NF-κBp50 and HDAC1 Interaction Is Implicated in the Host Tolerance to Infection Mediated by the Bacterial Quorum Sensing Signal 2-Aminoacetophenone

Some bacterial quorum sensing (QS) small molecules are important mediators of inter-kingdom signaling and impact host immunity. The QS regulated small volatile molecule 2-aminoacetophenone (2-AA), which has been proposed as a biomarker of Pseudomonas aeruginosa colonization in chronically infected human tissues, is critically involved in “host tolerance training” that involves a distinct molecular mechanism of host chromatin regulation through histone deacetylase (HDAC)1. 2-AA’s epigenetic reprogramming action enables host tolerance to high bacterial burden and permits long-term presence of P. aeruginosa without compromising host survival. Here, to further elucidate the molecular mechanisms of 2-AA-mediated host tolerance/resilience we investigated the connection between histone acetylation status and nuclear factor (NF)-κB signaling components that together coordinate 2-AA-mediated control of transcriptional activity. We found increased NF-κBp65 acetylation levels in 2-AA stimulated cells that are preceded by association of CBP/p300 and increased histone acetyltransferase activity. In contrast, in 2-AA-tolerized cells the protein–protein interaction between p65 and CBP/p300 is disrupted and conversely, the interaction between p50 and co-repressor HDAC1 is enhanced, leading to repression of the pro-inflammatory response. These results highlight how a bacterial QS signaling molecule can establish a link between intracellular signaling and epigenetic reprogramming of pro-inflammatory mediators that may contribute to host tolerance training. These new insights might contribute to the development of novel therapeutic interventions against bacterial infections

The Quorum Sensing Volatile Molecule 2-Amino Acetophenon Modulates Host Immune Responses in a Manner that Promotes Life with Unwanted Guests

Increasing evidence indicates that bacterial quorum sensing (QS) signals are important mediators of immunomodulation. However, whether microbes utilize these immunomodulatory signals to maintain infection remain unclear. Here, we show that the Pseudomonas aeruginosa QS-regulated molecule 2-amino acetophenone (2-AA) modulates host immune responses in a manner that increases host ability to cope with this pathogen. Mice treated with 2-AA prior to infection had a 90% survival compared to 10% survival rate observed in the non-pretreated infected mice. Whilst 2-AA stimulation activates key innate immune response pathways involving mitogen-activated protein kinases (MAPKs), nuclear factor (NF)-$\kappa B$, and pro-inflammatory cytokines, it attenuates immune response activation upon pretreatment, most likely by upregulating anti-inflammatory cytokines. 2-AA host pretreatment is characterized by a transcriptionally regulated block of c-JUN N-terminal kinase (JNK) and NF-$\kappa B$ activation, with relatively preserved activation of extracellular regulated kinase (ERK) 1/2. These kinase changes lead to CCAAT/enhancer-binding protein-$\beta$ $(c/EBP\beta)$ activation and formation of the $c/EBP\beta-p65$ complex that prevents NF-$\kappa B$ activation. 2-AA's aptitude for dampening the inflammatory processes while increasing host survival and pathogen persistence concurs with its ability to signal bacteria to switch to a chronic infection mode. Our results reveal a QS immunomodulatory signal that promotes original aspects of interkingdom communication. We propose that this communication facilitates pathogen persistence, while enabling host tolerance to infection

Identification of Anti-virulence Compounds That Disrupt Quorum-Sensing Regulated Acute and Persistent Pathogenicity

Etiological agents of acute, persistent, or relapsing clinical infections are often refractory to antibiotics due to multidrug resistance and/or antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes recalcitrant and severe acute chronic and persistent human infections. Here, we target the MvfR-regulated P. aeruginosa quorum sensing (QS) virulence pathway to isolate robust molecules that specifically inhibit infection without affecting bacterial growth or viability to mitigate selective resistance. Using a whole-cell high-throughput screen (HTS) and structure-activity relationship (SAR) analysis, we identify compounds that block the synthesis of both pro-persistence and pro-acute MvfR-dependent signaling molecules. These compounds, which share a benzamide-benzimidazole backbone and are unrelated to previous MvfR-regulon inhibitors, bind the global virulence QS transcriptional regulator, MvfR (PqsR); inhibit the MvfR regulon in multi-drug resistant isolates; are active against P. aeruginosa acute and persistent murine infections; and do not perturb bacterial growth. In addition, they are the first compounds identified to reduce the formation of antibiotic-tolerant persister cells. As such, these molecules provide for the development of next-generation clinical therapeutics to more effectively treat refractory and deleterious bacterial-human infections

