Hybrid-fuel bacterial flagellar motors in Escherichia coli

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H⁺ or Na⁺ ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H⁺- and Na⁺-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na⁺ concentration ([Na⁺]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na⁺ and H⁺ stator components expressed at high and medium levels, respectively, Na⁺ stator units dominate at high [Na⁺] and are replaced by H⁺ units when Na⁺ is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H⁺ and Na⁺ stator components sharing a common rotor.Molecular motors are tiny machines that perform a wide range of functions in living cells. Typically each motor generates mechanical work using a specific chemical or electrochemical energy source. Linear motors such as kinesin on microtubules or myosin on actin filaments and rotary motors such as F₁-ATPase, the soluble part of ATP-synthase, run on ATP, whereas the rotary bacterial flagellar motor embedded in the bacterial cell envelope is driven by the flux of ions across the cytoplasmic membrane (1–4). Coupling ions are known to be either protons (H⁺) or sodium ions (Na⁺) (5, 6).The bacterial flagellar motor consists of a rotor ∼50 nm in diameter surrounded by multiple stator units (7–10). Each unit contains two types of membrane proteins forming ion channels: MotA and MotB in H⁺ motors in neutrophiles (e.g., Escherichia coli and Salmonella) and PomA and PomB in Na⁺ motors in alkalophiles and Vibrio species (e.g., Vibrio alginolyticus) (1, 11). Multiple units interact with the rotor to generate torque independently in a working motor (9, 10, 12, 13). The structure and function of H⁺ and Na⁺ motors are very similar, to the extent that several functional chimeric motors have been made containing different mixtures of H⁺- and Na⁺-motor components (11). One such motor that runs on Na⁺ in E. coli combines the rotor of the H⁺-driven E. coli motor with the chimeric stator unit PomA/PotB, containing PomA from V. alginolyticus and a fusion protein between MotB from E. coli and PomB from V. alginolyticus (14).In most flagellated bacteria, motors are driven by ion-specific rotor–stator combinations. However, some species (e.g., Bacillus subtilis and Shewanella oneidensis) combine a single set of rotor genes with multiple sets of stator genes encoding both H⁺ and Na⁺ stator proteins, and it has been speculated that these stator components may interact with the rotor simultaneously, allowing a single motor to use both H⁺ and Na⁺. An appealing hypothesis that the mixture of stator components is controlled dynamically depending on the environment has arisen from the observation that the localization of both stator components depends upon Na⁺ (15). However, despite some experimental effort there is as yet no direct evidence of both H⁺ and Na⁺ stator units interacting with the same rotor (16).The rotation of single flagellar motors can be monitored in real time by light microscopy of polystyrene beads (diameter ∼1 μm) attached to truncated flagellar filaments (17). Under these conditions, the E. coli motor torque and speed are proportional to the number of stator units in both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB (17–19) motors. The maximum number of units that can work simultaneously in a single motor has been shown to be at least 11 by “resurrection” experiments, in which newly produced functional units lead to restoration of motor rotation in discrete speed increments in an E. coli strain lacking functional stator proteins (19). Stator units are not fixed permanently in a motor: Each dissociates from the motor with a typical rate of ∼2 min⁻¹, exchanging between the motor and a pool of diffusing units in the cytoplasmic membrane (20). Removal of the relevant ion gradient inactivates both H⁺ and Na⁺ stator units, most likely leading to dissociation from the motor into the membrane pool (2, 21, 22).Here we demonstrate a hybrid-fuel motor containing both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB stator components, sharing a common rotor in E. coli. We control the expression level of each stator type by induced expression from plasmids, and the affinity of Na⁺-driven stator units for the motor by external [Na⁺]. Units of each type compete for spaces around the rotor, and the motor torque is simply the sum of the independent contributions, with no evidence of direct interaction between units. Thus, we demonstrate the possibility of modularity in the E. coli flagellar motor, with ion selectivity determined by the choice of stator modules interacting with a common rotor. Our artificial hybrid motor demonstrates that species with multiple types of stator gene and a single set of rotor genes could contain natural hybrid motors that work on a similar principle (15, 16, 23).     

MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.Most bacteria move by flagella. The flagellar architecture is conserved and can be divided into the cytoplasmic C-ring, the basal body, the rod, and the exterior hook and filament structures (1). Bacterial species differ in the number and arrangement of their flagella (flagellation pattern) (2). However, the mechanisms that allow bacteria to establish their specific flagellation patterns reproducibly during each cell division are poorly understood. The protein FlhG (also known as “YlxH,” “MinD2,” “FleN,” or “MotR”) is essential for the correct flagellation pattern of polar- (3–5), lophotrichous- (6), amphitrichous- (7), and peritrichous-flagellated bacteria (8, 9). Deletion of flhG in polar-flagellated bacteria leads to hyperflagellation and impaired motility (3–5). In the amphitrichous-flagellated Campylobacter jejuni, ∼40% of the cells of a ΔflhG strain exhibited more than one flagellum at one pole and were impaired in motility (7). The peritrichous-flagellated bacterium Bacillus subtilis exhibits ∼26 flagellar basal bodies arranged symmetrically around midcell in a gridlike pattern (8). Furthermore, flagella are discouraged at the cell pole. Deletion of flhG does not result in swimming or swarming defects, although multiple flagella appear in tufts from constrained loci on the cell, and flagellar basal bodies often are aggregated (8). FlhG acts in concert with the signal recognition particle (SRP)-GTPase FlhF (10–14) that recruits the flagellar protein FliF to the cell pole in the polar-flagellated Vibrio cholerae (15). FlhG is predicted to belong to the MinD/ParA ATPase family (6, 16) whose characterized members act in orchestrated spatiotemporal processes (e.g., cell-division site determination and plasmid/chromosome partitioning (summarized in ref. 17). Together with MinC, MinD constitutes the conserved center of the Min system which regulates bacterial cell division by restricting cytokinetic Z-ring assembly to midcell (reviewed in ref. 18). MinD forms ATP-dependent homodimers (19) that interact with the inner membrane through a C-terminal amphipathic helix (membrane-targeting sequence, MTS) (20, 21). By this mechanism, MinD recruits MinC to the membrane where MinC inhibits polymerization of FtsZ into the Z-ring (22). Interestingly, Campylobacter jejuni does not contain a Min system, and FlhG is involved in flagellation pattern control and regulation of cell division (7). In contrast to MinD, the molecular framework in which the putative MinD-like ATPase FlhG controls flagellation is unknown. Also, it is enigmatic how conserved homologs of FlhG can control different flagellation patterns in different species. Here, we investigated the mechanism and function of FlhG in the Gram-positive, peritrichous-flagellated B. subtilis (Bs) and the Gram-negative, polar-flagellated Shewanella putrefaciens (Sp).     

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

The torque of the bacterial flagellum is generated by the rotor–stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomB_C) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomB_C for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121–153) and following the N-terminal two-thirds of α1(154-164) in PomB_C changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.Motility is a fundamental function for bacterial survival in environmental change. The bacterial flagellum is a large filamentous macromolecular assembly for motility. A reversible motor, which is embedded at the base of each filament and powered by the electrochemical gradient of the coupling ion (H⁺ or Na⁺), rotates the filament like a screw to drive the cell toward a favorable environment (1–3).The torque generation unit of the flagellar motor consists of a rotor and stators. A dozen stators surround the rotor and work together, although a single stator can generate torque. Stepwise drops and restorations of the rotational speed have been reported, suggesting that each stator is able to associate into or dissociate from the functioning motor (4–6). The fluorescent photo-bleaching study has revealed a rapid turnover of the stator in the rotating motor (7). The rotor is made up of the MS-ring and the C-ring. The C-ring is formed just beneath the MS-ring and is essential for torque generation and switching the direction of rotation.The stator is a hetero-hexameric complex of two membrane proteins, A and B, with a stoichiometry of A₄B₂ (8, 9). The stator consists of MotA and MotB for the H⁺-driven motor of Escherichia coli and Salmonella enterica, and PomA and PomB for the Na⁺-driven motor of Vibrio and Shewanella spp. (10). The torque is generated by the interaction between rotor protein FliG in C-ring and the stator A subunit coupled with the ion flow through the channel in the stator (1). The stator B subunit consists of a short cytoplasmic segment, a single transmembrane helix and a C-terminal periplasmic region (11) (Fig. 1A and Fig. S1A). The transmembrane helix has an aspartate residue essential for ion translocation across the cytoplasmic membrane, and forms an ion channel with the transmembrane region of the A subunits (12–14). The periplasmic region of the B subunit (B_C) shows a sequence similarity to OmpA-like proteins and contains a putative peptidoglycan binding motif (PGB motif), which is thought to anchor the stator to the peptidoglycan layer (PG layer) (15). Many nonmotile mutation sites were found within or contiguous to the PGB motif, suggesting that proper anchoring of the stator to the PG layer is important to the motor function (16, 17). Ion-conductivity of the stator is coupled to its installation in the motor. Overproduction of the stator does not arrest cell growth, indicating that the ion-conductivity of the stator is activated only when it is installed around the rotor (13). A short segment following the transmembrane region of the B subunit, called the plug, is thought to suppress the ion leakage until the stator is incorporated into the motor (18, 19). However, it is not clear how the activation of the ion channel is coupled to the stator incorporation.Open in a separate windowFig. 