Two essential light chains regulate the MyoA lever arm to promote Toxoplasma gliding motility

Key to the virulence of apicomplexan parasites is their ability to move through tissue and to invade and egress from host cells. Apicomplexan motility requires the activity of the glideosome, a multicomponent molecular motor composed of a type XIV myosin, MyoA. Here we identify a novel glideosome component, essential light chain 2 (ELC2), and functionally characterize the two essential light chains (ELC1 and ELC2) of MyoA in Toxoplasma. We show that these proteins are functionally redundant but are important for invasion, egress, and motility. Molecular simulations of the MyoA lever arm identify a role for Ca2+ in promoting intermolecular contacts between the ELCs and the adjacent MLC1 light chain to stabilize this domain. Using point mutations predicted to ablate either the interaction with Ca2+ or the interface between the two light chains, we demonstrate their contribution to the quality, displacement, and speed of gliding Toxoplasma parasites. Our work therefore delineates the importance of the MyoA lever arm and highlights a mechanism by which this domain could be stabilized in order to promote invasion, egress, and gliding motility in apicomplexan parasites

TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells

The phylum Apicomplexa comprises a group of obligate intracellular parasites of broad medical and agricultural significance, including Toxoplasma gondii and the malaria-causing Plasmodium spp. Key to their parasitic lifestyle is the need to egress from an infected cell, actively move through tissue, and reinvade another cell, thus perpetuating infection. Ca(2+)-mediated signaling events modulate key steps required for host cell egress, invasion and motility, including secretion of microneme organelles and activation of the force-generating actomyosin-based motor. Here we show that a plant-like Calcium-Dependent Protein Kinase (CDPK) in T. gondii, TgCDPK3, which localizes to the inner side of the plasma membrane, is not essential to the parasite but is required for optimal in vitro growth. We demonstrate that TgCDPK3, the orthologue of Plasmodium PfCDPK1, regulates Ca(2+) ionophore- and DTT-induced host cell egress, but not motility or invasion. Furthermore, we show that targeting to the inner side of the plasma membrane by dual acylation is required for its activity. Interestingly, TgCDPK3 regulates microneme secretion when parasites are intracellular but not extracellular. Indeed, the requirement for TgCDPK3 is most likely determined by the high K(+) concentration of the host cell. Our results therefore suggest that TgCDPK3's role differs from that previously hypothesized, and rather support a model where this kinase plays a role in rapidly responding to Ca(2+) signaling in specific ionic environments to upregulate multiple processes required for gliding motility.This work was supported by the National Health and Medical Research Council (NHMRC) Project Grant APP1025598. JMM is supported by a Australian Postgraduate Award, GGvD is supported by an Australian Research Council QEII Fellowship and CJT is supported by an NHMRC Career Development Award. This work was also made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Age-specific trends in cardiovascular mortality rates in Australia between 1980 and 2005

Aim: Recent analyses suggest the decline in coronary heart disease (CHD) mortality rates is slowing in younger age groups in countries such as the UK and US. We aimed to assess recent mortality rate trends in all circulatory disease and its subtypes in Australia. Methods: Annual all circulatory, CHD, and cerebrovascular disease mortality rates between 1980 and 2005 for Australia were analysed. Data were stratified by sex and ten-year age group (age 35 to 85+). The annual rate of change and significant changes in trends were identified using joinpoint Poisson regression. Results: Age standardised all circulatory disease mortality rates continue to decline in Australia, falling from 441 per 100,000 in 1980 to 145 per 100,000 in 2005 for males and from 264 per 100,000 to 96 per 100,000 for females. The rate of decline from both CHD and cerebrovascular disease appears to be stable or accelerating for individuals aged 55 years and over. However, the decline in young men and women aged 35-54 years is slowing for CHD and cerebrovascular disease mortality alike (except cerebrovascular disease mortality in males aged 35-44). For females aged 35-44 and 45-54 there has been no change in the cerebrovascular mortality rate since 1993 and 1999, respectively. Conclusions: In Australia, whilst in older adults the decline in cardiovascular mortality rates is generally accelerating, in younger adults it appears to be slowing. It will be important to identify the causes of these trends

Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite, and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We show that subtelomeric upsB var gene promoters carried on episomes are silenced by default, and that promoter activation is sufficient to silence all other family members. However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation. Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression. Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family

Potential for improvement of population diet through reformulation of commonly eaten foods

