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Infection and Immunity, March 2004, p. 1284-1290, Vol. 72, No. 3
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.3.1284-1290.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Purification and Analysis of a Phospholipase A2-Like Lytic Factor of Trichomonas vaginalis

Kirk J. Lubick and Donald E. Burgess*

Veterinary Molecular Biology, Montana State University, Bozeman, Montana

Received 14 August 2003/ Returned for modification 16 September 2003/ Accepted 10 December 2003

Trichomonas vaginalis produces soluble factors that have been reported to have the ability to damage target cells in vitro, and it has been hypothesized that these factors may play a role in the pathogenesis of human trichomoniasis. A lytic factor (LF) was purified from T. vaginalis, and the molecular characteristics of LF were determined. T. vaginalis extract was subjected to hydrophobic chromatography with a 10 to 60% N-propanol gradient in 0.1 M ammonium acetate, resulting in the elution of LF from the column at 30% N-propanol. Cytotoxicity assays revealed that LF was cytotoxic to WEHI 164 cells and bovine red blood cells, and inactivation of LF by treatment with trypsin suggested that the active component of LF was a protein. Size exclusion chromatography of LF produced two fractions at 144 and 168 kDa, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LF under reducing conditions revealed two subunits of 57 and 60 kDa. Results of a fluorescence assay of LF on carboxyfluorescein-labeled liposomes composed of phosphatidylcholine-cholesterol showed that liposomes were hydrolyzed, suggesting that LF had phospholipase activity. Thin-layer chromatography analysis of BODIPY (4,4-difluoro-3a,4adiaza-s-indacene)-labeled phosphatidylcholine treated with LF demonstrated products that migrated identically to the products produced by treatment with phospholipase A2 (PLA2). These results suggest that LF is a PLA2 and may be an important virulence factor of T. vaginalis mediating the destruction of host cells and contributing to tissue damage and inflammation in trichomoniasis.

* Corresponding author. Present address: ATCC, 10801 University Blvd., Manassas, VA 20110. Phone (703) 365-2722. Fax: (703) 265-2730. E-mail: dburgess{at}atcc.org.

Editor: W. A. Petri, Jr.

Infection and Immunity, March 2004, p. 1284-1290, Vol. 72, No. 3
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.3.1284-1290.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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