Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of Salmonella flagellin inside macrophages

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella. These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.

The innate immune system senses microbial pathogens through recognition of conserved entities collectively referred to as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). These entities interact with conserved pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), Nod-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), and C-type lectin receptors (CLRs) that are expressed by immune cells and other cell types. Activation of PRRs by PAMPs is dictated by the availability and expression levels of PAMPs at different stages of infection and results in host responses which are vital for inflammation and immunity against pathogens (1). However, some pathogens, including Salmonella spp., a facultative intracellular pathogen, have evolved the ability to use these host responses for their own replication and establishment of infection (2).Flagellin, the monomeric protein constituting bacterial flagella, is one of the key Salmonella effector molecules which binds and activates membrane-bound TLR-5 as well as the cytosolic sensor NLRC4 and plays a major role in generating inflammatory responses (3–5). In macrophages, flagellin as well as the rod protein PrgJ, which are inadvertently released into the host cytosol by the type III secretion system (T3SS), are detected by the NAIPs. In mice, seven NAIPs are present of which NAIP1 senses the T3SS needle protein, NAIP2 detects the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin (6–9). Humans however encode a single functional NAIP which has been recently shown to broadly detect multiple T3SS proteins and flagellin (10). Ligand binding to the NAIPs leads to recruitment and oligomerization of NLRC4 (11, 12). Activation of the NAIP-NLRC4 inflammasome by these effectors and activation of the NLRP3 inflammasome by an as yet unidentified aconitase-regulated Salmonella effector (13–15) results in caspase-1-dependent pyroptosis and production of active IL-1β which promotes clearance of the bacterium and protects the host against Salmonella (13, 16, 17). It is believed that as infection progresses, Salmonella circumvents this host-protective response by suppressing the expression of flagellin to lower than the resting levels usually expressed by bacteria in culture (18). Down-regulation of flagellin is essential for the bacterium to establish successful infection. Previous work has shown that a Salmonella Typhimurium strain modified to constitutively express flagellin (ST-FliC^ON) and therefore unable to naturally down-regulate flagellin expression is avirulent and cleared successfully from the host compared to its wild-type (WT) counterpart (17). Despite this central role of flagellin in Salmonella pathogenesis, the molecular mechanisms that regulate the physiological switch of Salmonella from a flagellin-high to a flagellin-low phenotype and aid in establishment of an intracellular niche within macrophages in vivo are incompletely understood.Upon entry into macrophages, Salmonella resides in a vacuole called the Salmonella-containing vacuole (SCV) where it shuts down expression of the Salmonella pathogenicity island 1 (SPI-1) and concomitantly switches on expression of Salmonella pathogenicity island 2 (SPI-2), which is activated by the PhoP/PhoQ two-component system (19) and encodes genes required for intracellular replication. Prior work has shown that shutdown of SPI-1 in growth media that mimic conditions associated with the SCV such as acidic pH and low Mg²⁺ is also accompanied by repression of flagellin (20, 21). This is because low pH and low Mg²⁺ activate the PhoP/PhoQ system (20, 22, 23) and activated PhoP is believed to suppress expression of flagellin (21). A noteworthy issue relating to these early studies is that effects on PhoP/PhoQ-regulated genes were examined only during in vitro culture of bacteria in growth medium and not in the context of S. Typhimurium residence within macrophages. Therefore, the physiological contribution of these mechanisms to flagellin repression of intracellular Salmonella remains debatable. For example, contrary studies have shown that the effect of low pH on flagellin protein expression is observed only at a very low pH (pH = 3) and not at pH 5 (20) which is close to the physiologically relevant pH of the SCV (24, 25). Likewise, neither variation of extracellular Mg²⁺ nor reduced Mg²⁺ in the SCV was found to play a role in PhoP activation by Salmonella inside macrophages (26). Consequently, the regulatory mechanisms conventionally thought to repress flagellin expression by Salmonella remain controversial and there is scarce evidence to suggest that these factors are responsible for down-regulation of flagellin by bacteria residing within macrophages. Moreover, the physiological mechanisms that regulate repression of flagellin in vivo are unknown.In this study we describe a host innate immune circuit that regulates expression of Salmonella flagellin during both the early/extracellular and the later/intracellular phases of macrophage infection with this pathogen. We find that during early infection of macrophages with S. Typhimurium, rapid NLRC4 inflammasome-dependent macrophage pyroptosis is necessary and sufficient for releasing a host lysophospholipid stimulus that promotes synthesis and release of flagellin from Salmonella. Unexpectedly, these host factors regulate not only the initial increase in flagellin production but also the later down-regulation of flagellin by Salmonella inside macrophages. This later effect is mediated by a natural type 1 IFN-dependent host negative feedback response that represses expression of NLRC4 and the lysophospholipid biosynthetic enzyme calcium-independent phospholipase A2 (iPLA2) within cells, causing a decline in intracellular lysophospholipids over time, which promotes eventual down-regulation of flagellin by intracellular bacteria. Our data identify host NLRC4 inflammasome activity as a temporal and biphasic regulator of expression of its own bacterial ligand, flagellin. We also describe a physiologically relevant type 1 IFN-mediated host mechanism that controls switching of Salmonella from a flagellin-high to a flagellin-low phenotype within macrophages in vivo. These findings have important implications for understanding the intricate evolutionary adaptations that shape host–pathogen cross-talk.     

