Diagram of the physical and cellular interactions that compose the blood-brain barrier.

<p><b>(A)</b> The blood-brain barrier (BBB) is composed of microvessels surrounded by an endothelial cell layer with tight junctions; pericytes surround the tight junctions and then astrocytic processes or endfeet provide the final layer. <b>(B)</b> <i>Toxoplasma gondii</i> has been postulated to have 3 mechanisms for crossing the BBB. <b>(1)</b> Paracellular entry, in which <i>T</i>. <i>gondii</i> migrates directly through the tight junctions of the endothelial cell layer, <b>(2)</b> transcellular entry, in which free parasites in the vascular compartment infect endothelial cells, replicate, and then egress out of the basolateral side of endothelial cell (lysing the host cell), <b>(3)</b> the “Trojan horse” method, whereby an infected immune cell infiltrates the CNS, after which the parasite egresses out of the immune cell and into the brain parenchyma.</p

A Nucleotide Sugar Transporter Involved in Glycosylation of the <i>Toxoplasma</i> Tissue Cyst Wall Is Required for Efficient Persistence of Bradyzoites

<div><p><i>Toxoplasma gondii</i> is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host <i>via</i> encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a <i>Toxoplasma</i> nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. <i>Toxoplasma</i> strains deleted for the <i>TgNST1</i> gene (Δ<i>nst1</i>) form cyst-like structures <i>in vitro</i> but no longer interact with lectins, suggesting that Δ<i>nst1</i> strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. <i>In vivo</i> infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δ<i>nst1</i> parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of <i>Toxoplasma</i> tissue cysts.</p></div

TgNST1 activity is not required for virulence during the acute stage of infection in mice.

<p>Mice were infected i.p. with 500 WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> tachyzoites. A) At the indicated days post-infection (pi), animals were injected i.p. with 200 µL of d-luciferin in PBS (150 mg substrate/kg body weight), anesthetized with isoflurane and imaged using the Xenogen IVIS200 charge-couple. Mice were imaged ventrally for 5 min. B) Infected animals shown in panel A were weighed at the indicated days post-infection and loss of weight was plotted as the percentage of the initial weight over time. Weights for animals infected with WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> parasites are shown with green, red and blue lines, respectively.</p

Design and verification of the strain deficient in NST1 and the corresponding complemented strain.

<p>A) Workflow of the generation of the wild type, Δ<i>nst1</i> and <i>Δnst1:NST1</i> strains. 5′- and 3′-FR indicates the 5′- and 3′-flanking regions of the TgNST1 gene used for the homologous recombination required for deletion of the gene. Primers used for PCR analysis are numbered. B) The engineered strains of panel A were analyzed by PCR using primers that specifically amplify the 5′- or 3′-flanking regions (primers 1+2 and 3+4, respectively), the NST1 coding region (primers 5+6), the HPT cassette (primers 7+8), or the 5′- to 3′- flanking regions (primers 9+10).</p

Δ<i>nst1</i> parasites are not defective in tropism to the brain.

<p>Mice were infected i.p. with 500 WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> tachyzoites.12 days post-infection, animals were sacrificed and the posterior left quarter of the brain was used to quantify the number of parasites present by real-time quantitative PCR detection of <i>Toxoplasma</i>'s B1 gene normalized to the detection levels of mouse actin (ACT1).</p

Deletion of <i>TgNST1</i> affects GRA2 mobility in a SDS-PAGE.

<p>Total parasite extracts from parental, control, <i>Δnst1</i> and <i>Δnst1:NST1</i> strains were separated by SDS-PAGE and proteins transferred to PVDF membrane. The mobility of GRA2 protein was analyzed using a mouse anti-GRA2 antibody (upper panel). The blot was stripped and reprobed with antibodies to SAG1 as a loading control (lower panel).</p

<i>Toxoplasma</i> TgNST1 is a nucleotide-sugar transporter that transports UDP-GlcNAc and UDP-GalNac.

<p>A) Analysis of protein expression by western blot. Total extracts from <i>Saccharomyces cerevisiae</i> strain PRY225 and the same strain transformed with the p426 vector encoding the <i>Toxoplasma</i> NST1HA protein (TgNST1) were resolved by SDS-PAGE followed by transfer to PVDF membrane. Detection of the recombinant protein was done using an anti-HA antibody. B) 1 mg of membrane vesicles from yeast transformed with an empty vector or the vector encoding the TgNST1HA protein were incubated with radioactive and unlabeled UDP-GlcNAc or UDP-GalNAc for 4 min. Final concentration of nucleotide-sugar is 2 µM per sample. Vesicles were separated by centrifugation and radioactivity within vesicles was measured in the pellet after acid precipitation. Transport activity is calculated as the difference of radioactive solutes in the vesicle pellet after incubation at 30°C vs 0°C and expressed as pmol of nucleotide sugar/mg protein/min.</p

<i>TgNST1</i> deletion interferes with CC2 antibody recognition but does not affect GRA2 secretion.

<p><i>In vitro</i> cysts were fixed in formaldehyde, permeabilized and stained using the rat monoclonal antibody CC2, specific for the tissue cyst wall (A) or the mouse monoclonal antibody GRA2, specific for the cyst wall and intravacuolar material surrounding the parasites (B). Both these antibodies were detected with Alexa-488-conjugated secondary antibodies and visualized by epifluroescence (part A) or confocal (part B) microscopy. Antibodies against the bradyzoite-specific antigen SRS9 and Alexa-647-conjugated goat-anti-rabbit were use to identify bradyzoites within cysts. For confocal images, 1 µm slice images were taken using a Zeiss confocal microscope and a single slice is shown. Scale bar represents 10 µm.</p

<i>TgNST1</i> deletion interferes with recognition by the DBA and WGA lectins.

<p>WT, <i>Δnst1</i>, or <i>Δnst1:NST1</i> parasites were grown under bradyzoite-inducing conditions for 4 days <i>in vitro</i>, fixed in formaldehyde, permeabilized and stained using the FITC-conjugated lectins DBA (A) or WGA (B). Antibodies against the bradyzoite-specific antigen SRS9 and Alexa 647-conjugated goat anti-rabbit antisera (pseudocolor red) were use to identify bradyzoites within cysts. Scale bar represents 10 µm. C) Mice infected with the WT, <i>Δnst1</i> and <i>Δnst1:NST1</i> strains were sacrificed 3 weeks post-infection, perfused with heparine/saline and brains were drop-fixed in paraformaldehyde, sucrose-embedded and cryosectioned for immunohistochemistry. 40 µm sections were stained using the FITC-DBA lectin and antibody against the bradyzoite-protein SRS9, as in parts A and B. 1 µm slice images were taken using a Zeiss confocal microscope and a single slice is shown. Scale bar represents 10 µm.</p

Mice infected with the Δ<i>nst1</i> strain show reduced numbers of cysts in the brain.

<p>Mice were infected i.p. with 500 WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> tachyzoites. 3 weeks post-infection, animals were sacrificed by perfusion and half the brain was homogenized for cyst enumeration using antibodies against the bradyzoite-specific antigen SRS9 to facilitate detection of cysts. The figure shows representative results from two independent experiments. Horizontal bars indicate the mean for each group. Asterisks indicate statistically significant differences (p = 0.05) between indicated groups.</p