Cell-cell Communication Mechanism Research Articles

Paracrine FGF1 signaling directs pituitary architecture and size

Organ architecture is established during development through intricate cell-cell communication mechanisms, yet the specific signals mediating these communications often remain elusive. Here, we used the anterior pituitary gland that harbors different interdigitated hormone-secreting homotypic cell networks to dissect cell-cell communication mechanisms operating during late development. We show that blocking differentiation of corticotrope cells leads to pituitary hypoplasia with a major effect on somatotrope cells that directly contact corticotropes. Gene knockout of the corticotrope-restricted transcription factor Tpit results in fewer somatotropes, with less secretory granules and a loss of cell polarity, resulting in systemic growth retardation. Single-cell transcriptomic analyses identified FGF1 as a corticotrope-specific Tpit dosage-dependent target gene responsible for these phenotypes. Consistently, genetic ablation of FGF1 in mice phenocopies pituitary hypoplasia and growth impairment observed in Tpit-deficient mice. These findings reveal FGF1 produced by the corticotrope cell network as an essential paracrine signaling molecule participating in pituitary architecture and size.

Do tunneling nanotubes drive chemoresistance in solid tumors and other malignancies?

Intercellular communication within the tumor microenvironment (TME) is essential for establishing, mediating, and synchronizing cancer cell invasion and metastasis. Cancer cells, individually and collectively, react at the cellular and molecular levels to insults from standard-of-care treatments used to treat patients with cancer. One form of cell communication that serves as a prime example of cellular phenotypic stress response is a type of cellular protrusion called tunneling nanotubes (TNTs). TNTs are ultrafine, actin-enriched contact-dependent forms of membrane protrusions that facilitate long distance cell communication through transfer of various cargo, including genetic materials, mitochondria, proteins, ions, and various other molecules. In the past 5-10 years, there has been a growing body of evidence that implicates TNTs as a novel mechanism of cell-cell communication in cancer that facilitates and propagates factors that drive or enhance chemotherapeutic resistance in a variety of cancer cell types. Notably, recent literature has highlighted the potential of TNTs to serve as cellular conduits and mediators of drug and nanoparticle delivery. Given that TNTs have also been shown to form in vivo in a variety of tumor types, disrupting TNT communication within the TME provides a novel strategy for enhancing the cytotoxic effect of existing chemotherapies while suppressing this form of cellular stress response. In this review, we examine current understanding of interplay between cancer cells occurring via TNTs, and even further, the implications of TNT-mediated tumor-stromal cross-talk and the potential to enhance chemoresistance. We then examine tumor microtubes, an analogous cell protrusion heavily implicated in mediating treatment resistance in glioblastoma multiforme, and end with a brief discussion of the effects of radiation and other emerging treatment modalities on TNT formation.

TempEasy 3D Hydrogel Coculture System Provides Mechanistic Insights into Prostate Cancer Bone Metastasis.

Patients diagnosed with advanced prostate cancer (PCa) often experience incurable bone metastases; however, a lack of relevant experimental models has hampered the study of disease mechanisms and the development of therapeutic strategies. In this study, we employed the recently established Temperature-based Easy-separable (TempEasy) 3D cell coculture system to investigate PCa bone metastasis. Through coculturing PCa and bone cells for 7 days, our results showed a reduction in PCa cell proliferation, an increase in neovascularization, and an enhanced metastasis potential when cocultured with bone cells. Additionally, we observed increased cell proliferation, higher stemness, and decreased bone matrix protein expression in bone cells when cocultured with PCa cells. Furthermore, we demonstrated that the stiffness of the extracellular matrix had a negligible impact on molecular responses in both primary (PCa cells) and distant malignant (bone cells) sites. The TempEasy 3D hydrogel coculture system is an easy-to-use and versatile coculture system that provides valuable insights into the mechanisms of cell-cell communication and interaction in cancer metastasis.

MzmL, a novel marine derived N-acyl homoserine lactonase from Mesoflavibacter zeaxanthinifaciens that attenuates Pectobacterium carotovorum subsp. carotovorum virulence.

