-responsive to SIF but, instead, are capable of being induced to stumpy-like forms by cell permeable, hydrolysable, cAMP/AMP analogues.B. A monomorphic RNAi library was exposed to 8pCPT cAMP/AMP. Those parasites that had a gene required in the cAMP/AMP response pathway depleted (red), remained slender and continued proliferating, whereas the others (green) underwent growth arrest. The resistant parasites eventually outgrew and predominated the population. DNA was extracted from the selected cells, the RNAi inserts amplified by PCR and then subjected to ion-torrent deep sequencing to identify the genes (A and B) involved in relaying the 8pCPT-cAMP/AMP signal. The identified genes were then validated through individual RNAi lines in pleomorphs to confirm their role in physiological SIF signalling (adapted from Mony et al., 2013).

Mentions: This ability of monomorphs to respond to 8-pCPT-cAMP/AMP and undergo growth arrest has been exploited in a genome-wide RNAi library screen that sought to select parasites that were unresponsive to 8-pCPT-cAMP/AMP after gene knockdown. The monomorphic library, capable of tetracycline-induced gene silencing on a genome-wide scale, was exposed to 8-pCPT-cAMP/AMP, following which, growth was monitored and compared with the uninduced set. The parasites that failed to undergo growth arrest were believed to continue proliferation due to the knockdown of a gene required for sensing the 8-pCPT-cAMP/AMP signal. Deep sequencing of the RNAi inserts enriched in the selected 8-pCPT-cAMP/AMP resistant parasite population divulged a collection of genes representing various steps of a typical signalling pathway and likely to be involved in the 8-pCPT-cAMP/AMP response (Fig. 2). Crucially, when many of these genes were individually knocked down in pleomorphs by RNAi, they conferred resistance to SIF in vivo thereby confirming their involvement in the biologically relevant QS signalling pathway. This demonstrated that the 8-pCPT-cAMP/AMP–mediated pathway intersected with the SIF signalling pathway at least to some extent, though inevitably molecules involved in the reception of the SIF signal were missing given the use of a cell permeable signal in the screen (Mony et al., 2013).


Assembling the components of the quorum sensing pathway in African trypanosomes.

Mony BM, Matthews KR - Mol. Microbiol. (2015)

In vitro induction of stumpy-like forms using 8pCPT-cAMP/AMP (A) and use of this response in a genome-wide RNAi screen to identify components of the stumpy induction pathway (B).A. Pleomorphic parasites are capable of responding to SIF, giving rise to stumpy forms. However, monomorphs are non-responsive to SIF but, instead, are capable of being induced to stumpy-like forms by cell permeable, hydrolysable, cAMP/AMP analogues.B. A monomorphic RNAi library was exposed to 8pCPT cAMP/AMP. Those parasites that had a gene required in the cAMP/AMP response pathway depleted (red), remained slender and continued proliferating, whereas the others (green) underwent growth arrest. The resistant parasites eventually outgrew and predominated the population. DNA was extracted from the selected cells, the RNAi inserts amplified by PCR and then subjected to ion-torrent deep sequencing to identify the genes (A and B) involved in relaying the 8pCPT-cAMP/AMP signal. The identified genes were then validated through individual RNAi lines in pleomorphs to confirm their role in physiological SIF signalling (adapted from Mony et al., 2013).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: In vitro induction of stumpy-like forms using 8pCPT-cAMP/AMP (A) and use of this response in a genome-wide RNAi screen to identify components of the stumpy induction pathway (B).A. Pleomorphic parasites are capable of responding to SIF, giving rise to stumpy forms. However, monomorphs are non-responsive to SIF but, instead, are capable of being induced to stumpy-like forms by cell permeable, hydrolysable, cAMP/AMP analogues.B. A monomorphic RNAi library was exposed to 8pCPT cAMP/AMP. Those parasites that had a gene required in the cAMP/AMP response pathway depleted (red), remained slender and continued proliferating, whereas the others (green) underwent growth arrest. The resistant parasites eventually outgrew and predominated the population. DNA was extracted from the selected cells, the RNAi inserts amplified by PCR and then subjected to ion-torrent deep sequencing to identify the genes (A and B) involved in relaying the 8pCPT-cAMP/AMP signal. The identified genes were then validated through individual RNAi lines in pleomorphs to confirm their role in physiological SIF signalling (adapted from Mony et al., 2013).
Mentions: This ability of monomorphs to respond to 8-pCPT-cAMP/AMP and undergo growth arrest has been exploited in a genome-wide RNAi library screen that sought to select parasites that were unresponsive to 8-pCPT-cAMP/AMP after gene knockdown. The monomorphic library, capable of tetracycline-induced gene silencing on a genome-wide scale, was exposed to 8-pCPT-cAMP/AMP, following which, growth was monitored and compared with the uninduced set. The parasites that failed to undergo growth arrest were believed to continue proliferation due to the knockdown of a gene required for sensing the 8-pCPT-cAMP/AMP signal. Deep sequencing of the RNAi inserts enriched in the selected 8-pCPT-cAMP/AMP resistant parasite population divulged a collection of genes representing various steps of a typical signalling pathway and likely to be involved in the 8-pCPT-cAMP/AMP response (Fig. 2). Crucially, when many of these genes were individually knocked down in pleomorphs by RNAi, they conferred resistance to SIF in vivo thereby confirming their involvement in the biologically relevant QS signalling pathway. This demonstrated that the 8-pCPT-cAMP/AMP–mediated pathway intersected with the SIF signalling pathway at least to some extent, though inevitably molecules involved in the reception of the SIF signal were missing given the use of a cell permeable signal in the screen (Mony et al., 2013).

Bottom Line: These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress.Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing 'slender retainer' and 'stumpy inducer' arms described.As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Charlotte Auerbach Road, Edinburgh, EH9 3FL, UK.

Show MeSH
Related in: MedlinePlus