Toxoplasma gondii

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• Toxoplasma gondii
• Member of the phylum Apicomplexa which also
   includes Plasmodium falciparum, Eimeria,
  Cryptosporidia, Babesia and Theileria
• Contain apicoplast, an essential organelle
  thought to  be involved in the synthesis of
  lipids
• Apical complex is central to invasion of host
  cells
• secretion from the apical complex supplies
  factors essential for attachment, invasion and
  formation of the parasitophorous vacuole

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Toxoplasma Gondii Life Cycle Review
Tachyzoites rapidly divide and invade neighboring
cells
Micro and macro gametes form (gametogenesis)
Gametes fuse to form oocyst
Excystation occurs and sporozoites released
Bradyzoites eventually sequestered in muscular
tissue cysts
Merizoites invade neighboring cells
Oocyst ingested by intermidiate host
Parasites multiply in inetestinal cells
Sporulation occurs
Bradyzoite ingested
Bradyzoites may exit cyst at random
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Toxoplasma MIC2 is a Major Determinant of
Invasion and Virulence My-Hang Huynh, Vern B.
Carruthers PLoS Pathogens August 2006, Vol. 2
Issue 8, pp 0753-0762
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Purpose To address the role of MIC2 in Toxplama
gondii Synopsis Generated a conditional MIC2
knockout mutant using a tetracycline regulatable
system. Show that MIC2-deficient parasites are
severely impaired in their ability to attach to
and invade host cells and are non-lethal in vivo.
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• MICs
• Micronemal proteins
• MIC2 is a transmembrane adhesin thought to play a
  role   in gliding motility and cell invasion
• Contains several extracellular adhesive domains
  for   receptor binding and a short cytosolic
  domain that   connnects it to the glydosome
• Member of the TRAP (thrombospondin-related
    anonymous protein) family
• MIC1 assembles with MIC4 and MIC6, soluble
  proteins   in micronemes
• MIC3 involved in host cell binding

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• Conditional Knockout System
• Transcriptional transactivator (tTA)
• Genes containing the operator cassettes are
  upstream of the transcription start site of gene
  of interest
• Genes are constitutively transcribed until
  addition of tetracycline
• tTA released from operator cassette preventing
  further transcription

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• Strains and Constructs
• tTA-dhfr reference strain containing inducible
  marker
• mic2i inducible MIC2 construct (transfected on
    episome)
• mic2e/mic2i has inducible and endogenous MIC2
• ?mic2e/mic2i endogenous MIC2 knocked out via
    homolgous recombination

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Supplementary Figure 1
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Relationship with M2AP MIC2 and M2AP intimately
associate soon after synthesis Absence of M2AP
causes mislocalization and reduced secretion of
MIC2 Underexpression of MIC2 abolishes
proteolytic maturation of M2AP from proform to
mature form
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Relationship with M2AP
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Red-Green Invasion Assay ExtracellularRed Intrace
llularGreen
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Correlation of number of invaded parasites with
expression and secretion of MIC2
Conclusion Absence of MIC2 causes, for one,
defect in attachment to host
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Time-Course Invasion Over 8 Hours
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Deletion of MIC2 has no effect on parasite
replication
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Gliding Trails and Tribulations
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Quantification of Gliding Motions
C
Circular GlideRed arrow Helical GlideRed
arrowhead Nonproductiveclosed black Twirlingopen
black
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Cytoskeletal Analysis
No difference in number of helical and straight
cytoskeletons
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Infectivity Studies
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Quantitative Real Time PCR to Monitor Parasite
Burden in Different Tissues
Dead by day 10
Infection cleared by day 15
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Cytokine Levels
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Conclusions Dramatic reduction in MIC2
production does not affect parasite replication
and has no effect on circular gliding
motions Extreme reduction of MIC2 causes
impairment in host attachment and decreases
helical and twirling motions ?mic2 parasites are
unable to establish an infection in mice up to
625X the normal lethal dose and can protect
against challenge with wild type