Comparative Protozoan Genomics Flashcards by Anna Safrankova

Give some reasons why you’d do it

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

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How much coding capacity does crypto have compared to trypanosoma 9000

Around 4000 because intracellular

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What are reads combined into scaffolds for

To map each chromosome of the organism

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Which repetitive genome difficult because too many contigs

Trichomonas (biggest genome)

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

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

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Under the protein functionalisation category of annotation what does this mean

Cell localisation, exp during lifecycle, domains, structure similarities

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Is the protein always annotated based on function

Not if there are no other similar proteins in databases

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

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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What are para, ortho or xenologues

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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Why is annotation dynamic when gene sequences are static overtime

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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Why is gene prediction an in exact science

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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Why would transcriptomics help this

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

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

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

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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What info would this give you

Evolutionary info eg if paralogs, xenologs etc

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What is the usefulness of this comparative genomics analysis

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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What 2 types of protein databases are there for annotation purposes

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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What are domain ones for

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

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Give example of how cell localisation can be dynamic of proteins

During cell cycle, SAV , environmental conditions

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Why is it important

To determine accessibility for drugs if they were to target them

Localisation indicates function especially during specific contexts eg transporters

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What bioinformatics tool is signalP and TMHMM and give example

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

predicts tmd eg on vsp

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Who usually has signalP

Secretory, membrane or surface proteins

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What is predgpi for

Predict gpi anchors eg vsg

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How can experimentation be used for annotating proteins

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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Which parasite has 74% introns meaning need transcriptomcis

Toxoplasma

Identifying and characterising vsp and vsg hemped what

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

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

VeupathDB

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

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

What was used before ngs to look at diversity of parasites

Micro satellites and snp sequencing

Explain the 4 infective stages of MICROSPORIDIA

1. Invasion of sporoplasm into cytoplasm via polar tube made of proteins being injected 2 Merogony (become merozoites and divide) 3. Sporogony (differentiate further into spores) 4. Release of environmentally resistant spores and killing of cells

Are they always monoxenous

No can also be heteroxenous with aquatic species but not common

What have they lost which makes them host dependant

Mitochondria Have mitosome instead Also no peroxisomes for glycolysis and lost ability for other metabolic paths for atp production, aa , nucleotides

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)

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

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

3266 vs around 2000 Half are from common ancestor

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

Sp signal

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

Likely needed for host interaction

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

72%

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)

1600

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

Adp/atp translocase Aka NTT

What in bacteria is it for

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

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

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

From bioinformatics THEN experiments where were they found

Cell surface (TMD) of spores for acquisition

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)

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

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

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

How many tmd does ntt have from bioinformatics

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

4 but 1 on cuniculi is mitosome surface not cell surface All 4 on cell surface in thom All able to transport atp

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

Atp in Adp out

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

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

What are the other 3

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

How has ntt evolved specifically for MICROSPORIDIA

Only atp, nad or gtp NOT utp or ttp

Symporter are for what

rna or dna synthesis via gtp or atp

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

Nope. Unknown

Where is ntt from

HGT from bacterial origin

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

Fe/s protein synthesis

APICOMPLEXA GENOMICS

Explain the losses of cryptosporidium

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

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

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

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

Also some plastid derived genes from secondary endosymbiosis in apicomplexan ancestor nuclear genes like UK-upRT for nucleotide metabolism too

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

Impdh for purine Ukuprt and tk for pyrimidine

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

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

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

Because they increase a. Muciniphila 2800 fold Mucin degrader suggesting it can lead to susceptibility to cryptosporidium?

What does this indicate on drug design

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

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

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

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

Have pfam13402 (also upreg in presence of mucins)

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

O-glycopeptidases

What is gh2

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

Other than galactosidase, what other enzymes linked to mucin degradation

Fucosidases,sialidases - both of which found in r gnavus strains for Ibd , GalNacdases (n-galactosaminodases (eg 109)

What does blast help you do and give example

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

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

Hypothetical proteins

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

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

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

32%

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

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

Which other pathogens have this enzyme

Intracellular like mycobacterium

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

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

Comparative Protozoan Genomics Flashcards

(74 cards)