Lecture 1 - Differentiation and Adaptation Flashcards
Trypansoma brucei life cycle
- Tsetse fly takes a blood meal (injects metacyclic trypomastigotes)
- Injected metacyclic tryptomastigotes
Life cycle and immune evasion of African trypanosomes
Upon reaching threshold density, long slender forms undergo differentiation to stumpy forms - characterised by cell cycle arrest as well as morphological changes
Differentiation from long slender to short stumpy form parasites
SIF production and reception (1-3 days)
Uncommitted slender form <-> SIF <-> Committed slender form
Committed slender form -> intermediate form
Intermediate form -> Stumpy form
Stumpy form -> Cell death
Monomorphic
Monomorphic (laboratory adapted strains) undergo uncontrolled proliferation - in mouse model they rapidly kill the host
Pleomorphic
Pleomorphic (i.e. different morphologies) parasites exhibit quorum sensing in the mammalian bloodstream.
Controlled infection
Laboratory adapted cells cannot respond to SIF but generate ‘stumpy-like’ forms in response to hydrolysable cell permeable cAMP/AMP analogues
Laboratory adapted cells cannot respond to SIF
Monomorphic slender -> Stumpy induction factor
Monomorphic slender -> Cell permeable cAMP analogues (non-hydrolysable)
Monomorphic slender -> Cell permeable cAMP/AMP analogues (hydrolysable) -> Stumpy like
Exposure of monomorphic slender cells to cell permeable cAMP/AMP analogues
(i) stop proliferating and increase expression of mRNAs enriched in stumpy forms
(iii) display increased capacity for differentiation into procyclic forms
NOT a cAMP-mediated response:
(i) Only hydrolysable cAMP analogues induce differentiation
(ii) Cell permeable AMP considerably more potent than cAMP
Genome-wide dissection of quorum sensing signalling pathway
Cells that continue to proliferate – RNAi knockdown of a gene encoding protein required for sensing cAMP/AMP signal
~30 genes identified – resemble components of nutritional starvation and quiescence pathways in other eukaryotes
Crucially, when many of these genes individually knocked down in pleomorphic cells by RNAi - conferred resistance to SIFin vivo
Confirmed involvement in biologically relevant QS signalling pathway
Proposed SIF-Signalling pathway in T. brucei
SIF -> SIF receptor
SIF receptor + Cell permeable cAMP, AMP -> Signal processing - enzymes cAMP/AMP-analogue processing e.g. adenylate kinase
SIF receptor -> Signal transduction
Signal transduction -> Effector molecules -> Inhibitor molecules
Strict maintenance of cellular energy
Catabolic processes:
ADP -> ATP using AMPK
Glucose metabolism, autophagy, lipid oxidation
Anabolic processes
ATP -> ADP
Lipid synthesis, gluconeogenesis/glycogen storage, protein synthesis
AMPK
Sensor of intracellular adenosine nucleotide levels and is activated upon energy stresess (i.e. high intracellular AMP and ADP)
Metabolic checkpoint
activates catabolic processes (generate more ATP) and inhibits anabolic processes – it achieves this (in part) by negatively regulating mTOR signalling
mTOR (mechanistic target of rapamycinO
Conserved Ser/Thr protein kinase belonging to phosphoinositide-3-kinase (PI3K)-related kinase family
Under conditions permissive for growth, mTOR is activated and promotes anabolic processes and represses catabolic processes
How do eukaryotic cells sense and respond to external stimuli?
Most organisms:
G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes
7 transmembrane domain proteins that allow cells to sense signals and (via trimeric G proteins) activate intracellular signalling pathways
Trypanosoma brucei
GPCRs NOT found in trypanosomes
However, ‘orphan’ GPCRs (9 TMDs) identified in many eukaryotes e.g. in plants a GPR89 family protein detects the phytohormone abscisic acid and in humans GPR89 acts as an anion channel protein
TbGPR89 protein homology modelling
GPR89 shows structural similarity to Proton-dependent oligopeptide transporters (POTs)
POT family transporters: encoded by a diversity of prokaryotes and eukaryotes and are linked to small molecule uptake
African Trypanosomes lack homologues to conventional POTs in the genome
How can oligopeptides arise in an infection?
Trypanosomes release serum-stable peptidases in vivo,
remain active and accumulates during an infection.
Coincides with appearance of stumpy forms.
Type I pyroglutamyl peptidase (TbPGP) – acts on substrates with N-terminal pyroglutamyl residue – released by lysed parasites during infections
Prolyl oligopeptidase (TbPOP) – cleaves after proline residues – secreted by parasites into blood
Secreted peptidases induce differentiation signal in mice
To determine if trypanosome-derived oligopeptidases could affect stumpy formation – pleomorphic T. brucei cell lines generated to overexpress TbPGP (modified by addition of a N-terminal signal to promote secretion) or TbPOP
Cells induced to express oligopeptidases in vivo arrested and differentiated into stumpy forms at lower parasitaemia
Mass spectrometry analysis of released trypanosome proteins
In order to identify proteins released by trypanosome parasites in the mammalian host, populations of slender or stumpy parasites were purified from mice infections
The detected 12 peptidases are released by bloodstream parasites.
Peptidases identified by mass spectrometry are released from intact trypanosomes
To assess whether any of the identified proteases contributed to the generation of the parasite QS signal, each of the released peptidases was engineered for doxycycline-inducible ectopic overexpression.
Ectopic expression of three peptidases enhances QS in vivo:
- MCPI
- Oligopeptidase B
- Peptidase I
Quorum sensing model in T. brunei
T. brunei releases peptidases
Peptidases degrade host proteins
Oligopeptides generated
TbGPR89 transport these
Oligopeptides trigger developmental program
Therapeutic implications?
In principle, chemical mimetics of SIF could induce premature differentiation of trypanosomes, reducing their number below the threshold needed to sustain transmission (and simultaneously reducing potential virulence)
Peptidomimetic-based drugs are a well-established therapeutic strategy for a large spectrum of diseases, from cardiovascular disease to metabolic disorder and cancer.
Caution is necessary because promoting premature differentiation
might select reduced sensitivity to the QS signal:
More virulent parasites, worsening outcomes for individual patients and disease spread.
