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Microbiota On Mucosal > Comparative Protozoan Genomics > Flashcards

Comparative Protozoan Genomics Flashcards

1
Q

Give some reasons why you’d do it

A

Study host-parasite interactions
Identify drug specific targets/diagnostics
Evolutionary biology of euk in general

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2
Q

How much coding capacity does crypto have compared to trypanosoma 9000

A

Around 4000 because intracellular

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3
Q

What are reads combined into scaffolds for

A

To map each chromosome of the organism

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4
Q

Which repetitive genome difficult because too many contigs

A

Trichomonas (biggest genome)

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5
Q

Annotation has many different levels. Genome annotation includes gene prediction as well as advanced functional characterisation/localisation, evolutionary origin. what does gene prediction mean?

A

Gene prediction is finding the location of the gene, promoters? Introns or exons?

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6
Q

Under the protein functionalisation category of annotation what does this mean

A

Cell localisation, exp during lifecycle, domains, structure similarities

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7
Q

Is the protein always annotated based on function

A

Not if there are no other similar proteins in databases

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8
Q

How can protein evolution be studied using annotation and what can taxonomic distribution tell you

A

Taxonomic and phylogenetic distribution
Are they orthologue? Paralogue? Xenologues?

If they’re found in particular organisms this potentially gives idea on functionality

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9
Q

What are para, ortho or xenologues

A

Para- genes derived from same gene but duplication event takes place. Can be same organism

Orthologue- 2 fenes in diff species with same function from commonancestor duplication

Xenologues- type of orthologue but gene from LGT

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10
Q

Why is annotation dynamic when gene sequences are static overtime

A

Dynamic because of new functional data/experimentation for example trancriptomics data, developing bioinformatics tools which are better, making it as detailed as possible for a hypothesis

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11
Q

Why is gene prediction an in exact science

A

Could have partial genome sets (not full scaffolds usually)

Don’t know the splice variants if there are any

Difficult to identify introns/exons which can lead to false predictions

Difficult to know where orf starts and ends

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12
Q

Why would transcriptomics help this

A

Removing introns as it’s only transcribed data
Also leads to alternative transcripts/splicing info

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13
Q

Which ways can transcriptomics work and how can this work together with proteomics/MAss spec

A

Microarray analysis
Or
Also converted to cDNA but use high throughput sequencing methods(NGS) (rna-seq)

Can even do RT-PCR to quantify

Could find that the different transcripts coincide with specific proteins found

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14
Q

How is in silico protein annotation by homology done and also for taxonomic analysis

A

Blast search requiring big databases full of annotated proteins - this is for homology
Compare with other proteins in database/ from different taxonomies

For info on para/etc you’d do read alignment and phylogenomic analysis

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15
Q

What info would this give you

A

Evolutionary info eg if paralogs, xenologs etc

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16
Q

What is the usefulness of this comparative genomics analysis

A

Can help establish if it is an ORF if not sure by comparing and if species have it then likely yes

Determine ortho,para etc (evolutionary origin of proteins)

Identify taxa-specific genes eg for their virulence

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17
Q

What 2 types of protein databases are there for annotation purposes

A

Whole Protein databases used after blast search eg ncbi - see known proteins with similar sequences

Domain/motif databases eg pfam,interpro

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18
Q

What are domain ones for

A

Group domains together to see functionality of proteins eg pfam13402 found to be m60 peptidase like

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19
Q

Give example of how cell localisation can be dynamic of proteins

A

During cell cycle, SAV , environmental conditions

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20
Q

Why is it important

A

To determine accessibility for drugs if they were to target them

Localisation indicates function especially during specific contexts eg transporters

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21
Q

What bioinformatics tool is signalP and TMHMM and give example

A

Predict signal peptide sequences on proteins using their aa sequence
Eg vsg n terminus

predicts tmd eg on vsp

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22
Q

Who usually has signalP

A

Secretory, membrane or surface proteins

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23
Q

What is predgpi for

A

Predict gpi anchors eg vsg

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24
Q

How can experimentation be used for annotating proteins

A

Info on function eg if they have enzyme activity
Host binding info
Cellular location (tagging)
When is it expressed (rnaseq)/translated during life cycle (tagging)

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25
Q

Which parasite has 74% introns meaning need transcriptomcis

A

Toxoplasma

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26
Q

Identifying and characterising vsp and vsg hemped what

A

Determine how they do monoallelic exp via experimentation eg deleting dicer or identified histone interactions with vsg

