ABSTRACT
Toxoplasma gondii has a broad host-range including man and a variety of warm-blooded animals. The ability to infect and survive in this wide spectrum of hosts suggests highly evolved mechanisms to handle the harsh environments encountered. Here we show that extracellular tachyzoites are resistant to milligram levels of trypsin and describe the presence of an inhibitor of trypsin associated with the surface of T. gondii, TgTI. TgTI has an estimated molecular mass of 37000 dalton and is encoded by the TgTI-gene which is found at low abundance as an expressed sequence tag (EST) in both the bradyzoite and tachyzoite stages. The inhibitory binding region was found to be in the N-terminus of TgTI where aminoacid-alignment to earlier described protease inhibitors demonstrates 75% similarity. In functional analysis, recombinant TgTI-protein inhibits the activity of trypsin approximately 10 times more efficiently than an inhibitor isolated from soybean. In contrast to other known trypsin inhibitors, TgTI also possesses a predicted membrane-binding region. Polyclonal antibodies raised against recombinant TgTI bind to the surface of the tachyzoite stage as seen both by immunofluorescence and immunoprecipitation of surface labelled parasite proteins. The high survival rate of the parasite in the upper gastrointestinal tract may be enhanced by the presence of the TgTI-molecule.
Subject(s)
Membrane Proteins/isolation & purification , Protozoan Proteins/isolation & purification , Toxoplasma/chemistry , Trypsin Inhibitors/isolation & purification , Amino Acid Sequence , Animals , Blotting, Western , Fluorescent Antibody Technique, Indirect , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Molecular Sequence Data , Precipitin Tests , Protozoan Proteins/genetics , Protozoan Proteins/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacology , Sequence Alignment , Toxoplasma/genetics , Toxoplasma/growth & development , Trypsin , Trypsin Inhibitors/genetics , Trypsin Inhibitors/pharmacologyABSTRACT
Kuratli, S., Lindh, J. G., Gottstein, B., Smith, D. F., and Connolly, B. 1999. Trichinella spp.: Differential expression of two genes in the muscle larva of encapsulating and nonencapsulating species. Experimental Parasitology 93, 153-159. The expression of the two genes tsmyd-1 and tsJ5 was studied in the muscle stage larva of three different species of Trichinella. T. spiralis and T. britovi are both encapsulating species, while T. pseudospiralis is a nonencapsulating species. Expression of tsJ5 is developmentally regulated in T. spiralis and has been shown in this study to be down-regulated in the T. pseudospiralis muscle larva compared with the other two species. Immunoblot analysis has also revealed that the relative abundance of the protein product of this gene, TSJ5, is lower in T. pseudospiralis muscle larvae. It has previously been shown that expression of tsmyd-1 is not developmentally regulated in T. spiralis (Connolly et al. 1996). In contrast, expression of this gene is slightly increased in the muscle larvae of T. pseudospiralis. Southern analysis of genomic DNA from the three Trichinella species shows that both genes are highly conserved.
Subject(s)
Gene Expression Regulation, Developmental , Genes, Helminth/genetics , Trichinella/genetics , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Conserved Sequence , DNA, Helminth , Down-Regulation , Female , Larva/genetics , Mice , Mice, Inbred ICR , Muscle, Skeletal/pathology , MyoD Protein/genetics , RNA, Helminth/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Development of the infective L1 larva of Trichinella spiralis occurs as an intracellular parasite of skeletal muscle and leads to the dedifferentiation of the host cell. A novel Trichinella gene, tsJ5, has been identified from a cDNA library screen for sequences encoding Trichinella proteins related to the myogenic bHLH factors. The tsJ5 gene is developmentally regulated, showing preferential expression in the infective muscle stage larva. The product of the tsJ5 gene is not a bHLH protein but represents a novel protein with properties in common with some myogenic repressors. A recombinant TsJ5 protein affects the formation of MyoD:DNA complexes in vitro.
