Charakterisierung der bradyzoitspezifisch exprimierten P-Typ Plasmamembran ATPase TgPMA1 in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that causes persistent infections in a broad range of warm-blooded vertebrates, including humans. A stage differentiation from tachyzoites to bradyzoites is a prerequisite for the persistence of the parasite in its hosts, as it is a mechanism for evasion of the host s immune response. During this differentiation event, a set of genes is down-regulated, while a new set of genes is switched on. The key genes responsible for this stage conversion have yet to be identified.In this work, we studied the expression and function of the bradyzoite-specific P-type proton ATPase TgPMA1. In plants, fungi and some protozoa, this class of transport proteins generates a proton gradient across the plasma membrane that is essential for nutrient uptake by secondary transporters. Furthermore, they have been correlated to the regulation of intracellular pH. Almost nothing is known about transport processes in bradyzoites. As changes in intracellular pH can initiate differentiation processes in several different cells, it was of interest to determine the role of TgPMA1 for stage differentiation of Toxoplasma gondii.TgPMA1 has an estimated molecular weight of 127 kDa and possesses all of the ten transmembrane segments and highly conserved sequence motifs characteristic for P-type ATPases. Highest amino acid homology can be seen with P-type proton ATPases of the red alga Cyanidium caldarium and the archaebacterium Methanococcus jannaschii. The open reading frame of this single-copy gene is interrupted by two introns approximately 350 nt in size, one of which is positioned immediately upstream and the other one downstream of the putative start codon. To localize the expression of the protein in the parasite, rabbit antiserum was raised against a small peptide of the C-terminus of TgPMA1. In an immunofluorescence assay, the protein could be visualized exclusively on the outer membrane of bradyzoites.To further characterize the gene, a T. gondii gene knock out mutant was generated by disruption of the tgpma1 open reading frame. To do this, a plasmid carrying the genomic locus of tgpma1 in which the open reading frame had been interrupted by deletion of a small fragment and insertion of a selectable marker was transfected into tachyzoites of the cyst-forming T. gondii strain Prugniaud/HX-. After cloning the parasites, one T. gondii clone could be isolated that carried a single copy of the targeting construct in its genome that had been integrated by homologous recombination. The identity of the gene knock out mutant was characterized and the lacking expression of TgPMA1 in bradyzoites was confirmed in an immunofluorescence assay.The knock out mutant did not show an altered growth rate as tachyzoites, during conversion to bradyzoites or during the spontaneous re-differentiation from bradyzoites to tachyzoites. A differentiation to bradyzoites could still be induced successfully in vitro, but the mutant parasites exhibited a markedly reduced expression of some bradyzoite-specific genes, when compared to the wild type strain. This difference in expression could be quantified to approximately the factor 10.Although the early events of differentiation to bradyzoites that were studied in vitro occur less efficiently in the knock out mutant, the ability to generate tissue cysts in vivo is not effected. When parasites of the knock out mutant were used to infect mice i. p., similar amounts of cysts could be found in the brains of these mice as were generated by the wild type. Of special interest was whether TgPMA1 as a proton transporter was responsible for the observed acid resistance of bradyzoites, thereby allowing survival of a stomach passage. This does not seem to be the case, though, as parasites remain viable and able to establish a successful infection cycle after oral infection of mice with cysts of the knock out strain.