Bacterial ATP Research Articles

Of Chloride and Quantal Size

The uptake into synaptic vesicles of glutamate—the major excitatory neurotransmitter in brain—depends on the proton electrochemical gradient generated by the vacuolar-type H + -ATPase and is modulated by extravesicular Cl – (see Schweizer). Extravesicular Cl – promotes the acidification of synaptic vesicles, a process that depends on its permeation into the vesicle, and Schenck et al . found that, whereas synaptic vesicles from mice lacking the CLC-3 Cl – channel acidified when exposed to ATP and Cl – , acidification was blunted in vesicles from mice lacking the major vesicular glutamate transporter (VGLUT1). Moreover, introduction of VGLUT1 into liposomes containing an electrogenic bacterial ATP synthase enabled their acidification by extravesicular Cl – . Thus, VGLUT1 itself mediates a major synaptic vesicle Cl – conductance. Like glutamate uptake into synaptic vesicles, glutamate uptake into VGLUT1-containing liposomes showed a biphasic dependence on external Cl – , with uptake greatest at low-mM external Cl – . Furthermore, experiments in which liposomes were preloaded with either 100 mM potassium chloride or 100 mM potassium gluconate revealed that luminal Cl – markedly enhanced ATP-dependent glutamate uptake. The promotion of glutamate uptake by luminal Cl – involved glutamate transport driven by the transmembrane electrical gradient (ΔΨ) generated by proton translocation; however, the transmembrane pH gradient contributed a component to glutamate uptake as well. Noting that the Cl – content of endocytosed synaptic vesicles depends on extracellular Cl – concentration, the authors propose that extracellular Cl – concentration may play a major role in regulating vesicular glutamate uptake and thus the size of individual quanta of neurotransmitter. S. Schenck, S. M. Wojcik, N. Brose, S. Takamori, A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles. Nat. Neurosci. 12 , 156–162 (2009). [PubMed] F. E. Schweizer, Exit chloride, enter glutamate. Nat. Neurosci . 12 , 111–112 (2009). [PubMed]

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams ( Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase α-chain ( rpoA), RecA protein ( recA), the α-subunit of bacterial ATP synthase ( atpA) and the uridine monophosphate (UMP) kinase ( pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA–DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9 T(=CECT 7244 T=LMG 24045 T).

Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA

Bacterial ATP binding cassette (ABC) exporters fulfill a wide variety of transmembrane transport roles and are homologous to the human multidrug resistance P-glycoprotein. Recent X-ray structures of the exporters MsbA and Sav1866 have begun to describe the conformational changes that accompany the ABC transport cycle. Here we present cryo-electron microscopy structures of MsbA reconstituted into a lipid bilayer. Using ATPase inhibitors, we captured three nucleotide transition states of the transporter that were subsequently reconstituted into helical arrays. The enzyme-substrate complex (trapped by ADP-aluminum fluoride or AMPPNP) crystallized in a different helical lattice than the enzyme-product complex (trapped by ADP-vanadate). Approximately 20A resolution maps were calculated for each state and revealed MsbA to be a dimer with a large channel between the membrane spanning domains, similar to the outward facing crystal structures of MsbA and Sav1866. This suggests that while there are likely structural differences between the nucleotide transition states, membrane embedded MsbA remains in an outward facing conformation while nucleotide is bound.

