Research project P6/15 (Research action P6)
The molecular mechanisms used by parasites to undergo developmental transformations and adapt to their hosts are poorly characterized. In direct continuation of results obtained during the previous collaborative work (P5/29), we propose to study African trypanosomes (prototype Trypanosoma brucei) as model organisms to investigate several aspects of the dialogue between the parasite and its hosts.
A. Studies on the parasite
1. Endocytosis of host macromolecules (TCD, ULB)
a- Characterization of the endocytic machinery. In T. brucei, the surface receptors and endocytic machinery are clustered in a specialized region of the cell surface, termed the flagellar pocket. We have initiated the characterization of these proteins by a proteomic approach of the cellular fraction containing or binding to the endocytic sorting signal identified under our previous IAP collaboration (linear chains of poly-N-acetyllactosamine), and propose to continue this work. We will particularly focus on components involved in the uptake of the trypanolytic factor apolipoprotein L-I (apoL-I).
b- Mechanism of resistance of T. b. gambiense to apoL-I (ULB)
T. b. gambiense is able to resist the trypanolytic activity of apoL-I, and the mechanism of this resistance differs from the one we have characterized for T. b. rhodesiense during phase V of our IAP collaboration. We propose to follow three approaches to investigate this question: (i) the evaluation of the possible involvement of a receptor-like protein that we have shown to be totally specific to the gambiense subspecies (TgsGP), (ii) the study of the process of apoL-I binding and uptake in T. b. gambiense, and (iii) the systematic screening for genes involved in resistance to apoL-I, by the use of a transposon-mediated mutagenesis.
c- Studies on apoL-I and apoLs (ULB, VUB,TCD)
In humans apoL-I belongs to a family of six members, and it is the only one of this family to be secreted extracellularly. The function of these proteins is presently unknown. Our previous work on the trypanolytic activity of apoL-I has uncovered the capacity of this protein to generate anionic pores in cellular membranes such as the lysosomal membrane of T. brucei. We propose to build on our acquired expertise to study the function of apoLs. Concerning apoL-I we will particularly investigate its putative interaction with haptoglobin-related protein (Hpr), and we will refine the development of trypanotoxins generated by the fusion of apoL-I with nanobodies (single domain antigen recognizing units) targeting the parasite surface. Concerning the intracellular apoLs we will study the phenotype of various types of human cells transfected with tagged versions of these recombinant proteins, either wild-type or mutated in order to probe the structure and function based on what we have found in apoL-I. From our preliminary results, we propose to detail the possible interplay between apoLs and apoptotic proteins of the Bcl-2 family. In this context, we plan to generate KO mice for the apoL gene cluster.
2. Cell signalling (LMUM, ULB)
Since cyclic AMP (cAMP) is a crucial player in trypanosome signalling, we propose to thoroughly investigate the mechanisms involved in the synthesis of this molecule (activation of adenylate cyclase), as well as the role played by cAMP in cellular proliferation and differentiation (involvement of protein kinase A?). In particular, the phenotype of different dominant negative mutants of adenylate cyclase, obtained under phase V of our IAP collaboration, will be analyzed in details since this phenotype suggests a key involvement of cAMP in the parasite escape to immune defences. In addition, we will try to determine if the motility phenotype observed upon underexpression of a protein kinase A-like protein is related to these processes. Finally, we propose to develop an RNAi screen and a FACS-based assay to identify genes important for the process of differentiation from bloodstream to procyclic forms.
3. Controls of gene expression (ULB)
The controls operating on the expression of the Variant Surface Glycoprotein (VSG) genes represent a paradigm of those responsible for cellular differentiation of T. brucei. As other genes, VSG genes are contained in polycistronic transcription units (termed here VSG expression sites, or ESs) whose individual level of mRNA production is controlled post-transcriptionally. But, in addition, a mono-allelic control only permits the transcription of a single ES at a time in bloodstream forms, whereas none of these sites is active in insect-specific procyclic forms. Finally, the transcription promoters of the ESs are of the ribosomal type, and recruit RNA polymerase I (RNA Pol I). Our major objective is to identify the mechanisms controlling RNA Pol I on the ESs. We intend to characterize the relevant transcription machinery and associated factors. In particular, we will analyze the multisubunit RNA elongation and DNA repair factor TFIIH, and study the functional relevance of the RNA Pol I-specific isoforms of the RPB5 and RPB6 subunits, discovered during our previous work in the IAP programme. Another protein of interest is PIE8, which seems to be part of a nuclear complex controlling the passage from mitosis to cytokinesis. We intend to characterize the proteins associating with PIE8.
