The Bacterial Quorum-Sensing Signal 2-Aminoacetophenone Rewires Immune Cell Bioenergetics through the PGC-1α/ERRα Axis to Mediate Tolerance to Infection.

How bacterial pathogens exploit host metabolism to promote immune tolerance and persist in infected hosts remains elusive. To achieve this, we show that Pseudomonas aeruginosa (PA), a recalcitrant pathogen, utilizes the quorum sensing (QS) signal 2-aminoacetophenone (2-AA). Here, we unveil how 2-AA-driven immune tolerization causes distinct metabolic perturbations in macrophages' mitochondrial respiration and bioenergetics. We present evidence indicating that these effects stem from a decrease in pyruvate transport into mitochondria. This reduction is attributed to decreased expression of the mitochondrial pyruvate carrier (MPC1), which is mediated by diminished expression and nuclear presence of its transcriptional regulator, estrogen-related nuclear receptor alpha (ERRα). Consequently, ERRα exhibits weakened binding to the MPC1 promoter. This outcome arises from the impaired interaction between ERRα and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Ultimately, this cascade results in diminished pyruvate influx into mitochondria and consequently reduced ATP production in tolerized macrophages. Exogenously added ATP in infected macrophages restores the transcript levels of MPC1 and ERR-α and enhances cytokine production and intracellular bacterial clearance. Consistent with the in vitro findings, murine infection studies corroborate the 2-AA-mediated long-lasting decrease in ATP and acetyl-CoA and its association with PA persistence, further supporting this QS signaling molecule as the culprit of the host bioenergetic alterations and PA persistence. These findings unveil 2-AA as a modulator of cellular immunometabolism and reveal an unprecedent mechanism of host tolerance to infection involving the PGC-1α/ERRα axis in its influence on MPC1/OXPHOS-dependent energy production and PA clearance. These paradigmatic findings paving the way for developing treatments to bolster resilience to pathogen-induced damage. Given that QS is a common characteristic of prokaryotes, it is likely that 2-AA-like molecules with similar functions may be present in other pathogens.

Multi-Biomarker Prediction Models for Multiple Infection Episodes Following Blunt Trauma.

Severe trauma predisposes patients to multiple independent infection episodes (MIIEs), leading to augmented morbidity and mortality. We developed a method to identify increased MIIE risk before clinical signs appear, which is fundamentally different from existing approaches entailing infections' detection after their establishment. Applying machine learning algorithms to genome-wide transcriptome data from 128 adult blunt trauma patients' (42 MIIE cases and 85 non-cases) leukocytes collected ≤48 hr of injury and ≥3 days before any infection, we constructed a 15-transcript and a 26-transcript multi-biomarker panel model with the least absolute shrinkage and selection operator (LASSO) and Elastic Net, respectively, which accurately predicted MIIE (Area Under Receiver Operating Characteristics Curve [AUROC] [95% confidence intervals, CI]: 0.90 [0.84-0.96] and 0.92 [0.86-0.96]) and significantly outperformed clinical models. Gene Ontology and network analyses found various pathways to be relevant. External validation found our model to be generalizable. Our unique precision medicine approach can be applied to a wide range of patient populations and outcomes.

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

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. aeruginosaIMPORTANCEPseudomonas 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.

Animal Models for Pseudomonas aeruginosa Quorum Sensing Studies.

Quorum sensing (QS) systems play global regulatory roles in bacterial virulence. They synchronize the expression of multiple virulence factors and they control and modulate bacterial antibiotic tolerance systems and host defense mechanisms. Therefore, it is important to obtain knowledge about QS modes of action and to test putative therapeutics that may interrupt QS actions in the context of infections. This chapter describes methods to study bacterial pathogenesis in murine acute and persistent/relapsing infection models, using the Gram-negative bacterial pathogen Pseudomonas aeruginosa as an example. These infection models can be used to probe bacterial virulence functions and in mechanistic studies, as well as for the assessment of the therapeutic potential of antibacterials, including anti-virulence agents.

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.

Polypharmacology Approaches against the Pseudomonas aeruginosa MvfR Regulon and Their Application in Blocking Virulence and Antibiotic Tolerance.

