1

Eukaryotic Pathogengenomes:

November 15Lecture by David Sullivan

dsulliva@jhsph.edu

Eukaryotes(after Baldauf et al., 2000)

2

Over 40 species of protozoa and over 100 helminths infect people.SubKingdom Protista-Protozoa

Phylum SarcomastigophoraSubphylum Mastigophora-flagellates-Giardia, Trichomonas,Kinetoplastida-Leishmania and TrypanosomaSubphylum Sarcodina-amebae move by pseudopodia- Entamoeba,

Phylum Apicomplexan-all obligate parasites with apical organellesPlasmodium, Toxoplasma, Cyptosporidium, Cyclospora, Babesia

Phylum Microspora-MicrosporidiaPhylum Ciliophora-ciliates Balantidium coli is the only parasite of humans

SubKingdom MetazoaPhylum Nematoda- round worms- hookworm, Ascaris, Strongyloides, pinworm & whip-worm, filiaria

Phylum Platyhelminthes-flatwormsClass Trematode-flukes and SchistososmaClass Cestode-tapeworms

Max Brodel’s 1896Cartoon of OslerChasing Away Microbes

Malaria infected RBC’s

3

Neglected Tropical diseases NEJM 357:1018-27 Sept 2007

The 10 Leading Causes of Life-YearsLost to Disability and Premature Death

48Amoebiasis

60 million0.3Human AfricanTrypanosomiasis

25 million9Chagas disease

350 million12Leishmaniasis

ND0.4Leprosy

590 million84Trachoma

ND0.01Dracunculiasis

90 million37Onchocerciasis

1.3 billion120Lymphaticfilariasis

779 million207Schistosomiasis

3.2 billion576Hookworm

3.2 billion604Trichuriasis

4.2 billion807Ascariasis

Populationat risk

GlobalPrevalence(millions)

Disease

Global impact of parasitesMore parasitic organisms than non parasitic organisms in the world. Most

parasitic infections cause chronic disease shortening lifespan and increasing sickdays and robbing nutrition from young children.

A hookworm consumes about 100 ul of blood a day multiplied by allinfections equals 130,000 liters of blood a day or the blood of 26,000 people that iscompletely sucked out each day by hookworms alone.

More than 80,000 tons of Ascaris eggs are shed in a year. "A hugenutritional beneficience bestowed upon ascaris by the human host" Stoll, Norman"This wormy world" J. Parasit. 1947 33:1-18. In 1950 over 2.2 billion helminthinfections for 2.1 billion people. 85% of all helminth infection are due to "ineffectiveinsulation from our excretory products".

Protozoan diseases-African sleeping sickness and Kala Azar are universallyfatal unless treated with present ineffective therapy. Many are severely disfiguringsuch as espundia-mucocutaneous leishmaniasis and elephantiasis-filiaria. Blinding byonchocerciasis affected most adults in African villages. Wild animal reservoirs forTrypanosoma prevent the use of 4.5 million acres of otherwise usable african land.

A single day of fever from malaria consumes 5,000 calaories or about twodays of hard labor. The caloric intake in endemic areas is only about 2,200. About30% of total energy yield of grain production is wasted on parasitic diseases. Theaverage bout of malaria prevents about 4 working days

4

Protozoans

Apicomplexans-http://www.apidb.org/apidb/

Toxoplasma, Cryptosporidium,Plasmodium,Babesia

Trypanosoma brucei and cruzi

Trichomonas

Entamoeba histolytica

Giardia lamblia

EupathDB

http://eupathdb.org/eupathdb/

Giardia lamblia

5

Individual eukaryotic genomes: Protozoans at the base of the tree

Giardia lamblia is a water-borne parasiteDisease relevance: giardiasis (causes diarrhea)Distinguishing features: lack of mitochondria, peroxisomes;

polyploid (has two nuclei in each cell)Genome size: 12 MbChromosomes: 5 (range 0.7 to >3 Mb)Website: http://www.giardiadb.org/giardiadb/(sequencing in progress)No extranuclear mitochondrial DNAThe genome has just three retrotransposons.Also, it appears to have a single intron (ferredoxin gene).Short intergenic regionsAbundant antisense transcripts-20% of cDNAs in librariesrepresent polyadenylated sterile (antisense) transcripts

Bidirectional transcription in Giardia

Nucleic Acids Res. 2007 April; 35(8): 2544–2553.

