Differentiation granules, a dynamic regulator of T. brucei development.

Adaptation to a change of environment is an essential process for survival, in particular for parasitic organisms exposed to a wide range of hosts. Such adaptations include rapid control of gene expression through the formation of membraneless organelles composed of poly-A RNA and proteins. The African trypanosome Trypanosoma brucei is exquisitely sensitive to well-defined environmental stimuli that trigger cellular adaptations through differentiation events that characterise its complex life cycle. The parasite has been shown to form stress granules in vitro, and it has been proposed that such a stress response could have been repurposed to enable differentiation and facilitate parasite transmission. Therefore, we explored the composition and positional dynamics of membraneless granules formed in response to starvation stress and during differentiation in the mammalian host between the replicative slender and transmission-adapted stumpy forms. We find that T. brucei differentiation does not reflect the default response to environmental stress. Instead, the developmental response of the parasites involves a specific and programmed hierarchy of membraneless granule assembly, with distinct components and regulation by protein kinases such as TbDYRK, that are required for the parasite to successfully progress through its life cycle development and prepare for transmission.

Developmental competence and antigen switch frequency can be uncoupled in Trypanosoma brucei.

African trypanosomes use an extreme form of antigenic variation to evade host immunity, involving the switching of expressed variant surface glycoproteins by a stochastic and parasite-intrinsic process. Parasite development in the mammalian host is another feature of the infection dynamic, with trypanosomes undergoing quorum sensing (QS)-dependent differentiation between proliferative slender forms and arrested, transmissible, stumpy forms. Longstanding experimental studies have suggested that the frequency of antigenic variation and transmissibility may be linked, antigen switching being higher in developmentally competent, fly-transmissible, parasites ("pleomorphs") than in serially passaged "monomorphic" lines that cannot transmit through flies. Here, we have directly tested this tenet of the infection dynamic by using 2 experimental systems to reduce pleomorphism. Firstly, lines were generated that inducibly lose developmental capacity through RNAi-mediated silencing of the QS signaling machinery ("inducible monomorphs"). Secondly, de novo lines were derived that have lost the capacity for stumpy formation by serial passage ("selected monomorphs") and analyzed for their antigenic variation in comparison to isogenic preselected populations. Analysis of both inducible and selected monomorphs has established that antigen switch frequency and developmental capacity are independently selected traits. This generates the potential for diverse infection dynamics in different parasite populations where the rate of antigenic switching and transmission competence are uncoupled. Further, this may support the evolution, maintenance, and spread of important trypanosome variants such as Trypanosoma brucei evansi that exploit mechanical transmission.

Non-linear hierarchy of the quorum sensing signalling pathway in bloodstream form African trypanosomes.

Trypanosoma brucei, the agents of African trypanosomiasis, undergo density-dependent differentiation in the mammalian bloodstream to prepare for transmission by tsetse flies. This involves the generation of cell-cycle arrested, quiescent, stumpy forms from proliferative slender forms. The signalling pathway responsible for the quorum sensing response has been catalogued using a genome-wide selective screen, providing a compendium of signalling protein kinases phosphatases, RNA binding proteins and hypothetical proteins. However, the ordering of these components is unknown. To piece together these components to provide a description of how stumpy formation arises we have used an extragenic suppression approach. This exploited a combinatorial gene knockout and overexpression strategy to assess whether the loss of developmental competence in null mutants of pathway components could be compensated by ectopic expression of other components. We have created null mutants for three genes in the stumpy induction factor signalling pathway (RBP7, YAK, MEKK1) and evaluated complementation by expression of RBP7, NEK17, PP1-6, or inducible gene silencing of the proposed differentiation inhibitor TbTOR4. This indicated that the signalling pathway is non-linear. Phosphoproteomic analysis focused on one pathway component, a putative MEKK, identified molecules with altered expression and phosphorylation profiles in MEKK1 null mutants, including another component in the pathway, NEK17. Our data provide a first molecular dissection of multiple components in a signal transduction cascade in trypanosomes.

The Cytological Events and Molecular Control of Life Cycle Development of Trypanosoma brucei in the Mammalian Bloodstream.

African trypanosomes cause devastating disease in sub-Saharan Africa in humans and livestock. The parasite lives extracellularly within the bloodstream of mammalian hosts and is transmitted by blood-feeding tsetse flies. In the blood, trypanosomes exhibit two developmental forms: the slender form and the stumpy form. The slender form proliferates in the bloodstream, establishes the parasite numbers and avoids host immunity through antigenic variation. The stumpy form, in contrast, is non-proliferative and is adapted for transmission. Here, we overview the features of slender and stumpy form parasites in terms of their cytological and molecular characteristics and discuss how these contribute to their distinct biological functions. Thereafter, we describe the technical developments that have enabled recent discoveries that uncover how the slender to stumpy transition is enacted in molecular terms. Finally, we highlight new understanding of how control of the balance between slender and stumpy form parasites interfaces with other components of the infection dynamic of trypanosomes in their mammalian hosts. This interplay between the host environment and the parasite's developmental biology may expose new vulnerabilities to therapeutic attack or reveal where drug control may be thwarted by the biological complexity of the parasite's lifestyle.