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Parasites and Infectious Diseases > Kinetoplastids - 1a > Flashcards

Kinetoplastids - 1a Flashcards

1
Q

Classification

A
  • 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
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2
Q

Kinetoplastids

Domain: Eukaryota

A
  • EUKARYOTES
  • NOT A VIRUS/BACTERIA
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3
Q

Kinetoplastids

Domain: Eukaryota

(bullets)

A
  • Eukaryota = true nucleus
  • glycosomes (glycolysis)
  • nucleus
  • endoplasmic reticulum/Golgi
  • single mitochondrion
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4
Q

Kinetoplastids

Phylum: Eugenozoa

A
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5
Q

Kinetoplastids

Phylum: Eugenozoa

(bullets)

A
  • Eugenozoa = flagellate
  • flagellar pocket → endo/exocytosis
  • flagellum → movement/attachment
    • signal transduction (sense envt)
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6
Q

Kinetoplastids

Class: Kinetoplastida

A
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7
Q

Kinetoplastids

Class: Kinetoplastida

(bullets)

A
  • kinetoplast → mitochondrial genome
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8
Q

Kinetoplastids

Order: Trypanosomatida

A
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9
Q

Kinetoplastids

Genus:

Leptomonas

Blastocrithidia

Crithidia

A
  • infect only insects (primarily digestive tract)
    • usually spread through feces
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10
Q

Kinetoplastids

Genus: Phytomonas

A
  • 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
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11
Q

Kinetoplastids

Genus: Trypanosoma and Leishmania

Species

A
  • 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
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12
Q

Kinetoplatids

Genus: Trypanosoma and Leishmania

cattle and horses

A
  • 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
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13
Q

Kinetoplastids

Genus: Trypanosoma and Leishmania

T. equiperdum

A
  • T. equiperdum
    • close relative of T. brucei
    • dourine (covering sickness) in equines
    • sexually transmitted
      • (only?) species not insesct transmitted
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14
Q

Trypanosoma and Leishmania

Complex life cycles =

A

different morphologies

(aka parasite forms)

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15
Q

Trypanosoma and Leishmania: basic morphology

Different morphologies (parasite forms) distinguished by:

A
  • 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)
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16
Q

Trypanosoma and Leishmania: basic morphology

Parasites can be found in different

A

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
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17
Q

Trypanosoma and Leishmania:

different parasite forms

A
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18
Q

Trypanosoma and Leishmania:

forms based on

A

front and back based on direction of motility

(direction flagellum takes it)

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19
Q

Trypanosoma and Leishmania

mastigote =

A

having a flagella

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20
Q

Trypanosoma and Leishmania

amastigote

(picture)

A
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21
Q

Trypanosoma and Leishmania

amastigote

(bullets)

A
  • “no flagella” but actually has one
    • not outside but within the flagella pocket
  • intracellular form of T. cruzi and Leishmania
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22
Q

Trypanosoma and Leishmania

Promastigote

(picture)

A
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23
Q

Trypanosoma and Leishmania

Promastigote

(bullets)

A
  • kinetoplast and flagella pocket toward anterior end compared to nucleus
  • flagella not attached by undulating membrane
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24
Q

Leshmania forms

(mastigote)

A

amastigote

promastigote

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25
Q

Trypanosoma and Leishmania

Epimastigote

(picture)

A
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26
Q

Trypanosoma and Leishmania

Epimastigote

(bullets)

A
  • 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
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27
Q

Trypanosoma and Leishmania

Trypomastigote

(picture)

A
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28
Q

Trypanosoma and Leishmania

Trypomastigote

(bullets)

A
  • 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
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29
Q

Trypanosoma and Leishmania: key features

A
  • mitochondrion
  • kinetoplast
  • relationship between kinetoplast and flagellum
  • kinetoplast and RNA editing
  • flagellum
  • flagellar pocket
  • glycosomes and glycolysis
  • redox metabolism
  • glycosylphosphaditylinositol (GPI) anchor
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30
Q

Trypanosoma and Leishmania

key feature - mitochondrion

A
  • 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
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31
Q

Trypanosoma and Leishmania:

mitochondrion - structure

A
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32
Q

Trypanosoma and Leishmania

mitochondrion - visualization using selective dyes and (eg Mitotracker) / confocal microscopy

A
  • 2 genomes
    • faint red = nuclear
    • dark red = kinetoplast
    • between 2 discs
33
Q

Trypanosoma and Leishmania

Mitochondrion - kDNA

A
  • 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
34
Q

Trypanosoma and Leishmania

Mitochondrion - function can alter during life cycle

eg bloodstream form of T. brucei ( in mammalian host)

A
  • lack cytochrome chains
  • lacks oxidative phosphorylation
  • relies on glycolysis for energy (ATP) productoin
  • effectively an anaerobic organism
35
Q

Trypanosoma and Leishmania

Mitochondrion - function can alter during life cycle

eg procyclic form of T. brucei (in insect vector)

A
  • contains cytochrome chains
  • has oxidatiave phosphorylation pathways
36
Q

