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.

Predation of oomycetes by myxobacteria via a specialized CAZyme system arising from adaptive evolution.

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized ß-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target ß-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain ß-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.

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.

Social Diversification Driven by Mobile Genetic Elements.

Social diversification in microbes is an evolutionary process where lineages bifurcate into distinct populations that cooperate with themselves but not with other groups. In bacteria, this is frequently driven by horizontal transfer of mobile genetic elements (MGEs). Here, the resulting acquisition of new genes changes the recipient's social traits and consequently how they interact with kin. These changes include discriminating behaviors mediated by newly acquired effectors. Since the producing cell is protected by cognate immunity factors, these selfish elements benefit from selective discrimination against recent ancestors, thus facilitating their proliferation and benefiting the host. Whether social diversification benefits the population at large is less obvious. The widespread use of next-generation sequencing has recently provided new insights into population dynamics in natural habitats and the roles MGEs play. MGEs belong to accessory genomes, which often constitute the majority of the pangenome of a taxon, and contain most of the kin-discriminating loci that fuel rapid social diversification. We further discuss mechanisms of diversification and its consequences to populations and conclude with a case study involving myxobacteria.

Conditional and Synthetic Type IV Pili-Dependent Motility Phenotypes in Myxococcus xanthus.

Myxobacteria exhibit a variety of complex social behaviors that all depend on coordinated movement of cells on solid surfaces. The cooperative nature of cell movements is known as social (S)-motility. This system is powered by cycles of type IV pili (Tfp) extension and retraction. Exopolysaccharide (EPS) also serves as a matrix to hold cells together. Here, we characterized a new S-motility gene in Myxococcus xanthus. This mutant is temperature-sensitive (Ts-) for S-motility; however, Tfp and EPS are made. A 1 bp deletion was mapped to the MXAN_4099 locus and the gene was named sglS. Null mutations in sglS exhibit a synthetic enhanced phenotype with a null sglT mutation, a previously characterized S-motility gene that exhibits a similar Ts- phenotype. Our results suggest that SglS and SglT contribute toward Tfp function at high temperatures in redundant pathways. However, at low temperatures only one pathway is necessary for wild-type S-motility, while in the double mutant, motility is nearly abolished at low temperatures. Interestingly, the few cells that do move do so with a high reversal frequency. We suggest SglS and SglT play conditional roles facilitating Tfp retraction and hence motility in M. xanthus.

Mapping the Influence of the Gut Microbiota on Small Molecules across the Microbiome Gut Brain Axis.

Microbes exert influence across the microbiome-gut-brain axis through neurotransmitter production, induction of host immunomodulators, or the release or induction of other microbial or host molecules. Here, we used mass spectrometry imaging (MSI), a label-free imaging tool, to map molecular changes in the gut and brain in germ-free, antibiotic-treated and control mice. We determined spatial distribution and relative quantification of neurotransmitters and their precursors in response to the microbiome. Using untargeted MSI, we detected a significant change in the levels of four identified small molecules in the brains of germ-free animals compared to controls. However, antibiotic treatment induced no significant changes in these same metabolites in the brain after 1 week of treatment. This work exemplifies the utility of MSI as a tool for the study of known and discovery of novel, mediators of microbiome-gut-brain axis communication.

Mesoscopic Structures and Coexisting Phases in Silica Films.

Silica films represent a unique two-dimensional film system, exhibiting both crystalline and vitreous forms. While much scientific work has focused on the atomic-scale features of this film system, mesoscale structures can play an important role for understanding confined space reactions and other applications of silica films. Here, we report on mesoscale structures in silica films grown under ultrahigh vacuum and examined with scanning tunneling microscopy (STM). Silica films can exhibit coexisting phases of monolayer, zigzag, and bilayer structures. Both holes in the film structure and atomic-scale substrate steps are observed to influence these coexisting phases. In particular, film regions bordering holes in silica bilayer films exhibit vitreous character, even in regions where the majority film structure is crystalline. At high coverages mixed zigzag and bilayer phases are observed at step edges, while at lower coverages silica phases with lower silicon densities are observed more prevalently near step edges. The STM images reveal that silica films exhibit rich structural diversity at the mesoscale.

Emergent Myxobacterial Behaviors Arise from Reversal Suppression Induced by Kin Contacts.