1.Structure of the C-terminal fragment of PomB. (A) Schematic representation of the full-length PomB, PomB_ΔL, and the PomB_C variants used for crystallization. PomB_ΔL is the smallest construct that forms a stable functional stator unit derived from a systematic deletion experiment (19). The PEM region is highlighted in blue. Asp-24, which is essential for ion translocation across the cytoplasmic membrane, is shown as a solid red circle. (B) Ribbon representation of PomB_C5 dimer. One subunit is color coded from blue to red from the N to the C terminus, and the other is painted magenta. (C) Structure of Salmonella MotB_C2 dimer (PDB ID code 2ZVY) (21). The two subunits are colored in cyan and magenta.A systematic deletion study of the periplasmic region of MotB demonstrated that a MotB mutant with the deletion of residues 51–100 still forms a stable active stator. Moreover, another MotB mutant with the deletion of residues 51–110 and 271–309 is also functional, although unstable (20), indicating that the essential periplasmic region for motor function is only the residues 111–270, named periplasmic region essential for motility (PEM). The deleted dispensable region seems to work as a linker that connects the transmembrane segment and the PEM. Recently, the structure of a periplasmic fragment of Salmonella MotB (St-MotB_C) that covers the PEM has been solved (21). St-MotB_C forms a homo-dimer, the subunit of which is composed of a single OmpA-like domain, which contains the putative PGB site, with N-terminal long and short helices. The distance between the PG layer and the surface of the hydrophobic core layer of the cytoplasmic membrane is about 100 Å (22), whereas the height of St-MotB_C is only 50 Å. Thus, a long extension of MotB_C is expected to anchor the stator to the PG layer, but it is still obscure what conformational changes occur.The sodium-driven Vibrio motor rotates up to 1,700 Hz (23), in contrast to the proton-driven motors of E. coli and Salmonella, which typically revolve up to 300 Hz (24). The basal body of the Vibrio motor has two unique ring structures, the T-ring and the H-ring (25, 26). These extra rings are thought to reinforce the motor to resist the high-speed rotation. Recent structural study demonstrated that FlgT acts as an assembly base or scaffold for both the ring structures (27). The T-ring is made up of MotX and MotY, and is located beneath the P-ring, which is a part of a bushing structure for the rod, thereby believed not to rotate. The T-ring is an essential component to incorporate the stator into the motor (25). The periplasmic region of PomB is likely to bind to MotX (28), and MotX is connected to the basal body through the N-terminal domain of MotY (29). Thus, the stator of the sodium-driven motor is tightly fixed not only to the PG layer but also to the basal body through the interaction between PomB and the T-ring. Despite the rigid anchoring structure, the stator of the sodium-driven motor still shows a dynamic behavior dependent on the binding of sodium ion to PomB (30). In this study, we purified and crystallized various fragments of PomB_C (Table S1) and solved the structure of PomB_C4 (121–315), which covers the PEM of PomB, and PomB_C5 (135–315) (Fig. 1A). The crystal structures, following in vivo disulfide cross-link studies and motility assay, revealed novel insights into the molecular mechanism of the stator assembly and activation of the sodium-driven flagellar motor.     

Switching dynamics of the bacterial flagellar motor near zero load

Switching dynamics of flagellar motors of Escherichia coli is commonly observed through markers attached to the flagellar filaments. To eliminate possible complications resulting from the conformational transitions of these filaments and to look at the output of motors more directly, we monitored motor rotation by attaching nanogold spheres to the hooks of cells lacking filaments. We observed exponentially distributed counterclockwise (CCW) and clockwise (CW) intervals and Lorentzian power spectra of the switching time series consistent with models that treat motor switching as a two-state Poisson process.Peritrichously flagellated bacteria such as Escherichia coli perform chemotaxis by a biased random walk comprising runs and tumbles, determined by the direction of rotation of the flagellar motors. When all of the motors on a cell rotate counterclockwise (CCW), the flagellar filaments form a helical bundle and the cell swims smoothly (runs); when one or more motors switch to clockwise (CW) rotation, the bundle is disrupted and the cell changes direction (tumbles) (1). The response regulator of the chemotaxis-signaling pathway, CheY-P, interacts with the motor switch protein FliM, increasing the probability that the motor spins CW (2–4). Switching dynamics of the motor has been observed by tethering single filaments to a glass slide and observing rotation of the cell body (5) or by attaching micrometer-sized beads to short (6, 7) or long (8, 9) filament stubs and watching the beads wobble. A recent model noted that conformational transitions of filaments can contribute to the observed dynamics (10). To remove this complication and observe the motor dynamics directly, we used the nanogold method developed to study motor behavior near zero load, in which 60- to 200-nm-diameter gold spheres are attached to the hooks of cells lacking flagellar filaments (11–13).     

