SummaryApicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

. Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 286: E568-E576, 2004. First published December 9, 2003 10.1152/ajpendo.00372.2003.-Glucose deprivation dramatically increases glucose transport activity in 3T3-L1 adipocytes without changing the concentration of GLUT1 in the plasma membrane (PM). Recent data suggest that subcompartments within the PM, specifically lipid rafts, may sequester selected proteins and alter their activity. To evaluate this possibility, we examined the distribution of GLUT1 in Triton X-100-soluble and -insoluble fractions. Our data show that 77% of the GLUT1 pool in PMs isolated from control 3T3-L1 adipocytes was extracted by 0.2% Triton X-100. After glucose deprivation for 12 h, only 56% of GLUT1 was extracted by detergent. In contrast, there was a twofold increase in the GLUT1 content of the detergent-resistant fraction. To evaluate whether GLUT1 interacts with a specific protein within lipid rafts, we focused on stomatin, recently shown to interact with and inhibit GLUT1 activity. Stomatin is distributed about equally between the PM and the biosynthetic compartments, and its expression is not affected by glucose deprivation. Nearly 90% of the PM pool of stomatin is in detergent-resistant lipid rafts. In normal 3T3-L1 adipocytes, we were unable to demonstrate an interaction between GLUT1 and stomatin in coimmunoprecipitation experiments. However, in stomatin-overexpressing cells, there was clear coprecipitation of stomatin with GLUT1 antibodies. Glucose deprivation increased this interaction threefold, which may reflect the increase of GLUT1 in lipid rafts. Despite this, there was little change in transport activity in glucosedeprived, stomatin-overexpressing cells vs. that in control cells. Thus GLUT1 interacts with stomatin in lipid rafts, but this interaction per se does not alter transport activity. Rather, stomatin may serve as an anchor for GLUT1 in lipid rafts, the environment of which favors activation.glucose transporter 1; lipid rafts; stomatin MOST CELLS ARE DEPENDENT ON GLUCOSE UPTAKE and metabolism as a source of energy. Although the number of members of the transporter family is expanding, GLUT1 is ubiquitously expressed and is responsible for constitutive uptake in mammalian cells. The regulation of GLUT1 expression and activity has been intensively studied. Our focus has been on the nutrient-dependent regulation of glucose transport in 3T3-L1 adipocytes. Specifically, we (and others) have shown that a decrease in extracellular glucose concentration increases transport activity in 3T3-L1 adipocytes in a protein synthesisdependent fashion (27,39,55,56). Although complete glucose deprivation increases transport activity 10-to 15-fold, a change of fourfold is observed within the range of 2.5 to 25 mM, the limits of circulating glucose concentration in diabetic patients, suggesting physiological relevance. Although 3T3-L1 adipocytes express both GLUT1 and GLUT4, the insulin-stimulated transporter, the effec...

ABSTRACTRapid clonal antigenic variation inBabesia bovisinvolves thevarianterythrocytesurfaceantigen-1 (VESA1) protein expressed on the infected-erythrocyte surface. Because of the significance of this heterodimeric protein for demonstrated mechanisms of parasite survival and virulence, there is a need to understand how expression of thevesmultigene family encoding this protein is controlled. As an initial step toward this goal, we present here initial characterization of thevespromoter driving transcription of VESA1a and -1b subunits. A series of transfection constructs containing various sequence elements from thein vivolocus of activevestranscription (LAT) were used to drive expression of the firefly luciferase gene in a dual luciferase-normalized assay. The results of this approach reveal the presence of two bidirectional promoter activities within the 434-bp intergenic region (IGr), influenced by putative regulatory sequences embedded within the flankingves1α andves1β genes. Repressor-like effects on the apposing gene were observed for intron 1 of bothves1α andves1β. This effect is apparently not dependent upon intronic promoter activity and acts only incis. The expression of genes within thevesfamily is likely modulated by local elements embedded withinvescoding sequences outside the intergenic promoter region in concert with chromatin modifications. These results provide a framework to help us begin to understand gene regulation during antigenic variation inB. bovis.

Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bb rad51 gene encoding the B . bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bb rad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bb rad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B . bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bb rad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

Babesia bovis, an intraerythrocytic parasite of cattle, establishes persistent infections of extreme duration. This is accomplished, at least in part, through rapid antigenic variation of a heterodimeric virulence factor, the variant erythrocyte surface antigen-1 (VESA1) protein.Previously, the VESA1a subunit was demonstrated to be encoded by a 1α member of the ves multigene family. Since its discovery the 1β branch of this multigene family has been hypothesized to encode the VESA1b polypeptide, but formal evidence for this connection has been lacking. Here, we provide evidence that products of ves1β genes are rapidly variant in antigenicity and size-polymorphic, matching known VESA1b polypeptides. Importantly, the ves1β-encoded antigens are co-precipitated with VESA1a during immunoprecipitation with antiVESA1a monoclonal antibodies, and antisera to ves1β polypeptide co-precipitate VESA1a. Further, the ves1β-encoded antigens significantly co-localize with VESA1a on the infectederythrocyte membrane surface of live cells. These characteristics all match known properties of VESA1b, allowing us to conclude that the ves1β gene divergently apposing the ves1α gene within the locus of active ves transcription (LAT) encodes the 1b subunit of the VESA1 cytoadhesion ligand. However, the extent and stoichiometry of VESA1a and 1b co-localization on the surface of individual cells is quite variable, implicating competing effects on transcription, translation, or trafficking of the two subunits. These results provide essential information facilitating further investigation into this parasite virulence factor.