Modeling non-inherited antibiotic resistance.

A mathematical model is presented for the increase and decrease of non-inherited antibiotic resistance levels in bacteria. The model is applied to experimental data on E. coli exposed to amoxicillin or tetracyclin in different concentrations. The parameters of the model are estimated using a Monte Carlo Markov Chain method. The model accurately describes build-up and decline of antibiotic resistance caused by physiological adaptations as long as no genetic changes have occurred. The main conclusion of the analysis is that short time periods are sufficient to re-obtain low MIC-values after long-lasting exposure to these antibiotics.

Transcriptome meets metabolome: hierarchical and metabolic regulation of the glycolytic pathway.

The fact that information flows from DNA to RNA to protein to function suggests that regulation is 'hierarchical', i.e. dominated by regulation of gene expression. In the case of dominant regulation at the metabolic level, however, there is no quantitative relationship between mRNA levels and function. We here develop a method to quantitate the relative contributions of metabolic and hierarchical regulation. Applying this method to the glycolytic flux in three species of parasitic protists, we conclude that it is rarely regulated by gene expression alone. This casts strong doubts on whether transcriptome and proteome analysis suffices to assess biological function.

Purification and specificity of two alpha-glucosidase isoforms of the parasitic protist Trichomonas vaginalis.

Two isoforms of alpha-glucosidase were purified from the parasitic protist Trichomonas vaginalis. Both consisted of 103 kDa subunits, but differed in pH optimum and substrate specificity. Isoform 1 had a pH optimum around 4.5 and negligible activity on glucose oligomers other than maltose, while isoform 2 with a pH optimum of 5.5 hydrolyzed also such substrates at considerable rates. Neither had activity on glycogen or starch. Isoform 1 had a specific activity for hydrolysis of maltose of 30 U/mg protein and isoform 2 101 U/mg protein. The Km values were 0.4 mM and 2.0 mM, respectively. Isoform 2 probably corresponds to the activity detected on the cell surface.

The length of the combined 3' untranslated region and poly(A) tail does not control rates of glyceraldehyde-3-phosphate dehydrogenase mRNA translation in three species of parasitic protists.

Experimental observations suggested that the length of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA 3' end has a role in regulating rates of translation in the parasitic protists Trypanosoma brucei, Leishmania donovani, and Trichomonas vaginalis. Using a PCR assay for poly(A) tail length, we measured the size of the RNA 3' end under different growth conditions in all three species. Our results showed that the combined 3' untranslated region and poly(A) tail of GAPDH mRNA do not vary with different rates of translation.

Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei.

The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic flux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the flux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the flux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions.

Regulation and adaptation of glucose metabolism of the parasitic protist Leishmania donovani at the enzyme and mRNA levels.

Adaptation of the glucose metabolism of Leishmania donovani promastigotes (insect stage) was investigated by simultaneously measuring metabolic rates, enzyme activities, message levels, and cellular parameters under various conditions. Chemostats were used to adapt cells to different growth rates with growth rate-limiting or excess glucose concentrations. L. donovani catabolized glucose to CO(2), succinate, acetate, and pyruvate in ratios that depended on growth rate and glucose availability. Rates of glucose consumption were a linear function of growth rate and were twice as high in excess glucose-grown cells as in glucose-limited organisms. The major end product was CO(2), but organic end products were also formed in ratios that varied strongly with growth conditions. The specific activities of the 14 metabolic enzymes measured varied by factors of 3 to 17. Two groups of enzymes adapted specific activities in parallel, but there was no correlation between the groups. The activities of only one group correlated with specific rates of glucose metabolism. Total RNA content per cellular protein varied by a factor of 6 and showed a linear relationship with the rate of glucose consumption. There was no correlation between steady-state message levels and activities of the corresponding enzymes, suggesting regulation at the posttranscriptional level. A comparison of the adaptation of energy metabolism in L. donovani and other species suggests that the energy metabolism of L. donovani is inefficient but is well suited to the environmental challenges that it encounters during residence in the sandfly, its insect vector.

Adaptation of metabolic enzyme activities of Trypanosoma brucei promastigotes to growth rate and carbon regimen.

