TgDrpC, an atypical dynamin‐related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division

Dynamin‐related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP‐2) complex subunit alpha‐1 and a homolog of the ezrin–radixin–moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re‐distribution of TgDrpC to the IMC during division is dependent on post‐Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division

The common parasite Toxoplasma gondii induces prostatic inflammation and microglandular hyperplasia in a mouse model

BACKGROUND: Inflammation is the most prevalent and widespread histological finding in the human prostate, and associates with the development and progression of benign prostatic hyperplasia and prostate cancer. Several factors have been hypothesized to cause inflammation, yet the role each may play in the etiology of prostatic inflammation remains unclear. This study examined the possibility that the common protozoan parasite Toxoplasma gondii induces prostatic inflammation and reactive hyperplasia in a mouse model. METHODS: Male mice were infected systemically with T. gondii parasites and prostatic inflammation was scored based on severity and focality of infiltrating leukocytes and epithelial hyperplasia. We characterized inflammatory cells with flow cytometry and the resulting epithelial proliferation with bromodeoxyuridine (BrdU) incorporation. RESULTS: We found that T. gondii infects the mouse prostate within the first 14 days of infection and can establish parasite cysts that persist for at least 60 days. T. gondii infection induces a substantial and chronic inflammatory reaction in the mouse prostate characterized by monocytic and lymphocytic inflammatory infiltrate. T. gondii-induced inflammation results in reactive hyperplasia, involving basal and luminal epithelial proliferation, and the exhibition of proliferative inflammatory microglandular hyperplasia in inflamed mouse prostates. CONCLUSIONS: This study identifies the common parasite T. gondii as a new trigger of prostatic inflammation, which we used to develop a novel mouse model of prostatic inflammation. This is the first report that T. gondii chronically encysts and induces chronic inflammation within the prostate of any species. Furthermore, T. gondii-induced prostatic inflammation persists and progresses without genetic manipulation in mice, offering a powerful new mouse model for the study of chronic prostatic inflammation and microglandular hyperplasia

How can healthcare professionals provide guidance and support to parents of adolescents? Results from a primary care-based study

Background: This study explored the rewards and difficulties of raising an adolescent and investigated parents' level of interest in receiving guidance from healthcare providers on parenting and adolescent health topics. Additionally, this study investigated whether parents were interested in parenting programs in primary care and explored methods in which parents want to receive guidance. Methods: Parents of adolescents (ages 12-18) who attended an outpatient pediatric clinic with their adolescent were contacted by telephone and completed a short telephone survey. Parents were asked open-ended questions regarding the rewards and difficulties of parenting and rated how important it was to receive guidance from a healthcare provider on certain parenting and health topics. Additionally, parents reported their level of interest in a parenting program in primary care and rated how they would like to receive guidance. Results: Our final sample included 104 parents, 87% of whom were interested in a parenting program within primary care. A variety of parenting rewards and difficulties were associated with raising an adolescent. From the list of parenting topics, communication was rated very important to receive guidance on (65%), followed by conflict management (50%). Of health topics, parents were primarily interested in receiving guidance on sex (77%), mental health (75%), and alcohol and drugs (74%). Parents in the study wanted to receive guidance from a pediatrician or through written literature. Conclusions: The current study finds that parents identify several rewarding and difficult aspects associated with raising an adolescent and are open to receiving guidance on a range of parenting topics in a variety of formats through primary care settings. Incorporating such education into healthcare visits could improve parents' knowledge. Healthcare providers are encouraged to consider how best to provide parenting support during this important developmental time period

The Natural Course of Adolescent Depression Treatment in the Primary Care Setting

Introduction: Little is known about how adolescents receive depression follow-up in primary care. The purpose of this study was to describe the rates of symptom assessment and depression treatment over time in a group of adolescents screening positive for moderate or severe depression in the primary care setting. Methods: Retrospective chart reviews were conducted to gather information related to symptom reassessments, antidepressant prescriptions, psychotherapy referrals, and treatment discontinuation. Descriptive statistics were calculated, and a qualitative content analysis was conducted to determine the reasons for treatment discontinuation. Results: Eighty records were reviewed (mean age = 15.3, 73% female, 59% Black). Treatment was initiated for 83% (n = 66) of patients, and 45% (n = 30) of patients discontinued treatment during the review period for a variety of reasons. Discussion: To improve adolescents' adherence to depression treatment, providers should address factors that contribute to treatment discontinuation and use tools to manage depression follow-up care

Lack of mitochondrial MutS homolog 1 in Toxoplasma gondii disrupts maintenance and fidelity of mitochondrial DNA and reveals metabolic plasticity.

