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Zpath term 2 > Apicoplasts - toxoplasma. > Flashcards

Apicoplasts - toxoplasma. Flashcards

1
Q

Toxoplasma genome

A

Mitochondrial, apicoplast and nuclear.

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2
Q

Toxoplasma mitochondrial genome.

A

Encodes only a few proteins: cytochrome B and some subunits of cytochrome c. Large and small subunit rRNAs.

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3
Q

Toxoplasma apicoplast genome.

A

Acts as a stripped down plastid. Retains some features such as energy generation and fatty acid synthesis.
Origin: secondary symbiotic event.
Maternally inherited
Target for chemotherapy.

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4
Q

Toxoplasma apicoplast genome. Origin a secondary symbiotic event.

A

Eukaryote engulfs red algae. Reduced 4 membrane plastid. Most plastid genes transferred to nuclear genome, resulting in apicomplexan parasites having mosaic genomes.
35 kb genome of algal origin.

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5
Q

Toxoplasma apicoplast genome. Origin a secondary symbiotic event. EVIDENCE.

A

Phylogenetic analysis of tufA suggests that the apicoplast is related to red algae.

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6
Q

Toxoplasma apicoplast genome. Target for chemotherapy

A

Differential drug sensitivity
Identifying parasite drug targets
Drugs affecting the apicoplast lead to delayed death kinetics.

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7
Q

Delayed death kinetics

A

Daughter parasites are the ones with damaged phenotype, not the parents that receive the drug. After 72 hours the toxoplasma (by now, daughter parasites) show decreased plastid/nuclear DNA ratio. Inheritance of a non-functional apicoplast.
Current apicoplast targets target DNA gyrases or transpeptidation.
Potential for herbicides.

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8
Q

Toxoplasma nuclear genome

A

14 chromosomes, lots of splicing to get mRNAs.

Can be targeted to other strucutures.

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9
Q

Toxoplasma nuclear genes with plastid origins.

A

Targetted to apicoplast with bipartite signal. Look for molecules in EST database, Identify cDNAs for sequence analysis or make GFP constructs.

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10
Q

Toxoplasma - epidemiology, population structure and evolution.

A

General distribution

Evolution

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11
Q

Toxoplasma - general distribution

A

All at risk
Lifecycle
3 major clonal lineages.

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12
Q

Toxoplasma lifecycle - key themes.

A

Infectious stages
Interconversion between stages.
Dissemination within host.

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13
Q

Toxoplasma lifecycle - infectious stages.

A

Sporozoites in oocysts
Bradyzoites in tissue cysts
Broken through transmission barrier of obligatory 2 host cycle.

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14
Q

Tg. Sporozoites in oocysts infect…

A

Herbivorous hosts, also give water-borne infection. Resistant to both physical and chemical destruction.

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15
Q

Tg. Bradyzoites

A

Infect carnivorous hosts.

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16
Q

Tg. Interconversion between lifecycle stages.

A

Is highly flexible.

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17
Q

Tg. Dissemination in the host

A

o Wide host cell range

o Tachyzoites penetrate tissues, actively migrating across biological barriers, and rapidly replicate by endodyogeny.

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18
Q

3 major clonal lineages

A

Almost identical within clades
Type I
Type II
Type III

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19
Q

Tg. Type I.

A

Poor at forming tissue cysts, is very virulent in mice, and has a bias for congenital infection.

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20
Q

Tg. Type II.

A

Less virulent, good at forming tissue cysts and has a particular bias for AIDS patients.

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21
Q

Tg. Type III.

A

Least virulent, good at forming tissue cysts and has a bias for livestock.

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22
Q

Tg evolution

A

General model
South American strains
World-wide

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23
Q

Tg evolution - general model.

A

Common ancestor. Mutation and recombination in cats leads to many types. Bottleneck means only 3 types now in North America and Europe, though more in South America.
Estimating time back to bottle neck.
Chromosome 1a
Expansion of clonal lineage.

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24
Q

Tg evolution - estimating the time to the bottleneck.

A

First identify which SNPs occurred since bottleneck, and which represent divergence since the last common ancestor of the clonal lineages.
T= (Number of mutations)/(mutation rate x number of nucleotides.) = 6-10, 000 years ago.

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25
Q

Tg probably cause of bottleneck

A

Probably coincided with domestication of crops and livestock and hence explosion in mus population, and increased opportunities for transmission between primary and intermediate hosts.

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26
Q

Formation of clonal groups after bottleneck

A

Extensive bi-allelism suggests clonal groups were formed from just a few genetic crosses early in evolution after bottleneck, after which clonal lineages expanded to populate different hosts.

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27
Q

South American strains

A

SNPs - see card.
Pocket of clonality observed.
Probably diverged from common ancestor, possibly in association with cat migration after reconnection of Panamanian land bridge.

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28
Q

Worldwide distribution of haplotypes.

