Bifidobacterium alters the gut microbiota and modulates the functional metabolism of T regulatory cells in the context of immune checkpoint blockade

Immune checkpoint-blocking antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe immune-related adverse events. Previously, we found that Bifidobacterium could mitigate intestinal immunopathology in the context of CTLA-4 blockade in mice. Here we examined the mechanism underlying this process. We found that Bifidobacterium altered the composition of the gut microbiota systematically in a regulatory T cell (Treg)-dependent manner. Moreover, this altered commensal community enhanced both the mitochondrial fitness and the IL-10–mediated suppressive functions of intestinal Tregs, contributing to the amelioration of colitis during immune checkpoint blockade.

Immune checkpoint blockade therapy has become a very successful cancer treatment. The first monoclonal antibody (mAb) approved for clinical use is specific for the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for melanoma treatment (1). However, the application of immune checkpoint inhibitors (ICIs) can cause various and even fatal autoimmune responses, of which diarrhea and colitis are among the most frequent and severe (2, 3).Components in the gut microbiota have been shown to regulate the host antitumor immune response (4–7), and several studies have implicated the function of the intestinal microbiota in modulating the efficacy of immune checkpoint blockade therapy (5, 8, 9). For example, the presence of Bifidobacterium can stimulate the host immune system to respond to anti–PD-L1 therapy in a CD8⁺ T cell-dependent manner (10).Although these studies have demonstrated a role for the microbiota in antitumor immunity, the underlying events related to checkpoint antibody-induced autoimmunity remain elusive. In the clinic, patients who experienced colitis after ICI treatment harbored gut bacteria compositions that are distinct from those of colitis-free patients (11). A recent study reported the first clinical case in which reconstituting the gut microbiota with fecal microbiota transplantation successfully rescued ICI-associated colitis (9). The baseline of gut microbiota was also shown to be related to that clinical response to ipilimumab, with enrichment of Faecalibacterium consistent with long-term clinical benefit and colitis (12). We previously reported that administration of Bifidobacterium attenuated intestinal inflammation without impairing the antitumor function of CTLA-4 in mice (13). Here we dissect the fundamental principles governing the relationship between the probiotic-induced microbiome optimization and the outcome of CTLA-4 blockade. We demonstrate that Bifidobacterium systematically alters the composition of the gut microbiota, profoundly increasing the other probiotic species, Lactobacillus. This microbiome optimization is dependent on the existence of regulatory T cells (Tregs). Furthermore, we found that both the metabolic and suppressive functions of intestinal Tregs are enhanced by this altered commensal community, contributing to maintaining regional immune homeostasis under the CTLA-4 blockade condition. Taken together, our observations reveal an immunologic principle governing the complex functions of microbiota dynamics, as well as a mechanism for the relay from Bifidobacterium to Lactobacillus in ameliorating immune checkpoint blockade-related colitis.     

CODY enables quantitatively spatiotemporal predictions on in vivo gut microbial variability induced by diet intervention

Microbial variations in the human gut are harbored in temporal and spatial heterogeneity, and quantitative prediction of spatiotemporal dynamic changes in the gut microbiota is imperative for development of tailored microbiome-directed therapeutics treatments, e.g. precision nutrition. Given the high-degree complexity of microbial variations, subject to the dynamic interactions among host, microbial, and environmental factors, identifying how microbiota colonize in the gut represents an important challenge. Here we present COmputing the DYnamics of microbiota (CODY), a multiscale framework that integrates species-level modeling of microbial dynamics and ecosystem-level interactions into a mathematical model that characterizes spatial-specific in vivo microbial residence in the colon as impacted by host physiology. The framework quantifies spatiotemporal resolution of microbial variations on species-level abundance profiles across site-specific colon regions and in feces, independent of a priori knowledge. We demonstrated the effectiveness of CODY using cross-sectional data from two longitudinal metagenomics studies—the microbiota development during early infancy and during short-term diet intervention of obese adults. For each cohort, CODY correctly predicts the microbial variations in response to diet intervention, as validated by available metagenomics and metabolomics data. Model simulations provide insight into the biogeographical heterogeneity among lumen, mucus, and feces, which provides insight into how host physical forces and spatial structure are shaping microbial structure and functionality.

