Bacterial ATP Research Articles

On the Mg2+ binding site of the ε subunit from bacterial F-type ATP synthases

F-type ATP synthases, central energy conversion machines of the cell synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane and, reversely, can also hydrolyze ATP to pump ions across the membrane, depending on cellular conditions such as ATP concentration. To prevent wasteful ATP hydrolysis, mammalian and bacterial ATP synthases possess different regulatory mechanisms. In bacteria, a low ATP concentration induces a conformational change in the ε subunit from the down- to up-states, which inhibits ATP hydrolysis. Moreover, the conformational change of the ε subunit depends on Mg2+ concentration in some bacteria such as Bacillus subtilis, but not in others. This diversity makes the ε subunit a potential target for antibiotics. Here, performing molecular dynamics simulations, we identify the Mg2+ binding site in the ε subunit from B. subtilis as E59 and E86. The free energy analysis shows that the first-sphere bi-dentate coordination of the Mg2+ ion by the two glutamates is the most stable state. In comparison, we also clarify the reason for the absence of Mg2+ dependency in the ε subunit from thermophilic Bacillus PS3, despite the high homology to that from B. subtilis. Sequence alignment suggests that this Mg2+ binding motif is present in the ε subunits of some pathogenic bacteria. In addition we discuss strategies to stabilize an isolated ε subunit carrying the Mg2+ binding motif by site directed mutagenesis, which also can be used to crystallize Mg2+ dependent ε subunits in future.

Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria

In this study we investigated the relationship between a rapid change in extracellular pH and the alteration of bacterial ATP concentration. This relationship is a key component of a hypothesis indicating that bacterial bioenergetics – the creation of ATP from ADP via a proton gradient across the cytoplasmic membrane – can be altered by the physiochemical charge-regulation effect, which results in a pH shift at the bacteria's surface upon adhesion to another surface. The bacterial ATP concentration was measured during a rapid change in extracellular pH from a baseline pH of 7.2 to pH values between 3.5 and 10.5. Experiments were conducted with four neutrophilic bacterial strains, including the Gram-negative Escherichia coli and Pseudomonas putida and the Gram-positive Bacillus subtilis and Staphylococcus epidermidis. A change in bulk pH produced an immediate response in bacterial ATP, demonstrating a direct link between changes in extracellular pH and cellular bioenergetics. In general, the shifts in ATP were similar across the four bacterial strains, with results following an exponential relationship between the extracellular pH and cellular ATP concentration. One exception occurred with S. epidermidis, where there was no variation in cellular ATP at acidic pH values, and this finding is consistent with this species’ ability to thrive under acidic conditions. These results provide insight into obtaining a desired bioenergetic response in bacteria through (i) the application of chemical treatments to vary the local pH and (ii) the selection and design of surfaces resulting in local pH modification of attached bacteria via the charge-regulation effect.

Role of ATP synthase as a molecular drug target against bacterial infections

BackgroundF1Fo ATP synthase is the main source of cellular energy in almost all organisms from bacteria to man through oxidative phosphorylation. Binding of ADP and Pi in the catalytic sites of ATP synthase is essential for the formation of ATP. Inhibition of ATP synthase deprives cells of required energy in the form of ATP resulting in cell death. Here, we propose that ATP synthase can be used as a molecular drug target against microbial infections in general and bacterial infections in particular. This can be achieved by targeting the bacterial ATP synthase. A wide variety of phytochemicals and peptides are known to potently in inhibit the ATP synthase. Thus, identification of natural phytochemicals or antibacterial peptides is an important goal towards using ATP synthase as a molecular drug target.MethodsPhytochemicals or peptide induced inhibition of wild type purified F1 or membrane bound F1Fo ATP synthase were performed. Many polyphenolic compounds were structurally modified to increase the extent of inhibition and potency on molar scale. Mutagenic analysis of polyphenol binding and peptide binding site was done to understand the mechanism of inhibition. Growth yield in limiting glucose and non‐fermentable carbon source succinate in presence or absence of inhibitors was done to prove the ATP synthase related cell death.ResultsLately, we found that structural medication of polyphenolic compound such as resveratrol resulted in ~60% additional inhibition of ATP synthase and caused complete abrogation of wild type E. coli cells while mutant cells along with null cells were not affected. Currently, we are studying the role of βDELSEED‐motif in peptide binding to elucidate the mechanism of inhibition of ATP synthase.

The perennial problem of variability in adenosine triphosphate (ATP) tests for hygiene monitoring within healthcare settings.

