Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii

ABSTRACT Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKG I is plasma membrane associated whereas PKG II is cytosolic in Toxoplasma gondii . To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii . By combining AID regulation with genome editing strategies, we determined that PKG I is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKG II is functionally insufficient and dispensable in the presence of PKG I . The difference in functionality mapped to the first 15 residues of PKG I , containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in T. gondii . IMPORTANCE Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii . We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii . </jats:p

REDHORSE-REcombination and Double crossover detection in Haploid Organisms using next-geneRation SEquencing data

BACKGROUND: Next-generation sequencing technology provides a means to study genetic exchange at a higher resolution than was possible using earlier technologies. However, this improvement presents challenges as the alignments of next generation sequence data to a reference genome cannot be directly used as input to existing detection algorithms, which instead typically use multiple sequence alignments as input. We therefore designed a software suite called REDHORSE that uses genomic alignments, extracts genetic markers, and generates multiple sequence alignments that can be used as input to existing recombination detection algorithms. In addition, REDHORSE implements a custom recombination detection algorithm that makes use of sequence information and genomic positions to accurately detect crossovers. REDHORSE is a portable and platform independent suite that provides efficient analysis of genetic crosses based on Next-generation sequencing data. RESULTS: We demonstrated the utility of REDHORSE using simulated data and real Next-generation sequencing data. The simulated dataset mimicked recombination between two known haploid parental strains and allowed comparison of detected break points against known true break points to assess performance of recombination detection algorithms. A newly generated NGS dataset from a genetic cross of Toxoplasma gondii allowed us to demonstrate our pipeline. REDHORSE successfully extracted the relevant genetic markers and was able to transform the read alignments from NGS to the genome to generate multiple sequence alignments. Recombination detection algorithm in REDHORSE was able to detect conventional crossovers and double crossovers typically associated with gene conversions whilst filtering out artifacts that might have been introduced during sequencing or alignment. REDHORSE outperformed other commonly used recombination detection algorithms in finding conventional crossovers. In addition, REDHORSE was the only algorithm that was able to detect double crossovers. CONCLUSION: REDHORSE is an efficient analytical pipeline that serves as a bridge between genomic alignments and existing recombination detection algorithms. Moreover, REDHORSE is equipped with a recombination detection algorithm specifically designed for Next-generation sequencing data. REDHORSE is portable, platform independent Java based utility that provides efficient analysis of genetic crosses based on Next-generation sequencing data. REDHORSE is available at http://redhorse.sourceforge.net/. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1309-7) contains supplementary material, which is available to authorized users

Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9

ABSTRACT Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excel-lent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special ge-netic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 con-tributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. IMPORTANCE Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. How-ever, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to suc

Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis

Circulating murine monocytes comprise two largely exclusive subpopulations that are responsible for seeding normal tissues (Gr-1−/CCR2−/CX3CR1high) or responding to sites of inflammation (Gr-1+/CCR2+/CX3CR1lo). Gr-1+ monocytes are recruited to the site of infection during the early stages of immune response to the intracellular pathogen Toxoplasma gondii. A murine model of toxoplasmosis was thus used to examine the importance of Gr-1+ monocytes in the control of disseminated parasitic infection in vivo. The recruitment of Gr-1+ monocytes was intimately associated with the ability to suppress early parasite replication at the site of inoculation. Infection of CCR2−/− and MCP-1−/− mice with typically nonlethal, low doses of T. gondii resulted in the abrogated recruitment of Gr-1+ monocytes. The failure to recruit Gr-1+ monocytes resulted in greatly enhanced mortality despite the induction of normal Th1 cell responses leading to high levels of IL-12, TNF-α, and IFN-γ. The profound susceptibility of CCR2−/− mice establishes Gr-1+ monocytes as necessary effector cells in the resistance to acute toxoplasmosis and suggests that the CCR2-dependent recruitment of Gr-1+ monocytes may be an important general mechanism for resistance to intracellular pathogens

Evaluating the impact of the internationalisation of urban planning on Saudi Arabian cities

The Kingdom of Saudi Arabia (KSA) has experienced change in the development of urban planning codes, the most recent being the 2010 planning codes. Every urban planning regulation and code is devised to implement the overall urban design paradigm most suited to the city in question. For instance, the demand for modern urban facilities, coupled with the need for better transport networks around cities, has resulted in the shift from limited storey heights for buildings to more liberal, limitless storey heights. From the 1970s to date, the Saudi Arabian government has been engaging internationally acclaimed designers and planners, such as Forster and Partners International Designers, and Henning Larsen Architects of Denmark, to work with Saudi Arabian experts in respect of the ‘internationalisation’ of urban planning. The main driver for the paradigm shift in the urban planning processes has been the population growth and economic development. This entails an increase in the demand for a new generation of high-tech and knowledge-oriented industries in order to spread public services more evenly and broaden the scope and effectiveness of development policies among regions. Given the potential of such an urban planning strategy to influence both the current and future urban form, this research assesses the impact of internationalisation of urban planning on Saudi Arabian cities. It concludes that planners themselves are not sufficiently appreciative of the requirement for urban infrastructure that accompanies the new planning regulations. Keywords: internationalisation, urban planning, urban form, sustainable urban centre, Saudi Arabia

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e., tachyzoites) and establishing chronic infection (i.e., bradyzoites). Calcium ion (Ca(2+)) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca(2+) signaling pathways in bradyzoites remain largely unexplored. Here, we show that Ca(2+) responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca(2+) responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca(2+) imaging demonstrated lower Ca(2+) basal levels, reduced magnitude, and slower Ca(2+) kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with downregulation of Ca(2+)-ATPases involved in intracellular Ca(2+) storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca(2+) rapidly restored their intracellular Ca(2+) and ATP stores, leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca(2+) signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility

How do older people with sight loss manage their general health? A qualitative study

Purpose: Older people with sight loss experience a number of barriers to managing their health. The purpose of this qualitative study was to explore how older people with sight loss manage their general health and explore the techniques used and strategies employed for health management. Methods: Semi-structured face-to-face interviews were conducted with 30 participants. Interviews were audio-recorded, transcribed verbatim and analysed using thematic analysis. Results: Health management challenges experienced included: managing multiple health conditions; accessing information; engaging in health behaviours and maintaining wellbeing. Positive strategies included: joining support groups, clubs and societies; using low vision aids; seeking support from family and friends and accessing support through health and social care services. Conclusion: Healthcare professionals need to be more aware of the challenges faced by older people with sight loss. Improved promotion of group support and charity services which are best placed to share information, provide fora to learn about coping techniques and strategies, and give older people social support to prevent isolation is needed. Rehabilitation and support services and equipment can only be beneficial if patients know what is available and how to access them. Over-reliance on self-advocacy in current healthcare systems is not conducive to patient-centred care. Implications for Rehabilitation Sight loss in older people can impact on many factors including health management. This study identifies challenges to health management and highlights strategies used by older people with sight loss to manage their health. Access to support often relies on patients seeking information for themselves. However, self-advocacy is challenging due to information accessibility barriers. Informal groups and charities play an important role in educating patients about their condition and advising on available support to facilitate health management