Changes in the human gut microbiome composition are connected with development of numerous diseases, like obesity, type-2 diabetes, and immune dysfunction (1–3). Quantitative understanding and predicting how microbial variations are determined are crucial for designing microbiome-directed therapies that target chronic metabolic diseases (4, 5). However, this remains challenging due to the temporal and spatial heterogeneity along the human gut resulting from a dynamic interplay among host, microbial, and environmental conditions (6, 7). Diet is recognized as a controllable and pivotal environmental factor in shaping longitudinal microbial landscape development (8, 9), such as early childhood colonization (10) and long-term adulthood stabilization (11). While profiling of fecal samples enables a snapshot of consequential changes of the fecal microbiota in response to different stimuli, e.g. dietary changes (12–14), it is still far from describing the intrinsic dynamics of how microbiome colonize in the gut. Recently, the spatial heterogeneity of microbial composition between lumen and mucus has been recognized in mice (15, 16), but similar studies in humans is impossible with current techniques. In addition, measurements of absolute abundance profiles are required to correct the artifacts associated with relative abundance that confound revealing the interplay between microbial variations and health (17). Therefore, methods that enable quantifying the absolute, temporal, and spatial variations of in vivo human gut microbiota are needed to understand how to maintain or restore healthy microbiota.Computational models are widely used to decipher microbial complexity and response to perturbations (18). Most existing models have limited usage as they only address specific elements of the multidimensional interaction mechanisms. For example, similarity-based (19) and rule-mining models (20) describe microbial–microbial interactions without considering temporal dependency. The dynamic Bayesian model enables incorporation of directed interactions and longitudinal dataset (21), while reliance on training dataset and difficulties in model selection render these stochastic models confining to specific statistic condition and predictions are therefore not consistent and generalizable (22). The generalized Lotka–Volterra model (18, 23, 24) represents a step forward to simulate dynamics via formulating microbial growth rate as a lumped term, but adherence to assumptions of pairwise interactions-driven community dynamics and constant environment limits their predictive power. Genome-scale models (GEMs) (25) provide a valuable resource for studying structured microbial metabolism. With GEMs, microbial metabolic capacity, microbe–microbe interactions (26–28), microbial–diet interactions (12), and structural changes of two-species cocultures (29) are characterized using flux balance analysis (FBA). With rare exceptions, FBA requires a priori knowledge of metabolite uptake fluxes distributed among community members, with current limitations on these resources, faces challenges with modeling multispecies communities in a dynamic manner (30). Therefore, in adapting a computational framework that can simulate microbiome dynamics along the human gut, one encounters three challenges: 1) endogenously, the intrinsic dynamics not only emerge from the large number of microbiota components but also through the intricate and dynamic ways they interact (31, 32); 2) exogenously, the microbiota is exposed to a series of host–microbe metabolic axes (33), such as colonic physical forces (34), nascent colonization, and nutrient conditions; and 3) spatial structure of the in vivo microbiota localization plays a significant role impacting 1 and 2 (24).Here, we bridge the current theoretical gap by developing a multiscale framework for COmputing the DYnamics of gut microbiota (CODY), which enables identification and quantification of spatiotemporal-specific variations of gut microbiome absolute and relative abundance profiles, without a prior knowledge of microbiome interactions. We evaluated CODY’s performance by comparing model simulations with longitudinal changes in the microbial composition in fecal samples and in plasma metabolomics of two cohorts: 1) long-term development of the gut microbiome in early infancy and 2) short-term variation patterns of the gut microbiome in obese adults experiencing diet intervention. Comparison of model simulations with experimental data demonstrated predictive strength of the CODY modeling framework and hence lays the foundation for performing design of microbiome-directed therapeutics or of precision nutrition based on CODY simulations. The source code of CODY is freely available together with full documentation at https://github.com/JunGeng-Sysbio-Chalmers/CODY1.0_SourceCode.     