To investigate the reliability of commercial ATP bioluminometers and to document precision and variability measurements using known and quantitated standard materials. Four commercially branded ATP bioluminometers and their consumables were subjected to a series of controlled studies with quantitated materials in multiple repetitions of dilution series. The individual dilutions were applied directly to ATP swabs. To assess precision and reproducibility, each dilution step was tested in triplicate or quadruplicate and the RLU reading from each test point was recorded. Results across the multiple dilution series were normalized using the coefficient of variation. The results for pure ATP and bacterial ATP from suspensions of Staphylococcus epidermidis and Pseudomonas aeruginosa are presented graphically. The data indicate that precision and reproducibility are poor across all brands tested. Standard deviation was as high as 50% of the mean for all brands, and in the field users are not provided any indication of this level of imprecision. The variability of commercial ATP bioluminometers and their consumables is unacceptably high with the current technical configuration. The advantage of speed of response is undermined by instrument imprecision expressed in the numerical scale of relative light units (RLU).

Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity

This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1–3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

Single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism.

ABSTRACTATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening.

In vitro assessment of the antimicrobial activity of wound dressings: influence of the test method selected and impact of the pH.

Antibacterial activity of dressings containing antimicrobials is mostly evaluated using in vitro tests. However, the various methods available differ significantly in their properties and results obtained are influenced by the method selected, micro-organisms used, and extraction method, the degree of solubility or the diffusability of the test-compounds. Here, results on antimicrobial activity of silver-containing dressings obtained by agar diffusion test (ADT), challenge tests (JIS L 1902, AATCC 100), and extraction-based methods (microplate laser nephelometry (MLN), luminescent quantification of bacterial ATP (LQbATP)) using Staphylococcus aureus and Pseudomonas aeruginosa were evaluated. Furthermore, the effect of the pH on antibacterial efficacy of these dressings was investigated. All silver-containing dressings exerted antimicrobial activity in all in vitro tests and results correlated considerably well. Differences were observed testing the agent-free basic materials. They did not exhibit any antimicrobial effects in the ADT, MLN or LQbATP, since these methods depend on diffusion/extraction of an active agent. However, they showed a strong antimicrobial effect in the challenge tests as they possess a high absorptive capacity, and are able to bind and sequester micro-organisms present. Therefore, it seems recommendable to choose several tests to distinguish whether a material conveys an active effect or a passive mechanism. In addition, it could be shown that release of silver and its antimicrobial efficacy is partially pH-dependent, and that dressings themselves affect the pH. It can further be speculated that dressings’ effects on pH and release of silver ions act synergistically for antimicrobial efficacy.

Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging.

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells.

Bedaquiline: a novel antitubercular agent for the treatment of multidrug-resistant tuberculosis.

Bedaquiline is a diarylquinoline antitubercular drug with a novel mechanism of action against Mycobacterium tuberculosis. Bedaquiline works by inhibiting bacterial adenosine triphosphate (ATP) synthase and represents the first novel class of antituberculosis agents in more than 40 years. Bedaquiline is indicated for the treatment of multidrug-resistant tuberculosis (MDR TB) in combination with at least three other antitubercular drugs when no other effective regimen is available. The recommended bedaquiline dosage is 400 mg orally once/day for 2 weeks followed by 200 mg orally 3 times/week for 22 weeks. Bedaquiline should be administered with food, which increases the bioavailability 2-fold. Bedaquiline is metabolized by cytochrome P450 isoenzyme 3A4 and is impacted by both inducers and inhibitors of this isoenzyme. Concentration-dependent bactericidal activity was observed in laboratory and murine studies. Accelerated approval was granted in the United States and European Union based on the results of two phase IIb clinical studies that used sputum culture clearance as a surrogate end point for clinical efficacy. These studies showed greater sputum culture clearance up to week 24 for the bedaquiline group compared with placebo. Common adverse events in clinical trials included nausea, arthralgia, and headache. Serious adverse events included elevated serum transaminase levels and rate-corrected QT-interval prolongation. Unexplained higher mortality was seen in patients receiving bedaquiline versus those receiving placebo. Bedaquiline is a novel agent with a unique mechanism of action and has the potential to meet a great need in patients with MDR TB who have no other treatment options. Due to safety concerns and limited clinical information, phase III trials are needed to fully determine its place in therapy.

