LB Agar Plates Research Articles

Sa1808 Endogenous Production of Antibiotics by Mesenchymal Stem Cells and the Potential Value in Crohn's Fistula Healing

Introduction Potent effects of human mesenchymal stem cells (MSCs) on immune responses have been demonstrated in vitro studies and animal models, encouraging further investigations into their clinical value. Studies of MSCs in fistula healing have emphasised the immunosuppressive mechanisms of action however, there has been little focus on their antibacterial activity in this setting. We report here on the antibacterial effects of MSCs against Adherent Invasive Escherichia coli (AIEC), reference strain LF82. Methods Cultured human bone marrow derived MSCs were plated at a density of 5×10 4 cells per square centimetre in 24 well plates and allowed to adhere overnight. Confluent MSCs were preincubated for 1-day with fresh culture medium alone or medium supplemented with 1 ng/ml TNFα. Cells of passage numbers 4–9 were co-cultured with a calculated 300 Colony Forming Units of AIEC strain LF82 and incubated for 6 h in a humidified CO 2 incubator. Aliquots of the infected medium were taken from each well and plated on LB-agar plates and colonies were counted after overnight incubation. Comparisons were made with aliquots from inoculated wells containing cells that were pre-stimulated with TNFα, in addition to wells that did not contain mesenchymal stem cells. Data analyses were performed using the SPSS statistical package (V.19.0). Results Each of the wells under test conditions was inoculated with 379±98 CFU at the start of the co-culture time period. In the absence of mesenchymal stem cells, the number of LF82 CFUs in mesenchymal stem cell culture medium had risen to a mean (±SD) of 1780±319 [n=3], after 6 h. In the presence of passage-4 MSCs, the number of LF82 CFU was significantly reduced by 81.2%, to 335±61 [n=3] (p Conclusion The antibacterial properties of MSCs alongside their tissue regenerative potential make them ideal candidates for use in therapeutic strategies for fistula healing. This study demonstrates that in a proinflammatory environment, MSCs enhance bacterial clearance of AIEC. These findings encourage further evaluation of the antimicrobial effector function of MSCs against other strains suspected to participate in the pathogenesis of Crohn9s disease. Competing interests None declared.

RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoSK173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes.

A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography

A new screening method for β-(1,3-1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.

Green synthesis of Silver nanoparticles through Calotropis gigantea leaf extracts and evaluation of antibacterial activity against Vibrio alginolyticus

Green synthesized silver nanoparticles by <em>Calotropis gigantea</em> leaf extract were used to study the inhibitory activity against pathogenic <em>Vibrio alginolyticus</em>, isolated from wild <em>Artemia franciscana</em> cysts. Silver nanoparticle synthesis was observed using UV-visible spectroscopy and the morphological characteristics were analyzed by atomic force microscope (AFM). In the present study, increasing concentrations of silver nanoparticles synthesized on LB agar plates effectively reduced the number of colonies of <em>V. alginolyticus</em>. A decrease in colonies (CFUs) was observed at 5 mg/mL of silver nanoparticle concentration and the complete inhibition of V. alginolyticus was observed at 20 mg/mL of silver nanoparticle concentration on LB agar plates. In vivo controlling efficiency of silver nanoparticles was tested in an <em>A. franciscana</em> hatching system. Effective control of <em>V. alginolyticus</em> in brine shrimp <em>A. franciscana</em> hatching units was achieved by experimental infection and treatment with silver nanoparticles. Experimental infection studies showed that <em>V. alginolyticus</em> infected <em>Artemia</em> nauplii treated with silver nanoparticles (10 mg/mL) had greater survival (>40%) than silver nanoparticles not treated with nauplii. Based on the findings of this study, it is recommended that low concentrations of green synthesized silver nanoparticles should be further investigated for other potential experimental models to control potential medical pathogens.