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Diet crucially affects the health of all animals (1). Most animals have a gut microbiota that can also affect host health both positively and negatively (2–6). However, understanding of the mechanisms underlying the effects of the gut microbiota remains a major challenge. This is because animals often consume complex or variable diets, and harbor large, multimember microbial communities that can result in many interactions that hinder identification of the factors responsible for particular host responses (2, 6–11). Metaanalyses and multiomic approaches can provide inferential insights on how diet–microbe or microbe–microbe interactions affect hosts (11–18), but functional support can be difficult to generate if proposed mechanisms cannot be studied experimentally (2, 14). Thus, study systems where hosts can be reared on defined diets with or without a microbiota of known composition can significantly advance mechanistic insights by providing the means to control and manipulate dietary, microbial, and environmental variables that potentially affect a given host response (19–21).Mosquitoes are best known as insects that blood feed on humans and other vertebrates. Only adult-stage female mosquitoes blood feed, which is required for egg formation by most species (22). Blood feeding has also led to several mosquitoes evolving into vectors that can transmit disease-causing microbes between hosts (22). In contrast, the juvenile stages of all mosquitoes are aquatic, with most species feeding on detritivorous diets (22–24). Larvae hatch from eggs with no gut microbiota but quickly acquire relatively low-diversity communities from the environment by feeding (25). Most gut community members are aerobic or facultatively anaerobic bacteria in four phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria), although other microbes, such as fungi and apicomplexans, have also been identified (25–39). Gut community composition also commonly varies within and between species as a function of where larvae develop, diet, and other variables (28–30, 32, 34, 40–42).Aedes aegypti has a worldwide distribution in tropical and subtropical regions, and is the primary vector of the agents that cause yellow fever, dengue fever, and lymphatic filariasis in humans (43). Preferentially living in urban habitats, females lay eggs in water-holding containers with microbial communities, and larvae molt through four instars before pupating and emerging as adults (30, 35, 41, 43). Conventionally reared cultures with a gut microbiota are usually maintained in the laboratory under conditions that mimic natural habitats with rearing temperatures of 25 to 28 °C and a 12- to 16-h light: 8- to 12-h dark photoperiod (44–46). Most insects that require microbial partners for survival live on nutrient-poor diets where microbes provision nutrients that cannot be synthesized or produced in sufficient abundance by the host (3). Mosquito larvae can experience resource limitations in the field (23–25), but in the laboratory are reared on undefined, nutrient-rich diets, such as rodent chow, fish food flakes, or mixtures of materials like liver powder, fish meal, and yeast extract (44–46). Nonetheless, our previous studies indicated that axenic A. aegypti as well as other species consume but fail to grow beyond the first instar when fed several diets that support the development of nonsterile, conventionally reared larvae (30, 47–49). Escherichia coli and several other bacteria identified as gut community members could colonize the gut (producing monoxenic, gnotobiotic larvae) and rescue development, but feeding axenic larvae dead bacteria could not (30, 35, 47). The presence of a gut microbiota in conventional and gnotobiotic but not axenic larvae was also associated with midgut hypoxia and activation of several signaling pathways with growth functions (50, 51). Finally, our own previous results using a strain of E. coli susceptible to ampicillin (50), and more recently a method for clearing an auxotrophic strain of E. coli from gnotobiotic larvae (52), both showed that the proportion of individuals that develop into adults correlates with the duration that larvae have living bacteria in their gut.Altogether, the preceding results suggested that A. aegypti and several other mosquitoes require a gut microbiota for development. In contrast, another recent study showed that axenic A. aegypti larvae develop into adults, albeit more slowly than larvae with a gut microbiota, when fed diets comprised of autoclaved bovine liver powder (LP) and brewer’s yeast (Saccharomyces cerevisiae) extract (YE) or autoclaved LP, YE, and E. coli (EC) embedded in agar (53). This latter finding suggests the undefined dietary components used provide factors larvae require for development into adults, whereas a gut microbiota was also required to provide these factors under the conditions in which our own previous studies were conducted. The goal of this study was to identify what these factors are. Toward this end, we first assessed the growth of axenic A. aegypti when fed diets containing autoclaved LP, YE, and EC under different conditions. We then used this information to develop a defined diet that allowed us to systematically manipulate nutrient, microbial, and environmental variables. We report that the instability of riboflavin is a key factor underlying why A. aegypti larvae require a gut microbiota under most conditions experienced in the laboratory and field.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.     

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles

Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.