Pseudomonas aeruginosa is an important nosocomial pathogen that is frequently recalcitrant to available antibiotics, underlining the urgent need for alternative therapeutic options against this pathogen. Targeting virulence functions is a promising alternative strategy as it is expected to generate less-selective resistance to treatment compared to antibiotics. Capitalizing on our nonligand-based benzamide-benzimidazole (BB) core structure compounds reported to efficiently block the activity of the P. aeruginosa multiple virulence factor regulator MvfR, here we report the first class of inhibitors shown to interfere with PqsBC enzyme activity, responsible for the synthesis of the MvfR activating ligands HHQ and PQS, and the first to target simultaneously MvfR and PqsBC activity. The use of these compounds reveals that inhibiting PqsBC is sufficient to block P. aeruginosa's acute virulence functions, as the synthesis of MvfR ligands is inhibited. Our results show that MvfR remains the best target of this QS pathway, as we show that antagonists of this target block both acute and persistence-related functions. The structural properties of the compounds reported in this study provide several insights that are instrumental for the design of improved MvfR regulon inhibitors against both acute and persistent P. aeruginosa infections. Moreover, the data presented offer the possibility of a polypharmacology approach of simultaneous silencing two targets in the same pathway. Such a combined antivirulence strategy holds promise in increasing therapeutic efficacy and providing alternatives in the event of a single target's resistance development.

A quorum-sensing signal promotes host tolerance training through HDAC1-mediated epigenetic reprogramming.

The mechanisms by which pathogens evade elimination without affecting host fitness are not well understood. For the pathogen Pseudomonas aeruginosa, this evasion appears to be triggered by excretion of the quorum-sensing molecule 2-aminoacetophenone, which dampens host immune responses and modulates host metabolism, thereby enabling the bacteria to persist at a high burden level. Here, we examined how 2-aminoacetophenone trains host tissues to become tolerant to a high bacterial burden, without compromising host fitness. We found that 2-aminoacetophenone regulates histone deacetylase 1 expression and activity, resulting in hypo-acetylation of lysine 18 of histone H3 at pro-inflammatory cytokine loci. Specifically, 2-aminoacetophenone induced reprogramming of immune cells occurs via alterations in histone acetylation of immune cytokines in vivo and in vitro. This host epigenetic reprograming, which was maintained for up to 7 days, dampened host responses to subsequent exposure to 2-aminoacetophenone or other unrelated pathogen-associated molecules. The process was found to involve a distinct molecular mechanism of host chromatin regulation. Inhibition of histone deacetylase 1 prevented the immunomodulatory effects of 2-aminoacetophenone. These observations provide the first mechanistic example of a quorum-sensing molecule regulating a host epigenome to enable tolerance of infection. These insights have enormous potential for developing preventive treatments against bacterial infections.

Bacterial-excreted small volatile molecule 2-aminoacetophenone induces oxidative stress and apoptosis in murine skeletal muscle.

Oxidative stress induces mitochondrial dysfunction and facilitates apoptosis, tissue damage or metabolic alterations following infection. We have previously discovered that the Pseudomonas aeruginosa (PA) quorum sensing (QS)-excreted small volatile molecule, 2-aminoacetophenone (2-AA), which is produced in infected human tissue, promotes bacterial phenotypes that favor chronic infection, while also dampening the pathogen­induced innate immune response, thus compromising muscle function and promoting host tolerance to infection. In this study, murine whole-genome expression data have demonstrated that 2-AA affects the expression of genes involved in reactive oxygen species (ROS) homeostasis, thus producing an oxidative stress signature in skeletal muscle. The results of the present study demonstrated that the expression levels of genes involved in apoptosis signaling pathways were upregulated in the skeletal muscle of 2-AA-treated mice. To confirm the results of our transcriptome analysis, we used a novel high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) method and observed increased levels of bisallylic methylene fatty acyl protons and vinyl protons, suggesting that 2-AA induces skeletal muscle cell apoptosis. This effect was corroborated by our results demonstrating the downregulation of mitochondrial membrane potential in vivo in response to 2-AA. The findings of the present study indicate that the bacterial infochemical, 2-AA, disrupts mitochondrial functions by inducing oxidative stress and apoptosis signaling and likely promotes skeletal muscle dysfunction, which may favor chronic/persistent infection.

Prediction of multiple infections after severe burn trauma: a prospective cohort study.

