Evaluating decline parameters of rotifer Brachionus plicatilis populations as an interstitial water toxicity bioassay

Population decline of the rotifer Brachionus plicatilis has been evaluated as a sensitive and reliable bioassay for assessing toxicity of marine sediment interstitial water. Three environmental conditions were examined using both interstitial and seawater cultures: (a) adverse effects from dissolved contaminant concentrations; (b) effects from particulate organic matter; and (c) increase or decrease of bacterial biomass from addition of mixtures of antibiotics. Three different parameters were measured to characterize decline: (i) time required for 50% of inoculated population to die (TL₅₀); (ii) curve of the decline rate of Brachionus plicatilis (µ _BP and (iii) graphical area contained below plot of the egg:female ratio curve against time (A _BPM ).Results indicated that, for each of the different assays, the three parameters of the Brachionus plicatilis population decline test were sensitive to concentrations of contaminants dissolved in interstitial waters (principally: total ammonia, Cu, Cr and alkylbenzenesulphonates) but not to particulate organic matter. Nevertheless, the presence or absence of mixed antibiotics with the contaminants may influence the Brachionus plicatilis population decline test, principally by retarding the hatching of eggs. Based on these results, Brachionus plicatilis is confirmed as an appropriate organism for use as an indicator of interstitial water contamination, using either decline rate, TL₅₀ or both parameters. The presence of particulate matter has no effect on these parameters, but the bacterial population may be an influence, although to a lesser extent than the toxicants.     

The ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency

The penetration ability of 12 antimicrobial agents, including antibiotics and biocides, was determined against biofilms of B. cereus and P. fluorescens using a colony biofilm assay. The surfactants benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB), and the antibiotics ciprofloxacin and streptomycin were of interest due to their distinct activities. Erythromycin and CTAB were retarded by the presence of biofilms, whereas ciprofloxacin and BAC were not. The removal and killing efficacies of these four agents was additionally evaluated against biofilms formed in microtiter plates. The most efficient biocide was CTAB for both bacterial biofilms. Ciprofloxacin was the best antibiotic although none of the selected antimicrobial agents led to total biofilm removal and/or killing. Comparative analysis of the results obtained with colony biofilms and microtiter plate biofilms show that although extracellular polymeric substances and the biofilm structure are considered a determining factor in biofilm resistance, the ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency. Also, the results reinforce the role of an appropriate antimicrobial selection as a key step in the design of disinfection processes for biofilm control.     

Prescreening bacterial colonies for bioactive molecules with Janus plates, a SBS standard double-faced microbial culturing system

Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method’s utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.     

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: Studies with extracellular adenosine 5′-triphosphate

The incubation of isolated rat hepatocytes with extracellular adenosine 5′-trihosphate (ATP) resulted in an inhibition of Ca²⁺ efflux. The ATP-induced Ca²⁺ accumulation as determined by the increase in phosphorylase a activity and the Ca²⁺ -sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis-[carboxymethyl] aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca²⁺, suggesting that the increased free Ca²⁺ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H-imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca²⁺ -calmodulin process may be involved in the initiation of cytotoxicity.     

Microbial Biomass,Respiration, and Decomposition of Hura crepitans L. (Euphorbiaceae) Leaves in a Tropical Stream1

The processing of leaves in temperate streams has been the subject of numerous studies but equivalent tropical ecosystems have received little attention. We investigated leaf breakdown of a tropical tree species (Hura crepitans, Euphorbiaceae), in a tropical stream using leaf bags (0.5 mm mesh) over a period of 24 days. We followed the loss of mass and the changes in adenosine triphosphate (ATP) concentrations and respiration rates associated with the decomposing leaves. The breakdown rate was fast (k=?0.0672/d, k_d=?0.0031/degree‐day), with 81 percent loss of the initial mass within 24 days. This high rate was probably related to the stable and high water temperature (22°C) favoring strong biological activity. Respiration rates increased until day 16 (1.1 mg O₂/h/g AFDM), but maximum ATP concentrations were attained at day 9 (725 nmol ATP/g AFDM) when leaf mass remaining was 52 percent. To determine the relative importance of fungi and bacteria during leaf decomposition, ATP concentrations, and respiration rates were determined in samples treated with antibiotics, after incubation in the stream. The results of the samples treated with the antifungal or the bacterial antibiotic suggest a higher contribution of the fungal community for total microbial biomass and a higher contribution of the bacterial community for microbial respiration rates, especially during the later stages of leaf decomposition. However, these results should be analyzed with caution since both antibacterial and antifungal agents did not totally eliminate microbial activity and biomass.     