Quorum sensing (QS) is a conserved cell-cell communication mechanism widely distributed in bacteria, and is oftentimes tightly correlated with pathogen virulence. Quorum quenching enzymes, which interfere with QS through degrading the QS signaling molecules, could attenuate virulence instead of killing the pathogens, and thus are less likely to induce drug resistance. Many Gram-negative bacteria produce N-acyl homoserine lactones (AHLs) for interspecies communication. In this study, we isolated and identified a bacterial strain, Mesoflavibacter zeaxanthinifaciens XY-85, from an Onchidium sp. collected from the intertidal zone of Dapeng Reserve in Shenzhen, China, and found it had strong AHL degradative activity. Whole genome sequencing and blast analysis revealed that XY-85 harbors an AHL lactonase (designated MzmL), which is predicted to have an N-terminal signal peptide and share the "HXHXDH" motif with known AHL lactonases belonging to the Metallo-β-lactamase superfamily. Phylogenetic studies showed MzmL was closest to marine lactonase cluster members, MomL and Aii20J, instead of the AiiA type lactonases. Ultra performance liquid chromatography-mass spectrometry analysis confirmed that MzmL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MzmL could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, and retained full bioactivity under a wide range of temperatures (28-100°C) and pHs (4-11). Furthermore, MzmL significantly reduced Pectobacterium carotovorum subsp. carotovorum virulence factor production in vitro, such as biofilm formation and plant cell wall degrading enzyme production, and inhibited soft rot development on potato slices. These results demonstrated that MzmL may be a novel type of AHL lactonase with good environmental stability, and has great potential to be developed into a novel biological control agent for bacterial disease management.

Comprehensive Analysis of Lung Adenocarcinoma and Brain Metastasis through Integrated Single-Cell Transcriptomics.

Lung adenocarcinoma (LUAD) is a highly prevalent and lethal form of lung cancer, comprising approximately half of all cases. It is often diagnosed at advanced stages with brain metastasis (BM), resulting in high mortality rates. Current BM management involves complex interventions and conventional therapies that offer limited survival benefits with neurotoxic side effects. The tumor microenvironment (TME) is a complex system where cancer cells interact with various elements, significantly influencing tumor behavior. Immunotherapies, particularly immune checkpoint inhibitors, target the TME for cancer treatment. Despite their effectiveness, it is crucial to understand metastatic lung cancer and the specific characteristics of the TME, including cell-cell communication mechanisms, to refine treatments. Herein, we investigated the tumor microenvironment of brain metastasis from lung adenocarcinoma (LUAD-BM) and primary tumors across various stages (I, II, III, and IV) using single-cell RNA sequencing (scRNA-seq) from publicly available datasets. Our analysis included exploring the immune and non-immune cell composition and the expression profiles and functions of cell type-specific genes, and investigating the interactions between different cells within the TME. Our results showed that T cells constitute the majority of immune cells present in primary tumors, whereas microglia represent the most dominant immune cell type in BM. Interestingly, microglia exhibit a significant increase in the COX pathway. Moreover, we have shown that microglia primarily interact with oligodendrocytes and endothelial cells. One significant interaction was identified between DLL4 and NOTCH4, which demonstrated a relevant association between endothelial cells and microglia and between microglia and oligodendrocytes. Finally, we observed that several genes within the HLA complex are suppressed in BM tissue. Our study reveals the complex molecular and cellular dynamics of BM-LUAD, providing a path for improved patient outcomes with personalized treatments and immunotherapies.

Exploring the mechanism of contact-dependent cell-cell communication on chemosensitivity based on single-cell high-throughput drug screening platform

High-throughput drug screening (HTDS) has significantly reduced the time and cost of new drug development. Nonetheless, contact-dependent cell-cell communication (CDCCC) may impact the chemosensitivity of tumour cells. There is a pressing need for low-cost single-cell HTDS platforms, alongside a deep comprehension of the mechanisms by which CDCCC affects drug efficacy, to fully unveil the efficacy of anticancer drugs. In this study, we develop a microfluidic chip for single-cell HTDS and evaluate the molecular mechanisms impacted by CDCCC using quantitative mass spectrometry-based proteomics. The chip achieves high-quality drug mixing and single-cell capture, with single-cell drug screening results on the chip showing consistency with those on the 96-well plates under varying concentration gradients. Through quantitative proteomic analysis, we deduce that the absence of CDCCC in single tumour cells can enhance their chemoresistance potential, but simultaneously subject them to stronger proliferation inhibition. Additionally, pathway enrichment analysis suggests that CDCCC could impact several signalling pathways in tumour single cells that regulate vital biological processes such as tumour proliferation, adhesion, and invasion. These results offer valuable insights into the potential connection between CDCCC and the chemosensitivity of tumour cells. This research paves the way for the development of single-cell HTDC platforms and holds the promise of advancing tumour personalized treatment strategies.