Thiol oxidation of mitochondrial FO-c subunits: A way to switch off antimicrobial drug targets of the mitochondrial ATP synthase

A primary goal in antimicrobial drug design is to find molecules which inhibit key proteins in bacteria without affecting mammalian homologues. To this aim, structural differences between eukaryotic and prokaryotic enzyme proteins involved in life processes are widely exploited. The membrane-bound enzyme complex ATP synthase synthesizes the energy currency molecule of the cell. Due to its bioenergetic role, it represents “the enzyme of life” of all living beings. The enzyme complex has the unique bi-functional property of exploiting either the electrochemical transmembrane gradient to make ATP or, conversely, the free energy of ATP hydrolysis to build an electrochemical gradient across the membrane. The catalytic mechanism of ATP synthesis/hydrolysis, based on the coupling between the two rotary sectors FO and F1 is shared by eukaryotes and prokaryotes. However slight structural differences distinguish prokaryotic ATP synthases, embedded in cell membrane, from eukaryotic ones localized in the mitochondrial inner membrane. In spite of its fundamental task in living organisms, up to now the ATP synthase has been poorly exploited as target in antibacterial therapy, mainly due to harmful effects on patients. Recent advances shoulder the use of drugs targeting the ATP synthase to fight mycobacteria and treat human tuberculosis. Macrolide antibiotics and other antimicrobial drugs specifically bind to the c-ring of the membrane-embedded FO domain, thus blocking ion translocation through FO which is essential for both ATP synthesis and ATP hydrolysis. Our findings show that, once bound to the ATP synthase, probably through different binding sites on a common binding region on FO, the macrolide antibiotics oligomycin, venturicidin and bafilomycin behave as enzyme inhibitors. Interestingly, the c subunits of mitochondrial ATP synthase contain conserved cysteine residues which are absent in bacteria. We pointed out that when these crucial cysteine thiols are oxidized, the common drug binding site of the enzyme is somehow destabilized, thus weakening the enzyme–drug interactions and making the ATP synthase insensitive to drug inhibition. On these bases we hypothesize that the selective oxidation of these cysteine thiols can be exploited to desensitize the mitochondrial ATP synthase to drugs which target FO and maintain their inhibitory potency on bacterial ATP synthases. According to our hypothesis, this strategy could represent an intriguing tool to prevent adverse effects of antimicrobial drugs in mammals, thus enhancing the number of natural and synthetic compounds which can be used in therapy. To this aim studies should be addressed to the identification and formulation of compounds and/or treatments able to selectively oxidize the crucial cysteine thiols of c-subunits without affecting the overall functionality of the mitochondrial ATP synthase and other thiol containing proteins.

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε's conformations is predominant. The assay measures kinetics of ε's binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε's conformational changes.

Nitrite produced by Mycobacterium tuberculosis in human macrophages in physiologic oxygen impacts bacterial ATP consumption and gene expression.

In high enough concentrations, such as produced by inducible nitric oxide synthase (iNOS), reactive nitrogen species (RNS) can kill Mycobacterium tuberculosis (Mtb). Lesional macrophages in macaques and humans with tuberculosis express iNOS, and mice need iNOS to avoid succumbing rapidly to tuberculosis. However, Mtb's own ability to produce RNS is rarely considered, perhaps because nitrate reduction to nitrite is only prominent in axenic Mtb cultures at oxygen tensions ≤1%. Here we found that cultures of Mtb-infected human macrophages cultured at physiologic oxygen tensions produced copious nitrite. Surprisingly, the nitrite arose from the Mtb, not the macrophages. Mtb responded to nitrite by ceasing growth; elevating levels of ATP through reduced consumption; and altering the expression of 120 genes associated with adaptation to acid, hypoxia, nitric oxide, oxidative stress, and iron deprivation. The transcriptomic effect of endogenous nitrite was distinct from that of nitric oxide. Thus, whether or not Mtb is hypoxic, the host expresses iNOS, or hypoxia impairs the action of iNOS, Mtb in vivo is likely to encounter RNS by producing nitrite. Endogenous nitrite may slow Mtb's growth and prepare it to resist host stresses while the pathogen waits for immunopathology to promote its transmission.

Quantifying bacterial biomass fixed onto biological activated carbon (PAC and GAC) used in drinking water treatment

Hybrid processes coupling the use of powder activated carbon (PAC) with membrane filtration for drinking water production are emerging as promising alternatives to conventional technologies due to their enhanced control of dissolved contaminants. The quantification of biomass colonizing PAC is crucial for modeling, designing control strategies and improving the overall performance of these processes. The aim of this study was to examine the applicability of several common methods developed for colonized granular activated carbon (GAC) to PAC. Six analytical methods (based on the measurement of proteins, polysaccharides, heterotrophic plate counts, potential glucose respiration, bacterial ATP and a potential acetate uptake rate, which is proposed herein) were compared. The results showed that the rates of glucose respiration and of acetate consumption could be used interchangeably. Proteins were also an interesting alternative for on-site measurements. It was concluded that biological PAC-based processes sustained a level of heterotrophic activity similar to or greater than that observed in GAC biofilters.

