Regulation of the Intracellular pH in the Protozoen Parasite Trypanosoma brucei brucei

126

Vacuolar proton pyrophosphatases (V-H؉ -PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H ؉ -PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H ؉ -PPase gene by using double-stranded RNA interference methodology to produce inducible V-H ؉ -PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H ؉ -PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 ؋ 10 5 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.Intracellular acidic vacuoles containing polyphosphate (polyP), 1 initially called volutin or polyP bodies, have been described in bacteria, algae, yeast, and protozoa (1). In trypanosomatids, these polyP vacuoles were called acidocalcisomes (2) and shown to be electron dense and contain large concentrations of PP i , calcium, magnesium, and other elements (3). Similar organelles have been identified in apicomplexan parasites (4, 5) as well as in the green algae, Chlamydomonas reinhardtii (6) and the slime mold, Dictyostelium discoideum (7). Acidocalcisomes were postulated to play an important role in the regulation of both cytosolic Ca 2ϩ concentration and intracellular pH (pH i ). For example, polyP hydrolysis (8) and activation of the Na ϩ /H ϩ antiporter (9, 10) were postulated to protect the cells against alkaline pH stress and increase the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ). In addition, these organelles possess a vacuolar-type H ϩ -translocating pyrophosphatase (V-H ϩ -PPase) (11), which is an electrogenic proton pump initially discovered in photosynthetic bacteria and plants (12). It has been shown to be associated with the plasma membrane or vacuoles in plants and with the chromatophore membranes of Rhodospirullum rubrum. The biochemical function of this enzyme in plants and unicellular eukaryotes is to couple hydrolysis of the high energy phosphate bond of PP i with H ϩ translocation from the cytosol to acidify the plant vacuole (tonoplast) or the acidocalcisome, respectively (10, 13). Working on isolated acidocalcisomes, Rodrigues et al. (14) demonstrated that the Trypanosoma brucei H ϩ -PPase was able to generate a membrane potential by PP i -dependent proton uptake. Moreover, Scott et al. (11,15) showed tha...

Section: Ca 2ϩ and Ph I Homeostasis In The Induced Pfivp1supporting

confidence: 85%

A Vacuolar-type H+-Pyrophosphatase Governs Maintenance of Functional Acidocalcisomes and Growth of the Insect and Mammalian Forms of Trypanosoma brucei

Lemercier¹,

Dutoya²,

Luo³

et al. 2002

126

“…The pK values of T. brucei thioredoxin, tryparedoxin, and trypanothione coincide with the intracellular pH of the parasites (46), which may contribute to their reactivity. As outlined by Gilbert (47), the second-order rate constants for thiol-disulfide exchange reactions exhibit an optimum when the thiol pK is equal to the surrounding pH.…”

Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othermentioning

confidence: 94%

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schmidt

Krauth‐Siegel

2003

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses a single thioredoxin that has an unusually high pI value of 8.5 and lacks a conserved aspartyl residue claimed to be involved in catalysis in other thioredoxins. Despite these peculiarities, T. brucei thioredoxin behaves like classical thioredoxins. It is reduced by thioredoxin reductases from different species, serves as donor of reducing equivalents for the ribonucleotide reductase of the parasite, and catalyzes the reduction of protein disulfides. The redox potential of ؊267 mV was obtained from protein-protein redox equilibration with Escherichia coli thioredoxin. The pK value of T. brucei thioredoxin was determined by two different methods. Carboxamidomethylation of the reduced protein yielded a pK value of 7.4 and generated mono-alkylated protein. The thiolate absorption at 240 nm resulted in a pK of 7.6 and, based on the extinction coefficient of 11.6 mM ؊1 cm ؊1 , there are two (or three) cysteines titrating with very similar pK values. A thioredoxin reductase has not yet been detected in any organism of the order Kinetoplastida. T. brucei thioredoxin is spontaneously reduced by trypanothione (bis(glutathionyl)spermidine). Obviously, a specific thioredoxin reductase is not required as thioredoxin reduction can be conducted by the parasite-specific trypanothione/ trypanothione reductase system. Trypanosomatids such as Trypanosoma brucei, the causative agent of African sleeping sickness, have a unique thiol metabolism in which the ubiquitous glutathione/glutathione reductase couple is replaced by trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine), and the flavoenzyme trypanothione reductase (1, 2). The parasite dithiol is much more reactive than glutathione. It is a spontaneous reductant of dehydroascorbate as well as the disulfide forms of glutathione and ovothiol (2). It reduces tryparedoxin, a 16-kDa dithiol protein with a CPPC active site motif, which distantly belongs to the thioredoxin protein family (3). The trypanothione/tryparedoxin couple is the donor of reducing equivalents for the detoxication of hydroperoxides catalyzed by a cascade of proteins that are composed of trypanothione reductase, trypanothione, tryparedoxin, and a peroxiredoxin-type tryparedoxin peroxidase. Tryparedoxin is also involved in the reduction of ribonucleotide reductase (4) and glutathione peroxidase-like parasite peroxidases (5, 6). The trypanothione metabolism is essential for the parasite. Down-regulation of trypanothione reductase in bloodstream T. brucei by more than 90% results in growth arrest and hypersensitivity toward hydrogen peroxide as well as the loss of infectivity in a mouse model (7).Besides tryparedoxin, T. brucei possesses a single classical thioredoxin with a M r of 12,000 and the conserved WCGPC catalytic site (8). The parasite protein is unusual in having a calculated pI value of 8.5 instead of the acidic pI found for most thioredoxins and a conserved functional aspartate (Asp-26 in Escherichia coli thioredoxin) (9, 10)...

“…However, colocalization studies performed with antibodies directed against p67 (43) and the T. brucei plant vacuolar-like proton pyrophosphatase (V-H ϩ -PPase) 2 did not show an association of TbKIFC1 with either lysosomes or acidocalcisomes (data not shown). Moreover, p67 containing vesicles and acidocalcisomes did not respond to similar alkaline treatment.…”

Section: Differential Expression and Localization Of Tbkifc1 Kinesin mentioning

confidence: 99%

“…The [Ca 2ϩ ] i and pH i were measured according to Scott et al (38) and Fraser-L'Hostis et al (2). Briefly, the cells were loaded with either fura2/AM or BCECF/AM (Molecular Probes) and resuspended in buffer A containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO 4 , 5.5 mM glucose, 50 mM Hepes, pH 7.4, and 1 mM EGTA for [Ca 2ϩ ] i determination.…”

Section: Spectrofluorometric Determinationsmentioning

confidence: 99%

“…To survive and develop in the distinctive environment of the mammalian host and the insect vector, Trypanosoma brucei must undergo morphological, biochemical, and physiological changes to adapt to the different environmental conditions. Cell viability requires mechanisms to control pH and Ca 2ϩ homeostasis (1,2). The level of endocytosis demonstrated by bloodstream forms (BF) 1 is among the highest described so far for eukaryotic cells (3).…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Novel C-terminal Kinesin Is Essential for Maintaining Functional Acidocalcisomes in Trypanosoma brucei

Dutoya¹,

Gibert²,

Lemercier³

et al. 2001

Kinesins are cytoskeletal motor proteins that play roles in a variety of fundamental cellular processes including cell division and the anterograde transport of vesicles and organelles. We purified, cloned, and functionally characterized in Trypanosoma brucei a new member of the C-terminal kinesin family, TbKIFC1. Kinetic constants of the recombinant motor domain of TbKIFC1 were estimated at 0.56 M for the microtubule dissociation constant (K d ) with a k cat of 0.2 s ؊1 . Immunolocalization analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH 4 Cl treatment, these structures were considered to be associated with acidic vesicles. To determine the role of the kinesin in vivo, we produced an inducible kinesin-deficient strain by double-stranded RNA interference methodology.