4. Metabolic changes during the parasite life-cycle
a- Glycosome turnover and autophagy (UCL, ULB, ITG)
Various metabolic systems of trypanosomes are sequestered inside peroxisome-like organelles called glycosomes. Using the model system developed under phase V of our IAP collaboration, preliminary indications were obtained that glycosome degradation during differentiation involves a special form of autophagy, termed pexophagy. We propose to pursue these studies and those on the biogenesis of new glycosomes, and to unravel the mechanistic details. Moreover, we will extend our studies on glycosome turnover to the various differentiation steps of the parasites when they move, within the tsetse fly, from the midgut to the proboscis and subsequently to the salivary glands, where they transform into the mammalian-infective stage.
b- Mitochondrial functions in the life-cycle (LMUM, UCL, ULB, ITG)
Complementary to the work on glycosome turnover, we will study the developmental regulation of mitochondrial energy metabolism and coordinate control of gene expression of the relevant enzymes. We aim at identification of signalling mechanisms that coordinate the biogenesis/degradation of the two energy producing organelles in the life cycle. We suggest that cytoplasmic aconitase and glycosomal isocitrate dehydrogenase are required for NADPH production and oxidative stress defence in glycosomes. The relevance of this pathway will be analysed upon fly passage of various pleomorphic knock out lines.
B. Studies on the host-parasite interactions
1. Mechanisms underlying pathology in mammalian hosts (VUB)
Under our previous IAP work we have shown that trypanotolerance and control of immunopathology both require the sequential expansion of classically activated myeloid cells (M1) followed by alternatively activated myeloid cells (M2) and regulatory T cells (Tregs). Accordingly, M1 control parasite growth and simultaneously contribute to host immunopathology, while M2 and Tregs protect the host against the immunopathology in an IL-10-dependent manner. We now propose to study, in liver and bone marrow compartments, the role of M1 or M2- and/or Treg -associated gene products in induction of/protection against immunopathology. This will be performed in animals that are naturally trypanotolerant or that are rendered trypanotolerant upon GPI treatment. On the other hand, we have also identified M1-associated genes and parasite compounds that may contribute to development of anemia, another pathological feature of African trypanosomiasis. The contribution of these factors to the induction of anemia will be investigated.
2. Myeloid cells – trypanosome interactions (VUB, ULB)
We will unravel the role of M1 and M2-inducing proteins (kinesin heavy chain, TSIF, GPI, immune complexes...) identified in the outcome of the disease during the previous phase of the IAP programme. Candidate genes for the functions of these immunomodulators will be selected using a myeloid cell activation state micro-array that we have recently developed to unravel their mechanism of action as well as their influence in the outcome of the disease.
3. Tsetse fly – trypanosome interactions (ITG, ULB, VUB,UCL)
In the final phase of the parasite development in the fly, trypanosomes tightly adhere to the epithelial surface of the insect salivary glands, re-activate binary multiplication and, after a complex cascade of cellular changes, free metacyclic forms are generated that are adapted to mammalian blood, where they are co-injected with the tsetse saliva. We will mainly focus our work on i) the tight tsetse-parasite junctional complex between the trypanosome epimastigote stage and the salivary gland epithelium, ii) the growth and differentiation of the parasite in the salivary glands and iii) the early development of the trypanosomes at the biting site in the mammalian host. By a mass spectrometry-based proteomic approach, epithelium-associated proteins that are candidates to be involved in the tsetse-parasite junctional complex will be identified. The gene silencing method for tsetse fly based upon RNAi, developed during our previous IAP work, will be exploited to assess the role of saliva proteins in the growth and differentiation of the trypanosome in the salivary glands. Genes encoding for saliva proteins with predicted functionality in cell growth stimulation or breakdown of ATP, ADP and nucleotides in purines will be given a priority. In the context of the interaction of tsetse fly saliva and trypanosome development in the mammalian host, the work will be focused on the unravelling of the transmission enhancing activity of saliva proteins during the early development of the trypanosome at the host inoculation site.