A wide range of biological systems, from microbial swarms to bird flocks, display emergent behaviors driven by coordinated movement of individuals. To this end, individual organisms interact by recognizing their kin and adjusting their motility based on others around them. However, even in the best-studied systems, the mechanistic basis of the interplay between kin recognition and motility coordination is not understood. Here, using a combination of experiments and mathematical modeling, we uncover the mechanism of an emergent social behavior in Myxococcus xanthus. By overexpressing the cell surface adhesins TraA and TraB, which are involved in kin recognition, large numbers of cells adhere to one another and form organized macroscopic circular aggregates that spin clockwise or counterclockwise. Mechanistically, TraAB adhesion results in sustained cell-cell contacts that trigger cells to suppress cell reversals, and circular aggregates form as the result of cells' ability to follow their own cellular slime trails. Furthermore, our in silico simulations demonstrate a remarkable ability to predict self-organization patterns when phenotypically distinct strains are mixed. For example, defying naive expectations, both models and experiments found that strains engineered to overexpress different and incompatible TraAB adhesins nevertheless form mixed circular aggregates. Therefore, this work provides key mechanistic insights into M. xanthus social interactions and demonstrates how local cell contacts induce emergent collective behaviors by millions of cells. IMPORTANCE In many species, large populations exhibit emergent behaviors whereby all related individuals move in unison. For example, fish in schools can all dart in one direction simultaneously to avoid a predator. Currently, it is impossible to explain how such animals recognize kin through brain cognition and elicit such behaviors at a molecular level. However, microbes also recognize kin and exhibit emergent collective behaviors that are experimentally tractable. Here, using a model social bacterium, we engineer dispersed individuals to organize into synchronized collectives that create emergent patterns. With experimental and mathematical approaches, we explain how this occurs at both molecular and population levels. The results demonstrate how the combination of local physical interactions triggers intracellular signaling, which in turn leads to emergent behaviors on a population scale.

Scalable Biosynthetic Production of Knotted Peptides Enables ADME and Thermodynamic Folding Studies.

Knotted peptides present a wealth of structurally diverse, biologically active molecules, with the inhibitor cystine knot/knottin class among the most ecologically common ones. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. Such compounds are often produced in low concentrations by intractable organisms, making structural and biological characterization challenging, which is frequently overcome by various expression strategies. Here, we sought to test a biosynthetic route for the expression and study of knotted peptides. We screened expression constructs for a biosynthesized knotted peptide to determine the most influential parameters for successful disulfide folding and used NMR spectroscopic fingerprinting to validate topological structures. We performed pharmacokinetic characterization, which indicated that the interlocking disulfide structure minimizes liabilities of linear peptide sequences, and propose a mechanism by which knotted peptides are cleared. We then developed an assay to monitor solution folding in real time, providing a strategy for studying the folding process during maturation, which provided direct evidence for the importance of backbone organization as the driving force for topology formation.

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.

Modular Lipoprotein Toxins Transferred by Outer Membrane Exchange Target Discrete Cell Entry Pathways.

Bacteria compete against related individuals by delivering toxins. In myxobacteria, a key delivery and kin discrimination mechanism is called outer membrane (OM) exchange (OME). Here, cells that display compatible polymorphic cell surface receptors recognize one another and bidirectionally transfer OM content. Included in the cargo is a suite of polymorphic SitA lipoprotein toxins. Consequently, OME between compatible cells that are not clonemates results in intoxication, while exchange between clonemates is harmonious because cells express a cognate repertoire of immunity proteins, which themselves are not transferred. SitA toxins belong to six nonhomologous families classified by sequence conservation within their N-terminal "escort domains" (EDs), while their C termini contain polymorphic nucleases that target the cytoplasmic compartment. To investigate how toxins delivered to the OM by OME translocate to the cytoplasm, we selected transposon mutants resistant to each family. Our screens identified eight genes that conferred resistance in a SitA family-specific manner. Most of these genes are predicted to localize to the cell envelope, and some resemble proteins that colicins exploit to gain cell entry. By constructing functional chimeric SitAs between families, we show that the ED determines the specificity of resistance. Importantly, a mutant that confers resistance to all six SitA families was discovered. This gene was named traC and plays an accessory role with traAB in OME. This work thus provides insight into the mechanism of kin discrimination in myxobacteria and provides working models for how SitA toxins exploit host proteins to gain cytoplasmic entry. IMPORTANCE Many bacterial species use diverse systems to deliver bacteriocins or toxins to neighboring competing cells. These systems are often selective in targeting cells that are related to themselves and therefore compete in the same niches for resources. How these systems specifically identify target cells and deliver toxins to particular cellular compartments is a fundamental question. This study uses the model social bacterium Myxococcus xanthus to unravel how its kin discrimination system, called outer membrane exchange, works. Along with the TraA polymorphic cell surface receptor that identifies related individuals with compatible receptors, this work discovered a new protein, called TraC, that functions in this discrimination system. Additionally, genetic screens identified host factors that are proposed to be involved in the cytoplasmic entry of lipoprotein toxins from the OM. This work complements and broadens our mechanistic understanding of how bacteria use transport systems to discriminate against related foes to build clonal populations.