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27
Q

What database covered euk parasites and hosts and can be modified with free access for analysis

A

VeupathDB

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28
Q

How was comparative genomics essential to find drug resistant and sensitive L.donovani strians differences genomically (17 diff strains from patients) - downing 2011

A

Compared and found 9 genetic loci different which could potentially be manipulated in future

For example CNVs varied between the strains, with mapk locus amplification

Suggestive of the selective pressures they are put under during drug policies

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29
Q

What was used before ngs to look at diversity of parasites

A

Micro satellites and snp sequencing

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30
Q

Explain the 4 infective stages of MICROSPORIDIA

A
  1. Invasion of sporoplasm into cytoplasm via polar tube made of proteins being injected
    2 Merogony (become merozoites and divide)
  2. Sporogony (differentiate further into spores)
  3. Release of environmentally resistant spores and killing of cells
31
Q

Are they always monoxenous

A

No can also be heteroxenous with aquatic species but not common

32
Q

What have they lost which makes them host dependant

A

Mitochondria
Have mitosome instead

Also no peroxisomes for glycolysis

and lost ability for other metabolic paths for atp production, aa , nucleotides

33
Q

T. Hominis is a type of MICROSPORIDIA with larger genome and gene coding capacity. What else does it have more of than others like E. cuniculi and intestinalis explaining why it has a larger genome. (Shows large variation in the MICROSPORIDIA lineage)

A

Larger non coding genome (not present in smaller MICROSPORIDIA)
And also larger repetitive elements including transposable elements

34
Q

How many protein coding genes does t Hominis have compared to cuniculi/intestinalis. And how many of these are from ancestor vs MICROSPORIDIA specific

A

3266 vs around 2000

Half are from common ancestor

35
Q

Analysis of the domains shows what common domain/function is present in MICROSPORIDIA specific genes

A

Sp signal

36
Q

Since this is more for secretory or membrane/cell surface proteins what does this mean

A

Likely needed for host interaction

37
Q

What % of proteins in microsporidia with sp are MICROSPORIDIA specific according to t. Hom genomics (show importance of sp proteins for virulence/adaptation)

A

72%

38
Q

Phylogenetic analysis across the opisthokonta lineage found a drastic loss of how many genes in a common ancestor which likely reflects the ic lifestyle adaptation??
Further losses down the tree identified between the diff MICROSPORIDIA , but also some GAINS!!
(Nakjang 2013)

A

1600

39
Q

Which transporters found first from bacteria origin like chlamydiae found MICROSPORIDIA had too (via blastP) - (nakjang 2013)

A

Adp/atp translocase

Aka NTT

40
Q

What in bacteria is it for

A

Transporting atp,utp,gtp,ctp,ttp (energy and nucleotides)

41
Q

Give the other transporters which were acquired by microsporidia since they can’t produce their own for growth and replication

A

Zip- for zinc transport
AAAP- amino acid permease
ABC- lipid transporter

42
Q

From bioinformatics THEN experiments where were they found

A

Cell surface (TMD) of spores for acquisition

43
Q

Rna seq data of t. Hominis allowed to identify whether these transporters. were actually expressed.
Also can study rna seq durinf infection cycle of up to 96hrs. What were the results from this study (polar tubes, aa transporters, ntt and zip)

A

Polar tubes were expressed highly at end of infection cycle
Amino acid transporters also, highest in sporogony except for thom2342 high throughout
Majority of ntt also highest in sporogony, with NTT4 expressed highly throughout whole cycle
Same with zip with most high at sporogony except for THOM2017 high throughout

44
Q

Is the reason why zip thom2017 and thom2342 aaap high known?

A

Not yet known why this one in particular relevant but points to one’s yoh need to focus on for experimentation

45
Q

How many tmd does ntt have from bioinformatics

A

12

46
Q

How many each in cuniculi and Hominis and where according to immunofluorescence. (After bioinformatics)
When transferred into ecoli, what do they all able to transport

A

4 but 1 on cuniculi is mitosome surface not cell surface
All 4 on cell surface in thom

All able to transport atp

47
Q

Which exchanger ntt 3 is at mitosome (what does it exchange)

A

Atp in
Adp out

48
Q

Since ntt4 is highly exp all infection cycle. What type is it a symporter or exchanger in t. Hominis

A

Symporter (in both H and atp/gtp) for rna/dna

49
Q

What are the other 3

A

Exchangers for atp in adp out OR NAD in some cases (for energy)

