Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxoplasma gondii.
BACKGROUND
Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T. gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T. gondii's etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion.


METHODS
We present here Laser Scanning Cytometer (LSC)-based assays of T. gondii invasion and attachment. The invasion assay involves automated counting of invaded and non-invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment.


RESULTS
The LSC-based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope-based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms.


CONCLUSIONS
The LSC-based assays represent useful new methods for quantitatively measuring attachment and invasion by T. gondii, and can be readily adapted to study similar processes in other host-pathogen systems.
Mital
Jeffrey
J
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
Schwarz
Janet
J
Taatjes
Douglas J
DJ
Ward
Gary E
GE
D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D005347:Fibroblasts / Q000469:parasitology*; D005456:Fluorescent Dyes; D006801:Humans; D047569:Laser Scanning Cytometry; D014122:Toxoplasma / Q000502:physiology*
D005456:Fluorescent Dyes