ATG

ATG

ATG

ATG

Three promoter sites have bidirtectional transcription

6

Serial analysis ofGene Expression(SAGE) tags weretabulated for senseor antisense for eachOpen reading Frame(ORF). The loactioanwas expressed asnumber of sense toantisense per ORF

Bidirectional transcription in Giardia

Nucleic Acids Res. 2007 April; 35(8): 2544–2553. Head to head orf orientation Head to tail orf orientation

7

Flask ulcer

Liver abscess

Neutrophil death Apoptosis of myeloid cells

liverabscess

8

Entamoeba histolyticahttp://www.sanger.ac.uk/Projects/E_histolytica/The genome of the protist parasite Entamoebahistolytica. Nature 2005;433;7028;865-8

Entamoeba histolytica is the causal agent ofamoebic dysentery and amoebic liver abscess,with an estimated 40 million cases worldwideand 100,000 deaths.Amitochondriate protozoaGenome size: 24 MbChromosomes: 14 (range 0.6 to 3.3 Mb)functionally tetraploidLateral gene transfer from bacteriaEpigenetic silencingno extranuclear mitochondrial DNA

tRNA genes in clustered arrays making upabout 10% of genome.4,500 copies (10 x that of human genome)Intergenic regions have simple sequencerepeats 7-12 bp but up to 44 bp.E. histolytica lacks mini/microsatelliteselsewhere.tRNA-linked E. histolytica short tandemrepeats (STRs) form part of a larger unit thatis itself tandemly arrayed

Molecular Biology and Evolution 2008 25(1):187-198

tRNA’s encoding amino acids

9

(A) The array unit organization involving the gene encoding tRNA ValTAC.(B) The array unit organization involving the genes encoding tRNAs SerGCT and SerTGA.

Molecular Biology and Evolution 2008 25(1):187-198

Molecular Biology and Evolution 2008 25(1):187-198

10

Intraspecific differences in STR organization in the Entamoeba histolytica N-K2intergenic region.

Molecular Biology and Evolution 2008 25(1):187-198

African Sleeping Sickness- Trypanosoma brucei rhodesienseTrypanosoma brucei gambiense

11

TseTse

Brain cuffing

African sleeping sickness

12

Visceral

Cutaneous

Leishmaniasis

Leishmaniasis

spleen

liver

Visceral LeishmaniasisKala-azar -black feverL. donovoni

Cutaneous LeishmaniasisMultiple species

Bone marrow macrophage

13

Individual eukaryotic genomes: Leishmania

Leishmania major causes leishmaniasisIntracellular pathogen of the immune system targetingmacrophages and dendritic cells. Affects the populations of88 counties worldwide with symptoms ranging fromdisfiguring cutaneous and muco-cutaneous lesions that cancause widespread destruction of mucous membranes tovisceral disease affecting the haemopoetic organs.Genome size: 34 MbChromosomes: 36 (range 0.3 to 2.5 Mb)Genes: about 9800 (all are being manually annotated)Website: http://www.sanger.ac.uk/Projects/L_major/Leishmania chromosome 1 has 79 protein-coding genes.The first 29 (from the left telomere) are all transcribed fromone strand, and the next 50 from the opposite strand.

14

Distribution of genes and domains among the kinetoplastid parasites and other organisms. (A) Genedistribution, calculated with the use of Jaccard-filtered COGs (2). (B) Domain distribution calculatedwith the use of TIGRFAMs and Pfam domains. The numbers indicate all domains that score abovethe trusted cutoff after manual curation with the three-way genome comparisons (2). (C) Pfamdomain distribution among the three kingdoms of life and the Tritryps. Numbers in small circlesindicate the number of domains that occur more than once in Tritryp parasite genomes. The numbersabove the small circles indicate Pfam domains that are not present in the Tritryps.

