Mitochondrial development in Trypanosoma brucei brucei transitional bloodstream forms.

Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F1F0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production.     

Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei

The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.     

Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits

The Trypanosoma brucei cytochrome c oxidase (respiratory complex IV) is a very divergent complex containing a surprisingly high number of trypanosomatid-specific subunits with unknown function. To gain insight into the functional organization of this large protein complex, the expression of three novel subunits (TbCOX VII, TbCOX X and TbCOX 6080) were down-regulated by RNA interference. We demonstrate that all three subunits are important for the proper function of complex IV and the growth of the procyclic stage of T. brucei. These phenotypes were manifested by the structural instability of the complex when these indispensible subunits were repressed. Furthermore, the impairment of cytochrome c oxidase resulted in other severe mitochondrial phenotypes, such as a decreased mitochondrial membrane potential, reduced ATP production via oxidative phoshorylation and redirection of oxygen consumption to the trypanosome-specific alternative oxidase, TAO. Interestingly, the inspected subunits revealed some disparate phenotypes, particularly regarding the activity of cytochrome c reductase (respiratory complex III). While the activity of complex III was down-regulated in RNAi induced cells for TbCOX X and TbCOX 6080, the TbCOX VII silenced cell line actually exhibited higher levels of complex III activity and elevated levels of ROS formation. This result suggests that the examined subunits may have different functional roles within complex IV of T. brucei, perhaps involving the ability to communicate between sequential enzymes in the respiratory chain. In summary, by characterizing the function of three hypothetical components of complex IV, we are able to assign these proteins as genuine and indispensable subunits of the procyclic T. brucei cytochrome c oxidase, an essential component of the respiratory chain in these evolutionary ancestral and medically important parasites.     

Expression of a Trypanosoma brucei brucei variant antigen in Escherichia coli

A cDNA library derived from antigenically homogeneous bloodstream stage Trypanosoma brucei brucei was screened with an antiserum directed against the variant surface antigen (VSA) using an enzyme-linked filter immunoassay. Several recombinant clones were detected and the clone giving the most intense reaction was further analyzed. It contained a VSA-specific cDNA insert and synthesized a protein of the expected molecular weight bearing VSA determinants. The nucleotide sequence of the insert was determined and shown to have the unusual codon bias characteristic of T. brucei VSAs, frequently employing codons specifying tRNAs rare in Escherichia coli. These results indicate that a codon bias very different from that of E. coli does not preclude the expression of a cloned sequence to detectable levels in this heterologous host.     

Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli.

Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei cleaves the glycosyl phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein (VSG) and other GPI structures. We have expressed this enzyme in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. We have purified large amounts of GPI-PLC from E. coli membranes, using a single step immunoaffinity technique. The expressed enzyme is identical to its trypanosome counterpart in enzymatic specificity, mobility on SDS-PAGE, and isoelectric point. Recombinant GPI-PLC is a membrane enzyme; it associates with E. coli membranes and, like the T. brucei GPI-PLC, partitions into the detergent phase in Triton X-114 phase separation experiments. The Michaelis constants for the two enzymes are similar (400 nM, with VSG as substrate). The turnover number (kcat, 72 min-1) of the recombinant enzyme (expressed from a. T. brucei rhodesiense WRATat 1.1 cDNA) is about one-tenth that of GPI-PLC from T. brucei brucei (ILTat 1.3).     

The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters.

Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the lambda phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the lambda PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5'-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the lambda PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the pho A promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.     

Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection

Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.     

Expression of a cloned lipopolysaccharide antigen from Neisseria gonorrhoeae on the surface of Escherichia coli K-12.

