Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn's disease associated adherent-invasive Escherichia coli infection.

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohn's disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNFα), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

Synthesis of 1H-isoindolin-1-ones via a simple photodecarboxylative addition of carboxylates to phthalimides and evaluation of their antibiotic activity.

A variety of 3-hydroxy-isoindolin-1-one derivatives were synthesized using the photodecarboxylative addition of carboxylates to phthalimide derivatives in aqueous media. Subsequent acid-catalyzed dehydration furnished 3-(alkyl and aryl)methyleneisoindolin-1-ones with variable E-diastereoselectivity in good to excellent overall yields. Noteworthy, the parent 3-phenylmethyleneisoindolin-1-one underwent isomerization and oxidative decomposition when exposed to light and air. Selected 3-hydroxy-isoindolin-1-one and 3-(alkyl and aryl)methyleneisoindolin-1-one derivatives showed moderate antibacterial activity that justifies future elaboration and study of these important bioactive scaffolds.

Two reasons to kill: predation and kin discrimination in myxobacteria.

Myxobacteria are social microbial predators that use cell-cell contacts to identify bacterial or fungal prey and to differentiate kin relatives to initiate cellular responses. For prey killing, they assemble Tad-like and type III-like secretion systems at contact sites. For kin discrimination (KD), they assemble outer membrane exchange complexes composed of the TraA and TraB receptors at contacts sites. A type VI secretion system and Rhs proteins also mediate KD. Following cellular recognition, these systems deliver appropriate effectors into target cells. For prey, this leads to cell death and lysis for nutrient consumption by myxobacteria. In KD, a panel of effectors are delivered, and if adjacent cells are clonal cells, resistance ensues because they express a cognate panel of immunity factors; while nonkin lack complete immunity and are intoxicated. This review compares and contrasts recent findings from these systems in myxobacteria.

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

Information acquisition and decision strategies in intertemporal choice.

Intertemporal decision models describe choices between outcomes with different delays. While these models mainly focus on predicting choices, they make implicit assumptions about how people acquire and process information. A link between information processing and choice model predictions is necessary for a complete mechanistic account of decision making. We establish this link by fitting 18 intertemporal choice models to experimental datasets with both choice and information acquisition data. First, we show that choice models have highly correlated fits: people that behave according to one model also behave according to other models that make similar information processing assumptions. Second, we develop and fit an attention model to information acquisition data. Critically, the attention model parameters predict which type of intertemporal choice models best describes a participant's choices. Overall, our results relate attentional processes to models of intertemporal choice, providing a stepping stone towards a complete mechanistic account of intertemporal decision making.

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

The Fluidity of the Bacterial Outer Membrane Is Species Specific: Bacterial Lifestyles and the Emergence of a Fluid Outer Membrane.

The outer membrane (OM) is an essential barrier that guards Gram-negative bacteria from diverse environmental insults. Besides functioning as a chemical gatekeeper, the OM also contributes towards the strength and stiffness of cells and allows them to sustain mechanical stress. Largely influenced by studies of Escherichia coli, the OM is viewed as a rigid barrier where OM proteins and lipopolysaccharides display restricted mobility. Here the discussion is extended to other bacterial species, with a focus on Myxococcus xanthus. In contrast to the rigid OM paradigm, myxobacteria possess a relatively fluid OM. It is concluded that the fluidity of the OM varies across environmental species, which is likely linked to their evolution and adaptation to specific ecological niches. Importantly, a fluid OM can endow bacteria with distinct functions for cell-cell and cell-environment interactions.

Self-identity barcodes encoded by six expansive polymorphic toxin families discriminate kin in myxobacteria.

Myxobacteria are an example of how single-cell individuals can transition into multicellular life by an aggregation strategy. For these and all organisms that consist of social groups of cells, discrimination against, and exclusion of, nonself is critical. In myxobacteria, TraA is a polymorphic cell surface receptor that identifies kin by homotypic binding, and in so doing exchanges outer membrane (OM) proteins and lipids between cells with compatible receptors. However, TraA variability alone is not sufficient to discriminate against all cells, as traA allele diversity is not necessarily high among local strains. To increase discrimination ability, myxobacteria include polymorphic OM lipoprotein toxins called SitA in their delivered cargo, which poison recipient cells that lack the cognate, allele-specific SitI immunity protein. We previously characterized 3 SitAI toxin/immunity pairs that belong to 2 families. Here, we discover 4 additional SitA families. Each family is unique in sequence, but share the characteristic features of SitA: OM-associated toxins delivered by TraA. We demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strikingly numerous and diverse, with most genomes possessing >30 and up to 83 distinct sitAI loci. Interestingly, all SitA protein families are serially transferred between cells, allowing a SitA inhibitor cell to poison multiple targets, including cells that never made direct contact. The expansive suites of sitAI loci thus serve as identify barcodes to exquisitely discriminate against nonself to ensure populations are genetically homogenous to conduct cooperative behaviors.