Food reformulation: Reformulation of foods is considered one of the key options to achieve population nutrient goals. The compositions of many foods are modified to assist the consumer bring his or her daily diet more in line with dietary recommendations. Initiatives on food reformulation: Over the past few years the number of reformulated foods introduced on the European market has increased enormously and it is expected that this trend will continue for the coming years. Limits to food reformulation: Limitations to food reformulation in terms of choice of foods appropriate for reformulation and level of feasible reformulation relate mainly to consumer acceptance, safety aspects, technological challenges and food legislation. Impact on key nutrient intake and health: The potential impact of reformulated foods on key nutrient intake and health is obvious. Evaluation of the actual impact requires not only regular food consumption surveys, but also regular updates of the food composition table including the compositions of newly launched reformulated foods

Elucidating the mitochondrial proteome of Toxoplasma gondii reveals the presence of a divergent cytochrome c oxidase

The mitochondrion of apicomplexan parasites is critical for parasite survival, although the full complement of proteins that localize to this organelle has not been defined. Here we undertake two independent approaches to elucidate the mitochondrial proteome of the apicomplexan Toxoplasma gondii. We identify approximately 400 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth. We demonstrate that one such protein, termed TgApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex. We identify numerous other apicomplexan-specific components of COX, and conclude that apicomplexan COX, and apicomplexan mitochondria more generally, differ substantially in their protein composition from the hosts they infect. Our study highlights the diversity that exists in mitochondrial proteomes across the eukaryotic domain of life, and provides a foundation for defining unique aspects of mitochondrial biology in an important phylum of parasites.This work was supported by a Discovery Grant and QEII fellowship from the Australian Research Council (ARC DP110103144) to GvD

How, why and when are delayed (back-up) antibiotic prescriptions used in primary care? A realist review integrating concepts of uncertainty in healthcare

Background: Antimicrobial resistance is a global patient safety priority and inappropriate antimicrobial use is a key contributing factor. Evidence have shown that delayed (back-up) antibiotic prescriptions (DP) are an effective and safe strategy for reducing unnecessary antibiotic consumption but its use is controversial. Methods: We conducted a realist review to ask why, how, and in what contexts general practitioners (GPs) use DP. We searched five electronic databases for relevant articles and included DP-related data from interviews with healthcare professionals in a related study. Data were analysed using a realist theory-driven approach – theorising which context(s) influenced (mechanisms) resultant outcome(s) (context-mechanism-outcome-configurations: CMOCs). Results: Data were included from 76 articles and 41 interviews to develop a program theory comprising nine key and 56 related CMOCs. These explain the reasons for GPs’ tolerance of risk to different uncertainties and how these may interact with GPs’ work environment, self-efficacy and perceived patient concordance to make using DP as a safety-net or social tool more or less likely, at a given time-point. For example, when a GP uses clinical scores or diagnostic tests: a clearly high or low score/test result may mitigate scientific uncertainty and lead to an immediate or no antibiotic decision; an intermediary result may provoke hermeneutic (interpretation-related) uncertainty and lead to DP becoming preferred and used as a safety net. Our program theory explains how DP can be used to mitigate some uncertainties but also provoke or exacerbate others. Conclusion: This review explains how, why and in what contexts GPs are more or less likely to use DP, as well as various uncertainties GPs face which DP may mitigate or provoke. We recommend that efforts to plan and implement interventions to optimise antibiotic prescribing in primary care consider these uncertainties and the contexts when DP may be (dis)preferred over other interventions to reduce antibiotic prescribing. We also recommend the following and have included example activities for: (i) reducing demand for immediate antibiotics; (ii) framing DP as an ‘active’ prescribing option; (iii) documenting the decision-making process around DP; and (iv) facilitating social and system support

Characterization of the ATP4 ion pump in Toxoplasma gondii

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+. Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.This work was supported by National Health and Medical Research Council Grant 1042272 (to K. K.) and Australian Research Council Discovery Project Grant DP150102883 (to K. K. and G. G. v. D.), Linkage Project Grant LP150101226 (to K. K.), Discovery Early Career Researcher Award DE160101035 (to A. M. L.), QEII Fellowship DP110103144 (to G. G. v. D.), and Future Fellowship FT120100164 (to C. J. T.). C. J. T. is grateful for institutional support from the Victorian State Government Operational Infrastructure Support Program and the National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme