Cyclic-di-GMP regulation promotes survival of a slow-replicating subpopulation of intracellular Salmonella Typhimurium.

Salmonella Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, S. Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of S Typhimurium virulence was due to overproduction of c-di-GMP-regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of S. Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for S. Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration.

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

Demolishing the great wall of biofilms in Gram-negative bacteria: To disrupt or disperse?

Bacterial infections lead to high morbidity and mortality globally. While current therapies against bacteria often employ antibiotics, most bacterial pathogens can form biofilms and prevent effective treatment of infections. Biofilm cells can aggregate and encased themselves in a self-secreted protective exopolymeric matrix, to reduce the penetration by antibiotics. Biofilm formation is mediated by c-di-GMP signaling, the ubiquitous secondary messenger in bacteria. Synthesis of c-di-GMP by diguanylate cyclases leads to biofilm formation via the loss of motility, increased surface attachment, and production of biofilm matrix, whereas c-di-GMP degradation by phosphodiesterases causes biofilm dispersal to new sites via increased bacterial motility and matrix breakdown. The highly variable nature of biofilm development and antimicrobial tolerance imposes tremendous challenges in conventional antimicrobial therapies, indicating an imperative need to develop anti-biofilm drugs against biofilm infections. In this review, we focus on two main emergent approaches-active dispersal and disruption. While both approaches aim to demolish biofilms, we will discuss their fundamental differences and associated methods. Active dispersal of biofilms involves signaling the bacterial cells to leave the biofilm, where resident cells ditch their sessile lifestyle, gain motility and self-degrade their matrix. Biofilm disruption leads to direct matrix degradation that forcibly releases embedded biofilm cells. Without the protection of biofilm matrix, released bacterial cells are highly exposed to antimicrobials, leading to their eradication in biofilm infections. Understanding the advantages and disadvantages of both approaches will allow optimized utility with antimicrobials in clinical settings.

A Two-Component System That Modulates Cyclic di-GMP Metabolism Promotes Legionella pneumophila Differentiation and Viability in Low-Nutrient Conditions.

During its life cycle, the environmental pathogen Legionella pneumophila alternates between a replicative and transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host, L. pneumophila further differentiates into the hardy cell type known as the mature infectious form (MIF). The second messenger cyclic di-GMP coordinates lifestyle changes in many bacterial species, but its role in the L. pneumophila life cycle is less understood. Using an in vitro broth culture model that approximates the intracellular transition from the replicative to the transmissive form, here we investigate the contribution to L. pneumophila differentiation of a two-component system (TCS) that regulates cyclic di-GMP metabolism. The TCS is encoded by lpg0278-lpg0277 and is cotranscribed with lpg0279, which encodes a protein upregulated in MIF cells. The promoter for this operon is RpoS dependent and induced in nutrient-limiting conditions that do not support replication, as demonstrated using a gfp reporter and quantitative PCR (qPCR). The response regulator of the TCS (Lpg0277) is a bifunctional enzyme that both synthesizes and degrades cyclic di-GMP. Using a panel of site-directed point mutants, we show that cyclic di-GMP synthesis mediated by a conserved GGDEF domain promotes growth arrest of replicative L. pneumophila, accumulation of pigment and poly-3-hydroxybutyrate storage granules, and viability in nutrient-limiting conditions. Genetic epistasis tests predict that the MIF protein Lpg0279 acts as a negative regulator of the TCS. Thus, L. pneumophila is equipped with a regulatory network in which cyclic di-GMP stimulates the switch from a replicative to a resilient state equipped to survive in low-nutrient environments.IMPORTANCE Although an intracellular pathogen, L. pneumophila has developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication of L. pneumophila from contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that support L. pneumophila persistence in low-nutrient environments can inform design and assessment of remediation strategies. Here we characterize a genetic locus that encodes a two-component signaling system (lpg0278-lpg0277) and a putative regulator protein (lpg0279) that modulates the production of the messenger molecule cyclic di-GMP. We show that this locus promotes both L. pneumophila cell differentiation and survival in nutrient-limiting conditions, thus advancing the understanding of the mechanisms that contribute to L. pneumophila environmental resilience.

Crystallographic snapshot of cellulose synthesis and membrane translocation.

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

AmrZ regulates cellulose production in Pseudomonas syringae pv. tomato DC3000.