The Poloxamer P85 Is Protective AgainstListeria monocytogenesInvasion

Listeria monocytogenes remains an important foodborne pathogen, and strategies designed to decrease the susceptibility of selected patient populations to foodborne pathogens are therefore desirable. Our objective was to determine if the poloxamer P85 was protective against L. monocytogenes infection. Caco-2 cells were treated with 0.1% (w/v) P85 and challenged with 10(7) L. monocytogenes EGD for 1 hour. A standard gentamicin protection assay was performed to determine invasion differences between the experimental groups. Effects of P85 on the pathogen were studied by measuring bacterial growth and ATP concentrations. In a murine model of listeriosis, FVB mice were administered 150 mg/kg P85 or vehicle control 45 minutes prior to intragastric inoculation of 10(7) L. monocytogenes. Dissemination of the pathogen from the gastrointestinal tract to the liver and spleen was determined 24 hours after bacterial challenge. Pretreatment of Caco-2 cells with P85 significantly decreased L. monocytogenes invasion compared to controls. Repletion of ATP reversed the protective effects of P85. No changes in bacterial ATP or growth profile were detected in P85-treated bacteria. Administration of P85 to mice prior to infection led to decreased dissemination to the liver and spleen compared to vehicle-treated mice. P85 is protective against L. monocytogenes infection when administered prior to bacterial challenge. Modulation of host ATP levels appears to be crucial for the protective effects of P85.

Alterations in Helicobacter pylori outer membrane and outer membrane vesicle-associated lipopolysaccharides under iron-limiting growth conditions*

Outer membrane vesicles (OMVs) shed from the gastroduodenal pathogen Helicobacter pylori have measurable effects on epithelial cell responses. The aim of this study was to determine the effect of iron availability, and its basis, on the extent and nature of lipopolysaccharide (LPS) produced on H. pylori OMVs and their parental bacterial cells. Electrophoretic, immunoblotting and structural analyses revealed that LPSs of bacterial cells grown under iron-limited conditions were notably shorter than those of bacteria and OMVs obtained from iron-replete conditions. Structural analysis and serological probing showed that LPSs of iron-replete cells and OMVs expressed O-chains of Lewis(x) with a terminal Lewis(y) unit, whereas Lewis(y) expression was notably reduced on bacteria and OMVs from iron-limiting conditions. Unlike the O-chain, the core oligosaccharide and lipid A moieties of iron-replete and iron-limited bacteria and their OMVs were similar. Quantitatively, shed OMVs from iron-replete bacteria were found to be LPSenriched, whereas shed OMVs from iron-limited bacteria had a significantly reduced content of LPS. These differences were linked to bacterial ATP levels. Since iron availability affects the extent and nature of LPS expressed by H. pylori, host iron status may contribute to H. pylori pathogenesis.

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

The F(1)F(0)-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the epsilon subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two alpha-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF(1)) interferes with the intrinsic rotational mechanism of the central gamma subunit and with the opening and closing of the catalytic beta-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF(1) protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F(1)F(0) mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F(1) portion. In addition, F(1)F(0) dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.

A Review of Interactions between Dietary Fiber and the Gastrointestinal Microbiota and Their Consequences on Intestinal Phosphorus Metabolism in Growing Pigs

Dietary fiber is an inevitable component in pig diets. In non-ruminants, it may influence many physiological processes in the gastrointestinal tract (GIT) such as transit time as well as nutrient digestion and absorption. Moreover, dietary fiber is also the main substrate of intestinal bacteria. The bacterial community structure is largely susceptible to changes in the fiber content of a pig's diet. Indeed, bacterial composition in the lower GIT will adapt to the supply of high levels of dietary fiber by increased growth of bacteria with cellulolytic, pectinolytic and hemicellulolytic activities such as Ruminococcus spp., Bacteroides spp. and Clostridium spp. Furthermore, there is growing evidence for growth promotion of beneficial bacteria, such as lactobacilli and bifidobacteria, by certain types of dietary fiber in the small intestine of pigs. Studies in rats have shown that both phosphorus (P) and calcium (Ca) play an important role in the fermentative activity and growth of the intestinal microbiota. This can be attributed to the significance of P for the bacterial cell metabolism and to the buffering functions of Ca-phosphate in intestinal digesta. Moreover, under P deficient conditions, ruminal NDF degradation as well as VFA and bacterial ATP production are reduced. Similar studies in pigs are scarce but there is some evidence that dietary fiber may influence the ileal and fecal P digestibility as well as P disappearance in the large intestine, probably due to microbial P requirement for fermentation. On the other hand, fermentation of dietary fiber may improve the availability of minerals such as P and Ca which can be subsequently absorbed and/or utilized by the microbiota of the pig's large intestine.