Science 15 July 2005: Vol. 309.

Individual eukaryotic genomes: trypanosomes

Trypanosoma brucei causes sleeping sickness (Africa)Distinguishing features: transmitted by tsetse fliesGenome size: 29 Mb (+/- 25% in various isolates) 11,000genesChromosomes: 11 (range 1 to >6 Mb); also has intermediate

chromosomes (200-700 kB) and 100 linearminichromosomes (30-150 kb)Chromosomes do not condense at metaphaseOnly a single intron in the poly (A) polymerase geneIntermediate and minichromosomes serve as repositories forVSGsTrypanosomes have kinetoplast DNA (circular rings ofmitochondrial DNA)(studied by Paul Englund’s lab here).

15

Fig. 1. EM showing a segment of a purified C.fasciculata kinetoplast DNA network. Small loops are the2.5 kb minicircles, and long strands threading through thenetwork interior are parts of the 38 kb maxicircles. EMby David Pérez-Morga

Morris et al., (2001) Int. J. Parasitol. 31, 453-458.

Guilbride & Englund (1998) J. Cell Science 111, 675-679.

Drew & Englund (2001) J. Cell Biol. 153, 735-743.

16

17

18

ApicomplexanPlasmodiumToxoplasmaCryptosporidium

19

trophozoite schizont

Merozoites in liver forseven to 10 days. P. vivax& ovale hypnozoite forMonths.

Gametocytes

Ingests 3 ul of bloodWith 100-300 gametes

Viable for 28 days

100’s

1,000’sreleasedPlasmodium

Life Cycle

Definitive hostmosquito

Sporozoites

Within hours invades 1-2 liver cells

Clinical signs and symptoms in erythro-cytic stages of ring,trophozoite and schizont

ring48-72 hrs

28 daysIn mosquitoOocysts on gut wal

Plasmodium vivax: Enlarged erythrocytesSource: CDC/Dr. Mae Melvin

Plasmodium ovale: Oval or comet shapeSource: CDC/Dr. Mae Melvin

Plasmodium malariae: Band formsSource: CDC/ Steven Glenn, Lab & Consultation Division

Plasmodium falciparum: High percent ringsSource: Thomas Spahr and David Sullivan JHMRI

20

Cat is definitive hostThe cat releases oocyst into feces.Cat litter infective for pregnantwoman. Infection by undercookedmeat with bradyzoite cyst.Tachyzoite in tissue invasive form.Toxoplasma gondii is singlespecies that invades all cell typesin all species from cats,humans,rodents, birds, sheep and pigs.

Toxoplasmosis

21

Cysts in brain-bradyzoite

Tachyzoites

Ocular(Retinal damage)

Broncho alveolar lavage from HIV + Patient

Cryptosporidium parvum

22

23

Individual eukaryotic genomes: malaria parasite Plasmodium falciparum

Plasmodium falciparum causes malaria, killing 2.7 millionpeople each year.

Distinguishing features: Four Plasmodium species infecthumans: P. falciparum, P. vivax, P. ovale, P. malariae.The life cycle is extremely complex.

Genome size: 22.8 Mb Chromosomes: 14 (range 0.6 to 3.3 Mb)Genes: 5268 (comparable to S. pombe)(1 gene/4300 bp)Website: http://www.plasmodb.org

P. falciparum has an adenine+thymine (AT) content of 80.6%.The P. yoelli yoelli genome was also sequenced (infects rats).

Individual eukaryotic genomes: malaria parasite Plasmodium falciparum

Bioinformatics approaches to Plasmodium falciparum:

-- The apicoplast (relic plastid; fatty acid, isoprene metabolism) is a potential drug target. Apicoplast signal sequences found.-- Comparative genomics defines some gene functions, identifies genes lacking in closely related species-- Genes implicated in antigenic variation and immune system evasion can be identified (e.g. 1000 copies of vir)-- Proteomics applied to four stages of the life cycle (sporozoites, merozoites, trophozoites, gametocytes)-- Atypical metabolic pathways may be exploited, e.g. use of 1-deoxy-D-xylulose 5-phosphate (DOXP) in isoprene biosynthesis.

