Healthy by law? The influence of labor and employment law on workers' health.
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Employment↗
Biomedical subjects
P N Ossorio
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Structure and function of the parasitophorous vacuole membrane surrounding Toxoplasma gondii.
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Animals↗
A soluble secretory protein of the intracellular parasite Toxoplasma gondii associates with the parasitophorous vacuole membrane through hydrophobic interactions.
The obligate intracellular parasite Toxoplasma gondii resides within a specialized, nonfusogenic vacuole termed the parasitophorous vacuole. We are exploring the hypothesis that parasite proteins contained in dense core granules and secreted into the vacuole after cell invasion contribute to the modification of the parasitophorous vacuole membrane (PVM). The dense granule protein GRA3 associates preferentially with the PVM. Here we show that GRA3 liberated from dense granules is a soluble protein. Following secretion, one-third or more of GRA3 in the parasitophorous vacuole becomes stably associated with the vacuole membrane as judged by sucrose flotation gradients and Triton X-114 phase partitioning experiments. GRA3 cannot be removed from the membrane by extraction with sodium carbonate at pH 11.0, nor can it be labeled in infected cells with [1-14C]acetate. In sucrose velocity sedimentation gradients, GRA3 from vacuolar membranes solubilized in Triton X-100 sediments as an oligomer. Membrane-associated GRA3 cross-links to itself, migrating as a ladder of bands in SDS-polyacrylamide gel electrophoresis. No other parasite or cell proteins co-precipitate with cross-linked GRA3. We conclude that a portion of GRA3 secreted from the parasite as a soluble protein directly inserts into the vacuole membrane in an oligomeric form. This is the first report in Toxoplasma or related parasites of a protein which inserts into the vacuole membrane for some purpose other than to lyse that membrane.
Animals↗
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.
Amino Acid Sequence↗
Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.
In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.
Amino Acid Sequence↗