The insect stage of Trypanosoma brucei adapted the activities of 16 metabolic enzymes to growth rate and carbon source. Cells were grown in chemostats with glucose, rate limiting or in excess, or high concentrations of proline as carbon and energy sources. At each steady state, samples were collected for measurements of substrate and end product concentrations, cellular parameters, and enzyme activities. Correlation coefficients were calculated for all parameters and used to analyze the data set. Rates of substrate consumption and end product formation increased with increasing growth rate. Acetate and succinate were the major nonvolatile end products, but measurable quantities of alanine were also produced. More acetate than succinate was formed during growth on glucose, but growth on proline yielded an equimolar ratio. Growth rate barely affected the relative amounts of end products formed. The end products accounted for the glucose consumed during glucose-limited growth and growth at high rates on excess glucose. A discrepancy, indicating production of CO2, occurred during slow growth on excess glucose and, even more pronounced, in cells growing on proline. The activities of the metabolic enzymes varied by factors of 2 to 40. There was no single enzyme that correlated with consumption of substrate and/or end product formation in all cases. A group of enzymes whose activities rigorously covaried could also not be identified. These findings indicate that T. brucei adapted the activities of each of the metabolic enzymes studied separately. The results of this complex manner of adaptation were more or less constant ratios of the end products and a very efficient energy metabolism.

Metabolic adaptation of Trichomonas vaginalis to growth rate and glucose availability.

The parasitic protist Trichomonas vaginalis adapted the specific activities of twelve of the enzymes involved in glucose metabolism to the growth rate an glucose availability. These changes in enzyme activities were induced by culturing T. vaginalis in chemostats with glucose, present in rate-limiting or excess concentrations, as carbon and energy source. The specific activities were measured in pelleted cells at each steady state, while metabolic end products were determined in filtered culture fluid. The specific activities were lower in cells grown on growth-rate-limiting concentrations of glucose and higher in organisms cultured in the presence of excess glucose. In both cases enzyme activities were higher at increasing growth rates. For most enzymes the difference between the highest and lowest activities was an order of magnitude. The specific activities of eleven of the enzymes were strongly correlated to each other (correlation coefficients 0.83-0.99), the exception being lactate dehydrogenase. The rates of production of the three major end products, lactate, acetate and glycerol, increased with increasing growth rates. Alanine was not formed in measurable quantities. The ratio of the end products formed was strongly influenced by the growth rates and glucose availability. The rates of formation of acetate and glycerol correlated best with the specific activities of the enzymes catalysing the final reactions of their respective pathways. This suggests that the production of acetate and glycerol is rate-limited by these final steps. In contrast, the formation of lactate did not correlate with the specific activity of lactate dehydrogenase but was determined by the rate of glucose consumption.

Maltose utilization by extracellular hydrolysis followed by glucose transport in Trichomonas vaginalis.

The amitochondriate parasitic protist Trichomonas vaginalis can utilize either glucose or maltose as carbon and energy source. The mechanisms of maltose utilization were explored with uptake experiments using radio-isotope labelled maltose in combination with the silicone-oil centrifugation technique and enzymatic assays measuring maltose hydrolysis. The uptake of maltose label became saturated after 2-3 h. The uptake of maltose as a function of the external maltose concentration was linear at low concentrations with no further increase at higher levels, kinetics characteristic of reactions obeying Michaelis-Menten kinetics preceded by a diffusion-limited step. Increased viscosity of the medium resulted in decreased maltose uptake, indicating an extracellular location of the diffusion-limited step. Most of the cellular alpha-glucosidase activity of T. vaginalis was detected on the cell surface, suggesting that maltose is hydrolysed to glucose outside the cell. Glucose interfered more with maltose uptake, and maltose less with glucose uptake, than would be expected if 1 mol of maltose were the equivalent of 2 mol of glucose. This pattern of interaction indicated that the interference occurs before the common metabolic pathway and even before the transport step, supporting the idea of extracellular maltose hydrolysis. We conclude that maltose is hydrolysed to glucose in the boundary layer of the cell, a process akin to membrane digestion in vertebrate enterocytes and on the teguments of helminths. The glucose formed is then transported by the glucose carrier of the organism.

Carbohydrate metabolism and physiology of the parasitic protist Trichomonas vaginalis studied in chemostats.

The parasitic protist Trichomonas vaginalis was cultured in chemostats with glucose or maltose as carbon and energy source. The maximum growth rate was about six divisions per day independent of the substrate, and the apparent Km for glucose was 0.375 mM. While growing on maltose, the growth rate depended linearly on the maltose concentration, indicating that in contrast to glucose metabolism a diffusion step is rate-limiting to maltose metabolism. Cultures were examined over a wide range of growth rates under four conditions: utilizing glucose or maltose as carbon and energy source, with the carbon source rate-limiting or present in excess. Cell density, cellular protein and carbohydrate content as well as residual substrate concentration in the culture fluid were measured at each steady state. The protein content was constant at 100 pg protein per cell except when T. vaginalis was cultured under glucose limitation; in the latter case, slow-growing cells had less protein than cells grown at high rates. When growing under glucose limitation T. vaginalis metabolism changed to become more energy efficient at growth rates exceeding about half the maximum rate. The maintenance energy at the low growth rates accounted for approximately half of the total carbon consumption, which is high in comparison to other micro-organisms. At low growth rates the yield on maltose exceeded that on glucose, when expressed in terms of carbon equivalents. The yields on maltose and glucose were equal, but much lower, when the carbon source was not rate-limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation of the carbon metabolism of Trichomonas vaginalis to the nature and availability of the carbon source.