24

25

26

Kooij et al. Nature Reviews Microbiology;published online 03 April 2006 | doi:10.1038/nrmicro1392

Kooij et al. Nature Reviews Microbiology;published online 03 April 2006 | doi:10.1038/nrmicro1392

27

Kooij et al. Nature Reviews Microbiology;published online 03 April 2006 | doi:10.1038/nrmicro1392

28

Proteomic validation of RNA detection

KL Olszewski et al. Nature 466, 774-778 (2010) doi:10.1038/nature09301

An integrated model for central carbon metabolism in P. falciparum.

29

KL Olszewski et al. Nature 466, 774-778 (2010) doi:10.1038/nature09301

Glutamine drives reverse flux through the TCA cycle.

http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0000005

The Transcriptome ofthe IntraerythrocyticDevelopmental Cycle ofPlasmodium falciparum

Zbynek Bozdech,Manuel Llin, Brian LeePulliam, Edith D. Wong,Jingchun Zhu, JosephL. DeRisi

(A) Giemsa stains of the majormorphological stages

(B-D) Example expression profiles for threegenes,

encoding EBA175, DHFR-TS, and ASL(E) MAL6P1.147, the largest predicted ORF in

the Plasmodium genome, is represented by 14 unique DNA

oligonucleotide elements. The location ofeach of the oligonucleotide elementswithin the predicted ORF and thecorresponding individual expressionprofiles are indicated (oligo 1ﾐ14).

30

Overview of the P. falciparum IDCTranscriptome

A phaseogram of the IDC transcriptomewas created by ordering the

transcriptionalprofiles for 2,712 genes by phase ofexpression along the y-axis. Thecharacteristic stages of intraerythrocyticparasite morphology are shown on the

left, aligned with thecorresponding phase of

peak gene expression.

Coregulation of Gene Expression alongthe Chromosomes of P. falciparum Is Rare,While Plastid Gene Expression Is HighlyCoordinatedExpression profiles for oligonucleotidesare shown as a function of location forChromosome 2 ([A], Oligo Map). Withthe exception of the SERA locus (B),coregulated clusters of adjacent ORFsare seldom observed, indicating thatexpression phase is largely independentof chromosomal position. (C) Incontrast to the nuclear chromosomes,the polycistronic expression of thecircular plastid genome is reflected inthe tight coregulation of geneexpression.

31

Temporal Distribution of theApicoplast-Targeted Proteinsand P. falciparum Proteases,Potential Antimalarial DrugCandidates

(A) The expression profiles of allputative plastid-targeted genesrepresented on our microarrayare shown. The yellow boxencompasses a highlysynchronized group of genes,which are in-phase with plastidgenome expression. The averageexpression profile for this in-phase group of genes is shownand includes most of the knownapicoplast-targeted genes aswell as many hypothetical genes.For reference, the averageexpression profile for the plastidgenome is shown (dashed grayline).

(B) Proteases represent an attractivetarget for chemotherapeuticdevelopment. The broad rangeof temporal expression forvarious classes of proteases andtheir putative functions aredisplayed.

Phaseogram of Putative VaccineTargetsThe similarity of all expressionprofiles to seven known vaccinecandidates (boxed) was calculated.The top 5% of similar profilescorrespond to 262 ORFs, 28 of whichhave been previously associated withplasmodial antigenicity and theprocess of merozoite invasion.

32

Pathport vbi vthttp://pathport.vbi.vt.edu/pathinfo/

http://pathport.vbi.vt.edu/pathinfo/pathogens/Plasmodium_falciparum_Info.shtml

http://pathport.vbi.vt.edu/pathinfo/pathogens/Cryptosporidium_parvum_Info.shtml

Websiteshttp://www.plasmodb.org/plasmo/home.jsphttp://www.apidb.org/apidb/http://www.ebi.ac.uk/parasites/parasite-genome.htmlCodon usage tablehttp://www.kazusa.or.jp/codon/