MYXO-CTERM sorting tag directs proteins to the cell surface via the type II secretion system.

Cells interact with their surrounding environment through surface proteins. However, knowledge gaps remain in understanding how these important types of proteins are transported and anchored on the cell surface. In the Gram-negative social bacterium, Myxococcus xanthus, a putative C-terminal sorting tag (MYXO-CTERM) is predicted to help direct 34 different proteins onto the cell surface. Here we investigate the sorting pathway for MYXO-CTERM proteins by using the TraA cell surface receptor as a paradigm. Deleting this motif from TraA abolishes the cell surface anchoring and results in extracellular secretion. Our findings indicate that conserved cysteines within the MYXO-CTERM are posttranslationally modified and are required for TraA cell surface localization and function. A region immediately upstream of these residues is predicted to be disordered and removing this motif caused a secretion defect and blocked cell surface anchoring. We further show that the type II secretion system is required for translocation across the outer membrane and that a cysteine-rich region directs TraA to the T2SS. Similar results were found with another MYXO-CTERM protein indicating our findings can be generalized. Further, we show the universal distribution of MXYO-CTERM motif across the Myxococcales order and provide a working model for sorting of these proteins.

Kin Recognition in Bacteria.

The ability of bacteria to recognize kin provides a means to form social groups. In turn these groups can lead to cooperative behaviors that surpass the ability of the individual. Kin recognition involves specific biochemical interactions between a receptor(s) and an identification molecule(s). Recognition specificity, ensuring that nonkin are excluded and kin are included, is critical and depends on the number of loci and polymorphisms involved. After recognition and biochemical perception, the common ensuing cooperative behaviors include biofilm formation, quorum responses, development, and swarming motility. Although kin recognition is a fundamental mechanism through which cells might interact, microbiologists are only beginning to explore the topic. This review considers both molecular and theoretical aspects of bacterial kin recognition. Consideration is also given to bacterial diversity, genetic relatedness, kin selection theory, and mechanisms of recognition.

Salmonella enterica Serovar Typhimurium Travels to Mesenteric Lymph Nodes Both with Host Cells and Autonomously.

Salmonella infection is a globally important cause of gastroenteritis and systemic disease and is a useful tool to study immune responses in the intestine. Although mechanisms leading to immune responses against Salmonella have been extensively studied, questions remain about how bacteria travel from the intestinal mucosa to the mesenteric lymph nodes (MLN), a key site for Ag presentation. In this study, we used a mouse model of infection with Salmonella enterica serovar Typhimurium (STM) to identify changes in intestinal immune cells induced during early infection. We then used fluorescently labeled STM to identify interactions with immune cells from the site of infection through migration in lymph to the MLN. We show that viable STM can be carried in the lymph by any subset of migrating dendritic cells but not by macrophages. Moreover, approximately half of the STM in lymph are not associated with cells at all and travel autonomously. Within the MLN, STM associates with dendritic cells and B cells but predominantly with MLN-resident macrophages. In conclusion, we describe the routes used by STM to spread systemically in the period immediately postinfection. This deeper understanding of the infection process could open new avenues for controlling it.

Conditional requirement of SglT for type IV pili function and S-motility in Myxococcus xanthus.

Myxobacteria exhibit complex social behaviors such as predation, outer membrane exchange and fruiting body formation. These behaviors depend on coordinated movements of cells on solid surfaces that involve social (S) motility. S-motility is powered by extension-retraction cycles of type 4 pili (Tfp) and exopolysaccharides (EPS) that provide a matrix for group cellular movement. Here, we characterized a new class of S-motility mutants in Myxococcus xanthus. These mutants have a distinctive phenotype: they lack S-motility even though they produce pili and EPS and the phenotype is temperature-sensitive. The point mutations were mapped to a single locus, MXAN_3284, named sglT. Similar to pilT mutants, sglT mutants are hyperpiliated and, strikingly, the temperature-sensitive phenotype is caused by null mutations. Our results indicate that SglT plays a critical role in Tfp function associated with pilus retraction and that the block in pili retraction is caused by a Tfp assembly defect in the absence of SglT at high-temperature growth.