SIK2 orchestrates actin-dependent host response upon Salmonella infection

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Salmonella enterica is a gram-negative, facultative intracellular human pathogen, annually causing more than 100 million food- and waterborne infections worldwide. Salmonella Typhimurium causes severe gastroenteritis, which could turn into a systemic infection in children, immune-compromised, or elderly people (1, 2). Concurrently, multidrug resistant bacteria are globally emerging and threatening our health systems, calling for a better understanding of the underlying virulence mechanism and host response.Pathogenic bacteria have evolved the inherent ability to infect and to establish their niche within host cells. For colonizing nonphagocytic cells such as epithelial cells, Salmonella uses a trigger mechanism–based entry mode. Bacterial virulence factors are then injected via a Type III-secretion system (T3SS) into the host cell to induce cytoskeletal rearrangements leading to membrane ruffling and macropinocytosis-driven internalization into a sealed phagosome (3, 4). This specialized compartment is referred to as the Salmonella-containing vacuole (SCV) and serves as the intracellular replicative niche by hiding the bacteria from the humoral and cell-autonomous immune response (5). Salmonella invasion requires a cooperative action of several bacterial effector proteins hijacking multiple host targets. One of the main targets forcing Salmonella´s uptake is the actin cytoskeleton by subverting the host Rho GTPases system. Bacterial effector proteins such as SopE/SopE2 mimic host nucleotide exchange factors (GEFs) to stimulate Rac1 and CDC42 activity (6, 7). Once GTP-activated, Rho GTPases stimulate downstream pathways to drive actin filament (F-actin) assembly and rearrangement.The actin cytoskeleton network is regulated by actin-binding proteins (ABPs), which orchestrate assembly and disassembly of actin in higher networks (8). Monomeric, globular actin (G-actin) is nucleated into new actin filaments, or the existing F-actin is elongated, stabilized, or disassembled by ABPs. The major actin nucleation factor is the multimeric Arp2/3 complex, which generates branched actin filament networks. Formins generate long unbranched actin filaments and represent another actin nucleation family. Together with actin nucleation-promoting factors, small Rho GTPases control ABPs in a spatiotemporal manner. Actin polymerization and membrane ruffling are necessary for Salmonella invasion. Following Salmonella internalization, the SCV undergoes SPI-1–dependent biogenesis and is transported to a juxtanuclear position at 1 to 2 h postinfection (pi). At later time-points (4 to 6 h pi), SPI-2–dependent effector proteins are expressed to further mature the SCV, allowing bacterial proliferation. Pioneering work described that, at later stages of the infection (≥6 h pi), an actin meshwork around the SCV stabilizes and protects the vacuolar niche (9–13).Here, we report SIK2 as a Salmonella resistance factor and a regulator of the actin cytoskeleton. SIK2 belongs to the AMPK kinase family and was named after its homolog SIK1, found to be expressed upon high-salt diet-induced stress in rats (14, 15). SIK2 has been implicated into multiple biological roles including melanogenesis, cancer progression, and gluconeogenesis (16–18). SIK2 depletion results in a loss of SCV integrity and bacterial escape into the host cytosol, causing intracellular Salmonella hyperproliferation. Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. Hence, SIK2 may represent a cellular safeguard, which controls the actin cytoskeleton and SCV integrity, thereby serving as a prime regulator of Salmonella proliferation subsequent to cellular internalization.     

