Effect of cetrimonium carrier micelles on bacterial membranes and extracellular DNA, an in silico study.

Microorganisms do not live as dispersed single cells but rather they form aggregates with extracellular polymeric substances at interfaces. Biofilms are considered efficient life forms because they shield bacteria from biocides and collect dilute nutrients. This is a big concern in industry since the microorganisms can colonize a wide range of surfaces, accelerating material deterioration, colonizing medical devices, contaminating ultrapure drinking water, increasing energy costs and creating focus of infection. Conventional biocides that target a specific component of the bacteria are not effective in the presence of biofilms. Efficient biofilm inhibitors are based on a multitarget approach interacting with the bacteria and the biofilm matrix. Their rationale design requires a thorough understanding of inhibitory mechanisms that are still largely lacking today. Herein we uncover via molecular modelling the inhibition mechanism of cetrimonium 4-OH cinnamate (CTA-4OHcinn). Simulations show that CTA-4OH micelles can disrupt symmetric and asymmetric bilayers, representative of inner and outer bacterial membranes, following three stages: adsorption, assimilation, and defect formation. The main driving force for micellar attack is electrostatic interactions. In addition to disrupting the bilayers, the micelles work as carriers facilitating the trapping of 4OH cinnamate anions within the bilayer upper leaflet and overcoming electrostatic repulsion. The micelles also interact with extracellular DNA (e-DNA), which is one of the main components of biofilms. It is observed that CTA-4OHcinn forms spherical micelles on the DNA backbone; which hinders their ability to pack. This is demonstrated by modelling the DNA along the hbb histone-like protein, showing that in the presence of CTA-4OHcinn, DNA does not pack properly around hbb. The abilities of CTA-4OHcinn to cause cell death through membrane disruption and to disperse a mature, multi-species biofilm are also confirmed experimentally.

Bacterial DNA promoting inflammation via the Sgk1/Nedd4L/Syk pathway in mast cells contributes to antihistamine-nonresponsive CSU.

Inflammation centered on non-IgE-mediated mast cell activation characterizes chronic spontaneous urticaria resistant to nonsedating H1-antihistamines. We recently uncovered a strong positive association between inflammation and the fecal Escherichia. To further explore the actions of bacterial DNA derived from Escherichia on mast cells, intestinal permeability of patients with chronic spontaneous urticaria with or without nonsedating H1-antihistamine resistance and healthy controls were determined, and LAD2 cells with knockdown of Syk, Nedd4L, or Sgk1 or with incubation of inhibitors GS9973, GSK650394, and MG132 were posttreated with btDNA. We found that (i) serum intestinal permeability indices and bacterial DNA markedly increased in patients with chronic spontaneous urticaria with nonsedating H1-antihistamine resistance compared with those without (all P < 0.001), and bacterial DNA positively correlated with the degree of inflammation; (ii) IL-6 and TNF-α levels were time- and dose-dependently upregulated in bacterial DNA-stimulated LAD2 cells, which relied on unmethylated CpG in bacterial DNA and Toll-like receptor 9 protein in cells; (iii) Syk knockdown or inhibition of Syk Tyr525/526 phosphorylation blocked bacterial DNA-initiated cytokine production; (iv) Nedd4L interacted with Tyr525/526-phosphorylated Syk, and inhibition of Nedd4L Ser448 phosphorylation induced by bacterial DNA-activated Sgk1 was mandatory for bacterial DNA's proinflammatory property; and (v) Sgk1 suppression showed an inhibitory effect on bacterial DNA-induced inflammation by ensuring Nedd4L-mediated ubiquitination of Tyr525/526-phosphorylated Syk. Collectively, we identified previously unknown contributory roles of bacterial translocation and serum bacterial DNA on the inflammation phenotype in patients with chronic spontaneous urticaria with nonsedating H1-antihistamine resistance and further uncovered a vital negative regulatory role for the Sgk1/Nedd4L/Syk pathway in bacterial DNA-induced inflammation in LAD2 cells.

Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles.