Abstract B083: Single-cell and spatial transcriptomic characterization of treatment resistance and immune dynamics in high-grade serous ovarian cancer

Abstract B083: Single-cell and spatial transcriptomic characterization of treatment resistance and immune dynamics in high-grade serous ovarian cancer

Stress in the metastatic journey - the role of cell communication and clustering in breast cancer progression and treatment resistance.

Breast cancer stands as the most prevalent malignancy afflicting women. Despite significant advancements in its diagnosis and treatment, breast cancer metastasis continues to be a leading cause of mortality among women. To metastasize, cancer cells face numerous challenges: breaking away from the primary tumor, surviving in the circulation, establishing in a distant location, evading immune detection and, finally, thriving to initiate a new tumor. Each of these sequential steps requires cancer cells to adapt to a myriad of stressors and develop survival mechanisms. In addition, most patients with breast cancer undergo surgical removal of their primary tumor and have various therapeutic interventions designed to eradicate cancer cells. Despite this plethora of attacks and stresses, certain cancer cells not only manage to persist but also proliferate robustly, giving rise to substantial tumors that frequently culminate in the patient's demise. To enhance patient outcomes, there is an imperative need for a deeper understanding of the molecular and cellular mechanisms that empower cancer cells to not only survive but also expand. Herein, we delve into the intrinsic stresses that cancer cells encounter throughout the metastatic journey and the additional stresses induced by therapeutic interventions. We focus on elucidating the remarkable strategies adopted by cancer cells, such as cell-cell clustering and intricate cell-cell communication mechanisms, to ensure their survival.

Promoter selectivity of the RhlR quorum-sensing transcription factor receptor in Pseudomonas aeruginosa is coordinated by distinct and overlapping dependencies on C4-homoserine lactone and PqsE.

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-β-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.

High-Throughput, Quantitative Screening of Quorum-Sensing Inhibitors Based on a Bacterial Biosensor.

Quorum sensing (QS) is a cell-cell communication mechanism by which bacteria synchronize social behaviors such as biofilm formation and virulence factor secretion by producing and sensing small molecular signals. Quorum quenching (QQ) by degrading signals or blocking signal transmissions has become a promising strategy for disrupting QS and preventing bacterial infection and biofilm formation. However, studies of high-throughput screening and identification approaches for quorum-sensing inhibitors (QSIs) are still inadequate. In this work, we developed a sensitive, high-throughput approach for screening QSIs based on the bacterial biosensor strain Agrobacterium tumefaciens N5 (pBA7P), which contains a traG gene promoter induced by QS signals fused with a promoterless β-lactamase gene reporter. Using this approach, we identified 31 QQ bacteria from ∼2000 soil bacterial isolates, some belonging to the genera Bosea, Cupriavidus, and Flavobacterium that have not been reported previously as QQ bacteria. We also identified four QS inhibitory compounds and one QS signal analogue from ∼5000 small-molecule compounds, which profoundly affected the expression of QS-regulated genes and phenotypes of the pathogenic bacteria. This high-throughput screening system is effective and sensitive for screening of both QQ microbes and small molecules, enabling the discovery of a wide variety of biocompatible compounds.

Deciphering the molecular components of the quorum sensing system in the fungus Ophiostoma piceae.