Cell-body rocking is a dominant mechanism for flagellar synchronization in a swimming alga

The unicellular green alga Chlamydomonas swims with two flagella that can synchronize their beat. Synchronized beating is required to swim both fast and straight. A long-standing hypothesis proposes that synchronization of flagella results from hydrodynamic coupling, but the details are not understood. Here, we present realistic hydrodynamic computations and high-speed tracking experiments of swimming cells that show how a perturbation from the synchronized state causes rotational motion of the cell body. This rotation feeds back on the flagellar dynamics via hydrodynamic friction forces and rapidly restores the synchronized state in our theory. We calculate that this “cell-body rocking” provides the dominant contribution to synchronization in swimming cells, whereas direct hydrodynamic interactions between the flagella contribute negligibly. We experimentally confirmed the two-way coupling between flagellar beating and cell-body rocking predicted by our theory.Eukaryotic cilia and flagella are long, slender cell appendages that can bend rhythmically and thus present a prime example of a biological oscillator (1). The flagellar beat is driven by the collective action of dynein molecular motors, which are distributed along the length of the flagellum. The beat of flagella, with typical frequencies ranging from 20–60 Hz, pumps fluids, for example, mucus in mammalian airways (2), and propels unicellular microswimmers such as Paramecia, spermatozoa, and algae (3). The coordinated beating of collections of flagella is important for efficient fluid transport (2, 4, 5) and fast swimming (6). This coordinated beating represents a striking example for the synchronization of oscillators, prompting the question of how flagella couple their beat. Identifying the specific mechanism of synchronization can be difficult because synchronization may occur even for weak coupling (7). Further, the effect of the coupling is difficult to detect once the synchronized state has been reached.Hydrodynamic forces were suggested to play a significant role for flagellar synchronization already in 1951 by Taylor (8). Since then, direct hydrodynamic interactions between flagella were studied theoretically as a possible mechanism for flagellar synchronization (9–12). Another synchronization mechanism that is independent of hydrodynamic interactions was recently described in the context of a minimal model swimmer (13–15). This mechanism crucially relies on the interplay of swimming motion and flagellar beating.Here, we address the hydrodynamic coupling between the two flagella in a model organism for flagellar coordination (16–19), the unicellular green alga Chlamydomonas reinhardtii. Chlamydomonas propels its ellipsoidal cell body, which has typical diameter of 10 μm, using a pair of flagella, whose lengths are about 10 μm (16). The two flagella beat approximately in a common plane, which is collinear with the long axis of the cell body. In that plane, the two beat patterns are nearly mirror-symmetric with respect to this long axis. The beating of the two flagella of Chlamydomonas can synchronize, that is, adopt a common beat frequency and a fixed phase relationship (16–19). In-phase synchronization of the two flagella is required for swimming along a straight path (19). The specific mechanism leading to flagellar synchrony is unclear.Here, we use a combination of realistic hydrodynamic computations and high-speed tracking experiments to reveal the nature of the hydrodynamic coupling between the two flagella of free-swimming Chlamydomonas cells. Previous hydrodynamic computations for Chlamydomonas used either resistive force theory (20, 21), which does not account for hydrodynamic interactions between the two flagella, or computationally intensive finite element methods (22). We employ an alternative approach and represent the geometry of a Chlamydomonas cell by spherical shape primitives, which provides a computationally convenient method that fully accounts for hydrodynamic interactions between different parts of the cell. Our theory characterizes flagellar swimming and synchronization by a minimal set of effective degrees of freedom. The corresponding equation of motion follows naturally from the framework of Lagrangian mechanics, which was used previously to describe synchronization in a minimal model swimmer (13, 15). These equations of motion embody the key assumption that the flagellar beat speeds up or slows down according to the hydrodynamic friction forces acting on the flagellum, that is, if there is more friction and therefore higher hydrodynamic load, then the beat will slow down. This assumption is supported by previous experiments that showed that the flagellar beat frequency decreases when the viscosity of the surrounding fluid is increased (23, 24). The simple force–velocity relationship for the flagellar beat employed by us coarse-grains the behavior of thousands of dynein molecular motors that collectively drive the beat. Similar force–velocity properties have been described for individual molecular motors (25) and reflect a typical behavior of active force generating systems.Our theory predicts that any perturbation of synchronized beating results in a significant yawing motion of the cell, reminiscent of rocking of the cell body. This rotational motion imparts different hydrodynamic forces on the two flagella, causing one of them to beat faster and the other to slow down. This interplay between flagellar beating and cell-body rocking rapidly restores flagellar synchrony after a perturbation. Using the framework provided by our theory, we analyze high-speed tracking experiments of swimming cells, confirming the proposed two-way coupling between flagellar beating and cell-body rocking.Previous experiments restrained Chlamydomonas cells from swimming, holding their cell body in a micropipette (17–19). Remarkably, flagellar synchronization was observed also for these constrained cells. This observation seems to argue against a synchronization mechanism that relies on swimming motion. However, the rate of synchronization observed in these experiments was faster by an order of magnitude than the rate we predict for synchronization by direct hydrodynamic interactions between the two flagella in the absence of any motion. In contrast, we show that rotational motion with a small amplitude of a few degrees only, which may result from either a residual rotational compliance of the clamped cell or an elastic anchorage of the flagellar pair, provides a possible mechanism for rapid synchronization, which is analogous to synchronization by cell-body rocking in free-swimming cells.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Novel serologic biomarkers provide accurate estimates of recent Plasmodium falciparum exposure for individuals and communities