A gonococcal gene bank maintained in Escherichia coli K-12 was screened by colony immunoblotting, and a transformant expressing a surface antigen reactive to anti-gonococcal outer membrane antiserum was isolated. The isolate carried a recombinant plasmid, pTME6, consisting of approximately 9 kilobases of Neisseria gonorrhoeae DNA inserted into the BamHI site of pBR322. Surface labeling of E. coli HB101(pTME6) confirmed that the antigen was expressed on the E. coli cell surface. The antigenic material was resistant to proteinase K digestion and sensitive to periodate oxidation, indicating that the material was carbohydrate. Purified lipopolysaccharide (LPS) from HB101(pTME6) produced a unique band on silver-stained polyacrylamide gels that contained immunoreactive material as seen on Western blots of LPS samples. Only two of three E. coli LPS mutant strains carrying pTME6 reacted with the antigonococcal antiserum, suggesting that a certain E. coli core structure is necessary for antigen expression. We conclude that pTME6 contains one or more gonococcal genes encoding an LPS core biosynthetic enzyme(s) which can modify E. coli core LPS to produce a gonococcuslike epitope(s).     

Novel invasion determinant of enteropathogenic Escherichia coli plasmid pLV501 encodes the ability to invade intestinal epithelial cells and HEp-2 cells.

An Escherichia coli K-12 transformant carrying 96.5-kb plasmid pLV501 from enteropathogenic E. coli (EPEC) strain K798 is able to produce the same characteristic attaching-effacing lesions in a rabbit ileal biopsy explant model as its parent strain. Cloned EcoRI-SalI DNA restriction fragments from this plasmid failed to reproduce the attaching-effacing lesions, but one recombinant plasmid, pLV527, containing 4.5 kb of pLV501 DNA, conferred on E. coli DH1 transformants the ability to invade enterocytes in the rabbit explant model. DH1(pLV527) was also able to adhere to and invade HEp-2 cells. The relative invasive ability of DH1(pLV527) was quantified by recovery of internalized bacteria following gentamicin treatment of infected HEp-2 monolayers. DH1(pLV527) was 1,000-fold more invasive than DH1 carrying pBR322 or a recombinant plasmid which had no physiological effect on ileal biopsy explants but was less invasive than an enteroinvasive E. coli strain or a transformant carrying the cloned invasion genes of Shigella flexneri. Invasion by DH1(pLV501) could also be detected but occurred at a level 30 times lower than that by DH1(pLV527). Colony-hybridization of the pLV527 insert against a panel of 49 EPEC and related strains revealed that only 11 contained pLV527-hybridizing sequences; thus, the invasion determinant is not an essential component of the attachment-effacement pathogenic mechanism. One pLV527-hybridizing strain displayed both attachment-effacement and invasiveness in the rabbit ileal biopsy explant model. No significant hybridization was observed to non-EPEC invasive pathogenic enteric bacteria, indicating that the invasion determinant encoded on pLV527 is distinct from those used by these organisms.     

The trypanosome alternative oxidase exists as a monomer in Trypanosoma brucei mitochondria

The bloodstream forms of African trypanosomes solely depend on trypanosome alternative oxidase (TAO), for respiration. Similar to alternative oxidases (AOXs) found in plants and fungi, TAO is a membrane-bound diiron protein. Here, we investigated if TAO exists as a dimer like plant AOXs, or as a monomer like that of fungi. We have found that TAO forms a homo-dimer on a regular SDS-PAGE in the absence of any reducing agent and exists as a monomer under reducing condition. However, TAO does not form a dimer upon treatment of mitochondria with diamide. TAO was found as a higher molecular mass complex on a Blue-native gel after solubilization with digitonin. In the detergent soluble form, TAO activity was stimulated under reducing and inhibited under oxidizing condition. However, these conditions have no effect on the TAO activity in the mitochondria. Moreover, chemical cross-linking analysis revealed that TAO could not be cross-linked when present in the mitochondria. Together, it suggests that like certain other hydrophobic membrane proteins, TAO forms a dimer or oligomer when solubilized with detergents, and the TAO-dimer is SDS-resistant. However, it exists as a monomer in Trypanosoma brucei mitochondria.     

Cyanide-resistant respiration in Streptomyces citreofluorescens

The capacity of Streptomycetes to carry out cyanide-resistant respiration was investigated. With the model strain, Streptomyces citreofluorescens, it was shown that deprivation of glucose followed by transition from exponential to stationary growth was coupled with declining sensitivity of cellular respiration to cyanide ions. Cyanide-resistant oxidase is located within the cytoplasmic membrane. The occurrence of the cyanide-resistant oxidase did not correspond to qualitative changes of cytochrome spectrum. Cytochrome d is involved neither in cyanide-sensitive nor in cyanide-resistant rspiratory chain.     