Self-identity reprogrammed by a single residue switch in a cell surface receptor of a social bacterium.

The ability to recognize close kin confers survival benefits on single-celled microbes that live in complex and changing environments. Microbial kinship detection relies on perceptible cues that reflect relatedness between individuals, although the mechanisms underlying recognition in natural populations remain poorly understood. In myxobacteria, cells identify related individuals through a polymorphic cell surface receptor, TraA. Recognition of compatible receptors leads to outer membrane exchange among clonemates and fitness consequences. Here, we investigated how a single receptor creates a diversity in recognition across myxobacterial populations. We first show that TraA requires its partner protein TraB to function in cell-cell adhesion. Recognition is shown to be traA allele-specific, where polymorphisms within TraA dictate binding selectivity. We reveal the malleability of TraA recognition, and seemingly minor changes to its variable region reprogram recognition outcomes. Strikingly, we identify a single residue (A/P205) as a molecular switch for TraA recognition. Substitutions at this position change the specificity of a diverse panel of environmental TraA receptors. In addition, we engineered a receptor with unique specificity by simply creating an A205P substitution, suggesting that modest changes in TraA can lead to diversification of new recognition groups in nature. We hypothesize that the malleable property of TraA has allowed it to evolve and create social barriers between myxobacterial populations and in turn avoid adverse interactions with relatives.

Search predicts and changes patience in intertemporal choice.

Intertemporal choice impacts many important outcomes, such as decisions about health, education, wealth, and the environment. However, the psychological processes underlying decisions involving outcomes at different points in time remain unclear, limiting opportunities to intervene and improve people's patience. This research examines information-search strategies used during intertemporal choice and their impact on decisions. In experiment 1, we demonstrate that search strategies vary substantially across individuals. We subsequently identify two distinct search strategies across individuals. Comparative searchers, who compare features across options, discount future options less and are more susceptible to acceleration versus delay framing than integrative searchers, who integrate the features of an option. Experiment 2 manipulates search using an unobtrusive method to establish a causal relationship between strategy and choice, randomly assigning participants to conditions promoting either comparative or integrative search. Again, comparative search promotes greater patience than integrative search. Additionally, when participants adopt a comparative search strategy, they also exhibit greater effects of acceleration versus delay framing. Although most participants reported that the manipulation did not change their behavior, promoting comparative search decreased discounting of future rewards substantially and speeded patient choices. These findings highlight the central role that heterogeneity in psychological processes plays in shaping intertemporal choice. Importantly, these results indicate that theories that ignore variability in search strategies may be inadvertently aggregating over different subpopulations that use very different processes. The findings also inform interventions in choice architecture to increase patience and improve consumer welfare.

Cell-cell recognition and social networking in bacteria.

The ability to recognize self and to recognize partnering cells allows microorganisms to build social networks that perform functions beyond the capabilities of the individual. In bacteria, recognition typically involves genetic determinants that provide cell surface receptors or diffusible signalling chemicals to identify proximal cells at the molecular level that can participate in cooperative processes. Social networks also rely on discriminating mechanisms to exclude competing cells from joining and exploiting their groups. In addition to their appropriate genotypes, cell-cell recognition also requires compatible phenotypes, which vary according to environmental cues or exposures as well as stochastic processes that lead to heterogeneity and potential disharmony in the population. Understanding how bacteria identify their social partners and how they synchronize their behaviours to conduct multicellular functions is an expanding field of research. Here, we review recent progress in the field and contrast the various strategies used in recognition and behavioural networking.

Tissue repair in myxobacteria: A cooperative strategy to heal cellular damage.

Damage repair is a fundamental requirement of all life as organisms find themselves in challenging and fluctuating environments. In particular, damage to the barrier between an organism and its environment (e.g. skin, plasma membrane, bacterial cell envelope) is frequent because these organs/organelles directly interact with the external world. Here, we discuss the general strategies that bacteria use to cope with damage to their cell envelope and their repair limits. We then describe a novel damage-coping mechanism used by multicellular myxobacteria. We propose that cell-cell transfer of membrane material within a population serves as a wound-healing strategy and provide evidence for its utility. We suggest that--similar to how tissues in eukaryotes have evolved cooperative methods of damage repair--so too have some bacteria that live a multicellular lifestyle.