OBJECTIVE: To develop predictive models for early triage of burn patients based on hypersusceptibility to repeated infections. BACKGROUND: Infection remains a major cause of mortality and morbidity after severe trauma, demanding new strategies to combat infections. Models for infection prediction are lacking. METHODS: Secondary analysis of 459 burn patients (≥16 years old) with 20% or more total body surface area burns recruited from 6 US burn centers. We compared blood transcriptomes with a 180-hour cutoff on the injury-to-transcriptome interval of 47 patients (≤1 infection episode) to those of 66 hypersusceptible patients [multiple (≥2) infection episodes (MIE)]. We used LASSO regression to select biomarkers and multivariate logistic regression to built models, accuracy of which were assessed by area under receiver operating characteristic curve (AUROC) and cross-validation. RESULTS: Three predictive models were developed using covariates of (1) clinical characteristics; (2) expression profiles of 14 genomic probes; (3) combining (1) and (2). The genomic and clinical models were highly predictive of MIE status [AUROCGenomic = 0.946 (95% CI: 0.906-0.986); AUROCClinical = 0.864 (CI: 0.794-0.933); AUROCGenomic/AUROCClinical P = 0.044]. Combined model has an increased AUROCCombined of 0.967 (CI: 0.940-0.993) compared with the individual models (AUROCCombined/AUROCClinical P = 0.0069). Hypersusceptible patients show early alterations in immune-related signaling pathways, epigenetic modulation, and chromatin remodeling. CONCLUSIONS: Early triage of burn patients more susceptible to infections can be made using clinical characteristics and/or genomic signatures. Genomic signature suggests new insights into the pathophysiology of hypersusceptibility to infection may lead to novel potential therapeutic or prophylactic targets.

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 ((1)H) nuclear magnetic resonance (NMR) metabolomics, in vivo (31)P 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 (1)H 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 (31)P 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.

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)-κB, 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-κB activation, with relatively preserved activation of extracellular regulated kinase (ERK) 1/2. These kinase changes lead to CCAAT/enhancer-binding protein-ß (c/EBPß) activation and formation of the c/EBPß-p65 complex that prevents NF-κB 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.

Activation of proinflammatory response in human intestinal epithelial cells following Vibrio cholerae infection through PI3K/Akt pathway.

Vibrio cholerae activates proinflammatory response in cultured intestinal epithelial cells. In this study, we demonstrate that V. cholerae O395 infection of intestinal epithelial cells results in the activation of Akt. Inhibition of Akt significantly decreases IL-1alpha, IL-6, and TNF-alpha production in V. cholerae infected Int407 cells. Analysis of the mechanisms of Akt influences on cytokine response demonstrates that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with virulence, adherence, or motility. Insertion mutants in the virulence genes coding for CtxA, ToxT, and OmpU of V. cholerae modulate the activation of PI3K/Akt signaling pathway, whereas an aflagellate non-motile mutant (O395FLAN) and a adherent and less motile mutant (O395Y3N/O395Y4N) of V. cholerae both show very significant down-regulation of Akt activity in Int407 cells. Together, these observations indicate that Akt promotes proinflammatory cytokine production by V. cholerae infected human intestinal epithelial cells through its influences on NF-kappaB.

Differential modulation of NF-kappaB-mediated pro-inflammatory response in human intestinal epithelial cells by cheY homologues of Vibrio cholerae.

Vibrio cholerae, the etiological agent of cholera, colonizes the small intestine, produces an enterotoxin and causes acute inflammatory response at intestinal epithelial surface. Chemotaxis and motility greatly influence the infectivity of V. cholerae although the role of chemotaxis genes in V. cholerae pathogenesis is less well understood. Four cheY genes are present in three clusters in the complete genome sequence of V. cholerae. A less motile and less adherent mutant was generated by inactivation of cheY-3 (O395Y3N) or cheY-4 (O395Y4N) whereas alterations in motility or adherence were not observed for cheY-1 (O395Y1N) or cheY-2 (O395Y2N) insertional mutants. In contrast to O395Y1N and O395Y2N, O395Y3N and O395Y4N showed reduced cholera toxin production compared to wild-type in vitro. Infection of the human intestinal epithelial cell line Int407 with O395Y3N and O395Y4N caused reduced secretion of interleukin (IL)-1a, IL-6, tumor necrosis factor (TNF-a) and monocyte chemotactic protein-1 (MCP-1) compared to wild-type and was associated with delayed activation of nuclear factor kappa B (NF-kappaB) p65 and its co-activator cAMP response element binding protein (CREB). Further, the absence of nuclear translocation of NF-kappaB p50 subunit upon infection with O395Y3N or O395Y4N and its reversal upon complementation indicates the involvement of cheY-3 and cheY-4 in V. cholerae-induced pro-inflammatory response in the INT407 cell line.