Quorum sensing (QS) is a complex cell-cell communication mechanism that coordinates population-level behaviors in microbes. In eukaryotes, this phenomenon has been extensively described in the dimorphic yeast Candida albicans as its main QS molecule, the sesquiterpene alcohol farnesol, is responsible for various phenotypic (i.e., inhibition of yeast-to-hyphae transition, biofilm formation, and, hence, pathogenesis) and metabolic (i.e., induction of oxidative stress and apoptosis) changes. Ophiostoma piceae CECT 20416 is a dimorphic saprotrophic ascomycete with biotechnological interest that also produces farnesol as QS molecule, but in this case, the alcohol promotes the morphological transition to the mycelial form, biofilm formation, enzyme secretion, and melanin production. Here, we characterized the physiological response of Ophiostoma piceae to farnesol, the molecular components of the QS system of this fungus have been investigated using a "multiomics" approach that involved genomic, transcriptomic, and proteomic analyses. Some genes identified in this work are proposed as key factors in farnesol transport and signaling. We have also cataloged the genes undergoing major transcriptional changes triggered by the presence of the autoinducer, such as cell-wall remodeling, reactive oxygen species (ROS) protection, and melanin biosynthesis, using self-organizing maps. This analysis could be useful for applications in the forestry industry, for enzymes production, and for the valorization of residues. Furthermore, it might as well help to investigate the QS mechanisms of clinically relevant fungi phylogenetically related to Ophiostoma. IMPORTANCE This manuscript presents a comprehensive study on the molecular mechanisms triggered by the quorum sensing (QS) molecule farnesol in the biotechnologically relevant fungus Ophiostoma piceae. We present for the first time, using a multiomics approach, an in-depth analysis of the QS response in a saprotroph fungus, detailing the molecular components involved in the response and their possible mechanisms of action. We think that these results are particularly relevant in the knowledge of the functioning of the QS in eukaryotes, as well as for the exploitation of these mechanisms.

Prediction of cell-cell communication patterns of dorsal root ganglion cells: single-cell RNA sequencing data analysis.

Abstract JOURNAL/nrgr/04.03/01300535-202406000-00042/inline-graphic1/v/2023-10-30T152229Z/r/image-tiff Dorsal root ganglion neurons transmit peripheral somatic information to the central nervous system, and dorsal root ganglion neuron excitability affects pain perception. Dorsal root ganglion stimulation is a new approach for managing pain sensation. Knowledge of the cell-cell communication among dorsal root ganglion cells may help in the development of new pain and itch management strategies. Here, we used the single-cell RNA-sequencing (scRNA-seq) database to investigate intercellular communication networks among dorsal root ganglion cells. We collected scRNA-seq data from six samples from three studies, yielding data on a total of 17,766 cells. Based on genetic profiles, we identified satellite glial cells, Schwann cells, neurons, vascular endothelial cells, immune cells, fibroblasts, and vascular smooth muscle cells. Further analysis revealed that eight types of dorsal root ganglion neurons mediated proprioceptive, itch, touch, mechanical, heat, and cold sensations. Moreover, we predicted several distinct forms of intercellular communication among dorsal root ganglion cells, including cell-cell contact, secreted signals, extracellular matrix, and neurotransmitter-mediated signals. The data mining predicted that Mrgpra3-positive neurons robustly express the genes encoding the adenosine Adora2b (A2B) receptor and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα-1). Our immunohistochemistry results confirmed the coexpression of the A2B receptor and GFRα-1. Intrathecal injection of the A2B receptor antagonist PSB-603 effectively prevented histamine-induced scratching behaviour in a dose-dependent manner. Our results demonstrate the involvement of the A2B receptor in the modulation of itch sensation. Furthermore, our findings provide insight into dorsal root ganglion cell-cell communication patterns and mechanisms. Our results should contribute to the development of new strategies for the regulation of dorsal root ganglion excitability.