Cell rejuvenation and social behaviors promoted by LPS exchange in myxobacteria.

Bacterial cells in their native environments must cope with factors that compromise the integrity of the cell. The mechanisms of coping with damage in a social or multicellular context are poorly understood. Here we investigated how a model social bacterium, Myxococcus xanthus, approaches this problem. We focused on the social behavior of outer membrane exchange (OME), in which cells transiently fuse and exchange their outer membrane (OM) contents. This behavior requires TraA, a homophilic cell surface receptor that identifies kin based on similarities in a polymorphic region, and the TraB cohort protein. As observed by electron microscopy, TraAB overexpression catalyzed a prefusion OM junction between cells. We then showed that damage sustained by the OM of one population was repaired by OME with a healthy population. Specifically, LPS mutants that were defective in motility and sporulation were rescued by OME with healthy donors. In addition, a mutant with a conditional lethal mutation in lpxC, an essential gene required for lipid A biosynthesis, was rescued by Tra-dependent interactions with a healthy population. Furthermore, lpxC cells with damaged OMs, which were more susceptible to antibiotics, had resistance conferred to them by OME with healthy donors. We also show that OME has beneficial fitness consequences to all cells. Here, in merged populations of damaged and healthy cells, OME catalyzed a dilution of OM damage, increasing developmental sporulation outcomes of the combined population by allowing it to reach a threshold density. We propose that OME is a mechanism that myxobacteria use to overcome cell damage and to transition to a multicellular organism.

Mechanism of Kin-Discriminatory Demarcation Line Formation between Colonies of Swarming Bacteria.

Swarming bacteria use kin discrimination to preferentially associate with their clonemates for certain cooperative behaviors. Kin discrimination can manifest as an apparent demarcation line (a region lacking cells or with much lower cell density) between antagonist strains swarming toward each other. In contrast, two identical strains merge with no demarcation. Experimental studies suggest contact-dependent killing between different strains as a mechanism of kin discrimination, but it is not clear whether this killing is sufficient to explain the observed patterns. Here, we investigate the formation of demarcation line with a mathematical model. First, using data from competition experiments between kin discriminating strains of Myxococcus xanthus and Proteus mirabilis, we found the rates of killing between the strains to be highly asymmetric, i.e., one strain kills another at a much higher rate. Then, to investigate how such asymmetric interactions can lead to a stable demarcation line, we construct reaction-diffusion models for colony expansion of kin-discriminatory strains. Our results demonstrate that a stable demarcation line can form when both cell movement and cell growth cease at low nutrient levels. Further, our study suggests that, depending on the initial separation between the inoculated colonies, the demarcation line may move transiently before stabilizing. We validated these model predictions by observing dynamics of merger between two M. xanthus strains, where one strain expresses a toxin protein that kills a second strain lacking the corresponding antitoxin. Our study therefore provides a theoretical understanding of demarcation line formation between kin-discriminatory populations, and can be used for analyzing and designing future experiments.

SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection.

Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors.

Central effects of insulin detemir on feeding, body weight, and metabolism in rats.

Insulin detemir (DET) is a basal insulin analog that, in contrast to other long-acting forms of insulin, has significant weight-gain-sparing effects in diabetic patients. We hypothesized that this effect of DET may be due to its enhanced catabolic action in the central nervous system. We investigated the long-term effects of single third ventricular (3V) microinjections of equimolar doses of DET and regular insulin in normal male rats on feeding, body weight, energy expenditure (EE), and respiratory quotient (RQ). Also, in acute testing, we assessed the ability of lower doses of DET to alter feeding, EE, and RQ when microinjected directly into the paraventricular nucleus (PVN). The anabolic peptide ghrelin served as a positive control in acute testing. 3V administration of both DET (0.5-2.0 mU) and regular insulin (2.0-8.0 mU) significantly reduced feeding and body weight over 48 and 120 h, respectively, with DET yielding greater inhibitory effects. DET also stimulated greater elevations of EE and reductions of RQ over 72 and 48 h postinjection, respectively. In acute (4 h) testing, microinjections of DET (0.5 mU) into the PVN reduced feeding, increased EE, and reduced RQ, while ghrelin (100 pmol) had the opposite effects. When administered sequentially into the PVN, DET (0.25 and 0.5 mU) reversed ghrelin-induced feeding, EE, and RQ effects. These data support the notion that the weight-sparing effect of DET is at least in part based on its central catabolic action and that enhanced EE and reduced RQ may participate in this effect.