Rapid Antimicrobial Susceptibility Determination of Uropathogens in Clinical Urine Specimens by Use of ATP Bioluminescence

We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

Review article: nitric oxide from dysbiotic bacterial respiration of nitrate in the pathogenesis and as a target for therapy of ulcerative colitis

Factors initiating human ulcerative colitis (UC) are unknown. Dysbiosis of bacteria has been hypothesized to initiate UC but, to date, neither the nature of the dysbiosis nor mucosal breakdown has been explained. To assess whether a dysbiosis of anaerobic nitrate respiration could explain the microscopic, biochemical and functional changes observed in colonocytes of UC. Published results in the gastroenterological, biochemical and microbiological literature were reviewed concerning colonocytes, nitrate respiration and nitric oxide in the colon in health and UC. A best-fit explanation of results was made regarding the pathogenesis and new treatments of UC. Anaerobic nitrate respiration yields nitrite, nitric oxide (NO) and nitrous oxide. Colonic bacteria produce NO and UC in remission has a higher lumenal NO level than control cases. NO with sulphide, but not NO alone, impairs beta-oxidation, lipid and protein synthesis explaining the membrane, tight junctional and ion channel changes observed in colonocytes of UC. The observations complement therapeutic mechanisms of those probiotics, prebiotics and antibiotics useful in treating UC. The prolonged production of bacterial NO with sulphide can explain the initiation and barrier breakdown, which is central to the pathogenesis of UC. Therapies to alter bacterial nitrate respiration and NO production need to evolve. The production of NO by colonic bacteria and that of the mucosa need to be separated to pinpoint the sequential nature of NO damage in UC.

A microfluidic ATP-bioluminescence sensor for the detection of airborne microbes

Airborne pathogenic microorganisms are hazardous bioaerosols which often cause serious respiratory diseases. To prevent airborne infectious disease, real-time detection and monitoring systems of airborne pathogens are needed. Since ATP (adenosine triphosphate) is a major biological energy source, the detection of ATP from aerosol reflects the existence of living microbes. Therefore, we developed a new biosensor to detect ATP from aerosols in real-time using an aerosol condensation system, a microfluidic channel, and an ATP-bioluminescence transducer. The condensation system enabled aerosol microbes (4 L) to be hydrosolized (0.2 ml) in 2 min. The bacterial intracellular ATP was then extracted in the passage through the microfluidic channel. The concentration of ATP could be determined by a bioluminescence sensor integrated in the channel. In this study, we used B. subtilis and E. coli JM110 as model airborne microbes. Our system can determine the existence of airborne microbes within 10 min. In the future, the application of our device will extend to the detection of fungi and consequently contribute to improving indoor air quality.

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F 1F o-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F 1F o-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F 1F o-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F 1F o-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F 1 catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F 1 to the lipid monolayer and the F o membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F o by electron microscopy and AFM.

Pannexin-1-Mediated Recognition of Bacterial Molecules Activates the Cryopyrin Inflammasome Independent of Toll-like Receptor Signaling

Cryopyrin is essential for caspase-1 activation triggered by Toll-like receptor (TLR) ligands in the presence of adenosine triphosphate (ATP). However, the events linking bacterial products and ATP to cryopyrin remain unclear. Here we demonstrate that cryopyrin-mediated caspase-1 activation proceeds independently of TLR signaling, thus dissociating caspase-1 activation and IL-1beta secretion. Instead, caspase-1 activation required pannexin-1, a hemichannel protein that interacts with the P2X(7) receptor. Direct cytosolic delivery of multiple bacterial products including lipopolysaccharide, but not flagellin, induced caspase-1 activation via cryopyrin in the absence of pannexin-1 activity or ATP stimulation. However, unlike Ipaf-dependent caspase-1 activation, stimulation of the pannexin-1-cryopyrin pathway by several intracellular bacteria was independent of a functional bacterial type III secretion system. These results provide evidence for cytosolic delivery and sensing of bacterial molecules as a unifying model for caspase-1 activation and position pannexin-1 as a mechanistic link between bacterial stimuli and the cryopyrin inflammasome.