A

• Neighbour network analysis of 138 strains described 6 clades and 15 haplogroups of T gondii. Study focussed on 3 in NA/E.
Most of the clonal lineages (all 3 of the NA/E ones, and several of the S. American ones seem to derive from just 4 ancestral genotypes.)

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29
Q

Tg. Chromosome 1a

A

Associated with clonality.
Common and recent origin.
Unique combination of alleles among genes on Chr1a was responsible for rapid reemergence of clonal lineages.
Chr1a associated with clonality even in South American strains: probably contains genes which enhance genetic fitness.

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30
Q

Expansion of clonal lineages.

A

Can be transmitted asexually by carnivorous/omnivorous feeding, or ingestion of infected water.
Very little sexual propagation occurs.
Genetic similarity very high.

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31
Q

Expansion of clonal lineages - little sexual propagation.

A

Given number of alleles at multiple loci, you would expect 1500 genotypes with random mating. Instead there are 20.
Recombinant strains do occur.

32
Q

Expansion of clonal lineages - little sexual propagation. EXPLANATIONS.

A

o Intermediate hosts develop strong immunity, so only ever have cysts of one type. Gametocyte formation is short in cat, so chances for genetic exchange are limited.
o Acquisition of direct oral transmission would free lineages from need for sexual cycle, ensuring expansion of these lineages and creating a selective genetic sweep.

33
Q

Expansion of clonal lineages - genetic similarity.

A

So high, that can be difficult to identify true mutations as rates of error of Taq polymerase and sequencing are higher.
Except at a few highly polymorphic microsatellite markers.

34
Q

Tg immunity

A

Immune response
Host variation
Parasite variation

35
Q

Tg immunity - immune response.

A 
TLRs activated by PAMPs.
GRA15 activates NFkB independenly.
--> proinflammatory gene transcription.
--> cytokines act on NK cells and T cells to cause IFNy release.
--> JAK-STAT pathway.
36
Q

Tg immune response. TLRs

A

TLR11 activation by TgPRF especially key.
TLR2 and TLR4 activation is important: act via MyD88 to activate NFkB pathway. Deletion of any TLR –> only modest effects on susceptibility –> lots of TLRs probably used.

37
Q

Tg immune response proinflammatory gene transcription.

A

IL-1B, IL-12 and IL-18, also iNOS and NLRs. Early production of IL-12 is vital for control.
Excess IL-1B and IL-18 especially can lead to host damage.

38
Q

Tg immune response proinflammatory gene transcription. Early IL-12 production.

A

Dendritic cells and inflammatory monocytes are important sources of these cytokines.

39
Q

Tg immune response - JAK/STAT1 pathway, effects

A
  • Expression of immunity related GTPases which cause vesiculation and rupture of the parasite vacuole, which leads to parasite digestion.
  • Alters tryptophan metabolism
  • Stimulates production of iNOS.
  • Autophagy
40
Q

Tg immunity - host variation.

A

Mice can be susceptible (C57BL) or resistant (BALB/c).

Relationship in congenital and AIDS outcome with HLA-DQ3 allele.

41
Q

Tg immunity, parasite variation

A

Due to differentces in enzymes or differeneces in regulation.
How to identify key genes.
ROP16, ROP18, ROP5, GRA15. NTPases, GRA4 and GRA6, ROP38.
ROP5 and ROP18 probably account for 90% of virulence differences.

42
Q

Tg immunity, parasite variation, identifying different genes.

A

Do pair-wise genetic crosses between types: Quantitative Trait Locus analysis. Identify key genetic regions by LOD score. Look for potential genes in these regions.

43
Q

ROP2 family of kinases

A

ROP18 and ROP5 belong to this.

44
Q

Identification of ROP18

A

Type I and type III genetic cross.

45
Q

Type II and type III genetic cross.

A

implicated 5 loci including ROP18, ROP5.

46
Q

ROP proteins

A

About 1/2 serine/threonine kinases, and 1/2 pseudokinases. Diverse family suggesting strong selective pressures.
ROP5 and ROP18 probably account for 90% of virulence difference between types.

47
Q

ROP16 basics

A

Targetted to host nucleus, alters induction of IL-12.

May also have a role in altering arginase metabolism and induction of alternatively activated immune cells (via STAT6)

48
Q

ROP16 mechanism

A

Acts on STATs. Leads to IL-12 downregulation.

Involves suppressor of cytokine signaling protien, SOCS.

49
Q

ROP16 mechanism - action on STATs.

A
  • STAT3 ( a known inhibitor of NFkB function) is initially stimulated independently of ROP16, but activation of STAT6 requires it.
  • ROP16I/III allows sustained STAT3/6 response. This occurs due to phosphorylation of tyrosines, despite sequence homology to S/T kinases.
50
Q

ROP16 mechanism - action on IL-12

A

Prolongued expression of ROP16I/III leads to IL-12 downregulation, and limitation of Th1 immune response. Eventually leads to suppression of IL-12 production. Less inflammation, better parasite survival. Partially mimics effect of IL-10.

