Implication of actin in the uptake of sucrose and valine in the tap root and leaf of sugar beet.

Actin microfilaments (F-actin) are major components of the cytoskeleton essential for many cellular dynamic processes (vesicle trafficking, cytoplasmic streaming, organelle movements). The aim of this study was to examine whether cortical actin microfilaments might be implicated in the regulation of nutrient uptake in root and leaf cells of Beta vulgaris. Using antibodies raised against actin and the AtSUC1 sucrose transporter, immunochemical assays demonstrated that the expression of actin and a sucrose transporter showed different characteristics, when detected on plasma membrane vesicles (PMVs) purified from roots and from leaves. The in situ immunolabeling of actin and AtSUC1 sites in PMVs and tissues showed their close proximity to the plasma membrane. Using co-labeling in protoplasts, actin and sucrose transporters were localized along the internal border and in the outermost part of the plasma membrane, respectively. This respective membrane co-localization was confirmed on PMVs and in tissues using transmission electronic microscopy. The possible functional role of actin in sucrose uptake (and valine uptake, comparatively) by PMVs and tissues from roots and leaves was examined using the pharmacological inhibitors, cytochalasin B (CB), cytochalasin D (CD), and phalloidin (PH). CB and CD inhibited the sucrose and valine uptake by root tissues in a concentration-dependent manner above 1 µM, whereas PH had no such effect. Comparatively, the toxins inhibited the sucrose and valine uptake in leaf discs to a lesser extent. The inhibition was not due to a hindering of the proton pumping and H+ -ATPase catalytic activity determined in PMVs incubated in presence of these toxins.

In vitro sensitivity of 30 anaerobic bacterial strains of the human intestinal core microbiota to antibiotics: Culture and LC-MS/MS approaches.

We have a vast knowledge on human intestinal microbiota but it can still be regarded incomplete. One of the objectives of scientists using so-called "omics" techniques is to be interested in the consequences that drugs can have on the composition of the intestinal microbiota and inversely. To date, few publications have reported the effects of drugs on the growth of bacteria composing this microbiota using a "culturomics" approach. We focused on antibiotics commonly prescribed for which the only published are the susceptibility of the pathogenic strains and not that of the commensal strains. The aim of our study was to determine the sensitivity of 30 strains considered to represent the intestinal core microbiota to 8 antibiotics and to study the possible modification of these molecules by bacteria. The 30 bacterial strains were cultured under anaerobic conditions in order to determine their sensitivity to the antibiotics. After 48 h of culture, the supernatants were also analyzed via UHPLC-MS/MS in order to determine if the antibiotics have been chemically modified. Under the current experimental conditions, cefpodoxime, metronidazole, erythromycin, sulfamethozaxole, trimethoprim and the trimethoprim/sulfamethozaxole combination have little impact on the core microbiota strain growth. On the contrary, moxifloxacin and amoxicillin inhibit the growth of numerous strains of our panel. Using UHPLC-MS/MS analyses, we have shown that some antibiotics can be modifed by the bacteria composing the intestinal core microbiome. The bacteria that make up the intestinal microbiota core are impacted by the antibiotics most commonly prescribed in clinics today and inversely.

Biocontrol of Biofilm Formation: Jamming of Sessile-Associated Rhizobial Communication by Rhodococcal Quorum-Quenching.

Biofilms are complex structures formed by a community of microbes adhering to a surface and/or to each other through the secretion of an adhesive and protective matrix. The establishment of these structures requires a coordination of action between microorganisms through powerful communication systems such as quorum-sensing. Therefore, auxiliary bacteria capable of interfering with these means of communication could be used to prevent biofilm formation and development. The phytopathogen Rhizobium rhizogenes, which causes hairy root disease and forms large biofilms in hydroponic crops, and the biocontrol agent Rhodococcus erythropolis R138 were used for this study. Changes in biofilm biovolume and structure, as well as interactions between rhizobia and rhodococci, were monitored by confocal laser scanning microscopy with appropriate fluorescent biosensors. We obtained direct visual evidence of an exchange of signals between rhizobia and the jamming of this communication by Rhodococcus within the biofilm. Signaling molecules were characterized as long chain (C14) N-acyl-homoserine lactones. The role of the Qsd quorum-quenching pathway in biofilm alteration was confirmed with an R. erythropolis mutant unable to produce the QsdA lactonase, and by expression of the qsdA gene in a heterologous host, Escherichia coli. Finally, Rhizobium biofilm formation was similarly inhibited by a purified extract of QsdA enzyme.