Bioluminescent assay of total bacterial contamination of drinking water

A bioluminescent assay of total bacterial contamination (TBC) of drinking water (DW) with a detection limit of approximately 1 CFU/mL and duration of less than 7 h has been developed. The protocol of the TBC assay comprises: incubation of water sample in nutrition broth supplemented with salts mixture, up to 6 h; filtration of bacterial suspension obtained through membrane filter (pore size 0.45 microm); release of bacterial ATP by dimethyl sulphoxide; determination of bacterial ATP concentration using highly sensitive ATP reagent based on recombinant Luciola mingrelica luciferase. To simplify the assay, special luminometer microcuvette Filtravette (New Horizons Diagnostics Corp., USA) are used. A good correlation (R=0.98) between ATP concentration measured after 6 h incubation and initial bacterial titre in DW was observed. Semi-quantitative TBC assay of DW is also available. The TBC value in DW is assessed by the fixation of incubation time required to detect a measurable bioluminescent signal: 3, 4 and 6 h corresponds to 100-1000, 10-100 and 1-10 CFU/mL, respectively.

Regulatory Interplay between Proton Motive Force, ADP, Phosphate, and Subunit ϵ in Bacterial ATP Synthase

ATP synthase couples transmembrane proton transport, driven by the proton motive force (pmf), to the synthesis of ATP from ADP and inorganic phosphate (P(i)). In certain bacteria, the reaction is reversed and the enzyme generates pmf, working as a proton-pumping ATPase. The ATPase activity of bacterial enzymes is prone to inhibition by both ADP and the C-terminal domain of subunit epsilon. We studied the effects of ADP, P(i), pmf, and the C-terminal domain of subunit epsilon on the ATPase activity of thermophilic Bacillus PS3 and Escherichia coli ATP synthases. We found that pmf relieved ADP inhibition during steady-state ATP hydrolysis, but only in the presence of P(i). The C-terminal domain of subunit epsilon in the Bacillus PS3 enzyme enhanced ADP inhibition by counteracting the effects of pmf. It appears that these features allow the enzyme to promptly respond to changes in the ATP:ADP ratio and in pmf levels in order to avoid potentially wasteful ATP hydrolysis in vivo.

Identification and transcriptional organization of aerobactin transport and biosynthesis cluster genes of Vibrio hollisae

We had previously reported that Vibrio hollisae produces aerobactin in response to iron starvation. In the present study, we identified in V. hollisae ATCC33564 the aerobactin system cluster which consists of eight genes, hatCDB, iucABCD and iutA. The hatCDB genes encode proteins homologous to components of bacterial ATP binding cassette transport systems for ferric aerobactin. The iucABCD and iutA orthologs code for aerobactin biosynthesis enzymes and the ferric aerobactin receptor, respectively. In accordance with their iron-regulated expression, putative Fur box sequences were found within the respective promoter regions of hatC, iucA and iutA. The monocistronic iutA transcript was detected by northern blotting. Moreover, phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions that were expected for the respective operons and genes on the basis of the homology search. The arrangement of the aerobactin gene clusters thus far found in Vibrio and enterobacterial species was compared and discussed from an evolutionary point of view.