Radioprotection Imparted by Four Spices in a Bacterial System

Phenolic substances derived from herbs and spices are potent antioxidants and exhibits anti-inflammatory, antimutagenic and anticarcinogenic activities in living systems. [1] These extracts may have the potential to modulate the genotoxic activity caused by harmful radiations and chemicals to which the human population is incessantly exposed to. The study of the modulation of genotoxic activity by some commonly used Indian spices will therefore be meaningful and interesting. As a first step to this, a number of such spices have been employed to study their potential role in the modulation of genotoxic activity induced by a harmful radiation viz. UVC, using Salmonella typhimurium as a model test system. The umu-gene expression assay as devised by Oda et al[2] in Salmonella typhimurium TA 1535/pSK1002 cells have been widely used and accepted as a short term bacterial assay system for the screening of genotoxic agents. In the above noted strain a umuC’-lacZ fusion gene is carried in a multi copy plasmid, thus improving the sensitivity of the system for the detection of umu gene expression (the gene that is believed to be responsible for induced mutagenesis). Measurement of the amount of β-galactosidase produced in these cells under different conditions would therefore reflect the extent of umu gene induction under those conditions. The present study aims at investigating on the modulation of DNA damage in TA 1535/pSK1002 cells induced by a fixed dose of UVC (which produces a significant amount of DNA damage) with varying doses of crude extracts of four commonly used Indian spices viz. Ajwain (Trachyspermum copticum), Black Pepper (Piper nigrum), Clove (Syzyquim aromaticum), and Allspice (Pimenta dioica). A study of the survival of the cells ABSTRACT: Introduction: A wide variety of substances derived from herbs and spices have been shown to possess potent antioxidant, anti-inflammatory, anti-mutagenic and anticarcinogenic activities. The study of the modulation of genotoxicity by the extracts of some such spices would thus be meaningful and interesting. Objective: Aqueous extracts of four extensively used spices viz. Trachyspermum copticum (Ajwain), Piper nigrum (Black pepper), Syzyquim aromaticum (Clove) and Pimenta dioica (Allspice) have been employed to study the modulation of genotoxicity, if any, induced by a potent DNA damaging agent viz. ultraviolet radiation (UV C), in Salmonella typhimurium cells. Methods: Induction of umu-gene was assayed in Salmonella typhimurium cells in presence or absence of varying amounts of the spice extract after a fixed dose of UV C exposure. The percentage survival of the Salmonella typhimurium cells exposed to UV C was computed by the study of the colony forming units on LB-Agar plates. Results: All the aqueous extracts of Ajwain, Black Pepper, Clove and Allspice was found to impart radioprotection to Salmonella typhimurium cells against UV C induced DNA damage in a dose–dependent manner. Clove has been found to impart a very high degree of radioprotection as compared to the other spice extracts and also at a much lower concentration. This has been reflected by both the assay methods mentioned above. Conclusions: Our results indicate that aqueous extracts of all four spices studied, impart radioprotection against UV induced DNA damage in Salmonella typhimurium cells in a dose-dependant manner. All the spice extracts mentioned above, excepting clove, presumably due to its antibacterial activity, were found to support the growth of the Salmonella typhimurium cells. This may be indicative that the extracts are imparting protection by effectively reducing the UV dose upon absorption of a part of UVC by the constituents of the spice extracts excepting clove.

A novel cold-active β-D-galactosidase from the Paracoccus sp. 32d - gene cloning, purification and characterization

Backgroundβ-D-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Cold-active β-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis.ResultsA genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the β-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-β-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C.ConclusionsA novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active β-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active β-D-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of Paracoccus sp. 32d β-D-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.

Characterization of chromosomal mediated cadmium resistance in Pseudomonas aeruginosa strain BC15

Cadmium (Cd) has been used extensively in metal plating, mining, paints and plastic generation etc. In this study, Cd resistance (cadR) gene was characterized from the environmental isolate Pseudomonas aeruginosa BC15. The cadR sequences showed high homology with P. aeruginosa FLH033011 (100%), P. aeruginosa PAO1 (99%), and P. aeruginosa UCBPP-PA14 (98%) respectively. Homology modeling of cadR was carried out by using swiss-prot server. Crystal structures of E. coli CueR for Cu (1q05) and ZntR (1q08) for Zn have been used as a template. The sequence identity of P. aeruginosa cadR shares 34% for CueR and 43% for ZntR. Fold recognition of P. aeruginosa cadR was created by using PHYRE web server. Transcriptional regulator CueR (1q06a) from E. coli was chosen as the template. CadR has 31% identity and the estimated precision was 100%. The cadR gene was cloned in pET30b and transformed into E. coli BL21. The molecular weight protein of cadR was estimated to be 25 kDa by SDS-PAGE. The recombinant E. coli cadR efficiently grow in the Cd supplemented LB medium and agar plate. The order of the resistance of E. coli cadR was Mn > Pb > Cu > Cd > Ni > Zn. These findings can lead to the use of P. aeruginosa BC15 for the remediation of Cd and other heavy metals present in the polluted environment.

