Dissemination of the apicomplexan parasite, Toxoplasma gondii

The parasitic protist Toxoplasma gondii is a common pathogen of rodents and felines that also infects humans. The most severe clinical manifestations of toxoplasmosis in humans derive from the systemic dissemination of T. gondii, during which the parasite penetrates biological barriers and accesses protected host compartments such as the central nervous system. T. gondii dissemination is enabled by the intrinsic gliding motility of extracellular parasites, which allows for travel to new host cells and tissues, and also powers the invasion of diverse host cells including migratory leukocytes. Dissemination is further advanced when migrating infected leukocytes shuttle intracellular parasites to new locations as they traffic throughout the host. All T. gondii gliding motility and host cell invasion was long presumed to be powered by the work of a parasite actin-myosin motor. The possibility of alternative gliding and invasion mechanisms was suggested by the development of inducible Cre-Lox technology that facilitated inducible disruption of genes thought to encode critical components of the T. gondii invasion machinery, including the parasite actin gene ACT1. To determine whether ACT1-independent invasion was likely, inducible Δact1 parasites were examined for uniformity of ACT1 protein depletion. Individual parasites with residual ACT1 protein persisted long after inducible ACT1 excision. Suggesting the residual ACT1 content of these parasites was functionally relevant, the invasion of Δact1 parasites was highly sensitive to an actin polymerization inhibitor. Parasite invasive ability was also found to negatively correlate with the length of time parasites were subjected to ACT1 depletion. Although the existence of ACT1-independent invasion mechanisms cannot be formally excluded, they do not appear to comprise robust alternatives to actin-dependent gliding and invasion in T. gondii. As the most abundantly infected circulating leukocyte during murine toxoplasmosis, monocytes have been theorized to be poised to deliver intracellular T. gondii across the blood-brain barrier and into the central nervous system. However, in vivo evidence supporting this theory was scarce. In vitro models had demonstrated that infection could alter the motility of monocytes when interacting with endothelial vasculature. However, whether infected monocytes could efficiently traverse the specialized endothelium that comprises the blood-brain barrier had not been tested, nor had the ability of infected monocytes to migrate through the tissue environments where T. gondii is first encountered. Models of peripheral and blood-brain barrier endothelium were used to show that infection markedly inhibited monocyte transendothelial migration. In contrast, infected monocytes and macrophages migrated through three-dimensional matrices in vitro and collagen-rich tissues in vivo with enhanced efficiency. Enhanced tissue migration relied on host Rho/ROCK and formin signaling, and the secreted T. gondii kinase ROP17. In a murine model, infection with Δrop17 parasites that fail to enhance tissue migration resulted in delayed dissemination and prolonged mouse survival. These results implicate monocytes in advancing the tissue spread of T. gondii during in vivo dissemination

Toxoplasma actin is required for efficient host cell invasion

ABSTRACT Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was re-cently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability ofact1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducibleact1 parasites as long as 5 days after ACT1 deletion. We further found that the longeract1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained inact1 parasites being responsi-ble for their invasive ability. Furthermore, invasion by theact1 parasites was also sensitive to the actin polymerization inhibi-tor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation byact1 para-sites. However,act1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducibleact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion. IMPORTANCE The prevailing model for apicomplexan invasion has recently been suggested to require major revision, based on studies where core components of the invasionmachinery were genetically disrupted using a Cre-Lox-based inducible knockout system. For the myosin component of the motor thought to power invasion, an alternative parasite myosin was recently demon

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii

Apicomplexan parasites such as Toxoplasma gondii possess a tubulin-rich structure called the conoid. Here, Long et al. identify a conoid protein that interacts with motor and structural proteins and is required for structural integrity of the conoid, parasite motility, and host cell invasion

Molecular mechanism of mRNA repression in by a ProQ-dependent small RNA.

