Acidification Asymmetrically Affects Voltage-dependent Anion Channel Implicating the Involvement of Salt Bridges

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to “gate” between an open and several “closed” states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pK_a values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.     

Determining different impact factors on the xylonic acid production using Gluconobacter oxydans DSM 2343

Xylonic acid is a promising compound for the substitution of gluconic acid. Gluconobacter oxydans DSM 2343 has proven to be a highly potent biocatalyst for the conversion of xylose to xylonic acid.In the present study, different nitrogen sources for the growth of G. oxydans and subsequent xylonic acid production were investigated for the first time with minimal medium. Application of 0.32 g/L glutamate supplemented with 0.15 g/L ammonium sulfate as a cheap nitrogen source enabled a xylonic acid productivity of 2.92 g/(Lh) which is similar to findings involving a complex medium (3.20 g/(Lh)). The study further investigated the impact of the xylose source on the growth and production of G. oxydans. Dose-response curves confirmed that G. oxydans is mainly insensitive towards the main inhibitory compounds, acetate and hydroxymethylfurfural, up to a concentration of 5 g/L and 2.5 g/L, respectively. However, batch investigations indicated that substitution of 25 % of the pure xylose with hemicellulosic xylose resulted in a xylonic acid yield of 90 % compared to the control approach without hemicellulosic xylose. The feeding of hemicellulosic xylose in a pulsed fed-batch mode even enabled the use of 50 g/L demonstrating that the proper selection of a feeding strategy for the hemicellulosic xylose greatly improves the production of xylonic acid.     

Production of xylonic acid byPseudomonas fragi

Summary Eleven microbial strains were tested for their ability to produce xylonic acid from xylose. The production of xylonic acid by one of the strains,Pseudomonas fragi ATCC 4973, was further studied in laboratory fermenter scale. The yield of xylonic acid was 92 % of original sugar. Xylonic acid production seemed to be growth associated and it was found to be very sensitive to the decrease of pH.     

Tetrathionate-Forming Thiosulfate Dehydrogenase from the Acidophilic,Chemolithoautotrophic Bacterium Acidithiobacillus ferrooxidans

Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg⁻¹) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg⁻¹) observed at pH 2.5 and 50°C.     

Human Tau Isoforms Assemble into Ribbon-like Fibrils That Display Polymorphic Structure and Stability

Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the “thin twisted” and “thin smooth” fibrils resemble flat ribbons (cross-section ∼10 × 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of ∼65–70 nm and thicknesses of ∼9–18 nm such as routinely reported for “paired helical filaments” but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of ∼17–19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures (∼2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility.     

Superoxide Triggers an Acid Burst in Saccharomyces cerevisiae to Condition the Environment of Glucose-starved Cells

Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this “acid burst” were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress.     

Oxidative d-xylose metabolism of Gluconobacter oxydans

Summary Gluconobacter oxydans subsp. suboxydans ATCC 621 oxidizes d-xylose to xylonic acid very efficiently, although it cannot grow on xylose as sole carbon source. The oxidation of xylose was found to be catalyzed by a membrane-bound xylose dehydrogenase. The xylono--lactone formed in the oxidation reaction is subsequently hydrolyzed to xylonic acid by a -lactonase. The complete oxidation pathway of d-xylose in G. oxydans is evidently located in the periplasmic space.     

Acid-induced Dissociation of VacA, the Helicobacter pylori Vacuolating Cytotoxin, Reveals Its Pattern of Assembly