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): Role of bacterial extracellular polymers

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV‐irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV‐dosage. Since UV energy crosslinks both intra‐ and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.     

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.     

Cloning, expression, and enzymatic activity of Acinetobacter baumannii and Klebsiella pneumoniae acetyl-coenzyme A carboxylases

Pathogenic Gram-negative bacteria are a major public health concern because they are causative agents of life-threatening hospital-acquired infections. Due to the increasing rates of resistance to available antibiotics, there is an urgent need to develop new drugs. Acetyl-coenzyme A carboxylase (ACCase) is a promising target for the development of novel antibiotics. We describe here the expression, purification, and enzymatic activity of recombinant ACCases from two clinically relevant Gram-negative pathogens, Acinetobacter baumannii and Klebsiella pneumoniae. Recombinant ACCase subunits (AccAD, AccB, and AccC) were expressed and purified, and the holoenzymes were reconstituted. ACCase enzyme activity was monitored by direct detection of malonyl-coenzyme A (malonyl-CoA) formation by liquid chromatography tandem mass spectrometry (LC–MS/MS). Steady-state kinetics experiments showed similar k_cat and K_M values for both enzymes. In addition, similar IC₅₀ values were observed for inhibition of both enzymes by a previously reported ACCase inhibitor. To provide a higher throughput assay suitable for inhibitor screening, we developed and validated a luminescence-based ACCase assay that monitors ATP depletion. Finally, we established an enzyme activity assay for the isolated AccAD (carboxyltransferase) subunit, which is useful for determining whether novel ACCase inhibitors inhibit the biotin carboxylase or carboxyltransferase site of ACCase. The methods described here could be applied toward the identification and characterization of novel inhibitors.     

Antibiotic‐Potentiating Activity of Phanostenine Isolated from Cissampelos sympodialis Eichler

Cissampelos sympodialis Eichler is well studied and investigated for its antiasthmatic properties, but there are no data in the literature describing antibacterial properties of alkaloids isolated from this botanical species. This work reports the isolation and characterization of phanostenine obtained from roots of C. sympodialis and describes for the first time its antimicrobial and antibiotic modulatory properties. Phanostenine was first isolated from Cissampelos sympodialis and its antibacterial activities were determined. Chemical structures of the alkaloid isolate were determined using spectroscopic and chemical analyses. Phanostenine was also tested for its antibacterial activity against standard strains and clinical isolates of Escherichia coli and Staphylococcus aureus. Minimal inhibitory concentration (MIC) was determined in a microdilution assay and for the evaluation of antibiotic resistance‐modifying activity. MIC of the antibiotics was determined in the presence or absence of phanostenine at sub‐inhibitory concentrations. The evaluation of antibacterial activity by microdilution assay showed activity for all strains with better values against S. aureus ATCC 12692 and E. coli 27 (787.69 mm ). The evaluation of aminoglycoside antibiotic resistance‐modifying activity showed reduction in the MIC of the aminoglycosides (amikacin, gentamicin and neomycin) when associated with phanostenine, MIC reduction of antibiotics ranging from 21 % to 80 %. The data demonstrated that phanostenine possesses a relevant ability to modify the antibiotic activity in vitro. We can suggest that phanostenine presents itself as a promising tool as an adjuvant for novel antibiotics formulations against bacterial resistance.     

Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.     