Type III secretion system and virulence markers highlight similarities and differences between human- and plant-associated pseudomonads related to Pseudomonas fluorescens and P. putida.

Pseudomonas fluorescens is commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the related Pseudomonas aeruginosa nosocomial species and the occurrence of this secretion system in plant-associated P. fluorescens raise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging to P. fluorescens were compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong to P. fluorescens but were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs from P. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged to P. fluorescens and harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associated P. fluorescens.

Effect of endocrine disruptors on bacterial virulence.

For several decades, questions have been raised about the effects of endocrine disruptors (ED) on environment and health. In humans, EDs interferes with hormones that are responsible for the maintenance of homeostasis, reproduction and development and therefore can cause developmental, metabolic and reproductive disorders. Because of their ubiquity in the environment, EDs can adversely impact microbial communities and pathogens virulence. At a time when bacterial resistance is inevitably emerging, it is necessary to understand the effects of EDs on the behavior of pathogenic bacteria and to identify the resulting mechanisms. Increasing studies have shown that exposure to environmental EDs can affect bacteria physiology. This review aims to highlight current knowledge of the effect of EDs on the virulence of human bacterial pathogens and discuss the future directions to investigate bacteria/EDs interaction. Given the data presented here, extended studies are required to understand the mechanisms by which EDs could modulate bacterial phenotypes in order to understand the health risks.

In vitro reciprocal interactions between yeasts from human cutaneous mycobiota and parabens used in cosmetics.

Parabens are substances with antifungal and antibacterial properties, suspected to be endocrine disruptors and widely used as preservatives in cosmetics. In this case, exposure to these compounds is mainly dermal and interactions may occur with skin components including cutaneous mycobiota. In this work, we have explored the in vitro reciprocal interactions between three parabens (methylparaben, ethylparaben, and propylparaben) and yeasts from the human cutaneous mycobiota (Candida parapsilosis, Cryptococcus uniguttulatus, and Rhodotorula mucilaginosa) by studying the effect of these parabens on fungal growth and the fungal ability to metabolize the tested compounds. Our results showed that, at the tested concentrations, the growth of three strains of C. parapsilosis was not influenced by the presence of parabens. Whereas, using the same parabens concentrations, growth of C. uniguttulatus and R. mucilaginosa was completely inhibited by ethylparaben since the first day of contact, whereas these same fungi were not sensitive to the two other parabens, even after seven days of incubation. The presence of a lamellar wall in these basidiomycete fungi as well as the physico-chemical properties of ethylparaben could explain this selective inhibition. Additionally, C. parapsilosis and R. mucilaginosa degraded 90% to 100% of propylparaben after seven days of incubation but had no effect on the other tested parabens. Thus, their enzymes seem to only degrade long chain parabens. In the same conditions, C. uniguttulatus did not degrade any paraben. This inability may be due to the absence of fungal enzymes able to degrade parabens or to the possible inaccessibility of intracellular enzymes due to the polysaccharide capsule. Our work has shown that parabens can act differently from one fungus to another within the cutaneous mycobiota. These preliminary results have evidenced that in vitro parabens, contained in cosmetic products, could be involved in the occurrence of a state of dysbiosis. The tested yeasts from the cutaneous mycobiota can also be involved in the degradation of parabens and thereby reduce, according to the produced metabolites and their activities, the risk of endocrine disruption they can induce.

