Adhesins of Trichomonas Vaginalis

Burgess, Donald

Abstract

The ability of extracellular parasitic protozoa to adhere to host cells correlates with tissue damage in diseases such as amoebiasis and trichomoniasis. The long term objective of this project is to identify adhesions in the human parasite, Trichomonas vaginalis, and explore their role in the pathogenesis of trichomoniasis.
Specific aims are as follow: 1. The libraries of T. vaginalis DNA will be prepared. 2. These libraries will be screened for sequences similar to those of known adhesion genes of the related trichomonad, T. foetus. 3. Putative adhesion genes of T. vaginalis will be characterized by sequencing and comparison of sequences to known adhesions (e.g., T. foetus, ICAMS, etc.). Nucleic acid probes prepared from sequences of adhesion genes of Tritrichomonas foetus will be used to search for similar genes and to evaluate mRNAs coding for these molecules in Trichomonas vaginalis. Nucleic acid probes designed from sequences of genes from a genomic library and/or cDNA library (in gamma gt 11) of T. foetus will be prepared by oligonucleotide synthesis or subcloning. Probes will be labeled (P) and used in Southern hybridizations with total genomic DNA from T. vaginalis to search for similar structural genes. A genomic library of T. vaginalis will also be constructed and screened with these probes. Clones selected from the T. vaginalis library will be subcloned into plasmid vectors (e.g., Bluescript, Stratagene) and sequenced. These sequences will be analyzed by comparing them to known adhesion gene sequences of T. foetus and other cells (ICAMS, etc.) to determine the degree of identity of sequences and the diversity of adhesions. Nucleic acid probes (selected T. vaginalis cDNA's) will be prepared and used to examine mRNA from several strains of T. vaginalis at different time points in the cell cycle to determine the kinetics of expression of adhesion messages. If time permits the polypeptides coded for by putative adhesion genes of T. vaginalis will be expressed and their antigenic and functional (i.e., adhesion to mammalian cells) characteristics established. Monospecific antisera will be prepared against these adhesion molecules and monoclonal antibodies specific for T. foetus adhesions (already available) will be assayed (dot blots; flow cytometry) for reactivity with adhesions of T. vaginalis. Lines (clones, distinct clinical isolates) of T. vaginalis that differ in their cytotoxicity toward mammalian target cells will be evaluated for adhesion, presence of adhesion gene(s) (Southern blots) and adhesion message (Northern blots) to determine the relationships of adhesion phenotype and adhesion genotype with cytotoxicity levels among these lines.