Phenotypic and genotypic characterisation of Burkholderia cenocepacia J2315 mutants affected in homoserine lactone and diffusible signal factor-based quorum sensing systems suggests interplay between both types of systems

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid ("Burkholderia diffusible signal factor'', BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (Delta cepI Delta BCAM0581, Delta cciI Delta BCAM0581 and Delta cepI Delta cciI) mutants and a triple (Delta cepI Delta cciI Delta BCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The Delta BCAM0581 mutant and the Delta cepI Delta BCAM0581 and Delta cciI Delta BCAM0581 double mutants produced significantly less AHL compared to the WT strain and the Delta cepI and Delta cciI single mutant, respectively. The expression of cepI and cciI in Delta BCAM0581, was approximately 3-fold and 7-fold (p < 0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the Delta BCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia </it>are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of <it>B. cenocepacia </it>J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.</p> <p>Results</p> <p>To investigate the contribution of efflux pumps to intrinsic drug resistance of <it>B. cenocepacia </it>J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes <it>BCAS0591</it>-<it>BCAS0593</it>, <it>BCAL1674</it>-<it>BCAL1676</it>, and <it>BCAL2822</it>-<it>BCAL2820</it>. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of <it>rnd-3 </it>also included <it>BCAL1672</it>, encoding a putative TetR regulator. The <it>B. cenocepacia rnd-3 </it>and <it>rnd-4 </it>mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the <it>rnd-3 </it>and <it>rnd-4 </it>mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the <it>rnd-1 </it>operon had no detectable phenotypes under the conditions assayed.</p> <p>Conclusion</p> <p>Two of the three inactivated RND efflux pumps in <it>B. cenocepacia </it>J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in <it>B. cenocepacia </it>is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.</p

S-CMC-Lys protective effects on human respiratory cells during oxidative stress.

The mucoactive drug S-carbocysteine lysine salt monohydrate (S-CMC-Lys) stimulates glutathione (GSH) efflux from respiratory cells. Since GSH is one of the most important redox regulatory mechanisms, the aim of this study was to evaluate the S-CMC-Lys effects on GSH efflux and intracellular concentration during an oxidative stress induced by the hydroxyl radical (xOH). Experiments were performed on cultured human respiratory WI-26VA4 cells by means of patch-clamp experiments in whole-cell configuration and of fluorimetric analyses at confocal microscope. xOH exposure induced an irreversible inhibition of the GSH and chloride currents that was prevented if the cells were incubated with S-CMC-Lys. In this instance, the currents were inhibited by the specific blocker CFTR(inh)-172. CFT1-C2 cells, which lack a functional CFTR channel, were not responsive to S-CMC-Lys, but the stimulatory effect of the drug was restored in LCFSN-infected CFT1 cells, functionally corrected to express CFTR. Fluorimetric measurements performed on the S-CMC-Lys-incubated cells revealed a significant increase of the GSH concentration that was completely hindered after oxidative stress and abolished by CFTR(inh)-172. The cellular content of reactive oxygen species was significantly lower in the S-CMC-Lys-treated cells either before or after xOH exposure. As a conclusion, S-CMC-Lys could exert a protective function during oxidative stress, therefore preventing or reducing the ROS-mediated inflammatory response

Transcription of Satellite III non-coding RNAs is a general stress response in human cells

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 104-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions

Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis

Serum Albumin Is Inversely Associated With Portal Vein Thrombosis in Cirrhosis

We analyzed whether serum albumin is independently associated with portal vein thrombosis (PVT) in liver cirrhosis (LC) and if a biologic plausibility exists. This study was divided into three parts. In part 1 (retrospective analysis), 753 consecutive patients with LC with ultrasound-detected PVT were retrospectively analyzed. In part 2, 112 patients with LC and 56 matched controls were entered in the cross-sectional study. In part 3, 5 patients with cirrhosis were entered in the in vivo study and 4 healthy subjects (HSs) were entered in the in vitro study to explore if albumin may affect platelet activation by modulating oxidative stress. In the 753 patients with LC, the prevalence of PVT was 16.7%; logistic analysis showed that only age (odds ratio [OR], 1.024; P = 0.012) and serum albumin (OR, -0.422; P = 0.0001) significantly predicted patients with PVT. Analyzing the 112 patients with LC and controls, soluble clusters of differentiation (CD)40-ligand (P = 0.0238), soluble Nox2-derived peptide (sNox2-dp; P &lt; 0.0001), and urinary excretion of isoprostanes (P = 0.0078) were higher in patients with LC. In LC, albumin was correlated with sCD4OL (Spearman's rank correlation coefficient [r(s)], -0.33; P &lt; 0.001), sNox2-dp (r(s), -0.57; P &lt; 0.0001), and urinary excretion of isoprostanes (r(s), -0.48; P &lt; 0.0001) levels. The in vivo study showed a progressive decrease in platelet aggregation, sNox2-dp, and urinary 8-iso prostaglandin F2 alpha-III formation 2 hours and 3 days after albumin infusion. Finally, platelet aggregation, sNox2-dp, and isoprostane formation significantly decreased in platelets from HSs incubated with scalar concentrations of albumin. Conclusion: Low serum albumin in LC is associated with PVT, suggesting that albumin could be a modulator of the hemostatic system through interference with mechanisms regulating platelet activation

Charcas y humedales antrópicos en canteras de áridos del Sudeste bonaerense

Los cambios ambientales ocurren naturalmente y son parte del resultado de múltiples ciclos e interacciones (Artiola et al., 2004). Sin embargo, cuando el ambiente es perturbado por actividades humanas, hay diversos factores adicionados que conducen a cambios que son de distinta escala temporal y espacial. Tal es el caso de la transformación que ocurre en áreas de explotación minera, en las que se dan alteraciones geomorfológicas y del paisaje, modificación altimétrica de los suelos, neo-formaciones de diversos tipos de suelos (por ej. tecnosoles, Osterreith et al., en este libro) y modificación de las comunidades biológicas que se desarrollan en el área (De Marco et al. 2008). En el caso particular de la explotación de rocas de aplicación (ortocuarcitas) como las que existen en el periurbano del partido de General Pueyrredon, un rasgo distintivo y evidente es el afloramiento del freático en la superficie de explotación. En los ambientes circunscriptos a las áreas de explotación se desarrollan neoformas (sensu Morello, 2000) tanto positivas (escombreras) como negativas (huecos y charcas, sensu Dangavs, 2005). Las negativas dan lugar al afloramiento freático. La ocurrencia de este hecho lleva a la culminación de la explotación y el eventual abandono del terreno por parte del propietario. El aporte freático, junto con el pluvial, confinan aguas en las oquedades resultantes de la explotación que no tienen posibilidad de fluir, motivo por el cual se conforman “charcas”, que dan paso al inicio de un proceso de sucesión ecológica secundaria, y que conducen a una transformación del ecosistema terrestre original a un conjunto de ecosistemas diversos: bañados, lagunas (de variada profundidad) y humedales, entre otros. Estos neo-ecosistemas constituyen una característica ambiental y paisajística típica en el área afectada. En este trabajo, se caracterizan las charcas y los humedales antrópicos generados por explotación de áridos desde el punto de vista fisicoquímico, biológico y ecofisiológico.Fil: de Marco, Silvia Graciela. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Bazzini, Sergio Martín. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Geología de Costas y del Cuaternario. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto de Geología de Costas y del Cuaternario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mallo, Juan Carlos. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Geología de Costas y del Cuaternario. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto de Geología de Costas y del Cuaternario; ArgentinaFil: Camino, Mariana. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Geología de Costas y del Cuaternario. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto de Geología de Costas y del Cuaternario; Argentin

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patchclamp techniques whether K1 channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskoline or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (Po) values for Ca21-activated K1 channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in Po of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (1025 M) was able to reduce Po when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the a subunit of largeconductance Ca21-activated K1 channels (BKCa) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments con- firm that these cells express the BK asubunit, which is located on the apical membrane. Other K1 channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA 1 ATP 1 cAMP (inside-out). These channels were insensitive to TEA1 and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca21- dependent, loperamide-sensitive K1 channels present on the apical membrane