51
Q

ROP18 basics

A

Polymorphic phosphorylation of IRGs. Also ATF6B.

Mostly active in mice. Alters IFNy response.

52
Q

ROP18 and IRGs.

A

Type I ROP18 phosphorylates IRGs. Phosphorylation prevents accumulation on PVM. Tachyzoites die in vacuolated PVMs. Probably not the only factor involved in preventing IRG loading.

53
Q

IRGs

A

Immunity related GTPases which vacuolate and disrupt the parasitophorous vacuole.

54
Q

Differences between types: ROP18.

A

Type I ROP18 phosphorylates and hence inactivates IRGs. Type II probably doesn’t because ROP18 works in concert with another ROP, not functionally expressed. Type III expression level is very low, 100 fold reduced.

55
Q

ROP18 and ATF6β.

A

Phosphorylates transcription factor ATF6β targetting to proteosome. ATF6β deficient mice have reduced survival. There are orthologues in humans. May have a role in MHCI pathway.

56
Q

ROP5 basics

A

Essential for mouse virulence. Sole quantitative trait locus for difference between type I and type II. Disruption attenuates virulence.
Activates other ROP kinases, including ROP18, probably via action as a scaffold protein. . Probably alters IFNy response.

57
Q

ROP5 differences between types.

A

Type I and III are virtually identical, but some Type II genes look frameshifted. Type I and III responsible for virulence dependent on genetic background.

58
Q

ROP5 action.

A

ROP5 locus is a polymorphic tandem array.
Probably does not have phosphotransferase activity, rather acting through binding proteins. But mechanism of action not fully elucidated.

59
Q

Polymorphic tandem array

A

tandem duplicated polymorphic genes.

60
Q

GRA15 basics

A

Alters induction of IL-12. Type II facilitates nuclear translocation of NFkB.
Leads to early conversion to bradyzoites and prevalence in chronic infections.

61
Q

Parasite proteins altering induction of IL-12.

A

ROP16, GRA15.

62
Q

Parasite proteins altering IFNy response.

A

ROP18, ROP5.

63
Q

GRA15 mechanism.

A

Type II GRA15 facilitates nuclear translocation of NFkB. Type I and type III don’t appear to do this. Molecular mechanism not entirely certain. Induction of NFkB seems to be TRAF6 and IKK dependent though. Leads to increased IL-12 expression.

64
Q

GRA4 and GRA6.

A

Promote cyst formation via nanotubular network in PVM.

65
Q

Effects of chronic T. gondii infection.

A
  • Age more quickly
  • Tail damage, piloerection, strange body position (poor balance)
  • Perivascular cuffing of lymphocytes, microglial cells cluster. Latter associated with neuronal death and neurodegenerative diseases.
66
Q

Murine models of T. gondii strains.

A

RH for Type I. LD100 of 1.
ME49 for Type II. LD100 of 1000-10,000.
VEG for Type III, LD100 of 10,000-100,000.

67
Q

Phosphorylation of STATs by ROP16.

A

STAT3 at tyrosine 705.

STAT6 at tyrosine 641.

68
Q

Type II ROP16

A

Amino acid change of leucine to serine at position 503, unable to activate STAT3.
Serine narrows kinase pocket weakening ROP16 binidng.
Only transiently activate STAT3/6, so early increase in IL-12.

69
Q

Type III low expression of ROP18

A

Stop ATG codon, 2.1 kbp upstream from start.

Expression of Type I ROP18 in type III parasite –» increased rep rate, and protection from IFNy mediated killing.

70
Q

ROP5 in virulence

A

Essential for virulence, Reese et al. Mice infected with ROP5 KO have 100% survival, while those with parental strain show 0% survival at 7 days post-infection.
Restoration with different paralogue combinations leads to differing virulences. Possibly have evolved for different hosts.

71
Q

GRA15 and ROP16

A

Account for 50% of difference in virulence between types II and III.

72
Q

ROP38

A

Possibly inhibits host cell transcription.

73
Q

NTPases

A

Implicated in control of infection by inflammasome or pyroptosome.
Current thinking: virulent strains reduce amount of ATP available to activate P2X7 receptor which triggers inflammasome mediated expulsion of the parasite.

74
Q

Identification of 3 clonal lineages.

A

o Originally french datashowed non-random association of isoforms of enzymes
suggesting discrete strains

o Genetic analysis confirmed and extended it to three

o Also a 4th in some wildlife in NA

75
Q

Bottlenecks

A

o 20mill ago – split from neospora
o 1-2 – single lineage expanded and accumulation of mutations
o 10k – bottle + clonal expansion

76
Q

Recombinant strains.

A

o In almost every case an allele is almost identical to one in type 1 or 3 but very rarely 2.

o Polymorphism between these alleles is limited to two – A and E (adam and eve)

o This is true even in exotic strains (thought to derive from French guyana)

77
Q

Number of ancestral lineages

A

8, by analysis of lineages of clades.

Zpath term 2 (14 decks)

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