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual’s recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86–0.93), whereas responses to six antigens accurately estimated an individual’s malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.Many countries have extensive programs to reduce the burden of Plasmodium falciparum (Pf), the parasite responsible for most malaria morbidity and mortality (1). Effectively using limited resources for malaria control or elimination and evaluating interventions require accurate measurements of the risk of being infected with Pf (2–15). To reflect the rate at which individuals are infected with Pf in a useful way, metrics used to estimate exposure in a community need to account for dynamic changes over space and time, especially in response to control interventions (16–18).A variety of metrics can be used to estimate Pf exposure, but tools that are more precise and low cost are needed for population surveillance. Existing metrics have varying intrinsic levels of precision and accuracy and are subject to a variety of extrinsic factors, such as cost, time, and availability of trained personnel (19). For example, entomological measurements provide information on mosquito to human transmission for a community but are expensive, require specially trained staff, and lack standardized procedures, all of which reduce precision and/or make interpretation difficult (19–22). Parasite prevalence can be measured by detecting parasites in the blood of individuals from a cross-sectional sample of a community and is, therefore, relatively simple and inexpensive to perform, but results may be imprecise, especially in areas of low transmission (19, 23), and biased by a number of factors, including immunity and access to antimalarial treatment (5, 6, 19, 23–25). The burden of symptomatic disease in a community can be estimated from routine health systems data; however, such data are frequently unreliable (5, 26–28) and generally underestimate the prevalence of Pf infection in areas of intense transmission. Precise and quantitative information about exposure at an individual level can be reliably obtained from cohort studies by measuring the incidence of asymptomatic and/or symptomatic Pf infection (i.e., by measuring the molecular force of infection) (29–35). Unfortunately, the expense of cohort studies limits their use to research settings. The end result is that most malaria-endemic regions lack reliable, timely data on Pf exposure, limiting the capabilities of malaria control programs to guide and evaluate interventions.Serologic assays offer the potential to provide incidence estimates for symptomatic and asymptomatic Pf infection, which are currently obtained from cohort studies, at the cost of cross-sectional studies (36–38). Although Pf infections are transient, a record of infection remains detectable in an individual’s antibody profile. Thus, appropriately chosen antibody measurements integrated with age can provide information about an individual’s exposure history. Antibodies can be measured by simple ELISAs and obtained from dried blood spots, which are easy to collect, transport, and store (39–41). Serologic responses to Pf antigens have been explored as potential epidemiological tools (42–45), and estimated rates of seroconversion to well-characterized Pf antigens accurately reflect stable rates of exposure in a community, whereas distinct changes in these rates are obtained from successful interventions (22, 39, 41, 46–53). However, current serologic assays are not designed to detect short-term or gradual changes in Pf exposure or measure exposure to infection at an individual level. The ability to calibrate antibody responses to estimates of exposure in individuals could allow for more flexible sampling of a population (e.g., not requiring age stratification), improve accuracy of exposure estimates from small sample sizes, and better characterize heterogeneity in exposure within a community.Different Pf antigens elicit antibody responses with different magnitudes and kinetics, providing a large and diverse set of potential biomarkers for exposure (38, 54–58). We hypothesized that new and more highly informative serologic biomarkers better able to characterize an individual’s recent exposure history could be identified by analyzing antibody responses to a large number of candidate Pf antigens in participants with well-characterized exposure histories. To test this hypothesis, we probed plasma from participants in two cohort studies in Uganda against a protein microarray containing 856 Pf antigens. The primary aim of this analysis was to identify responses to select antigens that were most informative of recent exposure using robust, data-adaptive statistical methods. Each participant’s responses to these selected antigens were used as predictors for two primary outcomes of their recent exposure to Pf: (i) days since last Pf infection and (ii) the incidence of symptomatic malaria in the last year. These individual-level estimates were then aggregated across a population to assess community-level malaria exposure. The selection strategy presented here identified accurate biomarkers of exposure for children living in areas of moderate to high Pf exposure and illustrates the utility of this flexible and broadly applicable approach.     

Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.The bacterium Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSIs), including cellulitis, furunculosis, and folliculitis (1–4), and a common etiologic agent of impetigo (5), erysipelas (6), and superinfection in atopic dermatitis (7). This bacterium is a significant cause of surgical or traumatic wound infections (8, 9), as well as decuibitus and diabetic skin lesions (10). Moreover, SSSI is an important risk factor for systemic infection. The skin is a key portal of entry for hematogenous dissemination, particularly in association with i.v. catheters. S. aureus is now the second most common bloodstream isolate in healthcare settings (11), and SSSI is a frequent source of invasive infections such as pneumonia or endocarditis (12, 13). Despite a recent modest decline in rates of methicillin-resistant S. aureus (MRSA) infection in some cohorts (13), infections due to S. aureus remain a significant problem (14, 15). Even with appropriate therapy, up to one-third of patients diagnosed with S. aureus bacteremia succumb—accounting for more attributable annual deaths than HIV, tuberculosis, and viral hepatitis combined (16).The empiric use of antibiotics in healthcare-associated and community-acquired settings has increased S. aureus exposure to these agents, accelerating selection of resistant strains. As a result, resistance to even the most recently developed agents is emerging at an alarming pace (17, 18). The impact of this trend is of special concern in light of high rates of mortality associated with invasive MRSA infection (e.g., 15–40% in bacteremia or endocarditis), even with the most recently developed antistaphylococcal therapeutics (19, 20). Moreover, patients who experience SSSI due to MRSA exhibit high 1-y recurrence rates, often prompting surgical debridement (21) and protracted antibiotic treatment.Infections due to MRSA are a special concern in immune-vulnerable populations, including hemodialysis (22), neutropenic (23, 24), transplantation (25), and otherwise immunosuppressed patients (26, 27), and in patients with inherited immune dysfunctions (28–31) or cystic fibrosis (32). Patients having deficient interleukin 17 (IL-17) or IL-22 responses (e.g., signal transduction mediators STAT3, DOCK8, or CARD9 deficiencies) exhibit chronic or “cold” abscesses, despite high densities of pathogens such as S. aureus (33, 34). For example, patients with Chronic Granulomatous Disease (CGD; deficient Th1 and oxidative burst response) have increased risk of disseminated S. aureus infection. In contrast, patients with Job’s Syndrome (deficient Th17 response) typically have increased risk to SSSI and lung infections, but less so for systemic S. aureus bacteremia (35, 36). This pattern contrasts that observed in neutropenic or CGD patients (37). These themes suggest efficacious host defenses against MRSA skin and invasive infections involve complementary but distinct molecular and cellular immune responses.From these perspectives, vaccines or immunotherapeutics that prevent or lessen severity of MRSA infections, or that enhance antibiotic efficacy, would be significant advances in patient care and public health. However, to date, there are no licensed prophylactic or therapeutic vaccine immunotherapies for S. aureus or MRSA infection. Unfortunately, efforts to develop vaccines targeting S. aureus capsular polysaccharide type 5 or 8 conjugates, or the iron-regulated surface determinant B protein, have not been successful thus far (38, 39). Likewise, passive immunization using monoclonal antibodies targeting the S. aureus adhesin clumping factor A (ClfA, tefibazumab) (40) or lipoteichoic acid (pagibaximab) (41) have not shown efficacy against invasive infections in human clinical studies to date. Moreover, the striking recurrence rates of SSSI due to MRSA imply that natural exposure does not induce optimal preventive immunity or durable anamnestic response to infection or reinfection. Thus, significant challenges exist in the development of an efficacious vaccine targeting diseases caused by S. aureus (42) that are perhaps not optimally addressed by conventional approaches.The NDV-3 vaccine reflects a new strategy to induce durable immunity targeting S. aureus. Its immunogen is engineered from the agglutinin-like sequence 3 (Als3) adhesin/invasin of Candida albicans, which we discovered to be a structural homolog of S. aureus adhesins (43). NDV-3 is believed to cross-protect against S. aureus and C. albicans due to sequence (T-cell) and conformational (B-cell) epitopes paralleled in both organisms (44). Our prior data have shown that NDV-3 is efficacious in murine models of hematogenous and mucosal candidiasis (45), as well as S. aureus bacteremia (46–48). Recently completed phase I clinical trials demonstrate the safety, tolerability, and immunogenicity of NDV-3 in humans (49).     