A rate threshold mechanism regulates MAPK stress signaling and survival

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

All cells employ signal transduction pathways to respond to physiologically relevant changes in extracellular stressors, nutrient levels, hormones, morphogens, and other stimuli that vary as functions of both concentration and rate in healthy and diseased states (1–7). Switch-like “instantaneous” changes in the concentrations of stimuli in the extracellular environment have been widely used to show that the strength of signaling and overall cellular response are dependent on the stimulus concentration, which in many cases needs to exceed a certain threshold (8, 9). Previous studies have shown that the rate of stimulation can also influence signaling output in a variety of pathways (10–17) and that stimulation profiles of varying rates can be used to probe underlying signaling pathway circuitry (4, 18, 19). However, it is still not clear how cells integrate information conveyed by changes in both the stimulation rate and concentration in determining signaling output. It is also not clear if cells require stimulation gradients to exceed a certain rate in order to commence signaling.Recent investigations have demonstrated that stimulation rate can be a determining factor in signal transduction. In contrast to switch-like perturbations, which trigger a broad set of stress-response pathways, slow stimulation rates activate a specific response to the stress applied in Bacillus subtilis cells (10). Meanwhile, shallow morphogen gradient stimulation fails to activate developmental pathways in mouse myoblast cells in culture, even when concentrations sufficient for activation during pulsed treatment are delivered (12). These observations raise the possibility that stimulation profiles must exceed a set minimum rate or rate threshold to achieve signaling activation. Although such rate thresholds would help cells decide if and how to respond to dynamic changes in stimulus concentration, the possibility of signaling regulation by a rate threshold has never been directly investigated in any system. Further, no study has experimentally examined how stimulation rate requirements impact cell phenotype or how cells molecularly regulate the stimulation rate required for signaling activation. As such, the biological significance of any existing rate threshold regulation of signaling remains unknown.The budding yeast Saccharomyces cerevisiae high osmolarity glycerol (HOG) pathway provides an ideal model system for addressing these issues (Fig. 1A). The evolutionarily conserved mitogen-activated protein kinase (MAPK) Hog1 serves as the central signaling mediator of this pathway (20–22). It is well established that instantaneous increases in osmotic stress concentration induce Hog1 phosphorylation, activation, and translocation to the nucleus (18, 21, 23–30). Activated Hog1 governs the majority of the cellular osmoadaptation response that enables cells to survive (23, 31, 32). Multiple apparently redundant MAPK phosphatases dephosphorylate and inactivate Hog1, which, along with the termination of upstream signaling after adaptation, results in its return to the cytosol (Fig. 1A) (23, 25, 26, 33–39). Because of this behavior, time-lapse analysis of Hog1 nuclear enrichment in single cells has proven an excellent and sensitive way to monitor signaling responses to dynamic stimulation patterns in real time (18, 27–30, 40, 41). Further, such assays have been readily combined with traditional growth and molecular genetic approaches to link observed signaling responses with cell behavior and signaling pathway architecture (27–29).Open in a separate windowFig. 1.Hog1 signaling and cell survival are sensitive to the rate of preconditioning osmotic stress application. (A) Schematic of the budding yeast HOG response. (B) Preconditioning protection assay workflow indicating the first stress treatments to a final concentration of 0.4 M NaCl (Left), high-stress exposure (Middle), and colony formation readout (Right). (C) High-stress survival as a function of each first treatment relative to the untreated first stress condition. Bars and errors are means and SD from three biological replicates. *Statistically significant by Kolmogorov–Smirnov test (P < 0.05). NS = not significant. (D) Treatment concentration over time. (E) Treatment rate over time for quadratic and pulse treatment. The rate for the pulse is briefly infinite (blue vertical line) before it drops to 0. (F) Hog1 nuclear localization during the treatments depicted in D and E. (Inset) Localization pattern in the quadratic-treated sample. Lines represent means and shaded error represents the SD from three to four biological replicates.Here, we use systematically designed osmotic stress treatments imposed at varying rates of increase to show that a rate threshold condition regulates yeast high-stress survival and Hog1 MAPK signaling. We demonstrate that only stimulus profiles that satisfy both this rate threshold condition and a concentration threshold condition result in robust signaling. We go on to show that the protein tyrosine phosphatase Ptp2, but not the related Ptp3 phosphatase, serves as a major rate threshold regulator. By expressing PTP2 under the control of a series of different enhancer–promoter DNA constructs, we demonstrate that changes in the level of Ptp2 expression can alter the stimulation rate required for signaling induction and survival. These findings establish rate thresholds as a critical and regulated component of signaling biology akin to concentration thresholds.     

Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

Eurasia is the largest landmass on Earth and has a rich demographic, cultural, and economic history. It was one of the birthplaces of agriculture and hosts the richest variety of crops, including vegetable crops (1–3). Eurasia is also the likely area of origin of many plant pathogens (4–6), but there have been few detailed studies of exactly when and where these pathogens emerged. Possibly owing to a lack of genetic sequence data, there also remains a poor understanding of the migration pathways of plant viruses and other plant pathogens in Eurasia.Turnip mosaic potyvirus (TuMV) is probably the most widespread and important plant virus and occurs throughout subtropical and temperate regions (7). It mainly damages dicotyledonous domestic brassica crops, which are important food vegetables and were probably developed from wild Brassica plants by plant breeders during the expansion of agriculture. First described from brassica crops in 1921 in the United States (8, 9), TuMV has been ranked behind only cucumber mosaic cucumovirus as the most important virus infecting field-grown vegetables (10, 11).Brassica vegetables, the main host plants of TuMV in modern agriculture (7, 10), mostly originated in the Mediterranean and Western Eurasia (12, 13). These economically important plants are commonly known as cabbages or mustard plants and include turnip, cauliflower, and broccoli. A possible wild ancestor of cabbage was originally found in Western and Southern Europe (14). Brassica vegetables then spread to East Asia, including Japan and Korea, while the first record of the cultivation of Chinese cabbage dates from the 15th century CE. The edible radish (Raphanus sativus) possibly originated from ancestral wild radish (Raphanus raphanistrum) in the Mediterranean region and was domesticated in Asia prior to Roman times (15).Within the genus Potyvirus (family Potyviridae), TuMV is closely related to viruses from monocotyledonous narcissus, scallion, wild onion, and yam to form the TuMV phylogenetic group (16–19). Potyviruses infect a wide range of flowering plants (16, 19, 20) and are spread by aphids in a nonpersistent manner and sometimes in seeds and infected living plant materials. They have a genome that is a single-stranded, positive-sense RNA of ∼10,000 nucleotides (nt). The genome has one major open reading frame (ORF), which is translated into one large polyprotein, and a small overlapping ORF, a “pretty interesting Potyviridae ORF” (21). The polyprotein is autocatalytically hydrolyzed into at least 10 mature proteins (19, 22).In terms of its evolution and epidemiology, the potyvirus TuMV is among the best studied of the plant-infecting RNA viruses. We have shown that it probably originated from a virus of wild orchids in Europe (7, 23). While adapting to wild and domestic Brassicaceae plants, TuMV spread from its center of emergence in Southern Europe, Asia Minor, and the Middle East to other parts of the world, including East Asia (24–27), Oceania (28), and the Americas (7, 29). However, the evolution and epidemiology of this virus in Eurasia remain poorly studied, leaving considerable uncertainty about its past migration pathways and its evolutionary dynamics and timescale.In this study, we collected TuMV isolates from locations throughout Eurasia, including countries neighboring the center of emergence. We examined the biological characteristics of viral isolates that had been sampled from these countries across half a century of growing seasons. By analyzing 579 whole genome sequences, we estimated the evolutionary rate and timescale of TuMV and inferred its phylodynamic and phylogeographic history. Our study presents one of the largest and most detailed evolutionary and epidemiological analyses of a plant pathogen, providing a comprehensive picture of the temporal and spatial spread of a major plant pathogen across Eurasia.     

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.     

GABAergic synapses suppress intestinal innate immunity via insulin signaling in Caenorhabditis elegans

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABA_AR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.

Innate immunity, an evolutionally conserved behavior, constitutes the first defense line of multiple organisms to prevent microbial infections (1). The nematode Caenorhabditis elegans has been used as a model host for human opportunistic pathogen Pseudomonas aeruginosa infection (2) to identify evolutionarily conserved mechanisms of innate immunity. Typically, p38/PMK-1 mitogen-activated protein kinases (MAPKs) (3) and insulin/insulin-like signaling (IIS)/DAF-2 signaling cascades are recognized as two key components of the C. elegans intestinal innate immune response upon P. aeruginosa strain PA14 infection (4), as they are in mammals (3, 4). Moreover, increasing evidence has revealed several neural mechanisms as also being involved in the regulation of innate immunity. For example, G protein–coupled receptor (GPCR) NPR-1– and soluble guanylate cyclase GCY-35–expressing sensory neurons actively suppress the immune response of nonneuronal tissues (5). Additionally, a putative octopamine GPCR, OCTR-1, which is expressed and functions in the C. elegans sensory neurons ASH and ASI (6), down-regulates the unfolded protein response genes pqn/abu to further suppress the immune response of nonneuronal tissues (5, 6).Recent studies demonstrate that dopaminergic signaling inhibits innate immunity (7) whereas neuronal acetylcholine stimulates muscarinic signaling in the epithelium and activates the epithelial canonical Wnt pathway to promote the ability to defend against bacterial infection (8). Moreover, insulin-like peptide INS-7 secreted by the nervous system functions in a cell-nonautonomous manner to activate the IIS/DAF-2 pathway and modulate the intestinal innate immunity of C. elegans (9).GABAergic signaling constitutes a major inhibitory neurotransmission system that plays crucial roles in the central nervous system, especially for maintaining the balance between excitation and inhibition of neuronal networks (10). Disruption of this balance is not only linked to several neuropsychiatric disorders including schizophrenia, autism, and epilepsy (11) but also implicated in autoimmune disease (12). Up to date, multiple lines of evidence have shown that GABAergic signaling cell-autonomously modulates the immune response in immune cells (13–15). However, the roles of GABAergic synapses in innate immunity remain unknown.Here, we found that the nematode C. elegans harboring a deficiency in GABAergic neuromuscular junctions (NMJs) exhibits enhanced resistance to pathogens. P. aeruginosa PA14 infection increases synaptic expression of GABAergic synaptic components at the nerve cord of worms and enhances the strength of GABAergic transmission. Moreover, we identified an insulin-like peptide, INS-31, acting downstream of GABAergic NMJs and in body wall muscle (BWM) to control intestinal innate immunity in a cell-nonautonomous manner. This work reveals a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.     

Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (S.Tm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin–dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.