Intestinal-derived ILCs migrating in lymph increase IFNγ production in response to Salmonella Typhimurium infection.

Innate lymphoid cells (ILCs) are enriched in mucosae and have been described as tissue-resident. Interestingly, ILCs are also present within lymph nodes (LNs), in the interfollicular regions, the destination for lymph-migratory cells. We have previously shown that LN ILCs are supplemented by peripheral tissue-derived ILCs. Using thoracic duct cannulations, we here enumerate the intestinal lymph ILCs that traffic from the intestine to the mesenteric LNs (MLNs). We provide, for the first time, a detailed characterisation of these lymph-migratory ILCs. We show that all ILC subsets migrate in lymph, and while global transcriptional analysis reveals a shared signature with tissue-resident ILCs, lymph ILCs express migration-associated genes including S1PRs, SELL (CD62L) and CCR7. Interestingly, we discovered that while Salmonella Typhimurium infections do not increase the numbers of migrating ILCs, infection changes their composition and cytokine profile. Infection increases the proportions of RORyt+ T-bet+ ILCs, levels of IFNγ, and IFNγ/GM-CSF co-expression. Infection-induced changes in migratory ILCs are reflected in colon-draining MLN ILCs, where RORyt+ T-bet+ ILCs accumulate and display corresponding increased cytokine expression. Thus, we reveal that ILCs respond rapidly to intestinal infection and can migrate to the MLN where they produce cytokines.

Kin recognition and outer membrane exchange (OME) in myxobacteria.

Myxobacteria conduct complex social traits that requires populations to be highly related and devoid of exploiters. To enrich for clonal cells in populations, they employ kin discrimination mechanisms. One key system involves a polymorphic cell surface receptor, TraA, which recognizes self by homotypic interactions with neighboring myxobacterial cells. Recent studies revealed that TraA and its partner TraB are fluid outer membrane proteins that coalesce into foci upon recognition of kin. The formation of foci leads to transient membrane fusion junctions and the bidirectional exchange of outer membrane components that facilitates cooperative behaviors. Additionally, expansive suites of polymorphic lipoprotein toxins are exchanged, which act as self-identity barcodes that exquisitely discriminate against nonself to assemble homogenous populations.

Rapid diversification of wild social groups driven by toxin-immunity loci on mobile genetic elements.

Many species form distinct social groups that provide fitness advantages to individuals. However, the evolutionary processes that generate new social groups are not well understood. Here we examined recently diverged natural isolates of the model social bacterium, Myxococcus xanthus, to probe the genetic mechanisms and evolutionary processes of kin discrimination that occurred naturally in soil. We show that social incompatibilities were formed from horizontal gene transfer of effectors belonging to three distinct polymorphic toxin systems; outer membrane exchange, type VI secretion and rearrangement hotspot systems. Strikingly, the unique toxin effectors and their respective immunity genes that are responsible for social incompatibilities reside on mobile genetic elements, which make up nearly all of the genotypic variation between isolates within clades. By disrupting these three toxin systems, we engineered social harmony between strains that were originally incompatible. In addition, a horizontal allele swap of a single kin recognition receptor changed social interactions and competition outcomes. Our results provide a case study for how horizontal gene transfer led to social diversification in a natural context. Finally, we show how genomic information of kin discriminatory loci can be used to predict social interactions.

Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection.

FoxP3+ regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are poorly understood. Th1, Th2, and Th17 cells can be identified by master transcription factors (TFs) T-bet, GATA3, and RORγT; Tregs also express these TFs. While T-bet+ Tregs can selectively suppress Th1 cells, it is unclear whether distinct Treg populations can alter Th bias. To address this, we used Salmonella enterica serotype Typhimurium to induce nonlethal colitis. Following infection, we observed an early colonic Th17 response within total CD4 T cells, followed by a Th1 bias. The early Th17 response, which contains both Salmonella-specific and non-Salmonella-specific cells, parallels an increase in T-bet+ Tregs. Later, Th1 cells and RORγT+ Tregs dominate. This reciprocal dynamic may indicate that Tregs selectively suppress Th cells, shaping the immune response. Treg depletion 1-2 days post-infection shifted the early Th17 response to a Th1 bias; however, Treg depletion 6-7 days post-infection abrogated the Th1 bias. Thus, Tregs are necessary for the early Th17 response, and for a maximal Th1 response later. These data show that Tregs shape the overall tissue CD4 T cell response and highlight the potential for subpopulations of Tregs to be used in targeted therapeutic approaches.

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.

Microbiome-derived carnitine mimics as previously unknown mediators of gut-brain axis communication.

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen-free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

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.