Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.
Summary 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.
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.
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.
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.
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