Antibacterial activity and synergy of antibiotics with sanguisorbigenin isolated from Sanguisorba officinalis L. against methicillin-resistant Staphylococcus aureus

The present study evaluated the antibacterial activity and the synergy of the sanguisorbigenin (SGB) from the dried root of Sanguisorba officinalis L. combined with β-lactam antibiotics against methicillin-resistant Staphylococcus aureus. A total of six strains of reference strain and clinical isolates were used to determine the antibacterial activity using a broth microdilution assay, and the synergistic effects were determined using a checkerboard assay. To analyse the mechanism of synergy, we conducted the level of penicillin-binding protein 2a by western blot. In addition, quantitative RT-PCR was performed to analyse the mecA gene expression. The minimal inhibitory concentration values of SGB against six strains of S. aureus were in the range of 12·5–50 μg ml⁻¹, and there were synergy, or partial synergy effects when SGB was combined with antibiotics. Furthermore, when treated with SGB, the level of penicillin-binding protein 2a and the expression of the mecA gene was reduced significantly. In conclusion, this study demonstrated that SGB is a potential natural antibacterial agent against methicillin-resistant S. aureus that represents a considerable burden on the healthcare system worldwide, and may an exceptionally modulator of β-lactam antibiotics.     

Evidence for the presence and role of tightly bound adenine nucleotides in phospholipid-free purifiedMicrococcus lysodeikticus adenosine triphosphatase

[³²P]-labeled ATPase was isolated in a highly purified state fromMicrococcus lysodeikticus strain PNB grown in medium supplemented with [³²P]orthophosphate. Selective extraction procedures allowed us to determine that at least 25% of the firmly bound label belonged to adenine nucleotides, ATP and ADP being present in equimolar amounts. However, no³²P label was found to be part of phospholipids. This was confirmed by purification of the ATPase from cells fed with [2—³H]glycerol. Using the luciferin-luciferase assay we estimated that ATPase freshly isolated by Sephadex chromatography (specific activity 10–14 µmole substrate transformed · min^–1 · mg protein^–1) contained 2 moles ATP/mole of enzyme. The ratio fell with the age of enzyme and its purification by gel electrophoresis and this was paralleled by a loss of ATPase activity. The endogenous nucleotides were readily exchanged by added ADP or ATP. This result suggests that the sites for tight binding of adenine nucleotides are equivalent, although ADP seems to have a higher affinity for them. The last properties represent a peculiar characteristic of this bacterial ATPase as compared with other bacterial and organelle energy-transducing proteins.     

Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase

The continuous bioluminescent assay of ATP has been adapted to the study of Mg²⁺-dependent ATPases, including the (Na⁺,K⁺) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg²⁺-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.     

The use of glass beads cultivation system to study the global effect of the <Emphasis Type="Italic">ppk</Emphasis> gene inactivation in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

The glass beads cultivation system developed in our laboratory for physiological studies of filamentous microorganisms supports differentiation and allows complete recovery of bacterial colonies and their natural products from cultivation plates. Here, we used this system to study the global effect of ppk gene disruption in Streptomyces lividans. The ppk encoding the enzyme polyphosphate kinase (P) catalyses the reversible polymerisation of gamma phosphate of ATP to polyphosphates. The resulting are phosphate and energy stock polymers. Because P activity impacts the overall energetic state of the cell, it is also connected to secondary metabolite (e.g. antibiotic) biosynthesis. We analysed the global effects of the disruption of this gene including its influence on the production of pigmented antibiotics, on morphological differentiation, on the levels of ATP and on the whole cytoplasmic protein expression pattern of S. lividans. We observed that the S. lividans ppk mutant produced antibiotics earlier and in greater amount than the wild-type (wt) strain. On the other hand, we did not observe any obvious effect on colony morphological development. In agreement with the function of Ppk, we detected much lower levels of ATP in ppk- mutant than in the wt strain. Proteomic analysis revealed that the genes that were influenced by ppk inactivation included enzymes involved in carbon or nitrogen metabolism, phosphate transport and components of the cell translational machinery. We showed that the synthesis of translation elongation factor Tu is during sporulation much higher in ppk- mutant than in wild-type strain.     

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes

Ecto-F₁-ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F₁-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y₁₃-mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F₁-ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F₁-ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F₁-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes.This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis.     

Enumeration of phagocytosed Staphylococcus aureus inside cells of the mouse macrophage cell line J774 by bioluminescence,fluorescence and viable counting techniques

In order to quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together to allow phagocytosis to occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase to remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence in a luminometer. This new method of enumerating intracellular bacteria was compared to the conventional method of viable counts and found to correlate (r = 0.78). The bioluminescence assay developed was found to be a relatively rapid alternative method to the techniques currently used to enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects of antimicrobial agents on intracellular pathogens.     