In Pseudomonas syringae pv. tomato DC3000, the second messenger c-di-GMP has been previously shown to stimulate pellicle formation and cellulose biosynthesis. A screen for genes involved in cellulose production under high c-di-GMP intracellular levels led to the identification of insertions in two genes, wssB and wssE, belonging to the Pto DC3000 cellulose biosynthesis operon wssABCDEFGHI. Interestingly, beside cellulose-deficient mutants, colonies with a rougher appearance than the wild type also arouse among the transposants. Those mutants carry insertions in amrZ, a gene encoding a transcriptional regulator in different Pseudomonas. Here, we provide evidence that AmrZ is involved in the regulation of bacterial cellulose production at transcriptional level by binding to the promoter region of the wssABCDEFGHI operon and repressing cellulose biosynthesis genes. Mutation of amrZ promotes wrinkly colony morphology, increased cellulose production and loss of motility in Pto DC3000. AmrZ regulon includes putative c-di-GMP metabolising proteins, like AdcA and MorA, which may also impact those phenotypes. Furthermore, an amrZ but not a cellulose-deficient mutant turned out to be impaired in pathogenesis, indicating that AmrZ is a key regulator of Pto DC3000 virulence probably by controlling bacterial processes other than cellulose production.

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli.

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.

Flagellar motility, extracellular proteases and Vibrio cholerae detachment from abiotic and biotic surfaces.

Vibrio cholerae of serogroups O1 and O139, the causative agent of Asiatic cholera, continues to be a major global health threat. This pathogen utilizes substratum-specific pili to attach to distinct surfaces in the aquatic environment and the human small intestine and detaches when conditions become unfavorable. Both attachment and detachment are critical to bacterial environmental survival, pathogenesis and disease transmission. However, the factors that promote detachment are less understood. In this study, we examine the role of flagellar motility and hemagglutinin/protease (HapA) in vibrio detachment from a non-degradable abiotic surface and from the suckling mouse intestine. Flagellar motility facilitated V. cholerae detachment from abiotic surfaces. HapA had no effect on the stability of biofilms formed on abiotic surfaces despite representing >50% of the proteolytic activity present in the extracellular matrix. We developed a balanced lethal plasmid system to increase the bacterial cyclic diguanylate (c-di-GMP) pool late in infection, a condition that represses motility and HapA expression. Increasing the c-di-GMP pool enhanced V. cholerae colonization of the suckling mouse intestine. The c-di-GMP effect was fully abolished in hapA isogenic mutants. These results suggest that motility facilitates detachment in a substratum-independent manner. Instead, HapA appears to function as a substratum-specific detachment factor.

Gut biofilm forming bacteria in inflammatory bowel disease.

Inflammatory bowel disease (IBD) symbolizes a group of intestinal disorders in which prolonged inflammation occur in the digestive tract (esophagus, large intestine, small intestine mouth, stomach). Both genetic and environmental factors (infections, stress, diet) are involved in the development of IBD. As we know that bacteria are found in the intestinal mucosa of human and clinical observations revealed bacterial biofilms associated with patients of IBD. Various factors and microbes are found to play an essential role in biofilm formation and mucosal colonization during IBD. Biofilm formation in the digestive tract is dependent on an extracellular matrix synthesized by the bacteria and it has an adverse effect on the immune response of the host. There is no satisfactory and safe treatment option for IBD. Therefore, the current research aims to disrupt biofilm in IBD and concentrates predominantly on improving the drug. Here, we review the literature on bacterial biofilm and IBD to gather new knowledge on the current understanding of biofilm formation in IBD, host immune deregulation and dysbiosis in IBD, molecular mechanism, bacteria involved in biofilm formation, current and future regimen. It is urgently required to plan new ways to control and eradicate bacteria in biofilms that will open up novel diagnostic and therapeutic avenues for IBD. This article includes the mechanism of signaling molecules with respect to the biofilm-related genes as well as the diagnostic methods and new technologies involved in the treatment of IBD.

GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.

In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain containing glycosyltransferase BcsA.

The Pseudomonas aeruginosa diguanylate cyclase GcbA, a homolog of P. fluorescens GcbA, promotes initial attachment to surfaces, but not biofilm formation, via regulation of motility.

