Development of new immunological tools for the study Development of new immunological tools for the study of Toxoplasma gondii

Furio SpanoDepartment of Infectious, Parasitic

and Immunomediated DiseasesIstituto Superiore di Sanità – Rome, Italy

The invasion process in the Apicomplexa

Toxoplasma

Plasmodium

Cryptosporidium

Mol. Biochem. Parasitol. 92: 147-62 (1998)

Mol. Biochem. Parasitol. 157: 98-101 (2008)

Int. J. Parasitol. 32: 121-31 (2002)

sporozoite

merozoite

Cry

pto

spo

rid

ium

par

vum

TRAP-C1

CpMIC1

SAG5

(unpublished)

sporozoite

tachyzoite

tachyzoite

Toxo

pla

sma

go

nd

ii

TgMIC14, TgMIC15

(unpublished)TgLCCL protein family

Identifying protein constituents of the oocyst wall of Toxoplasma gondii

phase UVproduction of oocyst-specific antibodies- coprodiagnosis- immunomagnetic separation (IMS)

basic knowledge

mice immunized with purified oocyst wallsA. Dumètre & M-L. DardéJ. Microbiol. Met. 61 (2005)Parasitol. Res. 101 (2007)

mAb 3G4 (anti-oocyst wall) - mAb 4B6 (anti-sporocys t wall)

mice immunized with purified oocyst walls

target proteins not identified

major limitations of developed IMS

Identification of putative oocyst wall proteins (OWPs)

Genecloning

Immunolocalization (IFA) with polyclonal antisera

Production of recombinant proteins in bacteria

Productionof mAbs

The coccidian oocyst wall (1): ultrastructure

Physical-chemical properties of the oocyst wall

Resistant to sodium hypochlorite (<10%)2% potassium dichromate2% sulphuric acidproteolytic enzymes

Impermeable to water-soluble substancesPermeable to lipid-soluble and gaseous substances

O/I layers proteins, lipids, carbohydrates

T. gondii Eimeria maxima

O/I layers proteins, lipids, carbohydratesT. gondii early oocyst

T. gondii unsporulated oocyst

Eiemeria maxima

Toxoplasma gondii

?

The coccidian oocyst wall (2): molecular structure

Eiemeria maxima

Cryptosporidium

parvum

Tyrosine-rich proteins: gam56 and gam82(dityrosine-protein crosslink)

Cysteine-rich proteins: COWP family

The oocyst wall protein (OWP) family of Toxoplasma gondii

20.m

25.m

27.m

41.m

(500) 0 3 0

(463) 0 24 0

(783) 0 4 0

(452) 0 5 1

50.m

59.m

76.m

(535) 0 23 0

(641) 0 3 0

(512) 0 5 1

Production of polyclonal sera against putative TgOWPs

20.m

25.m

27.m

41.m

(500)

(463)

(783)

(452)

**

50.m

59.m

76.m

(535)

(641)

(512)

= bacterially expressed recombinant protein fragment

**

* = synthetic peptide

Immunolocalization of putative TgOWPs

20.m 25.m

41.m27.m

Production of monoclonal Abs against select TgOWPs

20.m

25.m

27.m

41.m

(500)

(463)

(783)

(452)

**

50.m

76.m

(535)

(512)

59.m (641)

**

mAb 1B4mAb 1G12 IgM isotype

Reactivity of mAb 1B4

A

B

negative control

C

D

Conclusions

Toxoplasma gondii possesses a family of 7 cysteine-rich extracellular proteins structurally related to the Cryptosporidium oocyst wall protein COWP-1

The genes of 3 putative TgOWPs have been cloned and shown to be expressed in partially sporulated T. gondii oocysts

Polyclonal Abs raised against 6 of the putative TgOWPs were shown to react with the wall of T. gondii oocysts and/or sporocysts

Two monoclonal Abs specific for the protein 59.m were shown to recognize the outer wall of T. gondii oocysts

Future objectives

Cloning of the cDNAs encoding the remaining TgOWPs

Localization of individual TgOWPs by IFA and immunoEM

Production of monoclonal Abs directed against other members of the TgOWP family located in the outer layer of the oocyst wall

Assessment of the cross-reactivity of anti-TgOWPs mAbs with other Assessment of the cross-reactivity of anti-TgOWPs mAbs with other coccidia (Hammondia hammondi, H. heydorni, Neospora caninum)

Set up of an immunofluorescence test for the coprodiagnosis of toxoplasmosis

Development of an immunomagnetic separation technique for the captureof T. gondii oocysts from environmental samples