A Small Volatile Bacterial Molecule Triggers Mitochondrial Dysfunction in Murine Skeletal Muscle

Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) metabolomics, in vivo 31P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by 1H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-γ, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo 31P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections

Host-vibrio cholerae Interaction : Modulation of Cytokine Responses in Human Intestinal Epithelial Cells Following v. cholerae Infection

Human body is in continuous contact with microbes/pathogens. Although many microbes are beneficial or harmless to humans, pathogenic microbes are a threat and can cause severe infections. Mechanical shield against microbes is provided by rigid skin and sticky mucosal surfaces. Moreover, secreted antimicrobial compounds, enzymes and low pH offer further protection at these sites. However, these primary defense systems are often inadequate as, during the course of evolution, pathogenic microbes have developed means to overcome these barriers and invade. More efficient and powerful antimicrobial defense is provided by the immune system, which can be functionally divided into two parts. The innate immunity acts rapidly and nonspecifically towards foreign components. Adaptive immunity, on the other hand, requires time to develop but it is specific to foreign antigens and generates immunological memory

IL-1b Expression in Int407 is Induced by Flagellin of Vibrio cholerae through TLR5 Mediated Pathway

Vibrio cholerae, a noninvasive enteric bacterium, causing inflammatory diarrheal disease cholera, is associated with the secretion of proinflamammatory cytokines including IL-1b in cultured epithelial cells. Incubation of Int407 with live V. cholerae resulted in increased IL-1b mRNA expression as early as 2 h of infection, reached a peak at �3.5 h and decreased thereafter. The identity of the effector molecule(s) is largely unknown. The bacterial culture supernatant showed IL-1b stimulating activity. An engineered aflagellate V. cholerae flaA mutant (O395FLAN) resulted in highly reduced level of IL-1b expression in Int407. The crude flagellar protein of V. cholerae as well as recombinant FlaA induced IL-1b expression in Int407. Infection of Toll-like receptor 5 (TLR5) transfected HeLa cells with O395FLAN showed reduced expression of IL-1b compared to wild-type. Unlike wild-type V. cholerae, O395FLAN did not activate the NF-kB while the recombinant flagellin could activate NF-kB. Finally, the mitogen activated protein kinases (ERK1 and 2, p38) were phosphorylated in wild-type and recombinant flagellin treated Int407 cells and inhibition of the p38 and ERK pathways significantly decreased the IL-1b response induced by wild-type V. cholerae as well as recombinant flagellin. Our data clearly indicate that flagellin of V. cholerae could induce IL-1b expression by recognizing TLR5 that activate NF-kB and MAP kinase in Int407

Transcriptional Upregulation of Inflammatory Cytokines in Human Intestinal Epithelial Cells Following Vibrio cholerae Infection

Coordinated expression and upregulation of interleukin-1a, interleukin-1b, tumor necrosis factor-a, interleukin-6, granulocyte–macrophage colonystimulating factor, interleukin-8, monocyte chemotactic protein-1 (MCP-1) and epithelial cell derived neutrophil activator-78, with chemoattractant and proinflammatory properties of various cytokine families, were obtained in the intestinal epithelial cell line Int407 upon Vibrio cholerae infection. These proinflammatory cytokines also showed increased expression in T84 cells, except for interleukin-6, whereas a striking dissimilarity in cytokine expression was observed in Caco-2 cells. Gene expression studies of MCP- 1, granulocyte–macrophage colony-stimulating factor, interleukin-1a, interleukin- 6 and the anti-inflammatory cytokine transforming growth factor-b in Int407 cells with V. cholerae culture supernatant, cholera toxin, lipopolysaccharide and ctxA mutant demonstrated that, apart from cholera toxin and lipopolysaccharide, V. cholerae culture supernatant harbors strong inducer(s) of interleukin-6 and MCP-1 and moderate inducer(s) of interleukin- 1a and granulocyte–macrophage colony-stimulating factor. Cholera toxin- or lipopolysaccharide-induced cytokine expression is facilitated by activation of nuclear factor-jB (p65 and p50) and cAMP response elementbinding protein in Int407 cells. Studies with ctxA mutants of V. cholerae revealed that the mutant activates the p65 subunit of nuclear factor-jB and cAMP response element-binding protein, and as such the activation is mediated by cholera toxin-independent factors as well. We conclude that V. cholerae elicits a proinflammatory response in Int407 cells that is mediated by activation of nuclear factor-jB and cAMP response element-binding protein by cholera toxin, lipopolysaccharide and ⁄ or other secreted products of V. cholerae

