Kinetoplastids - 1a Flashcards
Classification
- Domain: Eukaryota
- Phylum: Eugenozoa
- Class: Kinetoplastida
- Genus: Phytomonas, Leptomonas, Blastocrithidia, Crithidia, Trypanosoma, Leishmamia
- T. brucei, T. cruzi, L. major, L. donovani, L. infantum, L. brazilenesis
- Genus: Phytomonas, Leptomonas, Blastocrithidia, Crithidia, Trypanosoma, Leishmamia
- Class: Kinetoplastida
- Phylum: Eugenozoa
Kinetoplastids
Domain: Eukaryota
- EUKARYOTES
- NOT A VIRUS/BACTERIA
Kinetoplastids
Domain: Eukaryota
(bullets)
- Eukaryota = true nucleus
- glycosomes (glycolysis)
- nucleus
- endoplasmic reticulum/Golgi
- single mitochondrion
Kinetoplastids
Phylum: Eugenozoa
Kinetoplastids
Phylum: Eugenozoa
(bullets)
- Eugenozoa = flagellate
- flagellar pocket → endo/exocytosis
- flagellum → movement/attachment
- signal transduction (sense envt)
Kinetoplastids
Class: Kinetoplastida
Kinetoplastids
Class: Kinetoplastida
(bullets)
- kinetoplast → mitochondrial genome
Kinetoplastids
Order: Trypanosomatida
Kinetoplastids
Genus:
Leptomonas
Blastocrithidia
Crithidia
- infect only insects (primarily digestive tract)
- usually spread through feces
Kinetoplastids
Genus: Phytomonas
- infects insects (digestive tract)
- transmtted via saliva
- infect plants (latex, phloem, fruit sap, seed albumen, nectar)
- transmitted via feeding habits of insect vector
- eg aphids saliva
- found essentially all over the plant
- transmitted via feeding habits of insect vector
Kinetoplastids
Genus: Trypanosoma and Leishmania
Species
- T. brucei
- human African trypanosoiasis (HAT)
- undulates, esp cattle → agricultural effect
- serious wasting disease (eg nagana and surra)
- T. cruzi
- chagas disease
- affects any animal
- Leishmania
- leishmaniasis
Kinetoplatids
Genus: Trypanosoma and Leishmania
cattle and horses
- serious wasting diseases
- nagana in cattle
- T. brucei
- T. congolense
- T. vivax
- surra in equines, pigs, sheep, goats
- T. evansi
- T. suis
→ agricultural effect (loss of ~$5bilion from food, plowing fields)
- 1 Trypanosoma species not insect transmitted - T. equiperdum
Kinetoplastids
Genus: Trypanosoma and Leishmania
T. equiperdum
- T. equiperdum
- close relative of T. brucei
- dourine (covering sickness) in equines
- sexually transmitted
- (only?) species not insesct transmitted
Trypanosoma and Leishmania
Complex life cycles =
different morphologies
(aka parasite forms)
Trypanosoma and Leishmania: basic morphology
Different morphologies (parasite forms) distinguished by:
- position of kinetoplast relative to nucleus
- position of flagella pocket relative to nucleus
- presence/size of flagellum
- eg intracellular forms of T. cruzi and Leishmania have stubby flagella
- eg T. brucei flagella throughout life cycle
- presence of undulating membrane
- (structure linking flagella to cell body)
Trypanosoma and Leishmania: basic morphology
Parasites can be found in different
cells, parts of the body
→
different morphological forms
- eg T. brucei found in fluids, extracellular parasite, found in blood stream and lymph system, cerebral-spinal fluid
- in midgut and saliva of insect vector
- in humans, T. cruzi intracellular pathogen - can invade any nucleated cell (also can invade any mammal)
- transition throughout whole gut of insect vector, different forms as it progresses through digestive tract
- Leishmania - intracellular, specialized, can only invade macrophages and neutrophils
- stays in midgut of insect vector before vomited up
- need different parasite forms to live in those different environments
Trypanosoma and Leishmania:
different parasite forms
Trypanosoma and Leishmania:
forms based on
front and back based on direction of motility
(direction flagellum takes it)
Trypanosoma and Leishmania
mastigote =
having a flagella