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

F₁F_o-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.F₁F_o-ATP synthases are ubiquitous, highly conserved energy-converting membrane protein complexes. ATP synthases produce ATP from ADP and inorganic phosphate (P_i) by rotary catalysis (1, 2) using the energy stored in a transmembrane electrochemical gradient. The ∼600-kDa monomer of the mitochondrial ATP synthase is composed of a soluble F₁ subcomplex and a membrane-bound F_o subcomplex (3). The main components of the F₁ subcomplex are the (αβ)₃ hexamer and the central stalk (4). The F_o subcomplex includes a rotor ring of 8–15 hydrophobic c subunits (5), the peripheral stalk, and several small hydrophobic stator subunits. Protons flowing through the membrane part of the F_o subcomplex drive the rotation of the c-ring (6–9). The central stalk transmits the torque generated by c-ring rotation to the catalytic head of the F₁ subcomplex, where it induces conformational changes of the α and β subunits that result in phosphate bond formation and the generation of ATP. The catalytic (αβ)₃ hexamer is held stationary relative to the membrane region by the peripheral stalk (10, 11). Several high-resolution structures of the F₁/rotor ring complexes have been solved by X-ray crystallography (12–16), and the structure of the complete assembly has been determined by cryoelectron microscopy (cryo-EM) (10, 17–20).In mitochondria, the ATP synthase forms dimers in the inner membrane. In fungi, plants, and metazoans, the dimers are V-shaped and associate into rows along the highly curved ridges of lamellar cristae (19–22). F_o subcomplexes of the two monomers in the dimer interact in the lipid bilayer via a number of hydrophobic stator subunits (20, 23–25). Coarse-grained molecular dynamics simulations have suggested that the V-shape of the ATP synthase dimers induces local membrane curvature, which in turn drives the association of ATP synthase dimers into rows (20). The exact role of the dimer rows is unclear, however rows of ATP synthase dimers have been proposed to promote the formation of lamellar cristae in yeast (20, 26).So far, all rows of ATP synthase dimers observed by electron cryotomography have been more or less straight (19–22, 27). However, an earlier deep-etch freeze-fracture study of mitochondria from the ciliate Paramecium multimicronucleatum revealed double rows of interdigitating 10-nm particles on helical tubular cristae (28). These particles were interpreted as ATP synthases, which, if correct, would suggest that the mitochondrial ATP synthase can assemble into rows that differ significantly from the standard geometry found in lamellar cristae (19, 21, 22).To investigate the helical rows in more detail, we performed electron cryotomography of isolated mitochondrial membranes from Paramecium tetraurelia. Using subtomogram averaging, we show that these helical rows do indeed consist of ATP synthase molecules, as suggested by Allen et al. (28). However, unlike the V-shaped dimers of metazoans, the ATP synthase of this species forms U-shaped dimers, which have new and unusual structural features. When assembled into the helical rows, the ATP synthase monomers interdigitate, whereas the U-shaped dimers align side by side. Thus, rows of ATP synthase dimers seem to be a universal feature of all mitochondria. We propose that the particular shape of the P. tetraurelia ATP synthase dimer induces its assembly into helical rows, which in turn cause the formation of the helical tubular cristae of ciliates.     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or “Cos-Seq”), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.Leishmaniasis is a neglected tropical disease causing significant morbidity and mortality worldwide (1). It is caused by parasites of the genus Leishmania that cycle between the flagellar promastigote form in the gut of the insect vector and the nonmotile amastigote stage in the vertebrate host. Given the lack of an effective antileishmanial vaccine, control of leishmaniasis relies mainly on chemotherapy. Only a few antileishmanial drugs are available, and their efficacy is severely limited by toxicity, cost, and drug resistance (2, 3). New methods for expediting the discovery of drug targets and resistance mechanisms in Leishmania would aid the reassessment of current therapies and the development of new, effective drugs.Next-generation sequencing (NGS) technologies have enabled the high-throughput and genome-scale screening of eukaryotic pathogens, and have been useful in identifying drug targets and elucidating drug resistance mechanisms (4, 5). The development of RNA interference (RNAi) target sequencing (RIT-Seq) in kinetoplastid parasites, such as Trypanosoma brucei (6), has revealed numerous genes associated with drug action (7); however, RNAi-based screening is not applicable to Leishmania, because most species lack functional RNAi machinery (8). In Leishmania spp., copy number variation and single-nucleotide polymorphism were detected in drug-resistant parasites using NGS (9, 10; reviewed in ref 11). Gain-of-function screening using genomic cosmid libraries is also a proven approach to studying drug resistance in Leishmania (12). Cosmid-based functional cloning was first implemented for studying lipophosphoglycan biosynthesis (13) and later successfully applied to study nucleoside transport (14, 15) and drug resistance (16–21). The technique has proven powerful, albeit with limitations; for instance, it is not easily amenable to high-throughput screening, because clones require individual characterization, and is biased toward the selection of cosmids conferring dominant phenotypes, leaving out less conspicuous candidates.In this study, such limitations were alleviated by combining genome-wide cosmid-based functional screening with NGS, a strategy that we term Cosmid Sequencing (or “Cos-Seq”). This method allows us to study the dynamics of cosmid enrichment under selective drug pressure and to isolate an unprecedented number of both known and previously unidentified antileishmanial targets and resistance genes.