The Effects of Combinatorial Treatments With Stress Inducing Molecules on Growth of E. coli Colonies

Stress inducing molecules affect both the mean behavior of bacterial growth and also variations in the growth. While the mechanisms that cause changes in the mean behavior are well understood, little is known about changes in the variation of the population. A true understanding of how organisms respond to stress must include an understanding of the mechanisms and purposes of changes in variation and the distribution not directly related to changes in the mean of the population. We have explored the results of combinatorial treatments using EDTA, copper sulfate, hydrogen peroxide, and hydrochloric acid as stress inducing molecules on bacterial colony formation and area on LB-agar plates. Three different combinations of X-gal and IPTG were used to create different background conditions. Some treatments alter the variation and/or the distribution of the area without having a significant effect on the mean, others affect the mean without altering the distribution, and yet others affect distribution and the mean.

Exopolysaccharide Production Is Influenced by Sugars,N-Acylhomoserine Lactone, and Transcriptional Regulators RcsA and RcsB, but Does Not Affect Pathogenicity in the Plant PathogenPantoea ananatis

Pantoea ananatis SK-1 produced EPS by AHL-mediated quorum sensing on an LB agar plate containing glucose, fructose, and sucrose. rcsA and rcsB mutants did not produce EPS with or without AHLs and with or without sugars, but they induced necrotic symptoms in onion leaves. These results indicate that EPS production does not relate to the pathogenicity of SK-1.

A mechanism for the effect of alginate on the gut microflora

IntroductionAlginates are hydrocolloids produced by brown seaweeds which are commonly used as gelling and thickening agents in the food industry. In addition in the pharmaceutical industry they are found in...

Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone

Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.

Development and Characterization of High Affinity Leptins and Leptin Antagonists

Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

Miller LB agar plates

Miller LB agar plates

Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.

Purification and characterization of highly thermostable α-amylase from thermophilic Alicyclobacillus acidocaldarius

In this study, the production of extracellular thermostable α-amylase by newly isolated thermophilic Alicyclobacillus acidocaldarius was detected on LB agar plates containing 1.0% soluble potato starch and incubated at 60°C. This extracellular α-amylase was purified to homogeneity by ammonium sulphate precipitation followed by Sephadex and ion-exchange chromatography. The α-amylase was purified to 8.138 fold homogeneity with a final recovery of 58% and a specific activity of 3,239 U/mg proteins. The purified α-amylase appeared as a single protein band on SDS-PAGE with a molecular mass of 94.5 kDa. Non-denaturing PAGE analysis showed one major band associated with enzyme activity, indicating the absence of isoenzymes. A TLC analysis showed maltose as major end product of the enzyme. The optimum assay temperature and pH for enzyme activity were 60°C and 6.0 respectively; however, the enzyme activity was stable over a wide range of pH and temperatures. The α-amylase retained its activity in the presence of the denaturing agents — SDS, Triton X-100, Tween-20, Tween-80, and was significantly inhibited by EDTA and urea. Calcium ions increased the enzyme activity, while Hg2+, Zn2+, and Co2+ had inhibitory effects. The K m and V max values were found to be 2.9 mg/mL and 7936 U/mL respectively.

Epigenetic Regulatory Mechanisms Distinguish Retinoic Acid-mediated Transcriptional Responses in Stem Cells and Fibroblasts

Retinoic acid (RA), a vitamin A metabolite, regulates transcription by binding to RA receptor (RAR) and retinoid X receptor (RXR) heterodimers. This transcriptional response is determined by receptor interactions with transcriptional regulators and chromatin modifying proteins. We compared transcriptional responses of three RA target genes (Hoxa1, Cyp26a1, RARbeta(2)) in primary embryo fibroblasts (mouse embryonic fibroblasts), immortalized fibroblasts (Balb/c3T3), and F9 teratocarcinoma stem cells. Hoxa1 and Cyp26a1 transcripts are not expressed, but RARbeta(2) transcripts are induced by RA in mouse embryonic fibroblasts and Balb/c3T3 cells. Retinoid receptors (RARgamma, RXRalpha), coactivators (pCIP (NCOA3, SRC3)), and p300 and RNA polymerase II are recruited only to the RARbeta(2) RA response element (RARE) in Balb/c3T3, whereas these proteins are recruited to RAREs of all three genes by RA in F9 cells. In F9, RA reduces polycomb (PcG) protein Suz12 and the associated H3K27me3 repressive epigenetic modification at the RAREs of all three genes. In contrast, in Balb/c3T3 cells cultured in the +/-RA, Suz12 is not associated with the Hoxa1, RARbeta(2), and Cyp26a1 RAREs, whereas slow levels of the H3K27me3 mark are seen at these RAREs. Thus, Suz12 is not required for gene repression in the absence of RA. Even though the Hoxa1 RARE and proximal promoter show high levels of H3K9,K14 acetylation in Balb/c3T3, the Hoxa1 gene is not transcriptionally activated by RA. In Balb/c3T3, CpG islands are methylated in the Cyp26a1 promoter region but not in the Hoxa1 promoter or in these promoters in F9 cells. We have delineated the complex mechanisms that control RA-mediated transcription in fibroblasts versus stem cells.