Exposure to endocrine disruptors promotes biofilm formation and contributes to increased virulence of Pseudomonas aeruginosa.

Anthropogenic activities contribute to the spread of chemicals considered as endocrine disruptors (ED) in freshwater ecosystems. While several studies have reported interactions of EDs with organisms in those ecosystems, very few have assessed the effect of these compounds on pathogenic bacteria. Here we have evaluated the impact of five EDs found in aquatic resources on the virulence of human pathogen P. aeruginosa. ED concentrations in French aquatic resources of bisphenol A (BPA), dibutyl phthalate (DBP), ethylparaben (EP), methylparaben (MP) and triclosan (TCS) at mean molar concentration were 1.13, 3.58, 0.53, 0.69, and 0.81 nM respectively. No impact on bacterial growth was observed at EDs highest tested concentration. Swimming motility of P. aeruginosa decreased to 28.4% when exposed to EP at 100 µM. Swarming motility increased, with MP at 1 nM, 10 and 100 µM (1.5-fold); conversely, a decrease of 78.5%, with DBP at 100 µM was observed. Furthermore, exposure to 1 nM BPA, DBP and EP increased biofilm formation. P. aeruginosa adhesion to lung cells was two-fold higher upon exposure to 1 nM EP. We demonstrate that ED exposure may simultaneously decrease mobility and increase cell adhesion and biofilm formation, which may promote colonisation and establishment of the pathogen.

Application of QSAR Approach to Assess the Effects of Organic Pollutants on Bacterial Virulence Factors.

The release of a wide variety of persistent chemical contaminants into wastewater has become a growing concern due to their potential health and environmental risks. While the toxic effects of these pollutants on aquatic organisms have been extensively studied, their impact on microbial pathogens and their virulence mechanisms remains largely unexplored. This research paper focuses on the identification and prioritization of chemical pollutants that increase bacterial pathogenicity, which is a public health concern. In order to predict how chemical compounds, such as pesticides and pharmaceuticals, would affect the virulence mechanisms of three bacterial strains (Escherichia coli K12, Pseudomonas aeruginosa H103, and Salmonella enterica serovar. Typhimurium), this study has developed quantitative structure-activity relationship (QSAR) models. The use of analysis of variance (ANOVA) functions assists in developing QSAR models based on the chemical structure of the compounds, to predict their effect on the growth and swarming behavior of the bacterial strains. The results showed an uncertainty in the created model, and that increases in virulence factors, including growth and motility of bacteria, after exposure to the studied compounds are possible to be predicted. These results could be more accurate if the interactions between groups of functions are included. For that, to make an accurate and universal model, it is essential to incorporate a larger number of compounds of similar and different structures.

Sensitivity of Legionella pneumophila to phthalates and their substitutes.

Phthalates constitute a family of anthropogenic chemicals developed to be used in the manufacture of plastics, solvents, and personal care products. Their dispersion and accumulation in many environments can occur at all stages of their use (from synthesis to recycling). However, many phthalates together with other accumulated engineered chemicals have been shown to interfere with hormone activities. These compounds are also in close contact with microorganisms that are free-living, in biofilms or in microbiota, within multicellular organisms. Herein, the activity of several phthalates and their substitutes were investigated on the opportunistic pathogen Legionella pneumophila, an aquatic microbe that can infect humans. Beside showing the toxicity of some phthalates, data suggested that Acetyl tributyl citrate (ATBC) and DBP (Di-n-butyl phthalate) at environmental doses (i.e. 10-6 M and 10-8 M) can modulate Legionella behavior in terms of motility, biofilm formation and response to antibiotics. A dose of 10-6 M mostly induced adverse effects for the bacteria, in contrast to a dose of 10-8 M. No perturbation of virulence towards Acanthamoeba castellanii was recorded. These behavioral alterations suggest that L. pneumophila is able to sense ATBC and DBP, in a cross-talk that either mimics the response to a native ligand, or dysregulates its physiology.

Catabolic pathway of gamma-caprolactone in the biocontrol agent Rhodococcus erythropolis.