Structural, biochemical and genetic characterization of dissimilatory ATP sulfurylase from Allochromatium vinosum.

ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 2−) with ATP to adenosine 5′-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes.

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

BackgroundF1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli. MethodsWe designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy. ResultsWe show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F1FO ATP synthases. ConclusionsOur system now allows performing previously impossible structural and functional studies on A. aeolicus F1FO ATP synthase. General significanceMore broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.

Extensively drug-resistant tuberculosis: early access to bedaquiline for a UK patient

To the Editor: The prevalence of drug resistance among tuberculosis (TB) cases is increasing in the UK [1] and worldwide [2], with increasing proportions of cases with multidrug- and extensively drug-resistant TB (MDR-/XDR-TB) being reported [1, 2]. Toxicity and poor efficacy of treatment are major problems and long durations with culture-positive disease increase the risk of transmission. This report describes difficulties in managing XDR-TB and the pre-license use of bedaquiline outside of clinical trials. Bedaquiline is an anti-tuberculous drug of the diarylquinoline class, which inhibits bacterial adenosine triphosphate synthase, and represents the first new class of anti-tuberculous agents for at least four decades. Its unique mechanism means that there is no cross-resistance with other drugs in current use. In phase II clinical trials, in combination with background MDR-TB regimens, bedaquiline resulted in reduced time to sputum culture conversion, reduced emergence of resistance to companion drugs and little additional toxicity compared with background regimens alone [3]. At present it is not yet licensed in Europe and the World Health Organization (WHO) has recently issued interim policy guidance for its use as part of combination therapy for adults with MDR-TB under specific conditions, based on currently available evidence [3]. A 28-year-old, HIV-negative Indian female with no prior history of TB treatment or contact was diagnosed with TB through screening on arrival in the UK. A pre-departure chest radiograph 3 months before assessment in the UK was reported to be normal. A bacille Calmette–Guerin scar was seen on examination. A tuberculin skin …

K+ Efflux Is the Common Trigger of NLRP3 Inflammasome Activation by Bacterial Toxins and Particulate Matter

The NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP, and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore, but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K⁺ and Na⁺. Notably, reduction of the intracellular K⁺ concentration was sufficient to activate NLRP3, whereas an increase in intracellular Na⁺ modulated but was not strictly required for inflammasome activation. These results provide a unifying model for the activation of the NLRP3 inflammasome in which a drop in cytosolic K⁺ is the common step that is necessary and sufficient for caspase-1 activation.

F1-ATPase of Escherichia coli

F1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ε, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F1·ε binding and dissociation, we show that formation of the extended, inhibitory conformation of ε (εX) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the εX state, and post-hydrolysis conditions stabilize it. We also show that ε inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ε is responsible for initial binding to F1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ε N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F1-bound ε suggest dynamic conformational and rotational mobility in F1 that is paused near the catalytic dwell position.

A critical study: Assessment of the effect of silica particles from 15 to 500 nm on bacterial viability

The current opinion on the toxicity of nanomaterials converges on a size-dependent phenomenon showing increasing toxicity with decreasing particle sizes. We demonstrate that SiO2 particles have no or only a mild effect on the viability of five bacterial strains, independently from the particle size. A two-hour exposure to 20 mg L−1 of 15, 50 and 500 nm sized SiO2 particles neither alters bacterial adenosine triphosphate (ATP) levels nor reduces the number of colony forming units (CFU). Additionally, we tested the effect of Al2O3-coated LUDOX®-CL (ACS 20) with a primary particle size of 20 nm. In contrast, these particles caused a significant reduction of ATP levels and CFU. Fluorescence microscopy revealed that ACS 20 induced a pronounced agglomeration of the bacteria, which led to underestimated counts in regard of CFU. Bactericide effects as indicated by decreased ATP levels can be explained by bactericide additives that are present in the ACS 20 suspension.

Glucose Triggers ATP Secretion from Bacteria in a Growth-Phase-Dependent Manner

ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that Enterococcus gallinarum, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci, Enterococcus mundtii secreted the greatest amount of ATP (>2 μM/10(8) cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from E. mundtii. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase E. mundtii and Enterococcus faecalis cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function.