The role of volatile and non-volatile antibiotics produced by Pseudomonas chlororaphis strain PA23 in its root colonization and control of Sclerotinia sclerotiorum

Pseudomonas chlororaphis strain PA23 has demonstrated excellent biocontrol in the canola phyllosphere. This bacterium produces the non-volatile antibiotics phenazine and pyrrolnitrin as well as the volatile antibiotics nonanal, benzothiazole and 2-ethyl-1-hexanol. In vitro experiments were conducted to study the effects of different mutations on the production of these three organic volatile antibiotics by PA23. In planta experiments in the greenhouse investigated the role of the non-volatile antibiotics on root colonization and biocontrol ability of PA23 against Sclerotinia sclerotiorum on sunflower. Analysis of phenazine- and pyrrolnitrin-deficient Tn mutants of PA23 revealed no differences in production of the three volatile antibiotics. On all sampling dates, PA23 applied alone or in combination with the mutants showed significantly higher (P = 0.05) root bacterial number and Sclerotinia wilt suppression (P = 0.05). Decline of the bacterial population seemed to be inversely proportional to/or negatively correlated with the number of antibiotics produced by PA23 but the relative importance of phenazine or pyrrolnitrin on root colonization and/or wilt suppression was not clear. In several cases, the strains producing at least one antibiotic maintained relatively higher bacterial numbers than non-producing strains. However, by 6 weeks after sowing, there was a rapid and significant (P = 0.05) increase in the proportion of introduced bacteria capable of producing at least one antibiotic over the total bacterial population. Furthermore, combining certain mutants with PA23 reduced the root colonization and biocontrol ability of PA23. Strain PA23-314 (gacS mutant) showed competitive colonization in comparison to the other mutants for most sampling dates.     

Physical Activity in Confined Conditions as an Indicator of Free‐Living Physical Activity

Objective: The main determinants of daily energy expenditure are body size and physical activity. Activity energy expenditure is the most variable component of total energy expenditure. It was assessed whether the physical activity level in confined conditions is an indicator of free‐living physical activity. Research Methods and Procedures: Activity energy expenditure was measured over 1 day in a confined environment of a respiration chamber (floor space, 7.0 m²), where activities were restricted to low‐intensity activities of daily living, and over 2 weeks in a free‐living environment using doubly labeled water. Subjects were 16 women and 29 men (age, 31 ± 10 years; BMI, 24.2 ± 2.7 kg/m²). Results: The free‐living activity level of the subjects, as a multiple of resting energy expenditure, was 1.76 ± 0.13. Activity energy expenditure in the chamber was 47 ± 13% of the value in daily life, and the two values were correlated (r = 0.50, p < 0.001; partial correlation corrected for age, gender, and BMI: 0.40, p < 0.01). The chamber value explained 25% of the total variance in free‐living activity energy expenditure. Discussion: The activity level of a subject under sedentary conditions is an indicator of activity energy expenditure in daily life, showing the importance of nonexercise activity for daily energy expenditure.     

dATP/ATP, a multifunctional nucleotide, stimulates bacterial cell lysis, extracellular DNA release and biofilm development

Background

Signaling by extracellular adenosine 5′-triphosphase (eATP) is very common for cell-to-cell communication in many basic patho-physiological development processes. Rapid release of ATP into the extracellular environment from distressed or injured eukaryotic cells due to pathogens or other etiological factors can serve as a “danger signal”, activating host innate immunity. However, little is known about how or whether pathogenic bacteria respond to this “danger signal”.

Methods and Principal Findings

Here we report that extracellular dATP/ATP can stimulate bacterial adhesion and biofilm formation via increased cell lysis and extracellular DNA (eDNA) release. We demonstrate that extracellular dATP/ATP also stimulates bacterial adherence in vitro to human bronchial epithelial cells.

Conclusions and Significance

These data suggest that bacteria may sense extracellular dATP/ATP as a signal of “danger” and form biofilms to protect them from host innate immunity. This study reveals a very important and unrecognized phenomenon that both bacteria and host cells could respond to a common important signal molecule in a race to adapt to the presence of one another. We propose that extracellular dATP/ATP functions as an “inter-domain” warning signal that serves to induce protective measures in both Bacterial and Eukaryotic cells.