T1784 An Inportant Role for Type III Interferon IL-28b in TLR Induced Inflammatory Reactions

BACKGROUND: Mesalamine (5ASA) has been used for many years as a primary IBD treatment, yet the mechanism of action is not fully understood. The drug is targeted to the distal gut, thereby exposing bacteria to high concentrations of 5ASA. We have previously shown, In Vivo, that 5ASA treatment is associated with changes in the major constituents of the GI microbiota in both mice and humans. We have also shown, In Vitro, that 5ASA has an effect on bacterial gene expression. Considering that Enterobacteriaceae are generally increased in patients with IBD and the specific association of adherent-invasive strains of Escherichia coli (AIEC) with this condition, we hypothesized that the therapeutic effects of 5ASA may be mediated through influences on E. coli. AIM: To determine whether 5ASA affects epithelial invasiveness of commensal E. coli and AIEC strains. METHODS: Gentamicin protection assays were performed using human colonic epithelial crypt-like T84 cells and two strains of E. coli: Non-invasive E. coli strain HB101 and adherent invasive E. coli strain LF82. These strains were pre-treated with therapeutic concentrations of 5ASA prior to infection. Gentamicin assays studying the effect of 5ASA on non-invasive E. coli used DNP (dinitrophenol) to metabolically stress monolayers prior to inoculation, mimicking epithelial stress in inflammation. All samples and dilutions were plated on LB agar plates and incubated for 24 hours. Bacteria were then enumerated and counts were presented as percent internalization (intracellular count/extracellular count). RESULTS: As expected, commensal strain HB101 shows minimal epithelial invasiveness, but under conditions of epithelial metabolic stress, HB101 was readily internalized. 5ASA pre-treatment of HB101 significantly reduced bacterial internalization by stressed epithelial cells. This response was increased with increasing doses of 5ASA. Similarly, AIEC strain LF82 invaded T84 epithelial cells and this was significantly decreased by 5ASA pre-treatment. This response was increased with increasing doses of 5ASA. CONCLUSIONS: 5ASA has direct effects on both non-invasive E. coli and AIEC strains, attenuating their interactions with the intestinal epithelium. These findings suggest that E. coli residing in the distal gut may be a target of 5ASA that is relevant to its therapeutic action in IBD.

Conformation Change in a Self-recognizing Autotransporter Modulates Bacterial Cell-Cell Interaction

Bacteria mostly live as multicellular communities, although they are unicellular organisms, yet the mechanisms that tie individual bacteria together are often poorly understood. The adhesin involved in diffuse adherence (AIDA-I) is an adhesin of diarrheagenic Escherichia coli strains. AIDA-I also mediates bacterial auto-aggregation and biofilm formation and thus could be important for the organization of communities of pathogens. Using purified protein and whole bacteria, we provide direct evidence that AIDA-I promotes auto-aggregation by interacting with itself. Using various biophysical and biochemical techniques, we observed a conformational change in the protein during AIDA-AIDA interactions, strengthening the notion that this is a highly specific interaction. The self-association of AIDA-I is of high affinity but can be modulated by sodium chloride. We observe that a bile salt, sodium deoxycholate, also prevents AIDA-I oligomerization and bacterial auto-aggregation. Thus, we propose that AIDA-I, and most likely other similar autotransporters such as antigen 43 (Ag43) and TibA, organize bacterial communities of pathogens through a self-recognition mechanism that is sensitive to the environment. This could permit bacteria to switch between multicellular and unicellular lifestyles to complete their infection.

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-kappaB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-kappaB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65(-/-) murine fibroblasts immortalize at considerably faster rates than RelA/p65(+/+) cells. The ability of RelA/p65(-/-) fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65(-/-) fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-kappaB. Altogether, our findings present a fresh perspective on the role of NF-kappaB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.