Trypanosoma and Leishmania

Key feature - kinetoplast

A
  • 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
37
Q

Trypanosoma and Leishmania

kinetoplast - easily visualized

A

by staining

  • cytological stain (eg Giemsa)
38
Q

Trypanosoma and Leishmania

kinetoplast - relationship between kinetoplast and flagellum

A

linked to parasite’s flagella basal body tightly bound to cytoskeleton

39
Q

Trypanosoma and Leishmania

kinetoplast - marker for…

A

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
40
Q

Trypanosoma and Leishmania

kinetoplast

1n - 1k

A

G1

41
Q

Trypanosoma and Leishmania

kinetoplast

1n - 2k

A

G2

42
Q

Trypanosoma and Leishmania

kinetoplast

2n - 2k

A

M

43
Q

Trypanosoma and Leishmania

kinetoplast

2n - 2k

A

M →

44
Q

Trypanosoma and Leishmania

kinetoplast

cell cycle - overall

A
45
Q

Trypanosoma and Leishmania

kinetoplast - composed of 2 classes of circular DNA that are

A

highly concatenated - individual circular DNA molecules interconnected to form a complex network

46
Q

Trypanosoma and Leishmania

kinetoplast 2 forms:

A
  • maxi-circles
  • mini-circles
47
Q

Trypanosoma and Leishmania

kinetoplast

maxi-circles

A
  • 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
      *
48
Q

Trypanosoma and Leishmania

kinetoplast

mini-circles

A
  • 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)​
49
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing

A
  • 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
50
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

modification performed by a multi-protein complex called the

A

editosome

51
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

editosome includes

A
  • endonuclease
  • exonucleases
  • ligases
  • nucleotide transferases
52
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

components of the editosome transcribed from

A

nuclear encoded genes

53
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

position of the nucleotide modification determined by

A

gRNA

54
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

gRNA complementary to

A

regions undergoing editing

55
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

adding

(picture)

A
56
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

adding

(descriptive)

A
  • 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
57
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

removing

(picture)

A
58
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

removing

(descriptive)

A
  • 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
59
Q

Trypanosoma and Leishmania

kinetoplast and RNA editing:

non-functional pre-mRNA converted to

A

functional mRNA

functional mRNA translated into functional protein

(chop off/add in U = convert defective to functional message to complete ORF → functional protein)

60
Q

Trypanosoma and Leishmania

key feature - flagellum

A
  • 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 (?)
61
Q

Trypanosoma and Leishmania

key feature - flagellum:

leaves body at

A

flagella pocket

62
Q

Trypanosoma and Leishmania

key feature - flagellum:

in some parasite forms

A
  • flagella remains attached to cell body for most of its length
  • flagella is free from body
63
Q

Trypanosoma and Leishmania

key feature - flagellum:

used for

A
  • motility
    • questionable in some life cycles
  • attachment
  • sensor
64
Q

Trypanosoma and Leishmania

key feature - flagellum

structure

A
65
Q

Trypanosoma and Leishmania

key feature - flagellum:

axoneme

(picture)

A
66
Q

Trypanosoma and Leishmania

key feature - flagellum:

axoneme

A
  • 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
67
Q

Trypanosoma and Leishmania

key feature - flagellum

lattice-like structure = PFR

A
  • paraflagellar rod
  • runs alongside axoneme
  • role unknown
  • essential for locomotion
    • without = no longer motile, form snail mutants
  • strengthening bar for rigidity
68
Q

Trypanosoma and Leishmania

key feature - flagellum

FAZ

A
  • flagellar attachment zone
  • attaches flagella to cell body
  • links PFR to cell cytoskeleton
69
Q

Trypanosoma and Leishmania

key feature - flagellar pocket

A
  • 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
70
Q

Trypanosoma and Leishmania

key feature - flagellar pocket

endocytosis

A
  • 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)
71
Q

Trypanosoma and Leishmania

key feature - flagellar pocket

exocytosis

A
  • recycling
  • transfer of newly synthesized material to cell surface
72
Q

Trypanosoma and Leishmania

key feature - glycosomes and glycolysis

A
  • other organisms, glycolysis occurs in cytosol
  • trypanosomes/leishmania occurs in specialized organelle - glycosome
73
Q

Trypanosoma and Leishmania

key feature - glycosome

A
  • modified peroxisome
  • bound by single membrane
    • low permeability to metabolites
  • protein-dense matrix
  • functions as typical peroxisome
    • breakdown of very long chain fatty acids
74
Q

Trypanosoma and Leishmania

key feature - glycosome

chemistry

A
  • contains several glycolytic enzymes
  • convertes glucose to 3-phosphoglycerate
  • remaining enzymes (3-phosphoglycerate to pyruvate) in cytosol
75
Q

Trypanosoma and Leishmania

key feature - glycosomes and glycolysis

why compartmentalize?

A
  • 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
76
Q

Trypanosoma and Leishmania

key feature - redox metabolism

A
  • 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
77
Q

Trypanosoma and Leishmania

key feature - redox metabolism

trypanosomes and leshmania are different

A
  • 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
78
Q

Trypanosoma and Leishmania

key feature - redox metabolism

dihydrotrypanothione

(reduced)

drives

A
  • peroxide detoxification
  • heavy metal detoxification
  • nucleotide reduction (DNA synthesis)
79
Q

Trypanosoma and Leishmania

key feature - GPI

A
  • glycosylphosphatidylinositol (GPI) anchor
  • post-translational modification
  • glycolipid attached to C-terminal of target protein
  • used to anchor protein into membranes

Parasites and Infectious Diseases (8 decks)

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