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BIOL314: Parasitology > Lecture 1: Differentiation and adaptation > Flashcards

Lecture 1: Differentiation and adaptation Flashcards

1
Q

What form are the T.brucei parasites in when they are injected into the mammalian blood from the salivary gland of Tsetse fly?

A

Dividing slender form

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2
Q

What form are the T.bruceii parasites in when they are taken by by Tsetse fly?

A

non-dividing stumpy form

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3
Q

True or false: T.bruceii differentiates from slender from to stumpy form within the mammalian host?

A

True

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4
Q

Describe the differentiation of the T.bruceii within the Tsetse fly

A

Taken up as stumpy form, differentiates into procyclic form and then into slender form in the salivary gland

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5
Q

What causes the cycles (peaks) in parasitaemia?

A
  • cell cycle arrest when threshold density is reached (parasites release SIFs that promote cell cycle arrest and differentiation into non-dividing stumpy form)
  • immune system response
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6
Q

True or false: Stumpy forms are cell cycle arrested in G0?

A

False, they are cell cycle arrested in G1.

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7
Q

What is PAD1 and what is its role?

A

PAD1 is a cell surface protein that is expressed by stumpy forms

It is required for the uptake of citrate or cis-aconitate found inside the tsetse fly vector in order to promote differentiation from the non-dividing stumpy form to the proliferative procyclic form.

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8
Q

What are monomorphic parasites?

A

laboratory adapted strains that have lost the capability to differentiate into the stumpy form

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9
Q

What is a pleomorphic parasite?

A

Is able to differentiate and adopt different morphologies

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10
Q

What is SIF?

A

Stumpy induction factor is released by slender forms when density threshold is reached (quorum sensing signal)

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11
Q

Why do monomorphic cells rapidly kill the host in mouse models?

A

They undergo uncontrolled proliferation and cannot response to the SIF quorum sensing signal so do not differentiate and arrest cell cycle

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12
Q

laboratory adapted
Monomorphic cells cannot respond to __(1)___ but can generate stumpy-like forms in response to ___(2)___?

A

(1) SIF
(2) hydrolysable cell permeable cAMP/AMP analogues

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13
Q

What resulted from the exposure of monomorphic slender cells to cell permeable cAMP/AMP analogues?

A
  • cells stopped proliferating and increased expression of mRNAs characteristic of the stumpy forms
  • also displayed increased capability to differentiate into the procyclic form (after the stumpy forms)
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14
Q

Outline the proposed SIF-signalling pathway in T.brucei for QS

A

SIF (Stumpy induction factor) produced by slender forms
- SIF receptor senses external SIF signal resulting in generation of AMP second messenger
- signal transduction involving kinases and phosphatases
- effector molecules that regulate gene expression such as RNA binding protein RBP7 and those that promote cell cycle exit and quiescence that lead to the stumpy formation

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15
Q

Explain how the quorum sensing signalling pathway was dissected from the genome of T. brucei

A
  • Generated an RNAi library that knockdown genes encoding proteins required for sensing cAMP/AMP signal in the differentiation pathway
  • induce differentiation of slender form monomorphs by hydrolysable cAMP/AMP to arrested stumpy-like forms
  • cells that continue to proliferate have become non-responsive to cAMP/AMP due to knockdown of sensing gene by RNAi so can identify the gene involved in this differentiation process (use PCR to amplify the RNAi inserts and sequence the RNAi targets)
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16
Q

How many genes were identified to be part of the SIF signalling pathway by induction of RNAi treated slender monomorphs with hydrolysable cAMP/AMP?

A

about 30 (RNAi of these gene targets resulted in slender forms that escaped differentiation)

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17
Q

What was found when the 30 genes identified in monomorphs by induction with hydrolysable cAMP/AMP and RNAi were knocked down by RNAi in pleomorphic cells in vivo?

A

many of these genes, when knocked down in pleomorphic cells by RNAi conferred resistance to SIF in vivo (QS signalling pathway inhibited)
- mice die due to parasite overgrowth (don’t differentiate and not cleared by immune system)

18
Q

Give two examples of SIF-signalling pathway inhibitors and how were they identified?

A

MAPK5
TOR4

Identified by RNAi, which when knocked down resulted in differentiation and promote stumpy formation

19
Q

How is the AMPK/TOR system involved in long slender to short stumpy differentiation?

A

SIF signal results in increase in AMP, which activates the intracellular sensor of adenosine nucleotide levels AMPK
- this promotes catabolic processes and inhibits anabolic processes
- phosphorylates and inhibits TOR4 (which is then degraded) in order to promote differentiation

AMPK by sensing AMP levels keeps cells either in the slender form or the stumpy form by inactivation and degradation of TOR4 when AMP levels are high

20
Q

What is the evidence that AMPK is involved in the differentiation pathway?