Viruses are intracellular parasites which co-opt cellular resources to produce abundant viral progeny. Positive-strand (+)RNA viruses replicate on subcellular membranes by forming viral replication organelles (VROs) (1–5). VROs sequester the viral proteins and viral RNAs together with co-opted host factors to provide an optimal subcellular environment for the assembly of numerous viral replicase complexes (VRCs), which are then responsible for robust viral RNA replication. VROs also spatially and temporally organize viral replication. Importantly, the VROs hide the viral RNAs from cellular defense mechanisms as well (5, 6). VROs consist of extensively remodeled membranes with unique lipid composition. How viruses achieve these membrane remodeling and lipid modifications and lipid enrichment is incompletely understood. Therefore, currently, there is a major ongoing effort to dissect the VRC assembly process and to understand the roles of viral and host factors in driving the biogenesis of VROs (1, 3, 7).Tomato bushy stunt virus (TBSV), a plant-infecting tombusvirus, has been shown to induce complex rearrangements of cellular membranes and alterations in lipid and other metabolic processes during infections (8–10). The VROs formed during TBSV infections include extensive membrane contact sites (vMCSs) and harbor numerous spherules (containing VRCs), which are vesicle-like invaginations in the peroxisomal membranes (8, 11–13). A major gap in our understanding of the biogenesis of VROs, including vMCSs and VRCs, is how the cellular lipid-modifying enzymes are recruited to the sites of viral replication.Tombusviruses belong to the large Flavivirus-like supergroup that includes important human, animal, and plant pathogens. Tombusviruses have a small single-component (+)RNA genome of ∼4.8 kb that codes for five proteins. Among those, there are two essential replication proteins, namely p33 and p92^pol, the latter of which is the RdRp protein and it is translated from the genomic RNA via readthrough of the translational stop codon in p33 open reading frame (14). The smaller p33 replication protein is an RNA chaperone, which mediates the selection of the viral (+)RNA for replication (14–16). Altogether, p33 is the master regulator of VRO biogenesis (3). We utilize a TBSV replicon (rep)RNA, which contains four noncontiguous segments from the genomic RNA, and it can efficiently replicate in yeast and plant cells expressing p33 and p92^pol (14, 17).Tombusviruses hijack various cellular compartments and pathways for VRO biogenesis (18). These include peroxisomes by TBSV or mitochondria (in the case of the closely related carnation Italian ringspot virus [CIRV]), the endoplasmic reticulum (ER) network, Rab1-positive COPII vesicles, and the Rab5-positive endosomes (8, 19–23). Tombusviruses also induce membrane proliferation, new lipid synthesis, and enrichment of lipids, most importantly phosphatidylethanolamine (PE), sterols, phosphatidylinositol-4-phosphate [PI(4)P], and phosphatidylinositol-3-phosphate [PI(3)P] phosphoinositides in peroxisomal or mitochondrial membranes for different tombusviruses (13, 24–27). This raised the question that how TBSV could hijack lipid synthesis enzymes from other subcellular locations that leads to enrichment of critical lipids in the large VROs in model yeast and plant hosts.The endosomal network (i.e., early, late, and recycling endosomes) is a collection of pleomorphic organelles which sort membrane-bound proteins and lipids either for vacuolar/lysosomal degradation or recycling to other organelles. With the help of the so-called retromer complex, tubular transport carriers formed from the endosomes recycle cargoes to the Golgi and ER or to the plasma membrane (28–31). The core retromer complex consists of three conserved proteins, Vps26, Vps29, and Vps35, which are involved in cargo sorting and selection. The retromer complex affects several cellular processes, including autophagy through the maturation of lysosomes (32), neurodegenerative diseases (33), plant root hair growth (34), and plant immunity (35).The cellular retromer is important for several pathogen–host interactions. For example, the retromer is targeted by Brucella, Salmonella, and Legionella bacteria (36–39) and the rice blast fungus (40). The retromer is also involved in the intracellular transport of the Shigella and Cholera toxins and the plant ricin toxin. The NS5A replication protein of hepatitis C virus (HCV) interacts with Vps35 and this interaction is important for HCV replication in human cells (41). The cytoplasmic tail of the Env protein of HIV-1 binds to the retromer components Vps35 and Vps26, which is required for Env trafficking and infectious HIV-1 morphogenesis (42). Moreover, the retromer complex affects the morphogenesis of vaccinia virus (43) and HPV16 human papillomavirus entry and delivery to the trans-Golgi network (44). Despite the importance of the retromer in pathogen–host interactions, the mechanistic insights are far from complete.In the case of tombusviruses, enrichment of PE and PI(3)P within VROs is facilitated by co-opting the endosomal Rab5 small GTPase and Vps34 PI3K (20, 24), suggesting that the endosome-mediated trafficking pathway might be involved in viral replication in host cells. However, the actual mechanism of how tombusviruses exploit the endosomal/endocytic pathway and induce lipid enrichment within VROs is not yet dissected. Therefore, in this work, we targeted the retromer complex, based on previous genome-wide screens using yeast gene-deletion libraries, which led to the identification of VPS29 and VPS35 as host genes affecting TBSV replication and recombination, respectively (45, 46). These proteins are components of the retromer complex (28–31). We found TBSV and the closely related CIRV co-opt the retromer complex for the biogenesis of VROs in yeast and plants. We observed that depletion of retromer proteins strongly inhibited TBSV and CIRV replication. The recruitment of the retromer is driven by the viral p33 replication protein, which interacts with Vps26, Vps29, and Vps35 retromer proteins. We show that the retromer helps delivering critical cargo proteins, such as Psd2 phosphatidylserine decarboxylase, Vps34 phosphatidylinositol 3-kinase (PI3K), and Stt4 phosphatidylinositol 4-kinase (PI4Kα-like). These co-opted cellular enzymes are then involved in de novo production and enrichment of PE phospholipid, PI(3)P, and PI(4)P phosphoinositides within the VROs. Altogether, these virus-driven activities create an optimized membrane microenvironment within VROs to support efficient tombusvirus replication.     