Research into post-transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ-dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode-of-action of RaiZ, a ProQ-dependent sRNA that is made from the 30 end of the mRNA encoding ribosome-inactivating protein RaiA. We show that RaiZ is a base-pairing sRNA that represses in trans the mRNA of histone-like protein HU-a. RaiZ forms an RNA duplex with the ribosome-binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU-a. Similarities and differences between ProQ- and Hfqmediated regulation will be discussed

Endogenous tagging and generation of knockouts in <i>T</i>. <i>gondii</i>.

<p><b>A</b>. Schematic of the CRISPR/Cas9 tagging system. Tagging plasmids were generated with various tags (green box) flanked by common ends (red and black boxes) and including a common stop codon (gray box) followed by the <i>HXGPRT</i> 3’ UTR (yellow box) and the selectable marker HXGPRT. Amplification of this central region with primers that contained short homology regions HR1 (purple box) and HR2 (blue box) together with the common flanks (red and black boxes) generated products for gene-specific tagging. Co-transfection of these amplicons with a CRISPR/Cas9 plasmid bearing the gene-specific single guide RNA (sgRNA3’) was used to add an epitope tag (green box) at the C-terminus of the endogenous locus. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#ppat.1006379.s006" target="_blank">S1 Fig</a> for more details. <b>B</b>. Localization of CaM1, CaM2 and CaM3 containing C-terminal 6HA tags. Detected with mouse anti-HA (green) and rabbit anti-GAP45 (red). Scale bar, 2 μM. <b>C</b>. Schematic of the double CRISPR/Cas9 gRNA system used for generation of clean knockouts using two sgRNAs matching the 5’ and 3’ ends of the coding sequence. The entire coding sequence was replaced by the DHFR marker flanked by short homology regions (HR3, red; HR2, blue). Primers (p) used for diagnostic PCR. <b>D</b>. Diagnostic PCR of knockouts compared to the parental ku80^KO line. <i>CDPK1</i>, PCR control. <b>E.</b> Plaque numbers formed by the knockouts compared to the parental ku80^KO line. ns, not significant, analyzed by one-way ANOVA.</p

Analysis of egress, invasion, and motility in parental and mutant lines.

<p><b>A</b>. Parasites grown for 30 hr ± IAA (500 μM vs 0.1% ethanol) were stimulated with 3 μM A23187 to simulate egress. Rabbit anti-GRA7 (red) and mouse anti-IMC1 (green) antibodies were used to distinguish intact vs. egressed vacuoles. *** <i>P ≤ 0</i>.<i>0001</i>, significant for the time points of 2, 5, 10 and 15 min, but not significant for 0 and 20 min. Scale bar, 5 μM. <b>B</b>. Quantitative analysis of invasion by parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) and used to challenge fresh HFF monolayers on coverslips for 20 min. Extracellular parasites (invaded) were distinguished from those that remained extracellular (attached) by differential IFA staining (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#sec002" target="_blank">methods</a>). *** <i>P</i> ≤ 0.0001. <b>C.</b> Evaluation of cell entry past the moving junction. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) were used to challenge fresh HFF monolayers on coverslips for 3 min, fixed and stained with rabbit anti-RON4 (green) and mouse anti-SAG1 (red) without permeabilization. Parasites with RON4 dots were considered to be apically attached (red column), and parasites with RON4 positive rings were classified as partially invaded (green column). *** <i>P ≤ 0</i>.<i>0001</i>. Scale bar, 2 μM. <b>D</b>. Parasite motility as monitored by video microscopy. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) were allowed to glide on serum-coated coverslips. Time-lapse video microscopy was used to score different motile behaviors. *** <i>P</i> ≤ 0.0001, the cam2^KOCaM1-AID line showed significant decrease in twirling and increase non-productive movement when grown in +IAA <i>vs</i>. -IAA or the TIR1 parental line, **, <i>P</i> ≤ 0.0001, the CaM3-AID line showed a significant decrease in twirling and increase in circling when grown in +IAA <i>vs</i>. -IAA or the TIR1 parental line. Panels <b>A</b>, <b>B</b>, <b>C</b>, <b>D</b> represent means ± S.D. from three independent experiments with triplicates for each (n = 9). Two-way ANOVA with Tukey’s multiple comparison test for <b>A</b>, <b>C</b> and <b>D</b>, and one-way ANOVA with Tukey’s multiple comparison test for <b>B</b>.</p

Analysis of parasite replication, conoid protrusion, apical organelle distribution and secretion in parental and mutant lines.