Gamma-caprolactone (GCL) is well-known as a food flavor and has been recently described as a biostimulant molecule promoting the growth of bacteria with biocontrol activity against soft-rot pathogens. Among these biocontrol agents, Rhodococcus erythropolis, characterized by a remarkable metabolic versatility, assimilates various γ-butyrolactone molecules with a branched-aliphatic chain, such as GCL. The assimilative pathway of GCL in R. erythropolis was investigated by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. This analysis suggests the involvement of the lactonase QsdA in ring-opening, a feature confirmed by heterologous expression in Escherichia coli. According to proteome analysis, the open-chain form of GCL was degraded by ß- and ω-oxidation coupled to the Krebs cycle and ß-ketoadipate pathway. Ubiquity of qsdA gene among environmental R. erythropolis isolates was verified by PCR. In addition to a previous N-acyl homoserine lactone catabolic function, QsdA may therefore be involved in an intermediate degradative step of cyclic recalcitrant molecules or in synthesis of flavoring lactones.

N-acyl homoserine lactones in diverse Pectobacterium and Dickeya plant pathogens: diversity, abundance, and involvement in virulence.

Soft-rot bacteria Pectobacterium and Dickeya use N-acyl homoserine lactones (NAHSLs) as diffusible signals for coordinating quorum sensing communication. The production of NAHSLs was investigated in a set of reference strains and recently-collected isolates, which belong to six species and share the ability to infect the potato host plant. All the pathogens produced different NAHSLs, among which the 3-oxo-hexanoyl- and the 3-oxo-octanoyl-L-homoserine lactones represent at least 90% of total produced NAHSL-amounts. The level of NAHSLs varied from 0.6 to 2 pg/cfu. The involvement of NAHSLs in tuber maceration was investigated by electroporating a quorum quenching vector in each of the bacterial pathogen strains. All the NAHSL-lactonase expressing strains produced a lower amount of NAHSLs as compared to those harboring the empty vector. Moreover, all except Dickeya dadantii 3937 induced a lower level of symptoms in potato tuber assay. Noticeably, aggressiveness appeared to be independent of both nature and amount of produced signals. This work highlights that quorum sensing similarly contributed to virulence in most of the tested Pectobacterium and Dickeya, even the strains had been isolated recently or during the past decades. Thus, these key regulatory-molecules appear as credible targets for developing anti-virulence strategies against these plant pathogens.

Development of an in vitro Model of Human Gut Microbiota for Screening the Reciprocal Interactions With Antibiotics, Drugs, and Xenobiotics.

Altering the gut microbiota can negatively affect human health. Efforts may be sustained to predict the intended or unintended effects of molecules not naturally produced or expected to be present within the organism on the gut microbiota. Here, culture-dependent and DNA-based approaches were combined to UHPLC-MS/MS analyses in order to investigate the reciprocal interactions between a constructed Human Gut Microbiota Model (HGMM) and molecules including antibiotics, drugs, and xenobiotics. Our HGMM was composed of strains from the five phyla commonly described in human gut microbiota and belonging to Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria. Relevantly, the bacterial diversity was conserved in our constructed human gut model through subcultures. Uneven richness distribution was revealed and the sensitivity of the HGMM was mainly affected by antibiotic exposure rather than by drugs or xenobiotics. Interestingly, the constructed model and the individual cultured strains respond with the same sensitivity to the different molecules. UHPLC-MS/MS analyses revealed the disappearance of some native molecules in the supernatants of the HGMM as well as in those of the individual strains. These results suggest that biotransformation of molecules occurred in the presence of our gut microbiota model and the coupled approaches performed on the individual cultures may emphasize new bacterial strains active in these metabolic processes. From this study, the new HGMM appears as a simple, fast, stable, and inexpensive model for screening the reciprocal interactions between the intestinal microbiota and molecules of interest.

Effect of Phthalates and Their Substitutes on the Physiology of Pseudomonas aeruginosa.