Dual Substrate and Reaction Specificity in Mouse Serine Racemase: Identification of High-Affinity Dicarboxylate Substrate and Inhibitors and Analysis of the β-Eliminase Activity

Mouse serine racemase (mSR) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the biosynthesis of the N-methyl-d-aspartate receptor coagonist d-serine in the brain. Furthermore, mSR catalyzes beta-elimination of serine and l-serine-O-sulfate into pyruvate. The biological significance of this beta-elimination activity and the factors influencing mSR substrate and reaction specificity, which are crucial for prospective inhibitor design, are poorly understood. Using a bacterial expression system and ATP-agarose affinity chromatography, we have generated a pure and active recombinant mSR and investigated its substrate and reaction specificity in vitro by analyzing a systematic series of compounds derived from l-Ser and l-serine-O-sulfate. The analysis revealed several competitive inhibitors of serine racemization including glycine (K(I) = 1.63 mM), several dicarboxylic acids including malonate (K(I) = 0.077 mM), and l-erythro-3-hydroxyaspartate (K(I) = 0.049 mM). The latter compound represents the most effective inhibitor of SR reported to date. A simple inversion of the beta-carbon configuration of the compound yields an excellent beta-elimination substrate l-threo-3-hydroxyaspartate. Inhibition analysis indicates that racemization and beta-elimination activities of mSR reside at the same active site. While the racemization activity is specific to serine, the beta-elimination activity has a broader specificity for l-amino acids with a suitable leaving group at the beta-carbon and optimal spatial orientation of the alpha-carboxyl and leaving groups. The possible implications of our observations for inhibitor design, regulation of activity, and function of mSR are discussed.

Rapid decrease of free vancomycin in dense staphylococcal cultures

Bacterial numbers in broth cultures were determined by bioluminescence assay of intracellular bacterial ATP. Broth MICs for strains of Staphylococcus epidermidis (ATCC 14990 and 35984) and Staphylococcus aureus (ATCC 25923, 29213 and 6538) were determined for cultures with different inocula (10(5)-10(8) bacteria/ml) after 24 h of incubation in supplemented Mueller-Hinton broth containing vancomycin. All of the tested strains except one were susceptible to methicillin, and all of the strains were susceptible to vancomycin. Free vancomycin concentrations in the broth cultures of all strains were determined with an agar well bioassay after 24 h of incubation. Free vancomycin concentrations and bacterial numbers of ATCC 35984 and ATCC 29213 were also determined after 0.5, 2, 4, and 8 h. In a low inoculum (10(5) bacteria/ml), the broth MICs were 1-4 microg/ml. In a high inoculum (approximately 10(8) bacteria/ml), the broth MICs increased two- to fourfold to 4-8 microg/ml. In dense inocula ( approximately 10(9)-10(10) bacteria/ml), the concentrations of free vancomycin in the broth were reduced, in most cases below the detection limit of the bioassay (</=0.5 microg/ml). This reduction of free vancomycin was fast, occurring in initially dense inocula in less than 30 min. No emergence of resistance was seen. These results show a rapid reduction of free vancomycin in the broth and a simultaneous increase in broth MICs in high inocula, without development of resistance. This indicates that the dosing regimen of vancomycin is of particular importance in staphylococcal infections with dense inocula, e.g. infective endocarditis.

Transcriptional Termination Control of a Novel ABC Transporter Gene Involved in Antibiotic Resistance in Bacillus subtilis