Endocytosis and endosomal trafficking to vacuoles play important roles in regulating the homeostasis of plasma membrane (PM) proteins in plant cells. FREE1 (FYVE domain protein required for endosomal sorting 1) is a plant-unique component of the ESCRT (endosomal sorting complex required for transport) machinery. In free1 mutant plants, PIN-FORMED 2 (PIN2)-GFP was found to mislocalize from the PM to the tonoplast. In this chapter, we describe a detailed protocol for studying vacuolar sorting and degradation of PIN2-GFP by using T-DNA insertional mutants, dexamethasone (DEX) inducible RNAi lines, and other tools, including Fei-Mao (FM) dye staining and dark treatment. By using these methods, we illustrate the endosomal trafficking and vacuolar degradation of PIN2-GFP in plants.

Decoy exosomes provide protection against bacterial toxins.

The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)1-3. It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding α-toxin4-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues2,5,6. Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against α-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins.

Bacterial DNA promotes Tau aggregation.

A hallmark feature of Alzheimer's disease (AD) and other tauopathies is the misfolding, aggregation and cerebral accumulation of tau deposits. Compelling evidence indicates that misfolded tau aggregates are neurotoxic, producing synaptic loss and neuronal damage. Misfolded tau aggregates are able to spread the pathology from cell-to-cell by a prion like seeding mechanism. The factors implicated in the initiation and progression of tau misfolding and aggregation are largely unclear. In this study, we evaluated the effect of DNA extracted from diverse prokaryotic and eukaryotic cells in tau misfolding and aggregation. Our results show that DNA from various, unrelated gram-positive and gram-negative bacteria results in a more pronounced tau misfolding compared to eukaryotic DNA. Interestingly, a higher effect in promoting tau aggregation was observed for DNA extracted from certain bacterial species previously detected in the brain, CSF or oral cavity of patients with AD. Our findings indicate that microbial DNA may play a previously overlooked role in the propagation of tau protein misfolding and AD pathogenesis, providing a new conceptual framework that positions the compromised blood-brain and intestinal barriers as important sources of microbial DNA in the CNS, opening novel opportunities for therapeutic interventions.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite.

OBJECTIVE: This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). METHODOLOGY: E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. RESULTS: CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). CONCLUSIONS: Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite

Abstract Objective This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). Methodology E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. Results CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Conclusions Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Pretreatment with bacterial components promotes DSS-injured colonic epithelial repair through the activation of STAT-3.

Bacterial protection and epithelial repair are important against inflammatory bowel disease (IBD). The present study was designed to examine the effects of different bacterial components on the repair of normal and dextran sodium sulfate (DSS)­treated colonic epithelial cells and the corresponding mechanisms. Human colonic epithelial cells (HT­29) were pretreated with various doses of LPS or CpG­dsDNA for 24 h and then treated with or without DSS for another 24 h. The epithelial repair was assessed by video analyses following mechanical injury. The epithelial expression of cluster of differentiation (CD)40 was assayed using flow cytometeric analysis. The production of interleukin (IL)­6 and tumor necrosis factor (TNF) in the cell culture medium were measured using ELISA. The expression of p38 mitogen­activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT)­3 were examined using western blot analysis and reverse transcription­quantitative polymerase chain reaction analysis. MAPK and STAT­3 inhibitors were also administrated to observe signaling­mediated repair. The results showed that pretreatment with lipopolysaccharide (LPS) or CpG­dsDNA promoted epithelial repair of the DSS­treated cells. The promoting effects were associated with the downregulation of CD40 molecules, inhibition of the p38 MAPK/TNFα pathway and activation of the STAT3/IL­6 pathway. The STAT3 inhibitor abrogated the protective effects of LPS and CpG­dsDNA on wound repair. These results demonstrated that LPS and CpG­dsDNA induced preadaptation to DSS injury. This preadaptation was accompanied by the activation of STAT­3. Thus, bacterial components may be used as a strategy for the therapeutic prevention of IBD.

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD.

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1-5 ß- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-ß1,4Araf-ß1,2Araf-ß1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its' product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

Induction of type I interferons in response to bacterial stimuli in large yellow croaker Larimichthys crocea.