Trypanosoma and Leishmania
amastigote
(picture)
Trypanosoma and Leishmania
amastigote
(bullets)
- “no flagella” but actually has one
- not outside but within the flagella pocket
- intracellular form of T. cruzi and Leishmania
Trypanosoma and Leishmania
Promastigote
(picture)
Trypanosoma and Leishmania
Promastigote
(bullets)
- kinetoplast and flagella pocket toward anterior end compared to nucleus
- flagella not attached by undulating membrane
Leshmania forms
(mastigote)
amastigote
promastigote
Trypanosoma and Leishmania
Epimastigote
(picture)
Trypanosoma and Leishmania
Epimastigote
(bullets)
- kinetoplast and flagella pocket toward anterior relative to nucleus
- closer to nucleus
- flagella pocket attached to body by undulating membrane
- form of T. cruzi cultured in lab
- in midgut form of T. brucei, in salivary glands
Trypanosoma and Leishmania
Trypomastigote
(picture)
Trypanosoma and Leishmania
Trypomastigote
(bullets)
- kinetoplast and flagella pocket toward posterior relative to nucleus
- flagella attached to body with undulating membrane
- bulk to T. brucei life cycle
- infectious stages of T. cruzi lifecycle
Trypanosoma and Leishmania: key features
- mitochondrion
- kinetoplast
- relationship between kinetoplast and flagellum
- kinetoplast and RNA editing
- flagellum
- flagellar pocket
- glycosomes and glycolysis
- redox metabolism
- glycosylphosphaditylinositol (GPI) anchor
Trypanosoma and Leishmania
key feature - mitochondrion
- 1 per cell
- has a complex lattice structure spread throughout cell
- readily visualized using selective dyes (eg Mitotracker) / confocal microscopy
- rod/bar shape in organelle
- kinetoplast or kDNA → constitues mitochondrial genome
- network of DNA found in defined region of mitochondrion
- function can alter during life cycle
Trypanosoma and Leishmania:
mitochondrion - structure
Trypanosoma and Leishmania
mitochondrion - visualization using selective dyes and (eg Mitotracker) / confocal microscopy
- 2 genomes
- faint red = nuclear
- dark red = kinetoplast
- between 2 discs
Trypanosoma and Leishmania
Mitochondrion - kDNA
- rod/bar shape in organelle = kinetoplast with kDNA
- kinetoplast or kDNA → constitutes mitochondrial genome
- network of DNA found in defined region of mitochondrion
- network of DNA molecules
Trypanosoma and Leishmania
Mitochondrion - function can alter during life cycle
eg bloodstream form of T. brucei ( in mammalian host)
- lack cytochrome chains
- lacks oxidative phosphorylation
- relies on glycolysis for energy (ATP) productoin
- effectively an anaerobic organism
Trypanosoma and Leishmania
Mitochondrion - function can alter during life cycle
eg procyclic form of T. brucei (in insect vector)
- contains cytochrome chains
- has oxidatiave phosphorylation pathways
Trypanosoma and Leishmania
Key feature - kinetoplast
- easily visualized by staining
- cytological stain (eg Giemsa)
- linked to parasites flagella by basal body tightly bound to cytoskeleton
- marker for cell cycle (along with flagellum)
- composed of 2 classes of circular DNA
- maxi-circles
- mini-circles
- highly concatenated
Trypanosoma and Leishmania
kinetoplast - easily visualized
by staining
- cytological stain (eg Giemsa)
Trypanosoma and Leishmania
kinetoplast - relationship between kinetoplast and flagellum
linked to parasite’s flagella basal body tightly bound to cytoskeleton
Trypanosoma and Leishmania
kinetoplast - marker for…
cell cycle (along with flagellum)
- 1kt/cell
- compare kinetoplast ratio to nucleus to see where dividing cell is within the cell cycle
- see where cells within a population are within the cell cycle
Trypanosoma and Leishmania
kinetoplast
1n - 1k
G1
Trypanosoma and Leishmania
kinetoplast
1n - 2k
G2
Trypanosoma and Leishmania
kinetoplast