Epithelial cells make up barriers that shield the interior of the body from the environment. In the homeostatic intestine, the surface of the epithelium is maximized to facilitate uptake of ingested nutrients, water, and electrolytes (1, 2). This large surface, however, makes the gut epithelium vulnerable to attack by pathogenic microorganisms. Pathogen onslaught and the ensuing inflammation causes death and loss of intestinal epithelial cells (IECs) (3, 4). This can be beneficial as damaged cells and intracellular pathogens are cleared from the mucosa. At the same time, cell loss may jeopardize epithelial integrity when insufficient IEC numbers remain to uphold the barrier. One way to prevent such an outcome would be to compact the epithelial layer in affected regions. It is well known that smooth muscle contraction results in shrinkage and compaction of the intestinal wall during inflammation (5), but whether and how the epithelium itself can alter its IEC packing upon infection appears less clear.As a system to sense mucosal intrusions, IECs express pattern recognition receptors (PRRs) [e.g., Toll-like receptors associated with cell membranes (6), and Nod-like receptors (NLRs) that form inflammasomes in the cytosol] (7–9). Epithelial recognition of pathogens through PRRs elicits a panel of countermeasures, including production of proinflammatory cytokines, chemokines, lipids (10–13), secretion of antimicrobial peptides (11, 14), and the death and expulsion of infected IECs into the lumen (12, 15, 16). An intricate cross-talk exists between IECs and immune cells residing in the underlying lamina propria (17). Epithelial defense signaling has in some cases also been shown to engage bystander epithelial cells surrounding a pathogen-infected IEC (18–20). Still, it remains poorly explored how PRR recognition of invading pathogens can instruct tissue-scale epithelial responses.Intestinal epithelial organoids (denoted “enteroids” when established from small intestinal crypts) provide a powerful experimental system to assess the behavior of untransformed epithelia in the absence of other mucosal cell types (21). Organoids can be grown as three-dimensional (3D) miniature organs within matrix domes (22, 23) or be disrupted to produce two-dimensional (2D) monolayers atop coated surfaces (24–27). By contrast to tumor cell lines, organoids maintain untransformed properties over time (28) and show reliable PRR expression patterns that mimic the intact gut epithelium (6, 9). Organoid models have for these reasons become attractive tools for physiological studies of gut infection (25, 29–32).In this work, we used time-lapse imaging to follow the tissue dynamicity of enteroid-derived mouse and human intestinal epithelia, placed atop pliable matrix supports. Upon infection with the prototypical enteropathogen Salmonella enterica serovar Typhimurium (S.Tm), we observed prompt and large epithelial contraction foci which preceded and could be uncoupled from IEC death and expulsion. Bacterial type-three secretion system (TTSS) and/or cytosolic flagellin triggered the epithelial NAIP/NLRC4 inflammasome, sublytic Gasdermin D pore formation, ion fluxes, and myosin-dependent contractions spreading from sensing events at focal epicentres. We show that this swift response allows the epithelium to increase its IEC packing at sites of infection, which may minimize the disruptive effects of subsequent cell death and expulsion.     

Molecular switch architecture determines response properties of signaling pathways

Many intracellular signaling pathways are composed of molecular switches, proteins that transition between two states—on and off. Typically, signaling is initiated when an external stimulus activates its cognate receptor that, in turn, causes downstream switches to transition from off to on using one of the following mechanisms: activation, in which the transition rate from the off state to the on state increases; derepression, in which the transition rate from the on state to the off state decreases; and concerted, in which activation and derepression operate simultaneously. We use mathematical modeling to compare these signaling mechanisms in terms of their dose–response curves, response times, and abilities to process upstream fluctuations. Our analysis elucidates several operating principles for molecular switches. First, activation increases the sensitivity of the pathway, whereas derepression decreases sensitivity. Second, activation generates response times that decrease with signal strength, whereas derepression causes response times to increase with signal strength. These opposing features allow the concerted mechanism to not only show dose–response alignment, but also to decouple the response time from stimulus strength. However, these potentially beneficial properties come at the expense of increased susceptibility to upstream fluctuations. We demonstrate that these operating principles also hold when the models are extended to include additional features, such as receptor removal, kinetic proofreading, and cascades of switches. In total, we show how the architecture of molecular switches govern their response properties. We also discuss the biological implications of our findings.