In this study, we describe the ultrastructural changes associated with acid activation of Helicobacter pylori vacuolating cytotoxin (VacA). Purified VacA molecules imaged by deep-etch electron microscopy form ~30-nm hexagonal “flowers,” each composed of an ~15-nm central ring surrounded by six ~6-nm globular “petals.” Upon exposure to acidic pH, these oligomeric flowers dissociate into collections of up to 12 teardrop-shaped subunits, each measuring ~6 × 14 nm. Correspondingly, glycerol density gradient centrifugation shows that at neutral pH VacA sediments at ~22 S, whereas at acidic pH it dissociates and sediments at ~5 S. Immunoblot and EM analysis of the 5-S material demonstrates that it represents ~90-kD monomers with 6 × 14–nm “teardrop” morphology. These data indicate that the intact VacA oligomer consists of 12 ~90-kD subunits assembled into two interlocked six-membered arrays, overlap of which gives rise to the flower-like appearance. Support for this interpretation comes from EM identification of small numbers of relatively “flat” oligomers composed of six teardrop-shaped subunits, interpreted to be halves of the complete flower. These flat forms adsorb to mica in two different orientations, corresponding to hexameric surfaces that are either exposed or sandwiched inside the dodecamer, respectively. This view of VacA structure differs from a previous model in which the flowers were interpreted to be single layers of six monomers and the flat forms were thought to be proteolysed flowers. Since acidification has been shown to potentiate the cytotoxic effects of VacA, the present results suggest that physical disassembly of the VacA oligomer is an important feature of its activation.     

Reconstitution of a Thermostable Xylan-Degrading Enzyme Mixture from the Bacterium Caldicellulosiruptor bescii

Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k_cat values of ∼8,000 and ∼4,500 s⁻¹, respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.     

木糖发酵生产高纯度D-乳酸大肠杆菌工程菌LHY02的构建

高效利用木糖发酵生产D-乳酸或其他生物质产品,是充分利用木质纤维素的一个关键问题。以高效利用木糖产L-乳酸的Escherichia coli WL204为出发菌株,采用RED基因置换技术将ldhL基因置换为ldhA基因,获得一株能利用木糖产D-乳酸的大肠杆菌工程菌株Escherichia coli LHY02,该菌株利用10%木糖发酵,D-乳酸产量达到84.4 g/L,产物光学纯度达到99.5%。此外,该菌株仍然具有较好的利用葡萄糖产D-乳酸的能力。     

Metabolite Proofreading in Carnosine and Homocarnosine Synthesis: MOLECULAR IDENTIFICATION OF PM20D2 AS β-ALANYL-LYSINE DIPEPTIDASE*

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a “metabolite repair” enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some “classic dipeptides” like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ∼100–200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

木糖高效生物转化的新出路

木糖的高效利用是影响木质纤维资源生物炼制经济效益的关键因素之一,也是构建其工业化生产体系的必要前提,但是木糖生物转化面临着重要的技术瓶颈,必须寻求新的思路。基于对木糖利用的现状及产业发展的综合分析,提出了木糖高效发酵制取木糖酸的新出路,论述了本领域首要的科学和技术问题是发酵抑制物的控制与消除;针对抑制物的问题,提出了细胞生理生化、代谢流分析及分子生物学的多层次和多尺度解析的研究方法;在此基础上,基于系统论的观点提出了菌种选育、原料预处理、抑制物控制与脱除、木糖酸高效发酵的技术集成的研究思路。     

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, ¹H and ¹³C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (∼1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative “transketolase” (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic “rheostat” controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11β-OHAD on the host or other gut microbes is currently unknown.     

Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli,Avoiding Dangers of Runaway Overreplication

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the “natural” CRC limit of ∼8 (cells divide more slowly); the “functional” CRC limit of ∼22 (cells divide extremely slowly); and the “tolerance” CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.     

Reexamination of the Species Assignment of Diacavolinia Pteropods Using DNA Barcoding

