Lecture 1

Question 1

What occurs when a threshold density is reached

Answer 1

Long slender trypomastigotes become short stumpy trypomastigotes

Characterised by cell cycle arrest/morphological changes

Question 2

Differentiation from long slender to short stumpy form

Answer 2

Day 1: Uncommitted slender form

Day 3: Committed slender form

Days 4-5 - Intermediate form

Day 6 - Stumpy form

Question 3

Procedures that occur in differentiation from long slender to short stumpy

Answer 3

Days 1-3 - Stumpy induction factor production and reception

Days 4-7 - Morphological transformation and Cell cycle exit after arrest in G1

Days 5-7 - PAD1 expression

Question 4

PAD proteins

Answer 4

Expressed in stumpy forms but not slender

Transducers of citate/cis-aconitate differentiation signal in T. brucei

Depletion of PAD proteins reduces differentiation competence

PAD1 and CCA initiate life-cycle development when transmitted to tsetse fly vector

Question 5

Monomorphic vs Pleomorphic

Answer 5

Monomorphic - Undergo controlled proliferation - rapidly kill mouse models

Pleomorphic - Exhibit quorum sensing in mammalian bloodstream - controlled infection

Question 6

Laboratory adapted cells and SIF pathway

Answer 6

Monomorphic slender -> SIF
OR
Cell permeable cAMP analogues (non-hydrolysable)
OR
Cell permeable cAMP/AMP analogues which can be hydrolysed -> Stumpy like

Question 7

Explain exposure of monomorphic slender cells to cell permeable cAMP/AMP analogues vs AMP analogues

Answer 7

1. Proliferation stops and stumpy form enriching mRNA expression increases
2. Increased capacity for differentiation to stumpy forms

In non cAMP analogues:

1. Only hydrolysable cAMP analogues induce differentiation
2. Cell permeable AMP more potent than cAMP

Question 8

Quorum sensing signalling pathway

Answer 8

Monomorph RNAi library
->
Induction to arrested
stumpy like forms via 8pCPT-cAMP/AMP
->
Non-responsive to drug cells outgrow and predominate
->
DNA extracted from enriched population
->
PCR amplification of RNAi inserts
->
Ion Torrent based deep sequencing to identify RNAi targets

Question 9

Dissection of quorum sensing

Answer 9

RNAi knockdown of still proliferating cells

30 genes identified - resemble components of nutritional starvation and quiescence pathways

Knockdown of individual genes in pleomorphs confers resistance to SIF

Question 10

SIF pathway

Answer 10

1. Signal processing using enzymes cAMP/AMP-analogue processing e.g. adenylate kinase
2. Signal transduction using kinases and phosphatase e.g. DYRkinase, AMPK, Protein phosphatase 2C
3. Effector molecules like RNA-binding protein 7
4. Inhibitor molecules like MAPkinase5 or target of rapamycin 4 (TOR4)

Question 11

Maintenance of cellular energy using AMPK

Answer 11

AMPK allows for catabolism (ADP->ATP) in glucose metabolism, autophagy and lipid oxidation

AMPK inhibits anabolism (ATP->ADP) in lipid synthesis, gluconeogenesis and protein synthesis

AMPK activated when AMP/ADP high and allows for stumpy formation

Question 12

mTOR

Answer 12

Mechanistic target of rapamycin is a conserved ser/thr protein kinase in PI3K family

mTOR activated for growth and promotes anabolism but inhibits catabolism

Inhibits stumpy formation

Question 13

AMPK/TOR system in differentation

Answer 13

AMP analogues activate TbAMPK1 and inhibit TbTOR4

Associated with stumpy form formation

Question 14

How do eukaryotes respond to external stimuli

Answer 14

GPCRs

7 transmembrane domain proteins that allow cells to sense signals and activate intracellular signalling pathways

Not found in trypanosomes

Question 15

GRP89 protein homology and function

Answer 15

Structural similarity to proton-dependent oligopeptide transporters (POTs)

Trypanosomes lack traditional homologues of POTs in genome

Promotes parasite differentiation:

TbGPR89 overexpression induces cell cycle arrest and differentiation

Question 16

Is TbGPR89 an oligopeptide transporter

Answer 16

TbGPR89 expressed in bacteria

Bacterial POT as + control

Mutation of one amino acid in GPR89 reduces uptake

TbGPR89 is therefore an oligopeptide transporter

Bacterial oligopeptide transporter induces differentiation in T. brucei

Bacterial YDJL induces cell cycle arrest and is located on T. brucei cell surface

Question 17

Explain how oligopeptides induce stumpy formation

Answer 17

• Using different concentrations of liver broth caused cell cycle arrest and differentiation
• PAD1 was stumpy marker

di/tri-peptides induce differentiation

• Tripeptides more potent, where tripeptides with Asn, Gln, His, Asp and Trp being most effective

Question 18

Oligopeptides in infection

Answer 18

Serum stable peptidases released by trypanosomes

Type I pyroglutamyl peptidase - acts on substrate with N-terminal pyroglutamyl residues - released when parasites lysed

Prolyl oligopeptidase - cleaves after proline residues where it’s secreted into blood

Arrested and differentiated into stumpy forms at lower parasitaemia

Question 19

Mass spectrometry analysis of released trypanosome proteins

Answer 19

12 peptidases released

Peptidases identified by MS released from intact trypanosomes

Peptidases engineered for doxycycline-inducible ectopic overexpression

MCPI, Oligopeptidase B and peptidase I enhance QS

Question 20

QS in T. brucei

Answer 20

1. Parasite-released peptidases cross plasma membrane where they digest host proteins
2. Oligopeptides form which bind TbGPR89
3. Unknown oligopeptide reception mechanism
4. Signal transduction and gene regulation occur
5. Stumpy formation