Several molecules involved in intracellular signaling pathways act as molecular switches. These are proteins that can be temporarily modified to transition between two conformations, one corresponding to an on (active) state and another to an off (inactive) state. Two prominent examples of such switches are proteins that are modified by phosphorylation and dephosphorylation and GTPases that bind nucleotides. For phosphorylation–dephosphorylation cycles, it is common for the covalent addition of a phosphate by a kinase to cause activation of the modified protein. A phosphatase removes the phosphate to turn the protein off. In the GTPase cycle, the protein is on when bound to guanosine triphosphate (GTP) and off when bound to guanosine diphosphate (GDP). The transition from the GDP-bound state to the GTP-bound state requires nucleotide exchange, whereas the transition from the GTP-bound to the GDP-bound state is achieved via hydrolysis of the γ phosphate on GTP. The basal rates of nucleotide exchange and hydrolysis are often small. These reaction rates are increased severalfold by Guanine Exchange Factors (GEFs) and GTPase Accelerating Proteins (GAPs), respectively (1, 2).A signaling pathway is often initiated upon recognition of a stimulus by its cognate receptor, which then activates a downstream switch. In principle, a switch may be turned on by three mechanisms: (a) activation, by increasing the transition rate from the off state to the on state; (b) derepression, by decreasing the transition rate from the on state to the off state; and (c) concerted activation and derepression. Examples of these three mechanisms are found in the GTPase cycles in different organisms. In animals, signaling through many pathways is initiated by G-protein-coupled receptors (GPCRs) that respond to a diverse set of external stimuli. These receptors act as GEFs to activate heterotrimeric G proteins (3–6). Thus, pathway activation relies upon increasing the transition rate from the off state to the on state. There are no GPCRs in plants and other bikonts; the nucleotide exchange occurs spontaneously, without requiring GEF activity (7–9). G proteins are kept in the off state by a repressor such as a GAP or some other protein that holds the self-activating G protein in its inactive state. In this scenario, the presence of a stimulus results in derepression, i.e., removal of the repressing activity (10–12). Concerted activation and dererpression occur in the GTPase cycle of the yeast mating-response pathway (13, 14), in which the inactive GPCRs recruit a GAP protein and act to repress, whereas active receptors have GEF activity and act to activate. Thus, perception of a stimulus leads to concerted activation and derepression by increasing GEF activity while decreasing GAP activity.These three mechanisms of signaling through molecular switches also occur in many other systems. For example, the activation mechanism described here is a simpler abstraction of a linear signaling cascade, a classical framework used to study general properties of signaling pathways (15–19), as well as to model specific signaling pathways (20–22). While derepression may seem like an unusual mechanism, it occurs in numerous important signaling pathways in plants (e.g., auxin, ethylene, gibberellin, and phytochrome), as well as gene regulation (23–27). In many of these cases, derepression occurs through a decrease in the degradation rate of a component instead of its deactivation rate. Concerted mechanisms are found in bacterial two-component systems, wherein the same component acts as kinase and phosphatase (28–35).Many previous studies have focused on the properties of a single switch mechanism without drawing comparisons between the three potential ways for initiating signaling. For example, the classical Goldbeter–Koshland model studied zero-order ultrasensitivity of an activation mechanism (15). Further analyses examined the effect of receptor numbers (36–38), feedback mechanisms (39, 40), and removal of active receptors via endocytosis and degradation (41, 42). Similarly, important properties of the concerted mechanism have been elucidated, such as its ability to perform ratiometric signaling (13, 14), to align dose responses at different stages of the signaling pathway (43), as well as its robustness (29, 44). The derepression mechanism is relatively less studied. Although there are models of G-signaling in Arabidopsis thaliana (45–47), these models have a large number of states and parameters and do not specifically examine distinct behaviors conferred by derepression.What are the evolutionary constraints that may favor activation over derepression and vice versa? Seminal studies have investigated this question for gene-regulatory networks (48–50). However, an analysis of differences in the functional characteristics of activation, derepression, and concerted mechanisms in the context of cell signaling is still lacking. To address this deficiency, we perform a systematic comparison of the three mechanisms using the following metrics: 1) dose–response, 2) response time, and 3) ability to suppress or filter stochastic fluctuations in upstream components. The rationale behind comparing dose–response curves is that they provide information about the input sensitivity range and the output dynamic range, both of which are of pharmacological importance. We supplement this comparison with response times, which provide information about the dynamics of the signaling activity. The third metric of comparison is motivated from the fact that signaling pathways are subject to intrinsic fluctuations that occur due to the stochastic nature of biochemical reactions (51–56).We construct and analyze both deterministic ordinary differential equation (ODE) models and stochastic models based on continuous-time Markov chains. We show that activation has the following two effects: It makes the switch response more sensitive than that of the receptor, and it speeds up the response with the stimulus strength. In contrast, derepression makes the switch response less sensitive than the receptor occupancy and slows down the response speed as stimulus strength increases. These counteracting behaviors of activation and derepression lead to intermediate sensitivity and intermediate response time for the concerted mechanism. In the special case of a perfect concerted mechanism (equal activation and repression), the dose–response curve of the pathway aligns with the receptor occupancy, and the response time does not depend upon the stimulus level. The noise comparison reveals that the concerted mechanism is more susceptible to fluctuations than the activation and derepression mechanisms, which perform similarly. We further show that these results qualitatively hold for more complex models, such as those incorporating receptor removal and proofreading. We finally discuss our findings to suggest reasons that might have led biological systems to evolve one of these mechanisms over the others, a question that has received considerable attention in the context of gene regulation (48–50).     

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as Mycobacterium tuberculosis accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. Escherichia coli K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by Dictyostelium discoideum or human macrophages. In contrast, Mycobacterium smegmatis and Mycobacterium tuberculosis accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by D. discoideum or macrophages. Adding extracellular polyphosphate increased E. coli survival after phagocytosis by D. discoideum and macrophages. Reducing expression of polyphosphate kinase 1 in M. smegmatis reduced extracellular polyphosphate and reduced survival in D. discoideum and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of D. discoideum and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed M. smegmatis or M. tuberculosis. D. discoideum cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed E. coli and M. smegmatis. Polyphosphate inhibited phagosome acidification and lysosome activity in D. discoideum and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation.