Mesoscale networks and corresponding transitions from self-assembly of block copolymers

A series of cubic network phases was obtained from the self-assembly of a single-composition lamellae (L)-forming block copolymer (BCP) polystyrene-block-polydimethylsiloxane (PS-b-PDMS) through solution casting using a PS-selective solvent. An unusual network phase in diblock copolymers, double-primitive phase (DP) with space group of Im3¯m, can be observed. With the reduction of solvent evaporation rate for solution casting, a double-diamond phase (DD) with space group of Pn3¯m can be formed. By taking advantage of thermal annealing, order–order transitions from the DP and DD phases to a double-gyroid phase (DG) with space group of Ia3¯d can be identified. The order–order transitions from DP (hexapod network) to DD (tetrapod network), and finally to DG (trigonal planar network) are attributed to the reduction of the degree of packing frustration within the junction (node), different from the predicted Bonnet transformation from DD to DG, and finally to DP based on enthalpic consideration only. This discovery suggests a new methodology to acquire various network phases from a simple diblock system by kinetically controlling self-assembling process.

From constituted molecules to polymers, finally ordered hierarchical superstructures, self-assembled solids cover a vast area of nanostructures where the characters of building blocks direct the progress of self-assembly (1, 2). In nature, fascinating periodic network structures and morphologies from different species are appealing in nanoscience and nanotechnology due to their superior properties, especially for photonic crystal structures (3–7). For gyroid, trigonal planar network with chirality demonstrates its potential as chiropitc metamaterial (8–10). Beyond the splendid colors, networks either in macroscale or mesoscale mechanically strengthen their skeletons and protect those fragile but vital organs from impact (11, 12). Inspired by nature, biomimicking materials with mesoscale network may exceed the limitation of the intrinsic properties (13). The topology of networks could further improve their adaptability, allowing extreme deformation for energy dissipation (14). Moreover, network materials from hybridization of self-assembled block copolymers (BCPs) have been exploited to the design of mesoscale quantum metamaterials (15, 16). With the desire to acquire network textures for biomimicking nanomaterials, BCPs with immiscible constituted segments covalently joined together give the accessibility to the formation of nanonetwork morphologies via balancing enthalpic penalty from the repulsive interaction of constituted blocks and entropic penalty from the stretching of polymer chains (17–21). By taking advantage of precise synthesis procedures, it is feasible to obtain the aimed network phases from the self-assembly of BCPs such as Fddd (O⁷⁰) (22–24), gyroid (Q²¹⁴, Q²³⁰) (20, 21, 25–27), and diamond (Q²²⁴, Q²²⁷) (28–31) experimentally and theoretically. On the basis of theoretical prediction, the junction points (nodes) in the network phases could be coordinated with three, four, or six neighbors in three-dimensional space, resulting in the enhancement of packing frustration (31). Topologically, all these phases match the coordination number to neighbors (n = 3, 4, 6), showing no special case of quasicrystal. Accordingly, an order–order transition from double-diamond phase (DD, tetrapod) to double-gyroid phase (DG, trigonal planar network) has been observed (29). Yet, there is no DP phase being found in simple diblock systems except for liquid crystals (32, 33) or organic–inorganic nanocomposites from the mixtures of BCP with inorganic precursors (34, 35). Searching the rare occurrence of network phases and the corresponding phase transitions among phases will be essential to the demands for application by considering the deliberate structuring effects on aimed properties but the approaches remain challenging (8, 36–40). For instance, viewing the narrow window for network morphologies in diblock copolymer phase diagram, it demands harsh requirements for syntheses (2, 41). Recently, by taking advantage of using selective solvent for solution casting, it is feasible to acquire DG phase and even inverted DG phase from the self-assembly of lamellae (L)-forming polystyrene-block-polydimethylsiloxane (PS-b-PDMS) (42). Apart from that, a triclinic DG phase was recently discovered from the PS-b-PDMS which is commonly believed nonexisting in the conventional phase diagram (43). As a result, the phase diagram of BCPs with high interaction parameter is worthy of study for searching the metastable phases with unique network textures (44). Herein, we aim to acquire network phases from a simple diblock system by kinetically controlling the transformation mechanisms of self-assembly. As exemplified by using the PS-b-PDMS for solution casting, with the use of a PS-selective solvent (chloroform), a DP phase and a DD phase could be formed through controlled self-assembly, giving unique network phases simply from solution casting. Moreover, a DG phase can be also acquired from phase transformation. Consequently, a series of network phases with hexapod, tetrapod, and trigonal planar building units could be successfully obtained by using a single-composition L-forming PS-b-PDMS for self-assembly. The corresponding order–order transitions among these network phases examined by temperature-resolved in situ small-angle X-ray scattering (SAXS) combining with electron tomography results provide insights of network phase formation and the corresponding phase transformation mechanisms in the self-assembly of BCPs.     

