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Silver in the linings The bacterium Shewanella oneidensis is well known to use extracellular electron sinks, metal oxides and ions in nature or electrodes when cultured in a fuel cell, to power the catabolism of organic material. However, the power density of microbial fuel cells has been limited by various factors that are mostly related to connecting the microbes to the anode. Cao et al . found that a reduced graphene oxide–silver nanoparticle anode circumvents some of these issues, providing a substantial increase in current and power density (see the Perspective by Gaffney and Minteer). Electron microscopy revealed silver nanoparticles embedded or attached to the outer cell membrane, possibly facilitating electron transfer from internal electron carriers to the anode. —MAF

Most microbes in the biosphere are attached to surfaces, where they experience mechanical forces due to hydrodynamic flow and cell-to-substratum interactions. These forces likely serve as mechanical cues that influence bacterial physiology and eventually drive environmental adaptation and fitness. Mechanosensors are cellular components capable of sensing a mechanical input and serve as part of a larger system for sensing and transducing mechanical signals. Two cellular components in bacteria that have emerged as candidate mechanosensors are the type IV pili (TFP) and the flagellum. Current models posit that bacteria transmit and convert TFP- and/or flagellum-dependent mechanical force inputs into biochemical signals, including cAMP and c-di-GMP, to drive surface adaptation. Here, we discuss the impact of force-induced changes on the structure and function of two eukaryotic proteins, titin and the human von Willebrand factor (vWF), and these proteins' relevance to bacteria. Given the wealth of understanding about these eukaryotic mechanosensors, we can use them as a framework to understand the effect of force on Pseudomonas aeruginosa during the early stages of biofilm formation, with a particular emphasis on TFP and the documented surface-sensing mechanosensors PilY1 and FimH. We also discuss the importance of disulfide bonds in mediating force-induced conformational changes, which may modulate mechanosensing and downstream biochemical signaling. We conclude by sharing our perspective on the state of the field and what we deem exciting frontiers in studying bacterial mechanosensing to better understand the mechanisms whereby bacteria transition from a planktonic to a biofilm lifestyle.

Antimicrobial peptides (AMPs) preferentially permeate prokaryotic membranes via electrostatic binding and membrane remodeling. Such action is drastically suppressed by high salt due to increased electrostatic screening, thus it is puzzling how marine AMPs work can possibly work. Here, we examine as a model system piscidin 1, a histidine-rich AMP, and show that ion-histidine interactions play unanticipated roles in membrane remodeling at high salt: Histidines can simultaneously hydrogen-bond to a phosphate and coordinate with an alkali metal ion to neutralize phosphate charge, thereby facilitating multidentate bonds to lipid headgroups in order to generate saddle-splay curvature, a prerequisite to pore formation. A comparison among Na(+), K(+), and Cs(+) indicates that histidine-mediated salt tolerance is ion specific. We conclude that histidine plays a unique role in enabling protein/peptide-membrane interactions that occur in marine environment. ((800–1000 characters with spaces, currently 950))

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that on soft agar, swarming motility can be repressed by cells that are nonmotile due to the absence of a flagellum or flagellar rotation. Mutants that lack either flagellum biosynthesis or rotation, when present at as little as 5% of the total population, suppressed swarming of wild-type cells. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. Flagellated cells required only type IV pili, but not Pel production, for their swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and nonmotile cells may enhance the formation of sessile communities, including those involving multiple genotypes, phenotypically diverse cells, and perhaps other species. IMPORTANCE Our study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of a larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and nonmotile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

Using glycopolymer surfaces, we have stimulated Shewanella oneidensis bacterial colonization and induced where the bacteria attach on a molecular pattern. When adherent bacteria were rinsed with methyl α-d-mannopyranoside, the glycopolymer-functionalized surfaces retained more cells than self-assembled monolayers terminated by a single mannose unit. These results suggest that the three-dimensional multivalency of the glycopolymers both promotes and retains bacterial attachment. When the methyl α-d-mannopyranoside competitor was codeposited with the cell culture, however, the mannose-based polymer was not significantly different from bare gold surfaces. The necessity for equilibration between methyl α-d-mannopyranoside and the cell culture to remove the enhancement suggests that the retention of cells on glycopolymer surfaces is kinetically controlled and is not a thermodynamic result of the cluster glycoside effect. The MshA lectin appears to facilitate the improved adhesion observed. Our findings that the surfaces studied here can induce stable initial attachment and influence the ratio of bacterial strains on the surface may be applied to harness useful microbial communities.