The importance of maintaining the fidelity of the mitochondrial genome is underscored by the presence of various repair pathways within this organelle. Presumably, the repair of mitochondrial DNA would be of particular importance in organisms that possess only a single mitochondrion, like the human pathogens Plasmodium falciparum and Toxoplasma gondii. Understanding the machinery that maintains mitochondrial DNA in these parasites is of particular relevance, as mitochondrial function is a validated and effective target for anti-parasitic drugs. We previously determined that the Toxoplasma MutS homolog TgMSH1 localizes to the mitochondrion. MutS homologs are key components of the nuclear mismatch repair system in mammalian cells, and both yeast and plants possess MutS homologs that localize to the mitochondria where they regulate DNA stability. Here we show that the lack of TgMSH1 results in accumulation of single nucleotide variations in mitochondrial DNA and a reduction in mitochondrial DNA content. Additionally, parasites lacking TgMSH1 function can survive treatment with the cytochrome b inhibitor atovaquone. While the Tgmsh1 knockout strain has several missense mutations in cytochrome b, none affect amino acids known to be determinants of atovaquone sensitivity and atovaquone is still able to inhibit electron transport in the Tgmsh1 mutants. Furthermore, culture of Tgmsh1 mutant in the presence atovaquone leads to parasites with enhanced atovaquone resistance and complete shutdown of respiration. Thus, parasites lacking TgMSH1 overcome the disruption of mitochondrial DNA by adapting their physiology allowing them to forgo the need for oxidative phosphorylation. Consistent with this idea, the Tgmsh1 mutant is resistant to mitochondrial inhibitors with diverse targets and exhibits reduced ability to grow in the absence of glucose. This work shows TgMSH1 as critical for the maintenance and fidelity of the mitochondrial DNA in Toxoplasma, reveals a novel mechanism for atovaquone resistance, and exposes the physiological plasticity of this important human pathogen

Sensitivity of <i>Tgmsh1</i> mutant strain to mitochondrial inhibitors.

<p>Parasites of the parental and MRC5 mutant strains were allowed to form plaques in presence of varying concentrations of myxothiazol (A), clopidol (B), antimycin A (C), pyrimethamine (D), or clindamycin (E). Percent survival represents the number of plaques formed in the presence of drug after 12 days of growth divided by the number of plaques formed in the presence of solvent. Data bars represent the average of three independent experiments, and error bars denote the standard deviation. Asterisks indicates significant statistical difference based on one-way ANOVA (*p<0.05, **p<0.005).</p

Monitoring of mitochondrial DNA in parasites lacking TgMSH1.

<p>A. Sequences of three mitochondrial fragments were monitored in clones of the parental (WT clone a), <i>Tgmsh1</i> mutant (MRC5 clones a, b and c), and complemented mutant (<i>c</i>MRC5 clones a, b, and c) strains after serial passage in culture. Horizontal arrows indicate passages in culture; solid ones indicate a known specific number of passages (psgs), which is indicated above. Black circle represents the establishment of clones of the particular strain by limiting dilution. The number of single nucleotide variants (SNV) indicated is in respect to the sequence of the parental strain. B. Schematics depict the emergence and relative position of SNVs in the MRC5 strain within the three fragments of mtDNA monitored, <i>Cox1</i>, <i>Cox3</i>, and <i>Cyb</i>. Tick marks represent relative position of SNV.</p

Sequencing of cytochrome b in parental, Tgmsh1 mutant and complemented strain after two years of culture.

<p>A. Total DNA was collected from parental, mutant and complemented strains before (WT, MRC5 and <i>c</i>MRC5) and after approximately 2 years of constant culture passage (WT<i>mic</i>, MRC5<i>mic</i> and (<i>c</i>MRC5)<i>mic</i>, where mic stands for “maintained in culture”). The entire coding region of the cytochrome b gene was amplified and sequenced. The number of single nucleotide variations identified is shown in the gray boxes below each strain. Arrows represent passage in culture over specified time. Line with black circle, indicates addition of TgMSH1 to generate the complemented MRC5 strain, <i>c</i>MRC5. B. Distribution of substitution types in the mitochondrial DNA of the <i>Tgmsh1</i> mutant strain. All 29 single nucleotide variations detected in the <i>Cox1</i>, <i>Cox3</i>, and <i>Cyb</i> fragments sequenced (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188040#pone.0188040.g001" target="_blank">1</a> and 2a) were cataloged and categorized based on the type of substitution.</p

Sensitivity to atovaquone treatment.

<p>Parasites of wildtype, MRC5 mutant, or MRC5 complemented strains were allowed to form plaques in media containing atovaquone or solvent control. Percent survival represents the number of plaques formed in the presence of atovaquone after 12 days of growth divided by the number of plaques formed in the presence of solvent control. Data bars represent the average of four independent experiments, and error bars denote the standard deviation. Asterisks denote statistically different from parental based on one-way ANOVA followed by post-hoc Tukey HSD test (* p<0.05, ** p<0.01). Pound sign denotes that cMRC5 is statistically different from MRC5 based on the same analysis (#p<0.05).</p

Atovaquone sensitivity of MRC5-AtovSel parasites maintained with and without atovaquone.

<p>A. The efficiency of plaque formation in atovaquone within five days was determined for MRC5 atovaquone selected parasites that were maintained in atovaquone (MRC5-AtovSel) and those that were maintained in the absence of atovaquone after the selection (MRC5-AtovSelRev). Percent survival represents the number of plaques formed in the presence of atovaquone after 5 days of growth divided by the number of plaques formed in the presence of solvent control, multiplied by 100. Data bars represent the average of three independent experiments, and error bars denote the standard deviation. B. The ability of MRC5 (dashed line), MRC5-AtoSel (solid line) and MRC5-AtovSelRev (dotted line) parasites to consume oxygen under standard conditions was measured every 10 minutes over 120 minutes as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188040#pone.0188040.g006" target="_blank">Fig 6</a>.</p