<p><b>A</b>. Parasite replication after 24 hr incubation with ± IAA (500 μM vs 0.1% ethanol). ns, not significant. <b>B</b>. Proportion of parasites with extruded conoid. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol), stimulated with 3 μM A23187 or DMSO vehicle control for 10 min. ns, not significant. <b>C and D</b>. Distribution of MIC2 (mouse anti-MIC2 (green) and ROP5 (rabbit annti-ROP5 (green) upon depletion of AID fusion proteins. Parasites grown ± IAA (500 μM vs 0.1% ethanol) for 24 hr in HFF monolayers and stained for IFA. Parasites were counterstained with mouse anti-IMC1 (red) or rabbit anti-GAP45 antibodies (green). Scale bar, 2 μM. <b>E.</b> Quantification of micronemal secretion using MIC2-GLuc-myc reporter lines. Parasites were grown for 2 days ±IAA (500 μM vs 0.1% ethanol), stimulated with 1% ethanol—1% BSA and secretion was monitored by releases of luciferase (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#sec002" target="_blank">methods</a>). Relative Luminescence Unit (RLU)<b>.</b> ns, not significant. <b>F and G.</b> Detection of rhoptry secretion by ROP1 staining. Parasites were grown for 2 days ± IAA (500 μM vs 0.1% ethanol), harvested and used to detect formation of evacuoles (arrows) on fresh monolayers of HFF cells in the presence of cytochalasin. Parasites were counted from triplicate samples on three separate experiments and ratios of parasites associated with evacuoles in were plotted. Scale bar, 5 μm. Panels <b>A</b>, <b>B</b>, <b>E</b>, <b>F, G</b> mean ± S.D. from three independent experiments with triplicates for each (n = 9). One-way ANOVA with Tukey’s multiple comparison test for <b>B</b> and <b>E</b> and two-way ANOVA with Tukey’s multiple comparison test for pair-wise multiple comparisons across each vacuole size for <b>A</b>, Man-Whitney non-parametric test for <b>F</b> and <b>G</b>.</p

Generation of AID tagged lines in the TIR parental line of <i>T</i>. <i>gondii</i>.

<p><b>A.</b> Western blot analysis using antibodies to detect CaM1-AID or CaM3-AID (mouse anti-HA to the AID-3HA tag), TIR1-3Flag (rat anti-Flag) and aldolase (rabbit anti-aldolase, ALD). <b>B and C.</b> Degradation of AID tagged proteins in cam2^KO<i>/</i>CaM1-AID (<b>B</b>) and CaM3-AID (<b>C</b>) lines after addition of auxin (500 μM IAA) for different time periods. Mock indicates parasites grown with 0.1% ethanol for 36 hr. CaM1-AID or CaM3-AID proteins were detected with mouse anti-HA and rabbit anti-aldolase (ALD) antibodies served as a loading control. Band intensities were analyzed by ImageJ, and ratios of anti-HA vs. anti-ALD signal were calculated (HA/ALD) and expressed as a percentage of the mock treatment (i.e. 100%). <b>D and E</b>. Degradation of AID tagged proteins in cam2^KO<i>/</i>CaM1-AID (D) and CaM3-AID (E) parasites after 24 hr incubation with 500 μM IAA (+IAA) or ethanol vehicle 0.1% (-IAA). CaM1-AID or CaM3-AID proteins were detected with mouse anti-HA (green) and rabbit GAP45 (red) antibodies served as a control to label the parasite. Scale bar, 2 μM. <b>F</b>. Plaque formation by parasites grown on HFF monolayers. Scale bar, 0.5 cm. Insert images in the CaM3-AID line, scale bar (red) = 1 mm. <b>G</b>. Measurement of plaque numbers and sizes for the CaM3-AID line treated with and without auxin. N≥ 25, ***, <i>P</i> < 0.0001. Mann Whitney non-parametric test.</p