Significance Here, we discover prototypical pacemaker neurons in the ancient cnidarian Hydra and provide evidence for a direct interaction of these neurons with the commensal microbiota. We uncover a remarkable gene-expression program conservation between the Hydra pacemaker neurons and pacemaker cells in Caenorhabditis elegans and the mammalian gut. We suggest that prototypical pacemaker cells emerged as neurons using components of innate immunity to interact with the microbial environment and ion channels to generate rhythmic contractions. The communication of pacemaker neurons with the microbiota represents a mechanistic link between the gut microbiota and gut motility. Our discoveries improve the understanding of the archetypical properties of the enteric nervous systems, which are perturbed in human dysmotility-related conditions., Pacemaker neurons exert control over neuronal circuit function by their intrinsic ability to generate rhythmic bursts of action potential. Recent work has identified rhythmic gut contractions in human, mice, and hydra to be dependent on both neurons and the resident microbiota. However, little is known about the evolutionary origin of these neurons and their interaction with microbes. In this study, we identified and functionally characterized prototypical ANO/SCN/TRPM ion channel-expressing pacemaker cells in the basal metazoan Hydra by using a combination of single-cell transcriptomics, immunochemistry, and functional experiments. Unexpectedly, these prototypical pacemaker neurons express a rich set of immune-related genes mediating their interaction with the microbial environment. Furthermore, functional experiments gave a strong support to a model of the evolutionary emergence of pacemaker cells as neurons using components of innate immunity to interact with the microbial environment and ion channels to generate rhythmic contractions.

Antimicrobial peptides (AMPs) are a class of molecules which generally kill pathogens via preferential cell membrane disruption. Chemokines are a family of signaling proteins that direct immune cell migration and share a conserved α–β tertiary structure. Recently, it was found that a subset of chemokines can also function as AMPs, including CCL20, CXCL4, and XCL1. It is therefore surprising that machine learning based analysis predicts that CCL20 and CXCL4’s α-helices are membrane disruptive, while XCL1’s helix is not. XCL1, however, is the only chemokine known to be a metamorphic protein which can interconvert reversibly between two distinct native structures (a β-sheet dimer and the α–β chemokine structure). Here, we investigate XCL1’s antimicrobial mechanism of action with a focus on the role of metamorphic folding. We demonstrate that XCL1 is a molecular “Swiss army knife” that can refold into different structures for distinct context-dependent functions: whereas the α–β chemokine structure controls cell migration by binding to G-Protein Coupled Receptors (GPCRs), we find using small angle X-ray scattering (SAXS) that only the β-sheet and unfolded XCL1 structures can induce negative Gaussian curvature (NGC) in membranes, the type of curvature topologically required for membrane permeation. Moreover, the membrane remodeling activity of XCL1’s β-sheet structure is strongly dependent on membrane composition: XCL1 selectively remodels bacterial model membranes but not mammalian model membranes. Interestingly, XCL1 also permeates fungal model membranes and exhibits anti-Candida activity in vitro, in contrast to the usual mode of antifungal defense which requires Th17 mediated cell-based responses. These observations suggest that metamorphic XCL1 is capable of a versatile multimodal form of antimicrobial defense.

During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact is still poorly understood, although mechanosensing is often invoked with little data supporting this claim. Using a combination of molecular genetics and single cell analysis, with biophysical, biochemical and genomics techniques we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain results in modestly lower surface adhesion forces, increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate TFP function in twitching from surface sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains, and its absence in the ~720 sequenced strains of P. aeruginosa PAO1, could contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1.ImportanceMost bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade are unclear. Here, we provide a key insight into this question: we show that the eukaryotic-like, vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that could account for differing surface colonization strategies.