In metazoans, cells such as macrophages use phagocytosis to obtain nutrients, remove cell debris, and engulf and kill pathogens (1). Phagocytosis begins by recognition of particles by cell-surface receptors and engulfment of the ingested particle to form a phagosome. Ingested material in the phagolysosome is then degraded by lysosomal acid, enzymes, and oxygen radicals (2, 3). Many successful pathogens, including Mycobacterium tuberculosis, Legionella pneumophila, Neisseria gonorrhoeae, and Streptococcus pyogenes, have evolved countermeasures to evade phago-lysosomal killing, allowing the pathogen to live in the arrested phagosome (4–8). One commonly used countermeasure is to inhibit phagosome acidification and fusion with lysosomes (9–11). How pathogens inhibit this process is poorly understood.Polyphosphate is a linear chain of phosphate residues present in all kingdoms of life (12). Polyphosphate metabolism is associated with the virulence of pathogens such as Burkholderia mallei, M. tuberculosis, Salmonella enterica, Shigella flexneri, and Pseudomonas aeruginosa (13–17). In a wide range of bacteria, including pathogens, polyphosphate is synthesized from ATP by an essential enzyme polyphosphate kinase (PPK), and polyphosphate levels are maintained by exopolyphosphatase (PPX), an enzyme that degrades polyphosphate by removing terminal phosphate residues (18–22). PPK mutants of many pathogens display decreased growth, reduced sensitivity to stress and starvation, decreased survival, reduced invasiveness, defects in quorum sensing, and defects in other features associated with virulence (13, 14, 23, 24). However, the role of PPK in the survival of pathogenic bacteria is not clearly understood.The eukaryotic social amoeba D. discoideum uses phagocytosis to uptake nutrients such as bacteria (25). D. discoideum cells accumulate extracellular polyphosphate, and, as the local cell density increases, the extracellular polyphosphate concentration increases (26). To anticipate starvation when a colony of cells is about to overgrow its food supply, the concomitant high extracellular polyphosphate concentration inhibits proliferation (26). The G-protein–coupled receptor glutamate receptor-like protein D (GrlD) binds, and helps cells sense, polyphosphate (26, 27).Since not digesting nutrients might be a way to inhibit D. discoideum proliferation, we examined whether polyphosphate might inhibit phagosome maturation in D. discoideum and human macrophages. In this report, we show that both exogenous polyphosphate and polyphosphate released from bacteria inhibit phagosome maturation in D. discoideum and that this effect is conserved in human macrophages.     

Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.

Interactions between pathogens cooccurring within a single host can have profound effects on infection outcomes, ranging from the severity of clinical disease in individual hosts to the rate of disease spread across populations (1–3). Because most hosts are commonly infected by more than one type of pathogen at a time (4), understanding the consequences of pathogen interactions during concurrent infection (or coinfection) is essential for effective disease management and control. While many studies focus on pathogen interactions that are the result of one pathogen responding to the simultaneous presence of another (5), two pathogens need not overlap in time to interact with one another. For example, heterologous immunity, where prior exposure or infection with one pathogen modifies the immune response to another, can drive both positive and negative interactions between pathogens (6). This phenomenon highlights how modifications of the host immune system by one pathogen that occur during the lifetime of a host (i.e., in ecological time) can shape future responses to secondary pathogens. Likewise, strong selection pressure imposed by pathogens on hosts, particularly on immune function (7), can result in modifications of the host immune system that occur over generations (i.e., in evolutionary time), a process which should also affect responses to secondary infections. In this case, a historical population-level response to selection by one pathogen may generate heritable differences among individuals in contemporary responses to another. Crucially, ecological- vs. evolutionary-scale interactions between pathogens may operate for different reasons, so distinguishing between the two is integral to understanding both the mechanistic basis and consequences of these interactions.Helminths, or parasitic worms, and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions (2, 8). Both pathogens affect approximately one-third of the world’s human population and are widespread in domestic and wild animals (9–11). Caused by bacteria in the Mycobacterium tuberculosis complex, including M. tuberculosis (Mtb), the causative agent of human TB, and Mycobacterium bovis (Mb), the causative agent of bovine TB, TB is responsible for 2 million human deaths (12) and 25% of all disease-related cattle deaths (13) annually. In humans, about 10% of individuals infected with Mtb progress to active pulmonary disease, but the mechanisms underlying progression to active TB are poorly defined (14). Accumulating evidence suggests that coinfection with worms may be a factor in TB disease progression (2, 15), although some studies do not support this link, highlighting the complex nature of worm–TB interactions (16). Interestingly, research in laboratory animals suggests that enhanced immunity (i.e., resistance) to worms can compromise a host’s ability to control TB even in the absence of active worm infection (17–20), implying that evolved defenses against worms may independently affect the response to TB. Considered in light of widespread worm resistance in human and animal populations (21, 22) and the broad geographic coincidence of worms and TB, worm–TB interactions may represent an illustrative case where variation in evolved resistance to one pathogen (worms) contributes to variable responses to another (TB).In this study, we tested the hypothesis that resistance to worms modifies the host response to TB. To do this, we monitored gastrointestinal (GI) worm (specifically strongyle nematode) and Mb infections in a cohort of wild African buffalo (Syncerus caffer) to assess the effects of natural variation in worm resistance on the incidence, severity, and progression of bovine TB (BTB). In previous work, we demonstrated the presence of an ecological interaction between worms and BTB in buffalo by showing that clearance of active worm infection via anthelmintic treatment reduces BTB-associated mortality (23). Thus, we took advantage of the fact that half of our study animals were subject to long-term deworming to compare the relative effects of worm coinfection vs. natural worm resistance on BTB outcomes. We found evidence of a genetic basis to worm resistance in buffalo and that buffalo with resistance to worms were more severely affected by BTB in terms of both mortality risk and disease progression. However, the mechanisms by which natural variation in the host response to worms was associated with BTB progression appeared to be distinct from the effects of anthelmintic treatment. Our results suggest that negative effects of worms on BTB outcomes occur as a result of both concurrent worm infection and genetically based differences in host responsiveness to worms. This discovery fundamentally alters our understanding of the timescales over which worms and TB interact in real-world populations.     

Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.

Human rhinoviruses (RVs) are ubiquitous respiratory pathogens composing a large group of antigenically diverse, positive-strand RNA viruses classified within the Enterovirus genus of the Picornaviridae family (1, 2). The most frequent cause of the common cold, RV infections among the young are associated with the development of asthma (3, 4). In older individuals, RV infections may also lead to acute exacerbations of asthma and chronic obstructive pulmonary disease and are a significant cause of lower respiratory tract disease (5, 6). RVs are grouped phylogenetically into three species, each containing multiple serotypes (2, 7). Unlike RV-A and RV-B, which have been recognized for decades and readily propagated in conventional cell cultures, RV-C was identified more recently and replicates in vitro only in cells engineered to express a critical entry factor, cadherin-related family member 3 (CDHR3) (8). RV-C is strongly associated with severe respiratory tract infections in young children and is more closely related to RV-A than to RV-B (2, 7, 9, 10). Nearly all hospital visits related to RV-triggered asthma are due to infections with RV-A or RV-C viruses, with RV-C associated with more severe symptoms (10–12).The molecular mechanisms underlying replication of these RNA viruses are only partially understood. Enteroviral RNAs are synthesized on the cytosolic surface of membranous cytoplasmic tubulovesicular structures (13–15). These replication organelles are derived from remodeled endoplasmic reticulum (ER) or Golgi membranes and contain multiple viral nonstructural proteins, including 2B, 2C, and an RNA-dependent RNA polymerase, 3D^pol (16). The formation of replication organelles is associated with a striking reordering of cellular lipid metabolism, with phosphatidylinositol 4-kinase-IIIβ (PI4Kβ) playing a key role. PI4Kβ is recruited to membranes at the site of replication by the viral 3A protein acting in concert with host acyl-CoA binding domain-containing 3 (ACBD3) (13, 17, 18). PI4Kβ mediates the enrichment of these membranes with phosphatidylinositol 4-phosphate (PI4P), leading to subsequent recruitment of oxysterol-binding protein 1 (OSBP1), which enhances cholesterol flux into the membranes (18). Thus, ACBD3, PI4Kβ, and OSBP1 are all crucial host factors for RV replication.The intracellular replication of poliovirus, a closely-related enterovirus, is also dependent on components of host autophagic signaling, including LC3 protein that associates with the membranes of replication organelles in a nonlipidated form (19, 20). Whether this is also true for rhinoviruses is uncertain. Unlike poliovirus, RV-A1a replication is not influenced by chemical compounds that promote or inhibit autophagy, rapamycin, and 3-methyadenine (3-MA) respectively, while similar studies of RV-A2 produced conflicting results (21, 22). These latter data show that even among closely related viruses in the same picornaviral genus, host factors involved in remodeling membranes and generating replication organelles may vary substantially. Here we describe a surprising requirement for the Stimulator of Interferon Genes (STING) protein in intracellular replication of RV-A and RV-C viruses. STING (also known as MITA, ERIS, or MPYS) is an essential adaptor protein downstream of cGMP-AMP synthase (cGAS) in the innate immune cytosolic DNA-sensing pathway, and thus is typically associated with antiviral rather than proviral effects (23–27). We show that RV-A16 replication organelles are enriched in STING, and that transfected subgenomic RV-A16 and RV-C15 RNA replicons fail to amplify in the absence of STING. Genetic evidence links STING to the nonstructural 2C protein of RV-A, which is known to play a crucial role in the formation of replication organelles.     

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.