Abstract 604: Hybrid quantification of SILAC-barcoded interacting tumor and T cells

Abstract Therapeutic interference of cell-cell communication mechanisms has allowed the clinical development of multiple cancer treatments. For example, the therapeutic intervention of T cell-tumor cell interactions of PD-1 and PD-L1, respectively, by immune checkpoint blockade (ICB) has improved the clinical outcome of cancer patients. However, only a minority of patients are currently benefiting from ICB. Thus, there is an urgent need to improve our understanding of the T cell-tumor cell interplay. Recently developed algorithms serve to deconvolute gene expression of physically interacting cells (PIC). However, such approaches are limited for proteomics (MS) and phosphoproteomics (pMS), which are required to get molecular insight into protein functionality. Here, we present a new method: Hybrid quantification of SILAC-barcoded interacting cells (HySic). Briefly, we use Stable Isotope Labelling by Amino acids in Cell culture (SILAC) to differentiate between two cell types of interest: T cells and tumor cells. We co-cultured the cells allowing to engage in functional interactions and without the need to separate them, and subsequently performed a label-free quantification (LFQ) LC-MS analysis. After initial bioinformatic analysis and validation with independent techniques, we used HySic to unravel protein mechanisms contributing to functional T cell-tumor cell interactions. We used established interaction mechanisms like those mediated by IFNγ signaling proteins to benchmark HySic, while this analysis also brought new protein pathways to light, which will be presented. In conclusion, we have established and validated HySIC as a new method to study protein signaling dynamics in physically interacting cells. Our data shows the effectiveness and applicability of HySIC to study cell-cell interactions, which could be easily extended to other biological systems. Citation Format: Sofía Ibáñez Molero, Alisha Atmopawiro, Alex Terry, Maarten Altelaar, Daniel Peeper, Kelly Stecker. Hybrid quantification of SILAC-barcoded interacting tumor and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 604.

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

In-Depth Proteomic Analysis of Blood Circulating Small Extracellular Vesicles.

Circulating small extracellular vesicles (sEVs), also called exosomes, are key players in the investigation of cell-cell communication mechanisms and in the identification of new potential biomarkers. These particles can carry proteins, DNA, mRNA, miRNA, lipids and metabolites that are transported all over the human body, potentially reaching all the cells. In particular, proteins, which are well-known biological actors in cell signalling, will be discussed in this context. In this article, we present a mass spectrometry approach for the in-depth characterization of the sEVs proteome. The protocols include strategies for the isolation and purification of sEVs, for the extraction of proteins and the purification of sEVs proteins by the immunodepletion of the most abundant plasmatic proteins. Finally, bioinformatic analysis for the extraction of the most important biological features associated with the proteomic content of sEVs is reported.

Non-canonical integrin signaling activates EGFR and RAS-MAPK-ERK signaling in small cell lung cancer.

Background: Small cell lung cancer (SCLC) is an extremely aggressive cancer type with a patient median survival of 6-12 months. Epidermal growth factor (EGF) signaling plays an important role in triggering SCLC. In addition, growth factor-dependent signals and alpha-, beta-integrin (ITGA, ITGB) heterodimer receptors functionally cooperate and integrate their signaling pathways. However, the precise role of integrins in EGF receptor (EGFR) activation in SCLC remains elusive. Methods: We analyzed human precision-cut lung slices (hPCLS), retrospectively collected human lung tissue samples and cell lines by classical methods of molecular biology and biochemistry. In addition, we performed RNA-sequencing-based transcriptomic analysis in human lung cancer cells and human lung tissue samples, as well as high-resolution mass spectrometric analysis of the protein cargo from extracellular vesicles (EVs) that were isolated from human lung cancer cells. Results: Our results demonstrate that non-canonical ITGB2 signaling activates EGFR and RAS/MAPK/ERK signaling in SCLC. Further, we identified a novel SCLC gene expression signature consisting of 93 transcripts that were induced by ITGB2, which may be used for stratification of SCLC patients and prognosis prediction of LC patients. We also found a cell-cell communication mechanism based on EVs containing ITGB2, which were secreted by SCLC cells and induced in control human lung tissue RAS/MAPK/ERK signaling and SCLC markers. Conclusions: We uncovered a mechanism of ITGB2-mediated EGFR activation in SCLC that explains EGFR-inhibitor resistance independently of EGFR mutations, suggesting the development of therapies targeting ITGB2 for patients with this extremely aggressive lung cancer type.

The alternate ligand Jagged enhances the robustness of Notch signaling patterns.