Significance It is well known that c-di-GMP concentration rises in surface-sensing bacteria and functions as a “molecular switch” for biofilm formation. Here, we provide an important recasting of this picture: Intracellular c-di-GMP signals do not just increase in surface-sensing bacteria; such signals are cooperatively broadcast across multiple generations of cells in a lineage with oscillations that undergo both amplitude and frequency modulation, which are controlled by the coupling between pili appendages and c-di-GMP synthesis machinery. The right “tuning” of these signals in terms of frequency and amplitude correlates ultimately to surface commitment. Amplitude and frequency modulation of c-di-GMP signals allows encoding of more complex instructions. Thus, our work provides a more nuanced understanding of how c-di-GMP signaling drives surface commitment., Work on surface sensing in bacterial biofilms has focused on how cells transduce sensory input into cyclic diguanylate (c-di-GMP) signaling, low and high levels of which generally correlate with high-motility planktonic cells and low-motility biofilm cells, respectively. Using Granger causal inference methods, however, we find that single-cell c-di-GMP increases are not sufficient to imply surface commitment. Tracking entire lineages of cells from the progenitor cell onward reveals that c-di-GMP levels can exhibit increases but also undergo oscillations that can propagate across 10 to 20 generations, thereby encoding more complex instructions for community behavior. Principal component and factor analysis of lineage c-di-GMP data shows that surface commitment behavior correlates with three statistically independent composite features, which roughly correspond to mean c-di-GMP levels, c-di-GMP oscillation period, and surface motility. Surface commitment in young biofilms does not correlate to c-di-GMP increases alone but also to the emergence of high-frequency and small-amplitude modulation of elevated c-di-GMP signal along a lineage of cells. Using this framework, we dissect how increasing or decreasing signal transduction from wild-type levels, by varying the interaction strength between PilO, a component of a principal surface sensing appendage system, and SadC, a key hub diguanylate cyclase that synthesizes c-di-GMP, impacts frequency and amplitude modulation of c-di-GMP signals and cooperative surface commitment.

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host-colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that under swarming conditions, swarming motility can be repressed by cells that are non-motile due to the absence of a flagellum or flagellar rotation. Non-swarming cells, due to mutations that prevent either flagellum biosynthesis or rotation, present at 5% of the total population suppressed swarming of wild-type cells under the conditions tested in this study. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. In contrast, flagellated cells required only type IV pili, but not Pel production, in order for swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and non-motile cells may enhance the formation of sessile communities including those involving multiple genotypes, phenotypically-diverse cells, and perhaps other species.ImportanceOur study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of the larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and non-motile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

Neutrophils are crucial for host defense but are notorious for causing sterile inflammatory damage. Activated neutrophils in inflamed tissue can liberate histone H4, which was recently shown to perpetuate inflammation by permeating membranes via the generation of negative Gaussian curvature (NGC), leading to lytic cell death. Here, we show that it is possible to build peptides or proteins that cancel NGC in membranes and thereby suppress pore formation, and demonstrate that they can inhibit H4 membrane remodeling and thereby reduce histone H4-driven lytic cell death and resultant inflammation. As a demonstration of principle, we use apolipoprotein A-I (apoA-I) mimetic peptide apoMP(1). X-ray structural studies and theoretical calculations show that apoMP(1) induces nanoscopic positive Gaussian curvature (PGC), which interacts with the NGC induced by the N-terminus of histone H4 (H4n) to inhibit membrane permeation. Interestingly, we show that induction of PGC can inhibit membrane-permeating activity in general and “turn off” diverse membrane-permeating molecules besides H4n. In vitro experiments show an apoMP(1) dose-dependent rescue of H4 cytotoxicity. Using a mouse model, we show that tissue accumulation of neutrophils, release of neutrophil extracellular traps (NETs), and extracellular H4 all strongly correlate independently with local tissue cell death in multiple organs, but administration of apoMP(1) inhibits histone H4-mediated cytotoxicity and strongly prevents organ tissue damage.

Microbial fuel cells (MFCs) can directly convert the chemical energy stored in organic matter to electricity and are of considerable interest for power generation and wastewater treatment. However, the current MFCs typically exhibit unsatisfactorily low power densities that are largely limited by the sluggish transmembrane and extracellular electron-transfer processes. Here, we report a rational strategy to boost the charge-extraction efficiency in

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world’s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.

International audience; The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

Bacteria are microscopic, single-celled organisms known for their ability to adapt to their environment. In response to stressful environmental conditions or in the presence of a contact surface, they commonly form multicellular aggregates called biofilms. Biofilms form on various abiotic or biotic surfaces through a dynamic stepwise process involving adhesion, growth, and extracellular matrix production. Biofilms develop on tissues as well as on implanted devices during infections, providing the bacteria with a mechanism for survival under harsh conditions including targeting by the immune system and antimicrobial therapy. Like pathogenic bacteria, members of the human microbiota can form biofilms. Biofilms formed by enteric bacteria contribute to several human diseases including autoimmune diseases and cancer. However, until recently the interactions of immune cells with biofilms had been mostly uncharacterized. Here, we will discuss how components of the enteric biofilm produced in vivo, specifically amyloid curli and extracellular DNA, could be interacting with the host's immune system causing an unpredicted immune response.