In members of one of the subfamilies of the bacterial ATP binding cassette (ABC) transporters, the two nucleotide binding domains are fused as a single peptide and the proteins have no membrane-spanning domain partners. Most of the ABC efflux transporters of this subfamily have been characterized in actinomycetes, producing macrolide, lincosamide, and streptogramin antibiotics. Among 40 ABC efflux transporters of Bacillus subtilis, five proteins belong to this subfamily. None of these proteins has been functionally characterized. We examined macrolide, lincosamide, and streptogramin antibiotic resistance in insertional disruptants of the genes that encode these proteins. It was found that only a disruptant of vmlR (formerly named expZ) showed hypersensitivity to virginiamycin M and lincomycin. Expression of the vmlR gene was induced by the addition of these antibiotics in growth medium. Primer extension analysis revealed that transcription of the vmlR gene initiates at an adenosine residue located 225 bp upstream of the initiation codon. From the analysis of the vmlR and lacZ fusion genes, a 52-bp deletion from +159 to +211 resulted in constitutive expression of the vmlR gene. In this region, a typical rho-independent transcriptional terminator was found. It was suggested that the majority of transcription ends at this termination signal in the absence of antibiotics, whereas under induced conditions, RNA polymerase reads through the terminator, and transcription continues to the downstream vmlR coding region, resulting in an increase in vmlR expression. No stabilization of vmlR mRNA occurred under the induced conditions.

Inhibition sites in F1-ATPase from bovine heart mitochondria

High-resolution crystallographic studies of a number of inhibited forms of bovine F1-ATPase have identified four independent types of inhibitory site: the catalytic site, the aurovertin B-binding site, the efrapeptin-binding site and the site to which the natural inhibitor protein IF1 binds. Hitherto, the binding sites for other inhibitors, such as polyphenolic phytochemicals, non-peptidyl lipophilic cations and amphiphilic peptides, have remained undefined. By employing multiple inhibition analysis, we have identified the binding sites for these compounds. Several of them bind to the known inhibitory sites. The amphiphilic peptides melittin and synthetic analogues of the mitochondrial import pre-sequence of yeast cytochrome oxidase subunit IV appear to mimic the natural inhibitor protein, and the polyphenolic phytochemical inhibitors resveratrol and piceatannol compete for the aurovertin B-binding site (or sites). The non-peptidyl lipophilic cation rhodamine 6G acts at a separate unidentified site, indicating that there are at least five inhibitory sites in the F1-ATPase. Each of the above inhibitors has significantly different activity against the bacterial Bacillus PS3 alpha3beta3gamma subcomplex compared with that observed with bovine F1-ATPase. IF1 does not inhibit the bacterial enzyme, even in the absence of the epsilon-subunit. An understanding of these inhibitors may enable rational development of therapeutic agents to act as novel antibiotics against bacterial ATP synthases or for the treatment of several disorders linked to the regulation of the ATP synthase, including ischaemia-reperfusion injury and some cancers.

Validation of a Rapid Diagnostic Strategy for Determination of Significant Bacterial Counts in Bronchoalveolar Lavage Samples

Bacterial cultures of bronchoscopic samples require 1 to 2 days for results to be available for use in clinical decisions. We developed a rapid diagnostic testing strategy that is highly sensitive for screening bacteria in bronchoalveolar lavage (BAL) samples, with results available within hours of collection. To validate the ability of a bacterial adenosine triphosphate (ATP) assay and routine Gram stain microscopy to detect significant bacterial counts in BAL samples. Four hundred seventy-seven BAL samples from 319 patients suspected of having pneumonia were tested using a rapid diagnostic strategy, consisting of Gram stain and a bacterial ATP assay. Rapid results were compared with quantitative cultures with a positive cutoff of 10(4) CFU/mL or higher. Significant bacterial counts were identified in 107 samples (22%). The most common etiologic agents were Staphylococcus aureus (25%), Haemophilus influenzae (17%), and Streptococcus pneumoniae (12%). The rapid test results were false negative in 5 cases (S aureus in 2, both Klebsiella pneumoniae and S aureus in 1, and Stenotrophomonas maltophilia and S pneumoniae in 1 case each). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the rapid diagnostic strategy were 95.3%, 54.9%, 37.9%, 97.6%, and 63.9%, respectively. A negative result with this rapid diagnostic testing strategy rules out significant bacterial counts in BAL samples with a high degree of certainty and may allow use of narrow-spectrum antimicrobial agents or withholding of empiric antimicrobial therapy in patients suspected of having ventilator-associated pneumonia.