The anaerobic parasitic protist Trichomonas vaginalis was adapted in chemostats to eight different conditions defined by different growth rates and carbon regimens. Glucose or maltose was used as carbon and energy source. Cells cultured under well-defined steady states were tested in short-term experiments. The kinetics of glucose and maltose uptake were determined and their glucokinase and alpha-glucosidase activities were measured. Uptake in 20 min was measured with radiolabelled glucose and maltose, rather than analogues, using the silicone oil centrifugation technique. Hence, the accumulated label represents both transport and metabolic activity. The total uptake of glucose was highest in organisms that had been starved for glucose during growth. The kinetics of glucose uptake can be understood by assuming rate-limitation by transport across the plasma membrane at low external concentrations and by the subsequent metabolism at concentrations exceeding a cross-over value. The specific glucokinase activity correlated in only four out of eight cases with the saturation uptake. The kinetics of maltose uptake indicated rate-limitation at low maltose concentrations by a diffusion-limited step and at higher levels by metabolic steps. The uptake of maltose was primarily affected by the growth rate during culture, the highest growth rates resulting in most uptake. Maltose uptake was determined only partially by the cellular alpha-glucosidase activity. The activities of both transport and metabolic enzymes changed due to the culture conditions suggesting that the control over glucose and maltose metabolism is shared by several steps in the pathway.

The kinetics of facilitated diffusion followed by enzymatic conversion of the substrate.

The uptake of a substrate that is transported into the cell by facilitated diffusion and subsequently converted in a series of enzymatic reactions, measured as a function of the external concentration, does not usually show the rectangular hyperbolic function characteristic of Michaelis-Menten kinetics. Instead, a seemingly biphasic curve is observed, consisting of Michaelis-Menten kinetics at low concentrations and no further uptake at higher levels. By combining the equations for facilitated diffusion and an enzymatic reaction, we have derived an equation that describes the overall rate of uptake and metabolism of a substrate that is transported across the plasma membrane by facilitated diffusion. Modelling based on this equation simulated the kinetics found experimentally, as long as the kinetic parameters of the carrier were chosen to render it asymmetric. The overall rate was influenced by the kinetics of both reactions over a wide range of concentrations, confirming the principles of the 'Control Analysis' theory in an independent manner.

Membrane-related processes and overall energy metabolism in Trypanosoma brucei and other kinetoplastid species.

An electrochemical proton gradient exists across the plasma membrane and the mitochondrial membrane of the bloodstream form of Trypanosoma brucei. The membrane potential across the plasma membrane and the regulation of the internal pH depend on the temperature. Leishmania donovani regulates its internal pH and maintains a constant electrochemical proton gradient across its plasma membrane under all conditions examined. The mitochondrion of the T. brucei bloodstream form is energized, even though the reactions taking place in it do not result in net ATP synthesis and the Kreb's cycle and the respiratory chain are absent. Glucose is transported across the plasma membrane of T. brucei by a facilitated diffusion carrier, that can transport a wider range of substrates than its mammalian counterparts. Pyruvate exits the cell via a facilitated diffusion transporter as well. Conflicting evidence exists for the mechanism of glucose transport in L. donovani; biochemical evidence suggests proton/glucose symport, while facilitated diffusion is indicated by physiological data.

Uptake and turnover of glucose in Leishmania donovani.

Glucose uptake and metabolism by Leishmania donovani promastigotes was studied using D-[14C]glucose in combination with the silicone oil centrifugation technique on organisms preadapted to different growth rates and glucose availability in the chemostat. The uptake step was differentiated from the subsequent metabolism by separation in time rather than by using non-metabolisable analogues. The uptake of glucose was measured as a function of time and/or the external glucose concentration on cells grown at high or low growth rate with glucose either as growth rate-limiting substrate, or present in excess. Glucose uptake as a function of its external concentration could be described as consisting of two components (1) a rapid equilibration owing to facilitated diffusion, followed by (2) a much slower uptake that involves an enzymatic component. This slower accumulation of label could be explained as the conversion of glucose into metabolites and a storage carbohydrate. Uptake experiments in the presence of inhibitors indicated that the conversion of glucose was an energy dependent process. These experiments indicate that the active uptake of glucose by L. donovani, as reported by others does not occur across the plasma membrane and should be reinterpreted as the intracellular conversion of glucose into metabolites and storage carbohydrate.