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.Pathogen-containing vacuoles (PVs) provide a safe haven to many intracellular bacterial and protozoan pathogens (1). Within the vacuolar enclosure of PVs, these pathogens can accumulate nutrients required for microbial growth. Moreover, life within the vacuolar niche shields microbes from cytoplasmic immune sensors that, once activated, can trigger proinflammatory and cell-autonomous immune responses (1). Accordingly, many intracellular pathogens such as the bacterium Chlamydia trachomatis and the protozoan Toxoplasma gondii have successfully adapted to a vacuolar lifestyle.For the host to successfully combat infections with PV-resident microbes, the innate immune system must target PVs and its inhabitants for destruction. Critical mediators of host-directed attacks on PVs are two families of IFNγ-inducible GTPases: immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) (2). Members of both GTPase families play roles in host-mediated lysis of PVs, a process resulting in the release of microbes into the host cell cytoplasm, subsequent killing of PV-expelled microbes, and host cell death (3–8). Additionally, GBPs help deliver cytosolic subunits of the antimicrobial NADPH oxidase NOX2 for assembly on phagosomal membranes, orchestrate the capture of PV-resident microbes inside degradative autophagolysosomes, and promote the activation of canonical and noncanonical inflammasome pathways (5, 8–12). As a critical first step underlying most if not all of these known GBP-controlled cell-autonomous immune responses, GBPs must locate to their intracellular microbial targets.GBPs belong to the dynamin superfamily of large GTPases (13). Similar to other members of the dynamin superfamiliy, GBPs can assemble as oligomers in a nucleotide-dependent fashion (13). Binding of GTP results in dimer formation; subsequent GTP hydrolysis prompts conformational changes that enable GBPs to assemble as tetramers (14, 15). Mutations in the G domain that reduce nucleotide binding affinities and hydrolytic activity block GBP oligomerization, constrain the localization of GBPs to the cytoplasm, and prevent GBPs from binding to PV membranes (9, 15–18). These observations support a model in which GBP monomers are diffusely distributed in the cytoplasm and GBP oligomers associate with membranes. However, these observations fail to account for the specificity with which oligomeric GBPs agglomerate on PV membranes.PVs formed by C. trachomatis and T. gondii recruit not only GBPs but also members of the IRG family of IFNγ-inducible GTPase (4, 19). The IRG protein family can be divided into two subgroups: IRGM and GKS proteins (20). Whereas GKS proteins feature the canonical glycine–lysine–serine (GKS) P-loop sequence, IRGM proteins have a substitution of a lysine for a methionine in their P-loop sequence (20). IRGM and GKS proteins also differ in their subcellular localization: IRGM proteins associate with endomembranes, whereas monomeric GDP-bound GKS proteins predominantly reside within the host cell cytoplasm (4, 17, 21, 22). Once GKS proteins transition into a GTP-bound active state, they can bind to PV membranes (21). IRGM proteins inhibit this activation step and thereby guard IRGM-decorated membranes against GKS protein targeting (17, 21). Because PV membranes surrounding either C. trachomatis or T. gondii are largely devoid of IRGM proteins, they are the preferred GKS binding substrate following a “missing-self” principle of immune targeting (17, 23). In IRGM-deficient cells, however, GKS proteins enter the active state prematurely, form protein aggregates, mislocalize, and thus fail to bind to PVs (17, 21). Although these previous observations help explain how IRGM proteins promote the delivery of GKS proteins to PVs, IRGM proteins also control the subcellular localization of GBPs through an uncharacterized mechanism (6, 17, 24–26).Here, we report a previously unidentified host-directed ubiquitination pathway involved in innate immunity. We demonstrate that Chlamydia- and Toxoplasma-containing vacuoles become ubiquitin-decorated upon IFNγ priming of their host cells. IFNγ-dependent association of ubiquitin with PVs requires IFNγ-inducible IRG proteins and the E3 ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). Experimental removal of the IFNγ-inducible ubiquitination pathway dramatically diminishes the p62-dependent delivery of GBPs to PVs and thereby renders host cells more susceptible to infections. Thus, our observations imply that ubiquitin serves as a host-induced pattern that marks intracellular structures as immune targets for members of the GBP family of host defense proteins.