Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans-kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs. Furthermore, genetic and molecular analyses have elucidated the functions of many effectors and key regulators of T3SS assembly and secretion hierarchy, which is the sequential order by which the protein substrates are secreted. As essential virulence factors, T3SSs are attractive targets for vaccines and therapeutics. This Review summarizes our current knowledge of the structure and function of this important protein secretion machinery. A greater understanding of T3SSs should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.

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. Ca2+-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 Ca2+ 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 Ca2+ signaling in specific ionic environments to upregulate multiple processes required for gliding motility.

Transmissible stages of Toxoplasma gondii store energy in the form of the carbohydrate amylopectin. Here, we show that the Ca(2+)-dependent protein kinase CDPK2 is a critical regulator of amylopectin metabolism. Increased synthesis and loss of degradation of amylopectin in CDPK2 deficient parasites results in the hyperaccumulation of this sugar polymer. A carbohydrate-binding module 20 (CBM20) targets CDPK2 to amylopectin stores, while the EF-hands regulate CDPK2 kinase activity in response to Ca(2+) to modulate amylopectin levels. We identify enzymes involved in amylopectin turnover whose phosphorylation is dependent on CDPK2 activity. Strikingly, accumulation of massive amylopectin granules in CDPK2-deficient bradyzoite stages leads to gross morphological defects and complete ablation of cyst formation in a mouse model. Together these data show that Ca(2+) signaling regulates carbohydrate metabolism in Toxoplasma and that the post-translational control of this pathway is required for normal cyst development.

Identifying equilibrium conformational exchange and characterizing conformational substates is essential for elucidating mechanisms of function in proteins. Site-directed spin labeling has previously been employed to detect conformational changes triggered by some event, but verifying conformational exchange at equilibrium is more challenging. Conformational exchange (microsecond-millisecond) is slow on the EPR time scale, and this proves to be an advantage in directly revealing the presence of multiple substates as distinguishable components in the EPR spectrum, allowing the direct determination of equilibrium constants and free energy differences. However, rotameric exchange of the spin label side chain can also give rise to multiple components in the EPR spectrum. Using spin-labeled mutants of T4 lysozyme, it is shown that high-pressure EPR can be used to: (i) demonstrate equilibrium between spectrally resolved states, (ii) aid in distinguishing conformational from rotameric exchange as the origin of the resolved states, and (iii) determine the relative partial molar volume (ΔV o ) and isothermal compressibility (Δβ T ) of conformational substates in two-component equilibria from the pressure dependence of the equilibrium constant. These volumetric properties provide insight into the structure of the substates. Finally, the pressure dependence of internal side-chain motion is interpreted in terms of volume fluctuations on the nanosecond time scale, the magnitude of which may reflect local backbone flexibility.P roteins undergo structural fluctuations that span a wide range of time scales. Among these motions are fast backbone fluctuations on the picosecond-nanosecond time scale and slower conformational fluctuations on the microsecond and longer time scale (1-3). Molecular flexibility on these time scales plays a central role in protein function (4). For example, in recognitionbinding sequences, dynamic disorder on the nanosecond-microsecond time scale may increase the rate of protein-protein interactions via a "fly casting" mechanism (5). An emerging disorder-to-order paradigm for interaction (6) can also give rise to promiscuity in binding that increases the size of the "interactome."Regulation of protein function is often linked to a conformational switch triggered by an interaction with a chemical or physical signal. One mechanistic interpretation of this event is provided by a "preequilibrium" model, which posits that all possible conformations of a protein exist at equilibrium with populations proportional to their relative energies (7). The exchange ("hopping") event between different conformers is characterized by lifetimes in the microsecond-millisecond range (1, 2, 8). In this model, a conformational switch is viewed as a shift in the relative populations of existing conformational states rather than the creation of a new state.To evaluate the above models and elucidate molecular mechanisms of protein function, it is essential to have experimental means for identifying dynamically disordered sequenc...

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The reduced scalar Helmholtz equation for a transversely inhomogeneous half-space supplemented with an outgoing radiation condition and an appropriate boundary condition on the initial-value plane defines a direct acoustic propagation model. This elliptic formulation admits a factorization and is subsequently equivalent to a first-order Weyl pseudodifferential equation which is recognized as an extended parabolic propagation model. Perturbation treatments of the appropriate Weyl composition equation result in a systematic development of approximate wave theories which extend the narrow-angle, weak-inhomogeneity, and weak-gradient ordinary parabolic (Schrödinger) approximation. The analysis further provides for the formulation and exact solution of a multidimensional nonlinear inverse problem appropriate for ocean acoustic and seismic studies. The wave theories foreshadow computational algorithms, the inclusion of range-dependent effects, and the extension to (1) the vector formulation appropriate for elastic media and (2) the bilinear formulation appropriate for acoustic field coherence.

ObjectiveThe Substitutable Medical Applications, Reusable Technologies (SMART) Platforms project seeks to develop a health information technology platform with substitutable applications (apps) constructed around core services. The authors believe this is a promising approach to driving down healthcare costs, supporting standards evolution, accommodating differences in care workflow, fostering competition in the market, and accelerating innovation.Materials and methodsThe Office of the National Coordinator for Health Information Technology, through the Strategic Health IT Advanced Research Projects (SHARP) Program, funds the project. The SMART team has focused on enabling the property of substitutability through an app programming interface leveraging web standards, presenting predictable data payloads, and abstracting away many details of enterprise health information technology systems. Containers—health information technology systems, such as electronic health records (EHR), personally controlled health records, and health information exchanges that use the SMART app programming interface or a portion of it—marshal data sources and present data simply, reliably, and consistently to apps.ResultsThe SMART team has completed the first phase of the project (a) defining an app programming interface, (b) developing containers, and (c) producing a set of charter apps that showcase the system capabilities. A focal point of this phase was the SMART Apps Challenge, publicized by the White House, using http://www.challenge.gov website, and generating 15 app submissions with diverse functionality.ConclusionKey strategic decisions must be made about the most effective market for further disseminating SMART: existing market-leading EHR vendors, new entrants into the EHR market, or other stakeholders such as health information exchanges.

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