Orthosteric–allosteric dual inhibitors of PfHT1 as selective antimalarial agents

Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a “selective starvation” strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure–activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum. Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.

Plasmodium falciparum is the deadliest species of Plasmodium, responsible for around 50% of human malaria cases and nearly all malarial death (1). Despite intensive malaria-eradication efforts to control the spread of this disease, malaria prevalence remains alarmingly high, with 228 million cases and a fatality tally of 405,000 in 2018 alone (2). The situation has become even more daunting as resistance to the first-line antimalarial agents has emerged and is rapidly spreading. For instance, artemisinin resistance, primarily mediated by P. falciparum Kelch13 (PF3D7_1343700) propeller domain mutations (3, 4), severely compromises the campaign of antimalarial chemotherapy (5–9). Novel antimalarial agents overcoming the drug resistance are therefore urgently needed (10).The blood-stage malaria parasites depend on a constant glucose supply as their primary source of energy (11). P. falciparum hexose transporter 1 (PfHT1; PF3D7_0204700) (12) is transcribed from a single-copy gene with no close paralogue (13) and has been genetically validated as essential for the survival of the blood-stage parasite (14). A possible approach to kill the parasite is to “starve it out” by the chemical intervention of the parasite hexose transporter (13, 15). The feasibility of this approach would depend on the successful development of selective PfHT1 inhibitors that do not affect the activities of human hexose transporter orthologs (e.g., human glucose transporter 1 [hGLUT1]).Previously, Compound 3361 (C3361) (15), a glucose analog, has been reported to moderately inhibit PfHT1 and suppress the growth of blood-stage parasites in vitro (16). Nonetheless, the modest potency and selectivity of C3361 had limited its further development. Structural determination of PfHT1 and human glucose transporters provides an unprecedented opportunity for rational design of PfHT1-specific inhibitors (17–20). While hGLUT1 is the primary glucose transporter in erythrocyte, its structure was determined only in the inward-open state (17). Fortunately, the neuronal glucose transporter hGLUT3, which shares over 80% sequence similarity with hGLUT1, was captured in both outward-open and outward-occluded conformations (18). A reliable homology model of outward-facing hGLUT1 could thus be generated based on the structure of hGLUT3.Comparing the structures of PfHT1 (19, 20) and hGLUT1, we identified an additional pocket adjacent to the substrate-binding site. Coadministration of allosteric and orthosteric drugs is generally applied to tackle drug resistance when these two pockets were spatially separated (21). However, this discovery led to a hypothesis that simultaneously targeting the orthosteric and allosteric sites by tethering a pharmacophore to the carbohydrate core might render selective inhibitors for PfHT1. Based on this hypothesis, we designed a class of small molecules containing a sugar moiety and an allosteric pocket-occupying motif connected by a flexible linker. Among them, TH-PF01, TH-PF02, and TH-PF03 have exhibited selective biophysical and antiplasmodial activities with moderate cytotoxicity. Furthermore, in silico computational simulations also confirmed their binding mode, lending further support to the dual-inhibitor design. Taken together, our studies validated an antimalaria development strategy that simultaneously targets the orthosteric and allosteric sites of PfHT1.