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 signal-regulated 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 tyrosine kinase (PTK) in upstream of MAPK and nuclear factor-kappaB (NF-kappaB) pathway. Inhibitor study of Ca(2+) and phospholipase-gamma (PLC-gamma) pathway suggested the possible involvement of Ca(2+) 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-kappaB signaling pathways. MAPK and NF-kappaB signaling pathway activation were also modulated by adherence and motility of V. cholerae. Studies with inhibitor of NF-kappaB, MAPK, PTK, PKA, PKC, Ca(2+) 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 mechanisms.

IL-1beta 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-1beta in cultured epithelial cells. Incubation of Int407 with live V. cholerae resulted in increased IL-1beta mRNA expression as early as 2h of infection, reached a peak at approximately 3.5h and decreased thereafter. The identity of the effector molecule(s) is largely unknown. The bacterial culture supernatant showed IL-1beta stimulating activity. An engineered aflagellate V. cholerae flaA mutant (O395FLAN) resulted in highly reduced level of IL-1beta expression in Int407. The crude flagellar protein of V. cholerae as well as recombinant FlaA induced IL-1beta expression in Int407. Infection of Toll-like receptor 5 (TLR5) transfected HeLa cells with O395FLAN showed reduced expression of IL-1beta compared to wild-type. Unlike wild-type V. cholerae, O395FLAN did not activate the NF-kappaB while the recombinant flagellin could activate NF-kappaB. 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-1beta response induced by wild-type V. cholerae as well as recombinant flagellin. Our data clearly indicate that flagellin of V. cholerae could induce IL-1beta expression by recognizing TLR5 that activate NF-kappaB 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-1alpha, interleukin-1beta, tumor necrosis factor-alpha, interleukin-6, granulocyte-macrophage colony-stimulating 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-1alpha, interleukin-6 and the anti-inflammatory cytokine transforming growth factor-beta 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-1alpha and granulocyte-macrophage colony-stimulating factor. Cholera toxin- or lipopolysaccharide-induced cytokine expression is facilitated by activation of nuclear factor-kappaB (p65 and p50) and cAMP response element-binding protein in Int407 cells. Studies with ctxA mutants of V. cholerae revealed that the mutant activates the p65 subunit of nuclear factor-kappaB 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-kappaB and cAMP response element-binding protein by cholera toxin, lipopolysaccharide and/or other secreted products of V. cholerae.

Human intestinal epithelial cell cytokine mRNA responses mediated by NF-kappaB are modulated by the motility and adhesion process of Vibrio cholerae.

Vibro cholerae, the etiological agent of cholera, colonizes the small intestine, produces an enterotoxin and causes acute inflammatory response at intestinal epithelial surface; the signals for such induction are still unknown. We determined the mRNA expression of proinflammatory and anti-inflammatory cytokines in Int407 cells following infection with V. cholerae or its mutants by semi-quantitaive and quantitative real-time RT-PCR. V. cholerae induces the coordinated expression and up-regulation of IL-1alpha, IL-6, GM-CSF and MCP-1 and down-regulation of TGF-beta in Int407 cells. While the pathogenecity of V. cholerae was found to be a possible determinant in modulation of IL-1alpha and TGF-beta, both IL-6 and MCP-1 OmpU might modulate induction. Significant reduction in IL-1alpha, GM-CSF and MCP-1 mRNA expression was observed upon infection with the less motile and less adherent strain O395YN. This association is supported by the absence of nuclear translocation of NF-kappaB (p50 subunit) upon infection with O395YN in contrast to wild-type. Moreover, TPCK treatment prior to V. cholerae infection indicated that proinflammatory cytokine gene expression in Int407 cells is NF-kappaB mediated. Thus, V. cholerae induces proinflammatory cytokine response in Int407 cells, which is mediated by NF-kappaB and is modulated, in part, by adherence or motility of this organism.

Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis.

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.