In addition to the crucial roles in coordinating antiviral immune responses, type I interferons (IFNs) also play a role in the host immunity against bacterial pathogens. Our previous study identified two type I IFNs from large yellow croaker Larimichthys croaea(Lc), LcIFNd and LcIFNh, and showed their strong induction by poly(I:C) and antiviral activities. In the present study, both LcIFNd and LcIFNh were found to be rapidly induced in head kidney and spleen by mixed bacteria of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas hydrophila. In the head kidney primary cells (HKCs), expression of these two LcIFN genes was increased by peptidoglycan (PGN) from Bacillus subtilis and lipopolysaccharide (LPS) from Escherichia coli. Consistently, Lc IFN-regulatory factor (LcIRF) 3 and LcIRF7, two key transcription factors of type I IFN expression, were also induced by these three bacteria, PGN, and LPS. These observations strongly suggested that large yellow croaker type I IFNs are involved in the immune response against bacterial infection. Luciferase assays showed that promoters of both LcIFNd and LcIFNh were activated by PGN, LPS, and genomic DNA of A. hydrophila, and A. hydrophila DNA was more potent than PGN and LPS in activating LcIFNd and LcIFNh promoters. Furthermore, the induction of LcIFNd promoter by these bacterial stimuli was further enhanced by the overexpression of LcIRF7 or LcIRF7 along with LcIRF3, while that of LcIFNh promoter was increased following the overexpression of LcIRF3 alone, suggesting that the induction of these two large yellow croaker IFNs by bacterial stimuli may be regulated via distinct manners. These results therefore revealed novel aspects of the functional regulation of teleost type I IFNs.

Biphasic Ccl20 regulation by Toll-like receptor 9 through the activation of ERK-AP-1 and non-canonical NF-κB signaling pathways.

BACKGROUND: Chemokines play key roles in immune homeostasis and inflammatory response. Considering the role of Ccl20 and Toll-like receptor 9 (TLR9) in gut homeostasis and inflammatory bowel disease (IBD), regulation of Ccl20 by bacterial DNA, the TLR9 ligand, merits in-depth studies. METHODS: We analyzed Ccl20 expression in various epithelial cell (EC) lines by q-PCR and ELISA. In-vivo expression was investigated in isolated murine colonocytes by immunoblotting. Transcriptional regulation of Ccl20 was studied by reporter assays, gene knock-down, electrophoretic mobility shift assay and chromatin immunoprecipitation. Activation of upstream kinases was checked by immunoblotting. RESULTS: We showed low levels of Ccl20 expression in mouse colonic ECs, but marked induction by in vivo treatment with bacterial DNA. This corroborated with persistent Ccl20 induction in different EC lines. We found involvement of MAP-kinases during the early hours after stimulation, and a novel AP-1site (-252bp) regulated the expression in colonic ECs. More importantly, mutually exclusive transcriptional regulation by AP-1 (cjun/cfos) and non-canonical NF-κB (RelB/p52) downstream of MEK-ERK and NIK-IKK-α-NF-κB2 (p100) phosphorylation, respectively was responsible for persistent Ccl20 expression in the colonic cells, while canonical NF-κB isoforms played no role. CONCLUSIONS: Persistent Ccl20 induction by TLR9 in colonic ECs involves early and delayed activation of two independent signaling pathways. This is the first report of non-canonical NF-κB activation and Ccl20 expression in the colonic ECs by TLR9. GENERAL SIGNIFICANCE: Our study will help to better understand immune regulation by Ccl20 in the intestine and may be exploited for future development of novel therapeutics against IBD.

Molecular Characterization and Epidemiologic Study of NDM-1-Producing Extensively Drug-Resistant Escherichia coli.

The emergence and dissemination of NDM-1 (New Delhi metallo-ß-lactamase-1)-producing Enterobacteriaceae have resulted in a worldwide public health risk. This study described a high incidence and endemic spread of NDM-1-producing extensively drug-resistant Escherichia coli in a teaching hospital in Zhejiang province, China. We recovered six nonduplicated NDM-1-producing E. coli isolates from May 2014 to August 2014 with positive modified Hodge test and EDTA synergistic test. These isolates were highly resistant to ß-lactam antimicrobials, aminoglycosides, and quinolones. PCR and DNA sequences analysis showed that all isolates carried the blaNDM-1, blaSHV-11, aac(6')-ib-cr, and qnrB. Several isolates also harbored blaCTX-M-1, blaCTX-M-9, rmtB, and qnrA. Southern blot confirmed that blaNDM-1 was located on the same ∼55 kb plasmid and conjugation experiments further proved the contransferable characteristic of blaNDM-1. The ompC sequences showed various mutations, which was related to multidrug resistance in E. coli. Pulsed-field gel electrophoresis identified four of six isolates that belonged to the same genotype. Multilocus sequence typing assigned them to ST2, except one strain that belonged to ST594. Our study demonstrated that the resistance-associated genes and the loss of the outer membrane proteins could account for high resistance of NDM-1-producing E. coli to multiple antimicrobial drugs. Both horizontal transfer of IncN and transmission of ST2 were responsible for the spread of drug resistance. These findings highlighted an urgent need to limit the further dissemination of NDM-1-producing E. coli in this region.