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and also promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC/membrane interactions and inhibit NEC-mediated buddingin vitro, providing a biophysical explanation for thein-vivophenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an “off” switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

Cathelicidins such as the human 37-amino acid peptide (LL-37) are peptides that not only potently kill microbes but also trigger inflammation by enabling immune recognition of endogenous nucleic acids. Here, a detailed structure-function analysis of LL-37 was performed to understand the details of this process. Alanine scanning of 34-amino acid peptide (LL-34) showed that some variants displayed increased antimicrobial activity against Staphylococcus aureus and group A Streptococcus. In contrast, different substitutions clustered on the hydrophobic face of the LL-34 alpha helix inhibited the ability of those variants to promote type 1 interferon expression in response to U1 RNA or to present U1 to the scavenger receptor (SR) B1 on the keratinocyte cell surface. Small-angle X-ray scattering experiments of the LL-34 variants LL-34, F5A, I24A, and L31A demonstrated that these peptides form cognate supramolecular structures with U1 characterized by inter-dsRNA spacings of approximately 3.5nm, a range that has been previously shown to activate toll-like receptor 3 by the parent peptide LL-37. Therefore, while alanine substitutions on the hydrophobic face of LL-34 led to loss of binding to SRs and the complete loss of autoinflammatory responses in epithelial and endothelial cells, they did not inhibit the ability to organize with U1 RNA in solution to associate with toll-like receptor 3. These observations advance our understanding of how cathelicidin mediates the process of innate immune self-recognition to enable inert nucleic acids to trigger inflammation. We introduce the term "innate immune vetting" to describe the capacity of peptides such as LL-37 to enable certain nucleic acids to become an inflammatory stimulus through SR binding prior to cell internalization.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation-driven tissue fibrosis and vasculopathy. CXCL4 is a chemokine overexpressed in SSc and represents an early serum biomarker of severe disease. CXCL4 likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we combine X-ray scattering, confocal microscopy, immune cell experiments, and analysis of SSc skin biopsies to elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc. We find that CXCL4 organizes “self” and microbial DNA into liquid crystalline immune complexes that drastically amplify Toll-like receptor 9 (TLR9)-mediated plasmacytoid dendritic cell (pDC) hyperactivation and interferon-α production. Surprisingly, this immunomodulatory activity is independent of CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) levels in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Taken together, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and identify a potential therapeutic opportunity to disrupt inflammation in SSc by inhibiting the self-assembly of CXCL4-DNA complexes. Our findings are consistent with an emerging general paradigm in which nucleic acid-scaffolding proteins like antimicrobial peptides, bacterial amyloids, and chemokines can drive autoimmunity by modulating innate immune receptors without being direct agonists.

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor., Physical Biology, 18 (5), ISSN:1478-3975, ISSN:1478-3967

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera., Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH. We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.

BACKGROUND & AIMS: Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection (CDI) via activation of TLR9. METHODS: We performed studies with human colonocytes and monocytes and macrophages from wildtype and TLR9-knockout mice, incubated with TcdA or its antagonist (ODN TTAGGG), or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with an ELISA. We studied a transduction domain of TcdA (TcdA(57–80)), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle x-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight:length ratios. A TLR9 antagonist was tested in mice infected with C. difficile. We incubated human colon with an antagonist of TLR9 and measured TcdA-induced production of cytokines. RESULTS: The TcdA(57–80) protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C. difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA(57–80) domain organized DNA into a geometrically ordered structure that activated TLR9. CONCLUSIONS: Rather than inactivating DNA’s ability to binding TLR9 via binding, TcdA can in fact chaperone and organize DNA into a highly pro-inflammatory spatially periodic structure. TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.

Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of Candida infections is limited by the paucity of antifungal drug classes. Naturally occurring antimicrobial peptides are promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis as well as the spectrum and mechanism of anti-Candida activity. In the mouse model of oropharyngeal candidiasis, application of wild-type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. CCL28 is effective against multiple clinical strains of C. albicans. Polyamine protein transporters are not required for CCL28 anti-Candida activity. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to that of clinical antifungal agents. Application of wild-type CCL28 to C. albicans results in membrane disruption as measured by solute movement, enzyme leakage, and induction of negative Gaussian curvature on model membranes. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results strongly suggest that CCL28 can exert antifungal activity in part via membrane permeation and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.

The initial pivotal phase of bacterial biofilm formation known as reversible attachment, where cells undergo a period of transient surface attachment, is at once universal and poorly understood. What is more, although we know that reversible attachment culminates ultimately in irreversible attachment, it is not clear how reversible attachment progresses phenotypically, as bacterial surface-sensing circuits fundamentally alter cellular behavior. We analyze diverse observed bacterial behavior one family at a time (defined as a full lineage of cells related to one another by division) using a unifying stochastic model and show that our findings lead to insights on the time evolution of reversible attachment and the social cooperative dimension of surface attachment in PAO1 and PA14 strains., What are bacteria doing during “reversible attachment,” the period of transient surface attachment when they initially engage a surface, besides attaching themselves to the surface? Can an attaching cell help any other cell attach? If so, does it help all cells or employ a more selective strategy to help either nearby cells (spatial neighbors) or its progeny (temporal neighbors)? Using community tracking methods at the single-cell resolution, we suggest answers to these questions based on how reversible attachment progresses during surface sensing for Pseudomonas aeruginosa strains PAO1 and PA14. Although PAO1 and PA14 exhibit similar trends of surface cell population increase, they show unanticipated differences when cells are considered at the lineage level and interpreted using the quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two surface colonization strategies. PAO1 lineages commit quickly to a surface compared to PA14 lineages, with early c-di-GMP-mediated exopolysaccharide (EPS) production that can facilitate the attachment of neighbors. PA14 lineages modulate their motility via cyclic AMP (cAMP) and retain memory of the surface so that their progeny are primed for improved subsequent surface attachment. Based on the findings of previous studies, we propose that the differences between PAO1 and PA14 are potentially rooted in downstream differences between Wsp-based and Pil-Chp-based surface-sensing systems, respectively.

All antibiotics have to engage bacterial amphiphilic barriers such as the lipopolysaccharide-rich outer membrane or the phospholipid-based inner membrane in some manner, either by disrupting them outright and/or permeating them and thereby allow the antibiotic to get into bacteria. There is a growing class of cyclic antibiotics, many of which are of bacterial origin, that exhibit activity against Gram-negative bacteria, which constitute an urgent problem in human health. We examine a diverse collection of these cyclic antibiotics, both natural and synthetic, which include bactenecin, polymyxin B, octapeptin, capreomycin, and Kirshenbaum peptoids, in order to identify what they have in common when they interact with bacterial lipid membranes. We find that they virtually all have the ability to induce negative Gaussian curvature (NGC) in bacterial membranes, the type of curvature geometrically required for permeation mechanisms such as pore formation, blebbing, and budding. This is interesting since permeation of membranes is a function usually ascribed to antimicrobial peptides (AMPs) from innate immunity. As prototypical test cases of cyclic antibiotics, we analyzed amino acid sequences of bactenecin, polymyxin B, and capreomycin using our recently developed machine-learning classifier trained on α-helical AMP sequences. Although the original classifier was not trained on cyclic antibiotics, a modified classifier approach correctly predicted that bactenecin and polymyxin B have the ability to induce NGC in membranes, while capreomycin does not. Moreover, the classifier was able to recapitulate empirical structure-activity relationships from alanine scans in polymyxin B surprisingly well. These results suggest that there exists some common ground in the sequence design of hybrid cyclic antibiotics and linear AMPs.

A common bioengineering strategy to add function to a given molecule is by conjugation of a new moiety onto that molecule. Adding multiple functions in this way becomes increasingly challenging and leads to composite molecules with larger molecular weights. In this review, we attempt to gain a new perspective by looking at this problem in reverse, by examining nature's strategies of multiplexing different functions into the same pleiotropic molecule using emerging analysis techniques such as machine learning. We concentrate on examples from the innate immune system, which employs a finite repertoire of molecules for a broad range of tasks. An improved understanding of how diverse functions are multiplexed into a single molecule can inspire new approaches for the deterministic design of multifunctional molecules.