50
Q

How has ntt evolved specifically for MICROSPORIDIA

A

Only atp, nad or gtp

NOT utp or ttp

51
Q

Symporter are for what

A

rna or dna synthesis via gtp or atp

52
Q

So is it known how they get pyrimidine if only get A or G not c or t

A

Nope. Unknown

53
Q

Where is ntt from

A

HGT from bacterial origin

54
Q

If mitosomes can’t generate atp using things like krebs or glycolysis, what do they do

A

Fe/s protein synthesis

55
Q

APICOMPLEXA GENOMICS

A
56
Q

Explain the losses of cryptosporidium

A

Loss of mt (have mitosomes with not atp gen) and loss of apicoplast except for some plastid derived genes in nuclear genome

57
Q

What HGT genes traced back to bacteria for nucleotide synthesis and what suggests these were 2 independent HGT events

A

Thymidine kinase TK
- more closely related phylogenetically to y-proteo like ecoli

and imp dehydrogenase - suggested to be from e-proteobac like campylo or helicobacter

58
Q

Why is cryptosporidium nucleotide metabolism termed chimeric/mosaic based from phylogenomic analysis

A

Also some plastid derived genes from secondary endosymbiosis in apicomplexan ancestor

nuclear genes like UK-upRT for nucleotide metabolism too

59
Q

Which enzyme from bacteria for purine salvage pathway (recycling) vs which for pyrimidine salvage pathway

A

Impdh for purine

Ukuprt and tk for pyrimidine

60
Q

Why are drugs targeting impdh specific and not toxic to human host

A

Because impdh homolog are too distant in sequences between human and crypto

61
Q

Testing drugs; Why were 2 identical impdh drugs inhibitors a110 and p131 but a110 actually increased oocysts in mice in vivo faeces

A

Because they increase a. Muciniphila 2800 fold

Mucin degrader suggesting it can lead to susceptibility to cryptosporidium?

62
Q

What does this indicate on drug design

A

Take into consideration Microbiota effects of drugs because could potentially lead to barrier disruption/dysbiosis

63
Q

Explain again why desai 2016 links to dysbiosis /diet and Ibd

A

Increased citrobacter induced colitis of mice ff due to increased AM, increased GH transcriptomics targeting o-glycans (gh109 and pfam13402)
and also increased b.caccae and a. Muciniphila specialists

64
Q

Why would b.caccae just like b.theta and A.M degrade mucins effectively

A

Have pfam13402 (also upreg in presence of mucins)

65
Q

What are pfam13402 containing mucin degraders like bt4244 in pul78 for o-glycan deg

A

O-glycopeptidases

66
Q

What is gh2

A

A b-galactosidase degrading mucin in periplasm (also increased in exp)

67
Q

Other than galactosidase, what other enzymes linked to mucin degradation

A

Fucosidases,sialidases - both of which found in r gnavus strains for Ibd

, GalNacdases (n-galactosaminodases (eg 109)

68
Q

What does blast help you do and give example

A

Look for proteins similar in sequence- can determine functionality eg pfam13402 in b theta and gluzincin like motif (metallopeptidase)

69
Q

What are proteins called which have no known function, structural similarity using blast search but Are sequenced?

A

Hypothetical proteins

70
Q

How is the cryptosporidium evolutionary origin of proteins/genes from Lgt important for drug development and one of the major reasons it’s studied

A

Because try find genes and proteins which are not present in the host for specificity

71
Q

From the opisthokonta phylogenetic analysis, what % of MICROSPORIDIA m gene families were shared only between micro and not other opisthokonts (nakjang 2013)

A

32%

72
Q

Give another major example of HGT from proteo bacteria in cryptosporidium and why this is important for immune evasion

A

Tryptophan synthase B

Found in intestinal targeting cryptosporidium and is important given the fact ifny is used to activate IDO which breaks down Trp in the parasitophorous vacuole into kyn. This starves crypto.

Has acquired this to produce Trp from Indole derived from the gut Microbiota = evade this killing

73
Q

Which other pathogens have this enzyme

A

Intracellular like mycobacterium

74
Q

Why did andreu Ballester 2013 associate MICROSPORIDIA with worsening outcomes of crohns potentially

A

Found that many Cd patients were actually microsporidia positive. This was correlated negatively with the amount of cd8 yd cells (IELs previously shown to be required for sufficient protection from micro)

Suggests that crohns don’t have the capacity to contain microsporidia (immunoincompetent) and therefore could worsen outcome

Microbiota On Mucosal (22 decks)

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