Protective role of dexmedetomidine in unmethylated CpG-induced inflammation responses in BV2 microglia cells.

Unmethylated CpG DNA, as a stimulatory molecule, has potent pro-inflammatory effects in the central nervous system (CNS). Dexmedetomidine (DEX) has been confirmed to exert anti-inflammatory effects in CNS. Our study was aimed to explore the effects of DEX on tumor necrosis factor-α (TNF-α) expression in unmethylated CpG DNA-challenged microglia. In vivo, after 3 d intracisternal injection of ODN1668, we evaluated the severity of meningitis with or without DEX via pathobiology method and detected the expression of TNF-α from molecular and protein levels. In vitro, we explored whether the ODN1668 could activate microglia to express TNF-α and the inhibition mechanism of DEX. Our results demonstrated that DEX could alleviate the severity of ODN1668-induced meningitis. And while BV2 microglia was stimulated by ODN1668 for different time, TNF-α was increased in mRNA and protein levels but the effect was attenuated by DEX via decreasing phosphorylated AKT and ERK.

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa.

Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.

Monoclonal Antibodies Against the Human Respiratory Syncytial Virus Obtained by Immunization with Epitope Peptides and CpG-DNA-liposome Complex.

Respiratory syncytial virus (RSV) is the major cause of pulmonary inflammation in infants, young children, and immunocompromised adults. However, the RSV vaccine is not yet available commercially. The RSV-F glycoprotein mediates virus-host cell fusion, leading to syncytial formation; therefore, the RSV-F glycoprotein has been a treatment target for prevention and therapy of RSV infection. To produce the RSV-F-protein epitope-specific monoclonal antibody (MAb), BALB/c mice were immunized with a complex consisting of epitope peptide and MB-ODN 4531(O), encapsulated in a phosphatidyl-ß-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex (Lipoplex(O)). Using conventional hybridoma technology, we obtained two clones able to produce antibodies reactive to two B-cell epitopes of RSV-F protein. Each anti-RSV-F glycoprotein MAb efficiently binds to each epitope. The F7-1A9D10 clone showed specific binding with RSV-F protein. There was no specific protein detected by Western blot analysis using F9 epitope-specific anti-RSV-F glycoprotein MAb (clone F9-1A6C8). However, based on confocal-image analysis, the antibody from the F9-1A6C8 clone showed specific binding with RSV-F protein. It is important that further study on possible applications for passive immunotherapy against RSV infection, such as therapeutic antibody production, is carried out.

Short communication: Antiproliferative effect of 8 different Lactobacillus strains on K562 cells.

Some strains of Lactobacillus genus have antiproliferative activities against cancer cells. However, until now, the exact effector molecules of Lactobacillus strains with anticancer activity have not been identified. The aim of the present study was to explore which fraction of the Lactobacillus cells exerts the highest antiproliferative effect. For this purpose, the heat-killed bacterial cells, bacterial cell wall extract, and genomic DNA of 8 Lactobacillus strains were prepared to assess their antiproliferative activities against human myeloid leukemia cell lines K562. The heat-killed bacterial cells of the 8 lactobacilli strains exerted antiproliferative effect on K562 cells, and the inhibition rates exerted by the heat-killed bacterial cells of the strains G15AL, M5AL, SB31AL, SB5AL, and T3AL were significantly higher than those exerted by the cell walls and genomic DNA of the strains. The bacterial DNA of G15AL exerted higher antiproliferative effect on K562 cells. The exact effector molecules and the effect mechanism of the strains should be further explored for the application of these strains as probiotic strains or bioactive probiotic molecules.

