An electrochemical method for measuring metabolic activity and counting cells

An express electrochemical method for determining the metabolic activity of live cells based on the possibility of an electron exchange between an electrode and elements of the biological electron transfer chain in the presence of a mediator is proposed. This method is useful for studying any live cells (animal, plant, and microbial), including anaerobic, dormant, and spore cells. The sample preparation and measurement itself does not take more than 30 min. The detection limit in a volume of 15 ml amounts to 10-5 cells/ml. The applicability of the assessment method of the metabolic activity level during the transition of the bacteria Mycobacterium smegmatis into an uncultivable dormant state was demonstrated. This method is of special value for medicine and environmental control, detecting latent forms of pathogens. An optimal combination of the methods for the express analysis of latent pathogens is proposed.     

Advantages of distinguishing the active fraction in bacterioplankton assemblages: some examples

The difficulty of distinguishing between active and dormant or dead bacterial cells is an important problem for the aquatic microbiologist.Active cells can be detected under the microscope by the presence of an intact electron transport system able to reduce the colourless INT [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride] to an optically dense intracellular deposit.An improvement of this method has been applied to Lake Geneva and to a fish pond in the Ivory Coast. The portion of INT-reducing bacterial cells ranged from 1 to 71%, depending on place, depth, season and time of the day. In all cases bacterial activity, determined by uptake of ³H Thymidine or ¹⁴C glucose, and frequency of dividing cells were better correlated with the number of INT-reducing cells than with the total number of cells. This means that counts of cells able to reduce INT have a better metabolic significance than total cell counts. Some examples are developed which show the advantages of applying this method in cases where it is useful to distinguish active cells in a bacterial assemblage.     

Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids

One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc²155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.     

Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Background

Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.

Methodology/Principal Findings

Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC₅₀ 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC₅₀. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.

Conclusions/Significance

NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.     

Dormant forms of <Emphasis Type="Italic">Micrococcus luteus</Emphasis> and <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> not platable on standard media

The colony-forming ability of long (3–9 months) incubated cystlike resting cells (CRC) of the nonspore-forming gram-positive bacteria Micrococcus luteus and Arthrobacter globiformis was studied in this work. The preservation of the CRC proliferative potential as assayed by plating on standard LB agar was shown to depend on the conditions of the formation of the dormant cells. In aged post-stationary cultures of micrococci and arthrobacters grown under carbon and phosphorus limitation the number of colony-forming units (CFU/ml) of CRC decreased in the course of 3–9 month incubation to the level of 10⁶–10⁷ CFU/ml. However, M. luteus CRC obtained under carbon and nitrogen limitation and A. globiformis CRC obtained under nitrogen limitation and starvation completely lost their ability to form colonies on standard solid medium after 4–6 months of incubation and turned into a ‘non-culturable’ (non-platable) state. In this case, the ratio of live cells in the population of M. luteus and A. globiformis ‘non-culturable’ CRCs (determined by the Live/Dead staining test) was 10–44% of the total cell number. To study the possible preservation of proliferative potential in non-platable CRCs, various methods of their reactivation were applied. Although preincubation of CRC suspensions in a buffer solution of 0.1 M K₂HPO₄ (pH 7.4) or in the presence of lysozyme (1 or 10 μg/ml) resulted in increased numbers of live cells (determined by the Live/Dead test) or in disruption of the cell conglomerates, it did not increase considerably the CFU titer on LB medium. Variations in the medium composition, such as addition of sodium pyruvate as an antioxidant or dilution of the medium, promoted the formation of macrocolonies by a small portion of nonplateable CRC of M. luteus (50?80 CFU/ml), whereas the number of the cells capable of microcolony formation (mCFU) was 1.8–6.8 × 10⁵ mCFU/ml, exceeding the CFU titers by four orders of magnitude. The application of semisolid agar and the most probable number (MPN) method was the most efficient for determination of the mCFU titer, and an almost complete reversion of ‘non-culturable’ micrococcal CRCs to microcolony formation was observed (up to 2.3 × 10⁷ mCFU/ml). The usefulness of diluted complete media for the restoration of the colony-forming ability of the dormant forms was confirmed in experiments with ‘nonculturable’ CRCs of A. globiformis. The development of special procedures and methods for determining actively proliferating cells not detected by ordinary methods is of great importance for advanced monitoring studies.     

Rpf proteins are the factors of reactivation of the dormant forms of actinobacteria

As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its par-ticipation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis – an infectious disease that affects one third of the World’s population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine–in diagnostics and in creation of antituberculosis subunit vaccines.     

Dormant forms of mycobacteria

Dormant states of bacteria with drastically decreased metabolic activity, enhanced resistance to harmful factors, and absence of cell division is a form for surviving unfavorable environmental conditions. This state does not necessarily imply formation of highly differentiated spores and cysts; it has been demonstrated for non-spore-forming bacteria, including pathogenic ones. The latency of a number of infectious diseases is generally believed to be related to the capacity of bacteria (including Mycobacterium tuberculosis, an infective agent of tuberculosis) to produce dormant forms. Indeed, some results of histological investigation and modeling of latent infections in animals, as well as results obtained with in vitro models, support the hypothesis of production of dormant forms by tuberculosis bacteria. In the present review, existing experimental models of dormant form production in mycobacteria are considered, as well as modern data concerning the mechanisms of their formation and their relation to the “nonculturable” state. The mechanisms of reversion to culturability and the role of extracellular factors in reactivation of dormant forms are discussed in detail.     

Excess exogenous pyruvate inhibits lactate dehydrogenase activity in live cells in an MCT1-dependent manner

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-¹³C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1–dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-¹³C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.     