Cyclic di-GMP is a conserved signaling molecule regulating the transitions between motile and sessile modes of growth in a variety of bacterial species. Recent evidence suggests that Pseudomonas species harbor separate intracellular pools of c-di-GMP to control different phenotypic outputs associated with motility, attachment, and biofilm formation, with multiple diguanylate cyclases (DGCs) playing distinct roles in these processes, yet little is known about the potential conservation of functional DGCs across Pseudomonas species. In the present study, we demonstrate that the P. aeruginosa homolog of the P. fluorescens DGC GcbA involved in promoting biofilm formation via regulation of swimming motility likewise synthesizes c-di-GMP to regulate surface attachment via modulation of motility, however, without affecting subsequent biofilm formation. P. aeruginosa GcbA was found to regulate flagellum-driven motility by suppressing flagellar reversal rates in a manner independent of viscosity, surface hardness, and polysaccharide production. P. fluorescens GcbA was found to be functional in P. aeruginosa and was capable of restoring phenotypes associated with inactivation of gcbA in P. aeruginosa to wild-type levels. Motility and attachment of a gcbA mutant strain could be restored to wild-type levels via overexpression of the small regulatory RNA RsmZ. Furthermore, epistasis analysis revealed that while both contribute to the regulation of initial surface attachment and flagellum-driven motility, GcbA and the phosphodiesterase DipA act within different signaling networks to regulate these processes. Our findings expand the complexity of c-di-GMP signaling in the regulation of the motile-sessile switch by providing yet another potential link to the Gac/Rsm network and suggesting that distinct c-di-GMP-modulating signaling pathways can regulate a single phenotypic output.

Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose.

Bacterial biofilms are a prevalent multicellular life form in which individual members can undergo significant functional differentiation and are typically embedded in a complex extracellular matrix of proteinaceous fimbriae, extracellular DNA, and exopolysaccharides (EPS). Bacteria have evolved at least four major mechanisms for EPS biosynthesis, of which the synthase-dependent systems for bacterial cellulose secretion (Bcs) represent not only key biofilm determinants in a wide array of environmental and host-associated microbes, but also an important model system for the studies of processive glycan polymerization, cyclic diguanylate (c-di-GMP)-dependent synthase regulation, and biotechnological polymer applications. The secreted cellulosic chains can be decorated with additional chemical groups or can pack with various degrees of crystallinity depending on dedicated enzymatic complexes and/or cytoskeletal scaffolds. Here, I review recent progress in our understanding of synthase-dependent EPS biogenesis with a focus on common and idiosyncratic molecular mechanisms across diverse cellulose secretion systems.

Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system.

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Coordinated cyclic-di-GMP repression of Salmonella motility through YcgR and cellulose.

Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.

Inactivation of cyclic Di-GMP binding protein TDE0214 affects the motility, biofilm formation, and virulence of Treponema denticola.

As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 µM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (∼6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola.

Genetic analysis of the role of yfiR in the ability of Escherichia coli CFT073 to control cellular cyclic dimeric GMP levels and to persist in the urinary tract.

During urinary tract infections (UTIs), uropathogenic Escherichia coli must maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states in E. coli. The yfiRNB locus in E. coli CFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion of yfiR yielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A double yfiRN mutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in the yfiR mutant. Expression of yhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiR suppressed the overproduction of curli fimbriae and cellulose and further verified that deletion of yfiR results in c-di-GMP accumulation. Additional deletion of csgD and bcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of the yfiR deletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between the yfiR mutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disable E. coli in the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenic E. coli in vivo.

The response threshold of Salmonella PilZ domain proteins is determined by their binding affinities for c-di-GMP.

c-di-GMP is a bacterial second messenger that is enzymatically synthesized and degraded in response to environmental signals. Cellular processes are affected when c-di-GMP binds to receptors which include proteins that contain the PilZ domain. Although each c-di-GMP synthesis or degradation enzyme metabolizes the same molecule, many of these enzymes can be linked to specific downstream processes. Here we present evidence that c-di-GMP signalling specificity is achieved through differences in affinities of receptor macromolecules. We show that the PilZ domain proteins of Salmonella Typhimurium, YcgR and BcsA, demonstrate a 43-fold difference in their affinity for c-di-GMP. Modulation of the affinities of these proteins altered their activities in a predictable manner in vivo. Inactivation of yhjH, which encodes a predicted c-di-GMP degrading enzyme, increased the fraction of the cellular population that demonstrated c-di-GMP levels high enough to bind to the higher-affinity YcgR protein and inhibit motility, but not high enough to bind to the lower-affinity BcsA protein and stimulate cellulose production. Finally, PilZ domain proteins of Pseudomonas aeruginosa demonstrated a 145-fold difference in binding affinities, suggesting that regulation by binding affinity may be a conserved mechanism that allows organisms with many c-di-GMP binding macromolecules to rapidly integrate multiple environmental signals into one output.

Comparison of the uptake of untargeted and targeted immunostimulatory nanoparticles by immune cells in the microenvironment of metastatic breast cancer.