Involvement of Intracellular Signaling Cascades in Inflammatory Responses in Human Intestinal Epithelial Cells following Vibrio Cholerae Infection

Vibrio cholerae, the etiological agent of cholera, leads to the induction of host cell nuclear responses and the activation of proinflammatory cytokines in the cultured intestinal epithelial cells. However, the host cell signaling pathway leading to proinflammatory response is not explored. In this study, we demonstrated that V. cholerae infection on intestinal epithelial cells results in the activation of extracellular signalregulated kinases1/2(ERK1/2) and p38 of the mitogen activated protein kinase (MAPK) family. V. cholerae induced intracellular pathways in Int407 cells leading to the activation of protein kinase A (PKA) and protein tyrosin kinase (PTK) in upstream of MAPK and nuclear factor-kappaB (NF-�B) pathway. Inhibitor study of Ca2+ and phospholipase-gamma (PLC-�) pathway suggested the possible involvement of Ca2+ signaling in the V. cholerae pathogenesis. V. cholerae culture supernatants as also insertional mutants of ctxA, toxR and toxT genes modulate the activation of MAPK and NF-�B signaling pathways. MAPK and NF-�B signaling pathway activation were also modulated by adherence and motility of V. cholerae. Studies with inhibitor of NF-�B, MAPK, PTK, PKA, PKC, Ca2+ and PLC pathways showed differential cytokine secretion in Int407 following V. cholerae infection. Therefore V. cholerae mediated induction of nuclear responses through signal transduction pathway and subsequent activation of proinflammatory cytokines in Int407 modulated by V. cholerae secretory factors, virulence, adhesion/motility which might explain some of its reactogenic mechanism

Pseudomonas aeruginosa Quorum Sensing Molecule Alters Skeletal Muscle Protein Homeostasis by Perturbing the Antioxidant Defense System

Pseudomonas aeruginosa, a bacterium that is resistant to treatment, causes serious acute, persistent, and relapsing infections in humans. There is increasing evidence that bacterial excreted small molecules play a critical role during infection. We have shown that a quorum sensing (QS)-regulated excreted small molecule, 2-AA, which is abundantly produced by P. aeruginosa, promotes persistent infections, dampens host inflammation, and triggers mitochondrial dysfunction in skeletal muscle. QS is a cell-to-cell communication system utilized by bacteria to promote collective behaviors. The significance of our study in identifying a mechanism that leads to skeletal muscle dysfunction, via the action of a QS molecule, is that it may open new avenues in the control of muscle loss as a result of infection and sepsis. Given that QS is a common characteristic of prokaryotes, it is possible that 2-AA-like molecules promoting similar effects may exist in other pathogens.Skeletal muscle function is compromised in many illnesses, including chronic infections. The Pseudomonas aeruginosa quorum sensing (QS) signal, 2-amino acetophenone (2-AA), is produced during acute and chronic infections and excreted in human tissues, including the lungs of cystic fibrosis patients. We have shown that 2-AA facilitates pathogen persistence, likely via its ability to promote the formation of bacterial persister cells, and that it acts as an interkingdom immunomodulatory signal that epigenetically reprograms innate immune functions. Moreover, 2-AA compromises muscle contractility and impacts the expression of genes involved in reactive oxygen species (ROS) homeostasis in skeletal muscle and in mitochondrial functions. Here, we elucidate the molecular mechanisms of 2-AA’s impairment of skeletal muscle function and ROS homeostasis. Murine in vivo and differentiated C2C12 myotube cell studies showed that 2-AA promotes ROS generation in skeletal muscle via the modulation of xanthine oxidase (XO) activity, NAD(P)H oxidase2 (NOX2) protein level, and the activity of antioxidant enzymes. ROS accumulation triggers the activity of AMP-activated protein kinase (AMPK), likely upstream of the observed locations of induction of ubiquitin ligases Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx), and induces autophagy-related proteins. The protein-level perturbation in skeletal muscle of silent mating type information regulation 2 homolog 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), and uncoupling protein 3 (UCP3) is rescued by the antioxidant N-acetyl-l-cysteine (NAC). Together, these results unveil a novel form of action of a QS bacterial molecule and provide molecular insights into the 2-AA-mediated skeletal muscle dysfunction caused by P. aeruginosa