Joseph D. Schwartzman

A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry.

The monoclonal antibody Tg49 both recognizes a Toxoplasma gondii rhoptry protein (ROP1) and inhibits penetration enhancing factor. The latter is a proteinaceous factor found in Toxoplasma lysates or conditioned media that increases the efficiency with which parasites invade host cells. Tg49 was used to screen a lambda gt11 cDNA library and the clone obtained was identified as the cognate gene for ROP1 by several criteria: (1) recombinant protein reacted with the monoclonal; (2) antiserum against the recombinant reacted with the same bands on Western blots as did Tg49; and (3) antiserum against the recombinant recognized a protein in the rhoptries. The ROP1 gene is a single copy gene with a message of approximately 2.1 kb. The predicted polypeptide sequence of ROP1 shows an unusual charge and amino acid asymmetry. There is a highly acidic, proline-rich domain in the amino-terminal portion of the predicted protein, followed by a strongly basic carboxy-terminal domain. An octapeptide repeat is found almost midway through the peptide sequence toward the end of the acidic domain. The ROP1 gene was expressed in a bacterial system, and the resulting polypeptide exhibited anomalous migration on polyacrylamide gel electrophoresis. Given that Tg49 inhibits penetration enhancing factor, it seems likely that the ROP1 protein is a component of that factor, and that the unusual sequence of this protein plays some role in host cell penetration by T. gondii.

Toxoplasma gondii: characterization of monoclonal antibodies that recognize rhoptries

We have previously reported on a series of monoclonal antibodies that recognize the rhoptries of Toxoplasma gondii and that interfere with the action of penetration enhancing factor. The antibodies immunoprecipitate several related antigens from [35S]methionine-labeled parasites that range in size from 60 to 43 kDa. By immunoblot, one of the antibodies reacts with the 60 kDa protein in the presence of protease inhibitors. Trypsin digestion of the antigen destroyed antigenic reactivity indicating that the 60 kDa antigen is a protein. The antigen was stable to periodate oxidation and failed to react with Schiffs reagent, indicating that the antigen contains little or no carbohydrate. Two-dimensional gel electrophoresis followed by immunoblot showed that the antigen recognized by Tg 49 was an acidic protein with an approximate pI of 5.8.

How Toxoplasma gondii Gets In and Out of Host Cells

The obligate intracellular parasite Toxoplasma gondii is responsible for a wide spectrum of disease affecting humans and many animals. While the parasite is in general well adapted to its hosts, usually causing an asymptomatic lifelong infection, it occasionally produces severe disease, especially when the host is immunoincompetent. Toxoplasma gondii has been known for years as an important congenital infection in humans (Stagno, 1980), and it is now a major cause of opportunistic infection in people with AIDS. There have been several recent reviews of advances in knowledge of the cell biology (McLeod et al., 1991), immunology, and molecular biology (Kasper and Boothroyd, 1991) of T. gondii. This chapter will review the progress of the last five years in understanding the mechanism of T. gondii invasion, since the review of Werk (1985).

Quantitative comparison of infection of neural cell and fibroblast monolayers by two strains of Toxoplasma gondii.

Abstract Disease caused by the coccidian Toxoplasma gondii can be confined to the central nervous system, although the parasite is capable of infecting all organ systems. To determine whether neural cells are differentially susceptible to infection and destruction by T. gondii, infection of neonatal mouse brain monolayers was compared to infection of human fibroblast monolayers under the same conditions with equal inocula of two parasite strains. In preliminary experiments there was no difference in total parasite yield or in plaques per monolayer between rodent and human cells. A standardized inoculum of T. gondii RH strain caused 35.6 ± 6.4 (SD) plaques per well in neural explant monolayers compared to 39.3 ± 12.5 plaques per well in fibroblasts. T. gondii P strain produced 35.6 ± 8.9 infected foci per well in neural cells compared to 32.6 ± 9.3 foci in fibroblasts. Intrinsic properties of neural cells do not appear to cause a higher rate of infection than that in nonneural cells.