To alter the immunosuppressive tumor microenvironment (TME), we developed an immunostimulatory nanoparticle (NP) to reprogram a tumor's dysfunctional and inhibitory antigen-presenting cells (APCs) into properly activated APCs that stimulate tumor-reactive cytotoxic T cells. Importantly, systemic delivery allowed NPs to efficiently utilize the entire microvasculature and gain access into the majority of the perivascular TME, which coincided with the APC-rich tumor areas leading to uptake of the NPs predominantly by APCs. In this work, a 60 nm NP was loaded with a STING agonist, which triggered robust production of interferon ß, resulting in activation of APCs. In addition to untargeted NPs, we employed 'mainstream' ligands targeting fibronectin, αvß3 integrin and P-selectin that are commonly used to direct nanoparticles to tumors. Using the 4T1 mouse model, we assessed the microdistribution of the four NP variants in the tumor immune microenvironment in three different breast cancer landscapes, including primary tumor, early metastasis, and late metastasis. The different NP variants resulted in variable uptake by immune cell subsets depending on the organ and tumor stage. Among the NP variants, therapeutic studies indicated that the untargeted NPs and the integrin-targeting NPs exhibited a remarkable short- and long-term immune response and long-lasting antitumor effect.

cGMP modulates responses to queen mandibular pheromone in worker honey bees.

Responses to social cues, such as pheromones, can be modified by genotype, physiology, or environmental context. Honey bee queens produce a pheromone (queen mandibular pheromone; QMP) which regulates aspects of worker bee behavior and physiology. Forager bees are less responsive to QMP than young bees engaged in brood care, suggesting that physiological changes associated with behavioral maturation modulate response to this pheromone. Since 3',5'-cyclic guanosine monophosphate (cGMP) is a major regulator of behavioral maturation in workers, we examined its role in modulating worker responses to QMP. Treatment with a cGMP analog resulted in significant reductions in both behavioral and physiological responses to QMP in young caged workers. Treatment significantly reduced attraction to QMP and inhibited the QMP-mediated increase in vitellogenin RNA levels in the fat bodies of worker bees. Genome-wide analysis of brain gene expression patterns demonstrated that cGMP has a larger effect on expression levels than QMP, and that QMP has specific effects in the presence of cGMP, suggesting that some responses to QMP may be dependent on an individual bees' physiological state. Our data suggest that cGMP-mediated processes play a role in modulating responses to QMP in honey bees at the behavioral, physiological, and molecular levels.

Calcium-Responsive Diguanylate Cyclase CasA Drives Cellulose-Dependent Biofilm Formation and Inhibits Motility in Vibrio fischeri.

The marine bacterium Vibrio fischeri colonizes its host, the Hawaiian bobtail squid, in a manner requiring both bacterial biofilm formation and motility. The decision to switch between sessile and motile states is often triggered by environmental signals and regulated by the widespread signaling molecule c-di-GMP. Calcium is an environmental signal previously shown to affect both biofilm formation and motility by V. fischeri. In this study, we investigated the link between calcium and c-di-GMP, determining that calcium increases intracellular c-di-GMP dependent on a specific diguanylate cyclase, calcium-sensing protein A (CasA). CasA is activated by calcium, dependent on residues in an N-terminal sensory domain, and synthesizes c-di-GMP through an enzymatic C-terminal domain. CasA is responsible for calcium-dependent inhibition of motility and activation of cellulose-dependent biofilm formation. Calcium regulates cellulose biofilms at the level of transcription, which also requires the transcription factor VpsR. Finally, the Vibrio cholerae CasA homolog, CdgK, is unable to complement CasA and may be inhibited by calcium. Collectively, these results identify CasA as a calcium-responsive regulator, linking an external signal to internal decisions governing behavior, and shed light on divergence between Vibrio spp. IMPORTANCE Biofilm formation and motility are often critical behaviors for bacteria to colonize a host organism. Vibrio fischeri is the exclusive colonizer of its host's symbiotic organ and requires both biofilm formation and motility to initiate successful colonization, providing a relatively simple model to explore complex behaviors. In this study, we determined how the environmental signal calcium alters bacterial behavior through production of the signaling molecule c-di-GMP. Calcium activates the diguanylate cyclase CasA to synthesize c-di-GMP, resulting in inhibition of motility and activation of cellulose production. These activities depend on residues in CasA's N-terminal sensory domain and C-terminal enzymatic domain. These findings thus identify calcium as a signal recognized by a specific diguanylate cyclase to control key bacterial phenotypes. Of note, CasA activity is seemingly inverse to that of the homologous V. cholerae protein, CdgK, providing insight into evolutionary divergence between closely related species.