Double-stranded RNA (dsRNA) induces production of pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) by specific binding to endosomal Toll-like receptor-3 (TLR3). Recently, it has been shown that hyperactivation of TLR3 in psoriatic keratinocytes by dsRNA can occur in the presence of human antimicrobial peptide (AMP) LL37. Here, we combine synchrotron X-ray scattering, microscopy, computer simulations, and measurements of NHEK cytokine production to elucidate a previously unanticipated form of specific molecular pattern recognition. LL37 and similar ⍺-helical AMPs can form proinflammatory nanocrystalline complexes with dsRNA that are recognized by TLR3 differently than dsRNA alone. dsRNA complexes that activate IL-6 production in NHEK and those that do not are both able to enter cells and colocalize with TLR3. However, the crystallinity of these AMP-dsRNA complexes, specifically the geometric spacing between parallel dsRNA and the repeat number of ordered dsRNA, strongly influence the level of TLR3 activation. Crystalline complexes that present dsRNA at a spacing that matches with the steric size of TLR3 can recruit and engage multiple TLR3 receptors, driving receptor clustering and immune amplification, whereas crystalline complexes that exhibit poor steric matching do not. Reverse-transcription quantitative PCR (RTqPCR) of IL-6 during siRNA knockdown of TLR3 confirms that cytokine production is due to TLR3: High levels of IL-6 transcription are observed for sterically-matched complexes without TLR3 knockdown, whereas such activity is abrogated with TLR3 knockdown.

Type I IFNs are a cytokine family essential for antiviral defense. More recently, type I IFNs were shown to be important during bacterial infections. In this article, we show that, in addition to known cytokine functions, IFN-β is antimicrobial. Parts of the IFN-β molecular surface (especially helix 4) are cationic and amphipathic, both classic characteristics of antimicrobial peptides, and we observed that IFN-β can directly kill Staphylococcus aureus. Further, a mutant S. aureus that is more sensitive to antimicrobial peptides was killed more efficiently by IFN-β than was the wild-type S. aureus, and immunoblotting showed that IFN-β interacts with the bacterial cell surface. To determine whether specific parts of IFN-β are antimicrobial, we synthesized IFN-β helix 4 and found that it is sufficient to permeate model prokaryotic membranes using synchrotron x-ray diffraction and that it is sufficient to kill S. aureus. These results suggest that, in addition to its well-known signaling activity, IFN-β may be directly antimicrobial and be part of a growing family of cytokines and chemokines, called kinocidins, that also have antimicrobial properties.

We design hybrid antibiotic peptide conjugates that can permeate membranes. Integration of multiple components with different functions into a single molecule is often problematic, due to competing chemical requirements for different functions and to mutual interference. By examining the structure of antimicrobial peptides (AMPs), we show that it is possible to design and synthesize membrane active antibiotic peptide conjugates (MAAPCs) that synergistically combine multiple forms of antimicrobial activity, resulting in unusually strong activity against persistent bacterial strains.

Background: Alpha-synuclein (αS) is a nerve cell protein associated with Parkinson disease (PD). Accumulation of αS within the enteric nervous system (ENS) and its traffic from the gut to the brain are implicated in the pathogenesis and progression of PD. αS has no known function in humans and the reason for its accumulation within the ENS is unknown. Several recent studies conducted in rodents have linked αS to immune cell activation in the central nervous system. We hypothesized that αS in the ENS might play a role in the innate immune defenses of the human gastrointestinal (GI) tract. Methods: We immunostained endoscopic biopsies for αS from children with documented gastric and duodenal inflammation and intestinal allograft recipients who contracted norovirus. To determine whether αS exhibited immune-modulatory activity, we examined whether human αS induced leukocyte migration and dendritic cell maturation. Findings: We showed that the expression of αS in the enteric neurites of the upper GI tract of pediatric patients positively correlated with the degree of acute and chronic inflammation in the intestinal wall. In intestinal allograft subjects who were closely monitored for infection, expression of αS was induced during norovirus infection. We also demonstrated that both monomeric and oligomeric αS have potent chemoattractant activity, causing the migration of neutrophils and monocytes dependent on the presence of the integrin subunit, CD11b, and that both forms of αS stimulate dendritic cell maturation. Interpretation: These findings strongly suggest that αS is expressed within the human ENS to direct intestinal inflammation and implicates common GI infections in the pathogenesis of PD.