Mass and Density Measurements of Live and Dead Gram-Negative and Gram-Positive Bacterial Populations

Monitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and dead Escherichia coli O157:H7 and Listeria innocua were measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 10⁵ and 10⁸ cells/ml, while growth was not observed with optical density measurements until the concentration was 10⁷ cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. Dead E. coli cells were found to have a larger density and smaller total mass than live E. coli cells. In contrast, density was the same for both live and dead L. innocua cells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.     

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time ^1-4.The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis ^5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression ^8-10 and reside growth-arrested in the patients'' bone marrow, blood and lymph nodes ^1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems. in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously ^1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies^15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.Download video file.^{(58M, mov)}     

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.     

Metabolic Responses of Primary and Transformed Cells to Intracellular Listeria monocytogenes

The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using ¹³C-labelled glucose or glutamine as carbon tracers. The ¹³C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.     

Effect of N,N'-dicyclohexylcarbodiimide (DCCD) on electron transport particles of Mycobacterium phlei

N,N′-dicyclohexylcarbodiimide (DCCD) was found to uncouple phosphorylation from oxidation with succinate and NAD⁺-linked substrates in the system from Mycobacterium phlei. However, in contrast to the effect of this agent in mammalian mitochondria, DCCD was found to stimulate oxidation with succinate as an electron donor and to inhibit the oxidation of NAD⁺-linked substrates. Furthermore, in the M. phlei system DCCD was found to inhibit the membrane bound latent ATP-ase but had no effect on this activity when the latent ATPase was removed from the membrane vesicles. Reconstitution with the fraction containing latent ATPase activity and the membrane vesicles resulted in inhibition of latent ATPase by DCCD. Studies of the effect of DCCD on the resolved system indicated that DCCD may be associated with membrane vesicles or causes secondary changes in conformation of membrane vesicles. Although DCCD inhibited membrane bound ATPase it did not prevent the addition of the solubilized ATPase to the membrane vesicles. DCCD was found to have no effect on purified succinic dehydrogenase activity but stimulated this activity in the electron transport particles.     

Evidence of a store of informational RNA in encysted embryos of Artemia salina

RNA prepared from dormant cysts and developmental stages of the brine shrimp Artemia salina stimulated the incorporation of ¹⁴C-leucine into polypeptide by a cell-free Escherichia coli system. Preparations from cysts were about as active as those from hatching embryos or nauplii. When analysed by density gradient centrifugation the activity of cyst RNA showed a heterodisperse distribution, not quantitatively related to the absorbance profile. These results and evidence from similar experiments with crude ribosome preparations indicated that the contribution of 18S and 28S ribosomal RNA to the template-like activity was fairly limited. The experiments suggest that RNA with latent messenger activity is present in Artemia cysts during the resting stage.     

Peptidoglycan fragments stimulate resuscitation of “non-culturable” mycobacteria

Resuscitation promoting factors (Rpfs), belonging to a family of secreted actinobacterial proteins with predicted peptidoglycan (PG) hydrolytic activities, participate in the reactivation of dormant cells. In the present study we demonstrate that a recombinant truncated form of Micrococcus luteus Rpf hydrolyzes isolated PG of Mycobacterium smegmatis and Mycobacterium tuberculosis liberating PG fragments of different size. These fragments possess stimulatory activity toward “non-culturable” dormant M. smegmatis and M. tuberculosis cells, similar to the activity of recombinant Rpf. Relatively large PG fragments (0.1–0.5 μm) obtained either by Rpf digestion or by PG ultrasonication revealed resuscitation activities when added in concentrations 0.1–0.2 μg/ml to the resuscitation medium. It is suggested that PG fragments could either directly activate the resuscitation pathway of dormant mycobacteria or serve as a substrate for endogenous Rpf, resulting in low molecular weight products with resuscitation activity. Whilst both suggestions are plausible, it was observed that PG-dependent resuscitation activity was suppressed by means of a specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate), which provides additional support for the second of these possibilities.     

Proteinbiosynthesen in dormanten und nachgereiften embryonen und samen von Agrostemma githago

Seed germination of Agrostemma githago is prevented by inhibitors of protein and RNA synthesis. Thus protein as well as RNA synthesis are essential prerequisites for germination. Early protein synthesis of Agrostemnia embryos can be completely inhibited by cycloheximide and cordycepin. During the aging of seeds there is a considerable decrease in germination capacity and protein synthesis. In dormant and afterripened embryos of Agrostemma githago¹⁴C-leucine and ¹⁴C-uracil are incorporated in protein and RNA respectively with nearly the same intensity, whereas RNA and protein synthesis of dormant seeds and embryos starts earlier than in those subjected to afterripening. ³H-uracil-labelled RNA from dormant and afterripened embryos are able to hybridize on oligo-dT-cellulose to the same extent. There is a similarity in the protein pattern of dormant and afterripened embryos revealed by electrophoresis in polyacrylamide gels of double-labelled proteins. According to these results dormancy of Agrostemma githago is not caused by a general but by a specific metabolic block.     

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant

Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm² in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins'' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s⁻¹. The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment.     

The model of resting forms of micobacteria for testing of chemodrugs for latent forms of tuberculosis

The new model of obtaining of ovoid resting forms of Mycobacterium smegmatis, which are morphologically different from vegetative (rod-like) cells, was developed. Ovoid forms were characterized by a drastically decreased level of metabolic activity, an increased stability to heat or antibiotics treatment, and also by prolonged (more than 2 months) storage time preserving colony-forming ability. Obtained resting forms of micobacteria may be used in test-systems for checking efficiency of new medical agents against latent forms of tuberculosis and determination of role of the genes in entering dormant state.