Phthalates are used in a variety of applications-for example, as plasticizers in polyvinylchloride products to improve their flexibility-and can be easily released into the environment. In addition to being major persistent organic environmental pollutants, some phthalates are responsible for the carcinogenicity, teratogenicity, and endocrine disruption that are notably affecting steroidogenesis in mammals. Numerous studies have thus focused on deciphering their effects on mammals and eukaryotic cells. While multicellular organisms such as humans are known to display various microbiota, including all of the microorganisms that may be commensal, symbiotic, or pathogenic, few studies have aimed at investigating the relationships between phthalates and bacteria, notably regarding their effects on opportunistic pathogens and the severity of the associated pathologies. Herein, the effects of phthalates and their substitutes were investigated on the human pathogen, Pseudomonas aeruginosa, in terms of physiology, virulence, susceptibility to antibiotics, and ability to form biofilms. We show in particular that most of these compounds increased biofilm formation, while some of them enhanced the bacterial membrane fluidity and altered the bacterial morphology.

Legionella spp. All Ears? The Broad Occurrence of Quorum Sensing Elements outside Legionella pneumophila.

Legionella spp. are ubiquitous bacteria principally found in water networks and ∼20 species are implicated in Legionnaire's disease. Among them, Legionella pneumophila is an intracellular pathogen of environmental protozoa, responsible for ∼90% of cases in the world. Legionella pneumophila regulates in part its virulence by a quorum sensing system named "Legionella quorum sensing," composed of a signal synthase LqsA, two histidine kinase membrane receptors LqsS and LqsT and a cytoplasmic receptor LqsR. To date, this communication system was only found in L. pneumophila. Here, we investigated 58 Legionella genomes to determine the presence of a lqs cluster or homologous receptors using TBlastN. This analysis revealed three categories of species: 19 harbored a complete lqs cluster, 20 did not possess lqsA but maintained the receptor lqsR and/or lqsS, and 19 did not have any of the lqs genes. No correlation was observed between pathogenicity and the presence of a quorum sensing system. We determined by RT-qPCR that the lqsA gene was expressed at least in four strains among different species available in our laboratory. Furthermore, we showed that the lqs genomic region was conserved even in species possessing only the receptors of the quorum sensing system, indicating an ancestral acquisition and various loss dynamics during evolution. This system could therefore function in interspecific communication as well.

Exploring the Individual Bacterial Microbiota of Questing Ixodes ricinus Nymphs.

Ixodes ricinus is the most common hard tick species in Europe and an important vector of pathogens of human and animal health concerns. The rise of high-throughput sequencing has facilitated the identification of many tick-borne pathogens and, more globally, of various microbiota members depending on the scale of concern. In this study, we aimed to assess the bacterial diversity of individual I. ricinus questing nymphs collected in France using high-throughput 16S gene metabarcoding. From 180 dragging-collected nymphs, we identified more than 700 bacterial genera, of which about 20 are abundantly represented (>1% of total reads). Together with 136 other genera assigned, they constitute a core internal microbiota in this study. We also identified 20 individuals carrying Borreliella. The most abundant species is B. afzelii, known to be one of the bacteria responsible for Lyme disease in Europe. Co-detection of up to four Borreliella genospecies within the same individual has also been retrieved. The detection and co-detection rate of Borreliella in I. ricinus nymphs is high and raises the question of interactions between these bacteria and the communities constituting the internal microbiota.

In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts.

Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K3]SHa, has been designed to improve those activities. SHa and [K3]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K3]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K3]SHa and exhibited low half maximal lethal concentrations (LC50). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments.

Prolactin stimulates prostate cell proliferation by increasing endoplasmic reticulum content due to SERCA 2b over-expression.

