Assembling the components of the quorum sensing pathway in African trypanosomes

Abstract

African trypanosomes, parasites that cause human sleeping sickness, undergo a density-dependent differentiation in the bloodstream of their mammalian hosts. This process is driven by a released parasite-derived factor that causes parasites to accumulate in G1 and become quiescent. This is accompanied by morphological transformation to 'stumpy' forms that are adapted to survival and further development when taken up in the blood meal of tsetse flies, the vector for trypanosomiasis. Although the soluble signal driving differentiation to stumpy forms is unidentified, a recent genome-wide RNAi screen identified many of the intracellular signalling and effector molecules required for the response to this signal. 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.

Figure 1

A schematic representation of the infection dynamics of T. brucei growth in the mammalian host. Upon reaching a threshold density (and SIF levels) slender forms undergo differentiation to stumpy forms, characterised by G1/G0 arrest as well as morphological changes. Thereafter, the parasitaemia declines, until the next wave emerges due to the proliferation of slender forms with a new VSG coat, this evading antibodies raised to parasites in the first peak. The stumpy forms play a crucial role in transmission, being pre-adapted for survival in the tsetse fly and differentiation to procyclic forms in the tsetse midgut. The growth arrest of stumpy forms is key to maintenance of infection chronicity, as curtailment of the parasitaemia ensures prolonged survival of the host.

Figure 2

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).

Figure 3

The balance between ‘slender retainers’ (SR) and ‘stumpy inducers’ (SI) controls stumpy formation. The slender cells remain proliferating as long as the levels of SR are high and SI are low. However, upon an increase in cell density, SIF accumulates, triggering a quorum sensing like response that induces the activation of SIs with a concomitant repression of the SRs. It is the combined action of ‘switching-off’ of the slender retention (SR) arm and ‘switching-on’ of the stumpy induction (SI) arm that ultimately drives the formation of stumpy cells.

Figure 4

A hypothetical framework for the molecular control of stumpy formation. The figure shows an integration of all known networks linked to starvation responses and genes identified in the genome-wide RNAi screen for drivers of stumpy formation. Many of the components included remain to be validated in independent RNAi or knockout lines and so their inclusion is speculative.