Thecosome pteropods (Mollusca, Gastropoda) are an ecologically important, diverse, and ubiquitous group of holoplanktonic animals that are the focus of intense research interest due to their external aragonite shell and vulnerability to ocean acidification. Characterizing the response of these animals to low pH and other environmental stressors has been hampered by continued uncertainty in their taxonomic identification. An example of this confusion in species assignment is found in the genus Diacavolinia. All members of this genus were originally indentified as a single species, Cavolinia longirostris, but over the past fifty years the taxonomy has been revisited multiple times; currently the genus comprises 22 different species. This study examines five species of Diacavolinia, including four sampled in the Northeast Atlantic (78 individuals) and one from the Eastern tropical North Pacific (15 individuals). Diacavolina were identified to species based on morphological characteristics according to the current taxonomy, photographed, and then used to determine the sequence of the “DNA barcoding” region of the cytochrome c oxidase subunit I (COI). Specimens from the Atlantic, despite distinct differences in shell morphology, showed polyphyly and a genetic divergence of <3% (K2P distance) whereas the Pacific and Atlantic samples were more distant (∼19%). Comparisons of Diacavolinia spp. with other Cavolinia spp. reveal larger distances (∼24%). These results indicate that specimens from the Atlantic comprise a single monophyletic species and suggest possible species-level divergence between Atlantic and Pacific populations. The findings support the maintenance of Diacavolinia as a separate genus, yet emphasize the inadequacy of our current taxonomic understanding of pteropods. They highlight the need for accurate species identifications to support estimates of biodiversity, range extent and natural exposure of these planktonic calcifiers to environmental variability; furthermore, the apparent variation of the pteropods shell may have implications for our understanding of the species’ sensitivity to ocean acidification.     

Immunoenzymological Evidence Suggesting a Change in Conformation of Adenylic Acid Deaminase and Creatine Kinase during Substrate Combination

The kinetics of inhibition of 5′-adenylic acid deaminase and creatine-ATP transphosphorylase by their respective antibodies are studied and rate constants of combination are ascertained. It is shown that the single substrate 5′-adenylic acid (AMP) of deaminase “protects” the enzyme against antibody inhibition. However, phosphate, a competitive inhibitor of the highly specific deaminase, enhances combination with antibody. With creatine kinase, however, addition of either of the substrates, alone or in combination with the required magnesium, each of which separately bind to the enzyme, does not prevent inhibition of the enzyme by its antibody. However, the “working” enzyme combined with all substrates is “protected” against antibody inhibition.     

Subtle Population Genetic Structure in Yelloweye Rockfish (Sebastes ruberrimus) Is Consistent with a Major Oceanographic Division in British Columbia,Canada

The boundaries between oceanographic domains often function as dispersal barriers for many temperate marine species with a dispersive pelagic larval phase. Yelloweye rockfish (Sebastes ruberrimus, YR) are widely distributed across the northeastern Pacific Ocean, inhabiting coastal rocky reefs from the Aleutian Islands in Alaska through southern California. This species exhibits an extended pelagic larval duration and has the capacity for long distance larval transport. We assayed 2,862 YR individuals from 13 general areas in the northeast Pacific Ocean for allelic variation at nine microsatellite loci. Bayesian model-based clustering analyses grouped individuals from the Strait of Georgia (SG) into a distinct genetic cluster, while individuals from outer coastal water locations (OCLs) were partitioned equally across two genetic clusters, including the cluster associated with the SG fish. Pairwise F_ST values were consistently an order of magnitude higher for comparisons between the SG and OCLs than they were for all OCL-OCL comparisons (∼0.016 vs. ∼0.001). This same pattern was observed across two time points when individuals were binned into an “old” and “young” group according to birth year (old: ∼0.020 vs. 0.0003; young: ∼0.020 vs. ∼0.004). Additionally, mean allelic richness was markedly lower within the SG compared to the OCLs (8.00 vs. 10.54–11.77). These results indicate that the Strait of Georgia “deep-basin” estuary oceanographic domain acts as a dispersal barrier from the outer coastal waters via the Juan de Fuca Strait. Alternatively, selection against maladapted dispersers across this oceanographic transition may underlie the observed genetic differentiation between the Georgia basin and the outer coastal waters, and further work is needed to confirm the SG-OCL divide acts as a barrier to larval dispersal.     

Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum

The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l(-1) with a yield of 0.38-0.59 g g(-1) xylose and a reactor productivity up to 3.19 g l(-1)h(-1). However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.     

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.