Extracellular DNA impedes the transport of vancomycin in Staphylococcus epidermidis biofilms preexposed to subinhibitory concentrations of vancomycin.

Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular d-Ala-d-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

TLR9 ligands induce S100A8 in macrophages via a STAT3-dependent pathway which requires IL-10 and PGE2.

S100A8 and S100A9 are highly-expressed calcium-binding proteins in neutrophils and monocytes, and in subsets of macrophages in inflammatory lesions. Unmethylated CpG motifs found in bacterial and viral DNA are potent activators of innate immunity via Toll-like receptor 9 (TLR9). S100A8, but not S100A9, mRNA and protein was directly induced by CpG-DNA in murine and human macrophages. Induction in murine macrophages peaked at 16 h. CpG-DNA-induced S100A8 required de novo protein synthesis; IL-10 and Prostaglandin E2 (PGE2) synergistically enhanced expression and promoted earlier gene induction. Inhibitors of endogenous IL-10, PGE2, and the E prostanoid (EP) 4 receptor strongly suppressed S100A8 expression, particularly when combined. Thus, S100A8 induction by E. coli DNA required both IL-10 and PGE2/EP4 signaling. The MAPKs, PI3K and JAK pathways were essential, whereas ERK1/2 appeared to play a direct role. S100A8 induction by CpG-DNA was controlled at the transcriptional level. The promoter region responsible for activation, either directly, or indirectly via IL-10 and PGE2, was located within a -178 to -34-bp region and required STAT3 binding. Because of the robust links connecting IL-10 and PGE2 with an anti-inflammatory macrophage phenotype, the induction profile of S100A8 strongly indicates a role for this protein in resolution of inflammation.

Differential protection from tobramycin by extracellular polymeric substances from Acinetobacter baumannii and Staphylococcus aureus biofilms.

We investigated biofilms of two pathogens, Acinetobacter baumannii and Staphylococcus aureus, to characterize mechanisms by which the extracellular polymeric substance (EPS) found in biofilms can protect bacteria against tobramycin exposure. To do so, it is critical to study EPS-antibiotic interactions in a homogeneous environment without mass transfer limitations. Consequently, we developed a method to grow biofilms, harvest EPS, and then augment planktonic cultures with isolated EPS and tobramycin. We demonstrated that planktonic cultures respond differently to being treated with different types of EPS (A. baumannii versus S. aureus) in the presence of tobramycin. By harvesting EPS from the biofilms, we found that A. baumannii EPS acts as a "universal protector" by inhibiting tobramycin activity against bacterial cells regardless of species; S. aureus EPS did not show any protective ability in cell cultures. Adding Mg(2+) or Ca(2+) reduced the protective effect of A. baumannii EPS. Finally, when we selectively digested the proteins or DNA of the EPS, we found that the protective ability did not change, suggesting that neither has a significant role in protection. To the best of our knowledge, this is the first study that demonstrates how EPS protects pathogens against antibiotics in a homogeneous system without mass transfer limitations. Our results suggest that EPS protects biofilm communities, in part, by adsorbing antibiotics near the surface. This may limit antibiotic diffusion to the bottom of the biofilms but is not likely to be the only mechanism of protection.

Cytosolic double-stranded DNA induces nonnecroptotic programmed cell death in trophoblasts via IFI16.

The mechanisms underlying the immune defense by trophoblasts against pathogens remain ill defined. We demonstrated that placental cell death was increased upon in vivo exposure to Listeria monocytogenes. The death of infected cells is an important host innate defense mechanism. Meanwhile, double-stranded DNA (dsDNA) derived from intracellular bacteria or dsDNA viruses is emerging as a potent pathogen-associated molecular pattern recognized by host cells. We sought to characterize trophoblast death in response to cytosolic dsDNA challenge. Our results showed that dsDNA induced caspase-dependent and -independent cell death in human trophoblasts. However, necroptosis, a cell death pathway independent of caspase, could not be induced by dsDNA treatment, even in the presence of exogenously expressed RIPK3. L. monocytogenes-derived genomic DNA triggered a similar cell death pattern. Moreover, the cell death in response to dsDNA was IFI16 dependent. These data suggest that cytosolic dsDNA induces nonnecroptotic cell death in trophoblasts via IFI16, and this could contribute to placental barrier against infection.