The Notch pathway, an example of juxtacrine signaling, is an evolutionary conserved cell-cell communication mechanism. It governs emergent spatiotemporal patterning in tissues during development, wound healing and tumorigenesis. Communication occurs when Notch receptors of one cell bind to either of its ligands, Delta/Jagged of the neighboring cell. In general, Delta-mediated signaling drives neighboring cells to have an opposite fate (lateral inhibition) whereas Jagged-mediated signaling drives cells to maintain similar fates (lateral induction). Here, by deriving and solving a reduced set of 12 coupled ordinary differential equations for the Notch-Delta-Jagged system on a hexagonal grid of cells, we determine the allowed states across different parameter sets. We also show that Jagged (at low dose) acts synergistically with Delta to enable more robust pattern formation by making the neighboring cell states more distinct from each other, despite its lateral induction property. Our findings extend our understanding of the possible synergistic role of Jagged with Delta which had been previously proposed through experiments and models in the context of chick inner ear development. Finally, we show how Jagged can help to expand the bistable (both uniform and hexagon phases are stable) region, where a local perturbation can spread over time in an ordered manner to create a biologically relevant, perfectly ordered lateral inhibition pattern.

Physical Properties and Shifting of the Extracellular Membrane Vesicles Attached to Living Bacterial Cell Surfaces.

Bacterial cells release nanometer-sized extracellular membrane vesicles (MVs) to deliver cargo molecules for use in mediating various biological processes. However, the detailed processes of transporting these cargos from MVs to recipient cells remain unclear because of the lack of imaging techniques to image nanometer-sized fragile vesicles in a living bacterial cell surface. Herein, we quantitatively demonstrated that the direct binding of MV to the cell surface significantly promotes hydrophobic quorum-sensing signal (C16-HSL) transportation to the recipient cells. Moreover, we analyzed the MV-binding process in the Paracoccus denitrificans cell surface using high-speed atomic force microscopy phase imaging. Although MV shapes were unaltered after binding to the cell surface, the physical properties of a group of single MV particles were shifted. Additionally, the phase shift values of MVs were higher than that of the cell's surfaces upon binding, whereas the phase shift values of the group of MVs were decreased during observation. The shifting physical properties occurred irreversibly only once for each MV during the observations. The decreasing phase shift values indicated alterations of chemical components in the MVs as well, thereby suggesting the dynamic process in which single MV particles deliver their hydrophobic cargo into the recipient cell. IMPORTANCE Compared to the increasing knowledge about MV release mechanisms from donor cells, the mechanism by which recipient cells receive cargo from MVs remains unknown. Herein, we have successfully imaged single MV-binding processes in living bacterial cell surfaces. Accordingly, we confirmed the shift in the MV hydrophobic properties after landing on the cell surface. Our results showed the detailed states and the attaching process of a single MV into the cell surface and can aid the development of a new model for MV reception into Gram-negative bacterial cell surfaces. The insight provided by this study is significant for understanding MV-mediated cell-cell communication mechanisms. Moreover, the AFM technique presented for nanometer-scaled mapping of dynamic physical properties alteration on a living cell could be applied for the analyses of various biological phenomena occurring on the cell surface, and it gives us a new view into the understanding of the phenotypes of the bacterial cell surface.

Structure of the RhlR-PqsE complex from Pseudomonas aeruginosa reveals mechanistic insights into quorum-sensing gene regulation

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P.aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P.aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthesized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical interaction with the enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand-bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR-mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor production in P.aeruginosa.

Regulatory Mechanisms between Quorum Sensing and Virulence in Salmonella.

Salmonella is a foodborne pathogen that causes enterogastritis among humans, livestock and poultry, and it not only causes huge economic losses for the feed industry but also endangers public health around the world. However, the prevention and treatment of Salmonella infection has remained poorly developed because of its antibiotic resistance. Bacterial quorum sensing (QS) system is an intercellular cell-cell communication mechanism involving multiple cellular processes, especially bacterial virulence, such as biofilm formation, motility, adherence, and invasion. Therefore, blocking the QS system may be a new strategy for Salmonella infection independent of antibiotic treatment. Here, we have reviewed the central role of the QS system in virulence regulation of Salmonella and summarized the most recent advances about quorum quenching (QQ) in virulence attenuation during Salmonella infection. Unraveling the complex relationship between QS and bacterial virulence may provide new insight into the therapy of pathogen infection.