2n - 2k
M
Trypanosoma and Leishmania
kinetoplast
2n - 2k
M →
Trypanosoma and Leishmania
kinetoplast
cell cycle - overall
Trypanosoma and Leishmania
kinetoplast - composed of 2 classes of circular DNA that are
highly concatenated - individual circular DNA molecules interconnected to form a complex network
Trypanosoma and Leishmania
kinetoplast 2 forms:
- maxi-circles
- mini-circles
Trypanosoma and Leishmania
kinetoplast
maxi-circles
- 20-50 copies per genome
- 20-38 kb in size
- encode for several genes found on mitochondrial genomes in other organisms
- rRNA genes
- genes encoding metabolic enzymes
- many pseudogenes
- defective genes
- with insertions/deletions, push ORFs out of frame, stop codons within genes
- no introns/exons
- translated directly to proteins
*
- translated directly to proteins
Trypanosoma and Leishmania
kinetoplast
mini-circles
- 5,000 - 10,000 copies per genome
- 0.5 - 1.5 kb in size
- heterogeneous high level of sequence variability
- encodes for specialized RNA molecules called guide RNA (gRNA)
Trypanosoma and Leishmania
kinetoplast and RNA editing
- cryptogenes/pseudogenes on maxi-circle DNA → transcribed to form pre-mRNA
- defective pre-mRNA → undergoes post-transcriptional modification
- modifications edit defective pre-mRNA
- modification performed by a multi-protein complex called the editosome
- editosome includes
- endonuclease
- exonucleases
- ligases
- nucleotide transferases
- components of editosome transcribed from nuclear encoded genes
- position of the nucleotide modification deterined by gRNA
- gRNA complementary to regions undergoing editing
Trypanosoma and Leishmania
kinetoplast and RNA editing:
modification performed by a multi-protein complex called the
editosome
Trypanosoma and Leishmania
kinetoplast and RNA editing:
editosome includes
- endonuclease
- exonucleases
- ligases
- nucleotide transferases
Trypanosoma and Leishmania
kinetoplast and RNA editing:
components of the editosome transcribed from
nuclear encoded genes
Trypanosoma and Leishmania
kinetoplast and RNA editing:
position of the nucleotide modification determined by
gRNA
Trypanosoma and Leishmania
kinetoplast and RNA editing:
gRNA complementary to
regions undergoing editing
Trypanosoma and Leishmania
kinetoplast and RNA editing:
adding
(picture)
Trypanosoma and Leishmania
kinetoplast and RNA editing:
adding
(descriptive)
- gDNA recognizes a specific, homologous region of pre-mRNA to undergo editing
- gRNA binds to defective pre-mRNA because complementary
- an endonuclease cleaves the sugar-phosphate backbone of the pre-mRNA
- uridylyl transferase (TUTase) guides insertion of uridine(s) at cleavage site
- adds predeterined number of uridines
- determined by gRNA
- DNA ligase re-joins the 2 RNA strands
Trypanosoma and Leishmania
kinetoplast and RNA editing:
removing
(picture)
Trypanosoma and Leishmania
kinetoplast and RNA editing:
removing
(descriptive)
- gDNA recognizes a specific, homologous region of pre-mRNA to undergo editing
- an endonuclease cleaves the sugar-phosphate backbone of the pre-mRNA
- uridine specific exonuclease (ExoUase) removes uridine(s) adjacent to cleavage site
- DNA ligate re-joins 2 RNA strands
Trypanosoma and Leishmania
kinetoplast and RNA editing:
non-functional pre-mRNA converted to
functional mRNA
functional mRNA translated into functional protein
(chop off/add in U = convert defective to functional message to complete ORF → functional protein)
Trypanosoma and Leishmania
key feature - flagellum
- 1 per cell
- leaves cell body at flagella pocket
- in some parsite forms
- flagella remains attached to cell body for most of length
- flagella is free from cell body
- used in:
- motility (questionable in certain life cycle stages)
- attachment
- sensor (?)