Interaction between facilitated diffusion of glucose across the plasma membrane and its metabolism in Trichomonas vaginalis.

The parasitic protist Trichomonas vaginalis transports glucose across the plasma membrane by facilitated diffusion. The Km of the transporter for glucose was 1.6 mM. The uptake of labelled glucose in a minimal medium not allowing growth reached saturation only after 2.5 h, indicating the turnover of storage carbohydrate. Organisms grown on glucose showed higher activities both of the transporter and of the subsequent metabolic pathway than organisms grown on maltose. At low external glucose concentrations the transport step was rate limiting, at higher levels a subsequent enzymatic step. The uptake mechanism for glucose of T. vaginalis resembled that of parasitic kinetoplastid protists and Entamoeba histolytica.

Impairment of growth of Leishmania donovani by Trypanosoma brucei during co-culture.

Cells of Leishmania donovani in co-culture with Trypanosoma brucei, were severely affected in their growth, resulting in swelling and subsequent lysis. Similar effects were also observed when Crithidia luciliae or Phytomonas sp. were co-cultured with T. brucei. Direct contact between the cells under investigation and T. brucei was necessary because T. brucei did not hamper the growth of the other trypanosomatids, when separated by a filter with 0.2 microns pore size. Examination of this phenomenon at the ultrastructural level, in a co-culture of L. donovani and T. brucei, suggests that the plasma membrane permeability is increased in the former, as a result of a close cellular contact between the two cell types.

The electrochemical proton gradient in the bloodstream form of Trypanosoma brucei is dependent on the temperature.

The membrane potential and pH gradient over the plasma membrane of the protozoan parasite Trypanosoma brucei were measured with radioactive indicators in combination with the silicone oil centrifugation technique over a range of temperatures. At 37 degrees C a small membrane potential and pH gradient of similar magnitude, but of opposite polarity, were measured. The resulting electrochemical proton gradient was almost zero. However, when the temperature was lowered from 37 degrees C to 22 degrees C, the internal pH was kept constant independent of the external pH and a membrane potential of between -100 and -150 mV was measured, depending on the external pH. Measurements at various temperatures between 15 degrees C and 37 degrees C revealed that above 26 degrees C the membrane potential collapsed and that this collapse correlated with a sudden increase in membrane fluidity. The uptake of 2-deoxy-D-glucose and of pyruvate, which are both mediated by facilitated diffusion carriers in the plasma membrane of the trypanosome, were also affected by this sudden increase in fluidity of the membrane. The overall rate of the conversion of glucose into its metabolites, which is independent of the plasma membrane, varied only gradually. We conclude (i) that major changes occur in the plasma membrane of T. brucei around 26 degrees C, that affect all membrane related processes; (ii) that the electrochemical proton gradient plays a minor role in the energy metabolism of T. brucei when it resides in the bloodstream of the mammalian host at 37 degrees C; and (iii) that below 26 degrees C an electrochemical proton gradient is maintained over the plasma membrane.

A chemostat study on proline uptake and metabolism of Leishmania donovani.

Leishmania donovani grew in the chemostat on proline as its sole carbon and energy source at a maximum growth rate of 1.39 divisions per day. The efficiency of proline metabolism decreased with increasing external proline concentration. The internal concentration of proline and its intracellular metabolites was low when proline was the growth rate limiting substrate and high when proline was available in excess. In time-course experiments proline uptake leveled off after 30 min, independent of the culture conditions prior to the experiment. Proline uptake depended on the external proline concentration in a manner that is best described as the combination of an enzymatic and a diffusion component. Adaptation to different proline concentrations did not occur and no evidence was found that proline is actively transported by L. donovani.

Comparative physiology of two protozoan parasites, Leishmania donovani and Trypanosoma brucei, grown in chemostats.

Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate. L. donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T. brucei is 1.06 day-1 and the Ks is 0.06 mM. At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity. L. donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP). Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium. L. donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted. T. brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates. Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism. The Ks is constant as long as glucose is the rate-limiting substrate. The results of this study demonstrate that L. donovani and T. brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts.

Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier.

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.