A

AMP analogues activate AMPK and negatively regulate TOR4

AMPK activation and RNAi-mediated depletion of TOR4 promotes stumpy formation

AMPK inhibition reduces stumpy formation, whilst TOR4 activity maintains proliferation and slender forms

21
Q

True or false: GPCRs and G-proteins are found in trypanosomes and are responsible for sensing and responding to external stimuli?

A

False, but do have orphan GPCRs, GPRs, that have 9 TMDs (as opposed to the 7 TMDs and associated G-proteins of a conventional GPCR)

22
Q

The TbGPR89 showed structural homology to what?

A

Proton-dependent oligopeptide transporters (POTs)

23
Q

conventional POT homologues are absent in the genomes of which trypanosomes? what does this potentially suggest about the role of the GPR89?

A

African Trypnosomes (E.g. brucei, vivax, congolense, gambiense)

Since GPR89 shows structural homology to the POTs, and these POTs are absent in trypanosomes, GPR89 may complement the activity of these transporters.

24
Q

which trypanosome form expresses the TbGPR89 (GPCR-like family)?

A

The slender form (GPR89 is degraded in the stumpy form)

25
Q

What was observed from overexpression of TbGPR89?

A

induction of cell-cycle arrest and differentiation to stumpy form

26
Q

How was it shown that TbGPR89 was an oligopeptide trasnporter (like the bacterial POTs)?

A

expression of TbGPR89 in bacteria showed it was able to transport fluorescent dipeptide into bacteria

Point mutation in GPR89 resulted in decreased oligopeptide uptake into bacteria

27
Q

What happened when the bacterial POT was expressed in T. brucei?

A

this induced cell cycle arrest and differentiation
–> good indication that the oligopeptide transporters GPR89 might be involved in the differentiation process from slender to stumpy form

28
Q

True or false: oligopeptides can induce stumpy formation in vitro?

A

True - incubation of cells with oligopeptides showed arrest of cell cycle (decrease in parasites/% growth) and increase in the % of PAD+ cells (PAD1 only expressed on stumpy forms) which indicates differentiation

29
Q

What is the link between peptidase activity during T. brucei infection and induction of stumpy formation?

A
  • activity of peptidases increases in the blood during infection due to release of serum-stable peptidases in vivo into serum of host
  • increase in oligopeptide concentration
  • induction of differentiation into stumpy form

(the release of parasite peptidases coincides with appearance of stumpy forms)

30
Q

What are the two parasite peptidases and how do they appear in the host blood?

A

Type I pyroglutamyl peptidase (TbPGP) = released by lysed parasites during infections

Prolyl oligopeptidase (TbPOP) = secreted by parasites into the blood

31
Q

What was the effect of overexpression of secreted peptidases TbPGP or TbPOP in pleomorphic T. brucei cell lines?

A

arrest of parasite in the blood and induction of differentiation into stumpy forms at lower parasitaemia

32
Q

How were additional parasite-released peptidases identified?

A

Cultivation of slender and stumpy forms in vitro and subject growth media to mass spectrometry to identify proteins secreted by the parasites into the media

33
Q

How many peptidases were identified by mass spec of the growth media?

A

12

34
Q

Of the 12 peptidases identified to be released by, how were those involved in generation of the parasite QS signal identified?
and what were the three peptidases that were found to be important in the generation of QS SIF signal in vivo?

A

ectopic overexpression in pleotrophic cells in vivo

Three peptidases:
- MCPI
- Oligopeptidase B
- Peptidase I

35
Q

What are the results of knock down of MCPI peptidase gene? does the cell die - why/why not?

A

higher parasitaemia in blood before differentiation, meaning these cells are not as effective at generating the differentiation signal (less signal)

the cell does not die because there are two other peptidases being secreted to generate the signal in the end

36
Q

True or false: the combined activity of peptidases contributes to the parasite’s capacity for QS and generation of the differentiation signal? how do we know this?

A

True - knockout of individual peptidases results in differentiation occurring at a higher parasitaemia because it takes longer for the signal to reach the threshold level for differentiation

37
Q

True or false: QS involves paracrine signalling?

A

True - peptidases released from one parasites digest host proteins that produce oligopeptides that induce changes in nearby cells

38
Q

What is the SIF receptor?

A

GPR89

39
Q

What are some therapeutic implications of uncovering the biochemistry behind differentiation to stumpy form and the SIF signalling pathway?

A
  • could give chemical mimetics of SIF oligopeptides (induce premature differentiation of trypanosomes to stumpy forms an elimination by immune cells to reduce their number below the threshold needed to sustain transmission)
  • peptidomimetic-based drugs
40
Q

What may be a potential issue with the use of SIF mimetics to treat trypanosome infections?

A

might end up with parasites that become blind to the differentiation signal and kill the host
- overexposure to QS might result in reduced sensitivity of the parasite to the QS signal (like how laboratory monomorphs are generated)
–> resulting in more virulent parasites, worsening outcomes for patients and disease spread

BIOL314: Parasitology (10 decks)

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