Prolactin (PRL) has been shown to be involved in the differentiation and proliferation of numerous tissues, including the prostate gland. Moreover, variations in [Ca2+]ER (calcium concentration within the endoplasmic reticulum) may play a role in cell growth. However, few studies have focused on the regulation of calcium homoeostasis by prolactin. The present study evaluates the regulation of calcium pools as well as the possible role of [Ca2+]ER variations as a signal for growth modulation by PRL. We show that PRL stimulates the proliferation of normal SV40 immortalized epithelial prostate (PNT1A) cells with a maximum effect at a dose of 100 ng/ml. We also show that 100 ng/ml PRL increases the [Ca2+]ER when measured either by indirect quantification with Fura-2AM after application of 1 mM thapsigargin or by direct quantification with Mag-Fura-2AM within the endoplas-mic reticulum. Western blot analysis shows that the SERCA 2b (sarcoendoplasmic calcium ATPase 2b) is over-expressed in PNT1A cells treated with 100 ng/ml PRL for 24 h. A small inter-fering RNA SERCA 2a/b, used to down-regulate endogenous SERCA 2b expression, reduced both PNT1A cell proliferation and [Ca2+]ER. We thus identify [Ca2+]ER and SERCA 2b as protagonists in PRL-induced proliferation.

The aliphatic amidase AmiE is involved in regulation of Pseudomonas aeruginosa virulence.

We have previously shown that the eukaryotic C-type natriuretic peptide hormone (CNP) regulates Pseudomonas aeruginosa virulence and biofilm formation after binding on the AmiC sensor, triggering the amiE transcription. Herein, the involvement of the aliphatic amidase AmiE in P. aeruginosa virulence regulation has been investigated. The proteome analysis of an AmiE over-producing strain (AmiE+) revealed an expression change for 138 proteins, including some that are involved in motility, synthesis of quorum sensing compounds and virulence regulation. We observed that the AmiE+ strain produced less biofilm compared to the wild type, and over-produced rhamnolipids. In the same line, AmiE is involved in P. aeruginosa motilities (swarming and twitching) and production of the quorum sensing molecules N-acyl homoserine lactones and Pseudomonas Quinolone Signal (PQS). We observed that AmiE overproduction reduced levels of HCN and pyocyanin causing a decreased virulence in different hosts (i.e. Dictyostelium discoideum and Caenorhabditis elegans). This phenotype was further confirmed in a mouse model of acute lung infection, in which AmiE overproduction resulted in an almost fully virulence decrease. Taken together, our data suggest that, in addition to its role in bacterial secondary metabolism, AmiE is involved in P. aeruginosa virulence regulation by modulating pilus synthesis and cell-to-cell communication.

Bcl-2-dependent modulation of swelling-activated Cl- current and ClC-3 expression in human prostate cancer epithelial cells.

Cell shrinkage is an integral part of apoptosis. However, intimate mechanisms linking apoptotic events to the alterations in cell volume homeostasis remain poorly elucidated. We investigated how overexpression of Bcl-2 oncoprotein, a key antiapoptotic regulator, in lymph node carcinoma of the prostate (LNCaP) prostate cancer epithelial cells interferes with the volume-regulated anion channel (VRAC), a major determinant of regulatory volume decrease. Bcl-2 overexpression resulted in the doubling of VRAC-carried swelling-activated Cl(-) current (I(Cl,swell)) and weakened I(Cl,swell) inhibition by store-operated Ca(2+) channel (SOC)-transported Ca(2+). This also was accompanied by substantial up-regulation of ClC-3 protein, a putative molecular candidate for the role of VRAC. ClC-3-specific antibody suppressed I(Cl,swell) in the wild-type and Bcl-2-overexpressing LNCaP cells. Epidermal growth factor treatment of wild-type LNCaP cells, promoting their proliferation, resulted in the enhancement of endogenous Bcl-2 expression and associated increases in ClC-3 levels and I(Cl,swell) magnitude. We conclude that Bcl-2-induced up-regulation of I(Cl,swell), caused by enhanced expression of ClC-3 and weaker negative control from SOC-transported Ca(2+), would strengthen the ability of the cells to handle proliferative volume increases and thereby promote their survival and diminish their proapoptotic potential.

Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation.

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.