Trypanosoma and Leishmania
key feature - flagellum:
leaves body at
flagella pocket
Trypanosoma and Leishmania
key feature - flagellum:
in some parasite forms
- flagella remains attached to cell body for most of its length
- flagella is free from body
Trypanosoma and Leishmania
key feature - flagellum:
used for
- motility
- questionable in some life cycles
- attachment
- sensor
Trypanosoma and Leishmania
key feature - flagellum
structure
Trypanosoma and Leishmania
key feature - flagellum:
axoneme
(picture)
Trypanosoma and Leishmania
key feature - flagellum:
axoneme
- 9 pairs + 2 cytoskeletal arrangement
- 9 outer pairs around 2 in the center
- attached
- dyneins attach microtubule pairs together
- ATP driven process
- dyneins flex such that adjacent microtubule pairs move
- generates wave-like motion
Trypanosoma and Leishmania
key feature - flagellum
lattice-like structure = PFR
- paraflagellar rod
- runs alongside axoneme
- role unknown
- essential for locomotion
- without = no longer motile, form snail mutants
- strengthening bar for rigidity
Trypanosoma and Leishmania
key feature - flagellum
FAZ
- flagellar attachment zone
- attaches flagella to cell body
- links PFR to cell cytoskeleton
Trypanosoma and Leishmania
key feature - flagellar pocket
- deep invagination of plasma membrane
- located at base of flagellum
- one end connects (posterior), other end thickens (anterior)
- posterior of flagellar pocket associated with flagellar basal body/kinetoplast
- anterior of flagellar pocket spearated form external environment by desmosome-like thickening
- junctional complex - may restrict flow of material into/ot of flagellar pocket
- site of endocytosis/exocytosis
Trypanosoma and Leishmania
key feature - flagellar pocket
endocytosis
- occurs via clathrin coated vesicles
- material trafficked via endosomal system
- EE - early endosome
- L - lysosome
- LE - late endosome
- taken up material
- transferred to cytosol
- degraded via proteases
- recycled back to flagella pocket (exocytosis)
Trypanosoma and Leishmania
key feature - flagellar pocket
exocytosis
- recycling
- transfer of newly synthesized material to cell surface
Trypanosoma and Leishmania
key feature - glycosomes and glycolysis
- other organisms, glycolysis occurs in cytosol
- trypanosomes/leishmania occurs in specialized organelle - glycosome
Trypanosoma and Leishmania
key feature - glycosome
- modified peroxisome
- bound by single membrane
- low permeability to metabolites
- protein-dense matrix
- functions as typical peroxisome
- breakdown of very long chain fatty acids
Trypanosoma and Leishmania
key feature - glycosome
chemistry
- contains several glycolytic enzymes
- convertes glucose to 3-phosphoglycerate
- remaining enzymes (3-phosphoglycerate to pyruvate) in cytosol
Trypanosoma and Leishmania
key feature - glycosomes and glycolysis
why compartmentalize?
- high rate of glycolysis in some parasite forms
- high concentrations of enzymes/metabolites in specialized organelle promotes high rate
- low membrane permeability allows metabolites to be segregated
- segregation may prevent metabolic interference between competing pathways
Trypanosoma and Leishmania
key feature - redox metabolism
- ROS (superoxide anions, H2O2) are:
- by-products of aerobic metabolism
- highly reactive and toxic
- damage DNA, RNA, proteins, lipids, etc
- cells evolved defense mechanisms to detoxify ROS
- most organisms,glutathione (thil) plays a key role in detoxification
- trypanothione synthesis
- trypanothione reductase
- trypanothione-dependent enzymes are unique to parasites
→ all potential drug targets
- trypanothione is essential for parasite viability
Trypanosoma and Leishmania
key feature - redox metabolism
trypanosomes and leshmania are different
- do have glutathione but
- major thiol is trypanothione (specific to these parasites)
- consists of 2 glutathione molecules inked by spermidine
trypanothione disulphide
- oxidized
- disulphide bond
Trypanothione reductase
NADPH → NADP+
dihydrotrypanothione
- reduced
Trypanosoma and Leishmania
key feature - redox metabolism
dihydrotrypanothione
(reduced)
drives
- peroxide detoxification
- heavy metal detoxification
- nucleotide reduction (DNA synthesis)
Trypanosoma and Leishmania
key feature - GPI
- glycosylphosphatidylinositol (GPI) anchor
- post-translational modification
- glycolipid attached to C-terminal of target protein
- used to anchor protein into membranes
