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

Demographic and Psychological Factors Associated with Feelings of Antibiotic Entitlement in New Zealand

Patients’ expectations of being prescribed antibiotics can have an important influence on inappropriate prescribing. Therefore, it is important to understand the drivers of patients’ antibiotic expectations. The 2015/16 New Zealand Attitudes and Values Study measured sense of entitlement to antibiotics in a nationally representative sample of New Zealanders (n = 13,484). Participants were asked to rate their agreement with the statement “If I go to my doctor/GP with a minor illness (e.g., sore throat, cough, runny nose, etc.), I think that I should be prescribed antibiotics by default.” Eighty percent of participants showed low feelings of antibiotic entitlement, while 18.5% exhibited moderate and 3.7% high feelings of entitlement. People of ethnic minority, lower socio-economic status, and with diabetes expressed higher expectations of being prescribed antibiotics. This may be partially based on a higher risk of rheumatic fever or other complications. Men, religious people, those with lower educational attainment and self-rated health, but greater psychological distress and feelings of control over their health exhibited higher feelings of antibiotic entitlement. Those high on Extraversion, Conscientiousness, and Narcissism, but low on Agreeableness and Openness, also showed greater feelings of entitlement. Our findings help identify key characteristics of those more likely to express inappropriate expectations of antibiotic prescription

Congenital Toxoplasmosis in France and the United States: One Parasite, Two Diverging Approaches

International audienc

Congenital Toxoplasmosis in France and the United States: One Parasite, Two Diverging Approaches

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Psychological predictors of COVID-19 vaccination in New Zealand

Is it possible to predict COVID-19 vaccination status prior to the existence and availability of COVID-19 vaccines? Here, we present a logistic model by regressing decisions to vaccinate in late 2021 on lagged sociodemographic, health, social, and political indicators from 2019 in a sample of New Zealand adults aged between 18 and 94 (Mage = 52.92, SD = 14.10; 62.21% women; N = 5324). We explain 31% of the variance in decision making across New Zealand. Significant predictors of being unvaccinated were being younger, more deprived, reporting less satisfaction with general practitioners, lower levels of neuroticism, greater levels of subjective health and meaning in life, higher distrust in science and in the police, lower satisfaction in the government, as well as political conservatism. Additional cross-sectional models specified using the same, and additional COVID-19-specific factors are also presented. These findings reveal that vaccination decisions are neither artefacts of context nor chance, but rather can be predicted in advance of the availability of vaccines

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