The effect of down-regulation of mitochondrial RNA-binding proteins MRP1 and MRP2 on respiratory complexes in procyclic Trypanosoma brucei

MRP1 and MRP2 are multifunctional mitochondrial RNA-binding proteins with a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Silencing of MRP1 and/or MRP2 by RNA interference affected the assembly and functionality of respiratory complexes. The absence of several subunits of complexes I, III and IV resulted in their disintegration and subsequent decrease of specific activities and also caused a significant decrease of membrane potential. The overall respiration in the interfered cells decreased by only about 20%, since the trypanosome alternative oxidase effectively replaced the missing cytochromes and became the principal terminal oxidase.     

Evidence for a branched electron transport chain in Trypanosoma brucei

The flow of electrons the terminal oxidases present in the bloodstream and procyclic trypomastigotes of Trypanosoma brucei LUMP 1026 has been investigated by the use of salicylhydroxamic acid (SHAM) and cyanide. Respiration in bloodstream trypomastigotes was completely inhibited by 0.5 mM SHAM with a Ki below 10 microM. The Ki for SHAM in procyclic trypomastigotes was 70 microM. In procyclic trypomastigotes there are at least three terminal oxidases of which the two major ones are cytochrome aa3 oxidase, sensitive to cyanide inhibition, and alpha-glycerophosphate oxidase (GPO), sensitive to SHAM inhibition. These two oxidases contribute 60 and 30%, respectively, to total cell respiration. Inhibition of the cytochrome system with cyanide causes an increase in the flow of electrons through the GPO system, and inhibition of the GPO system with SHAM stimulates electron flow in the cytochrome system. Succinate oxidation in the mitochondrial fraction is partially inhibited by SHAM and this SHAM-sensitive respiration is not inhibited by antimycin A. The kinetic data of respiration by procyclic trypomastigotes fit a model proposed by Bahr and Bonner to determine the maximum rates of two competing electron transport pathways. It is concluded that the electron transport chain in T. brucei is branched.     

Insight into the mechanism of trypanosome lytic factor-1 killing of Trypanosoma brucei brucei.

It has been known for almost a century that normal human serum can lyse the extracellular blood parasite Trypanosoma brucei brucei. This process is a result of a non-immune killing factor in human sera known as trypanosome lytic factor (TLF). In this work, we demonstrate that killing of T. b. brucei by trypanosome lytic factor-1 (TLF-1) in vitro is inhibited by the lipophyllic iron chelator, LI, the lipophyllic antioxidant DPPD, and the protease inhibitors antipain and E64. Thus TLF-1 killing likely requires iron, oxidants, and serine and cysteine proteases. Furthermore, we demonstrate that TLF-1 mediated lysis causes measurable peroxidation in T. brucei lipids via a reaction that is inhibited by DPPD, weak bases, and human haptoglobin. We hypothesize that TLF-1 lysis requires intracellular factors within the trypanosome including high intracellular H2O2 and high polyenoic lipid concentrations, lysosomal acidification and proteases, and intracellular iron sources. The data presented supports the hypothesis that the combination of these factors with TLF-1 inside the lysosome results in lysosomal membrane breakdown, release of the lysosomal contents, and subsequent autodigestion of the cell.     

Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei

Mitochondrial protein translocation machinery in the kinetoplastid parasites, like Trypanosoma brucei, has been characterized poorly. In T. brucei genome database, one homolog for a protein translocator of mitochondrial inner membrane (Tim) has been found, which is closely related to Tim17 from other species. The T. brucei Tim17 (TbTim17) has a molecular mass 16.2kDa and it possesses four characteristic transmembrane domains. The protein is localized in the mitochondrial inner membrane. The level of TbTim17 protein is 6-7-fold higher in the procyclic form that has a fully active mitochondrion, than in the mammalian bloodstream form of T. brucei, where many of the mitochondrial activities are suppressed. Knockdown of TbTim17 expression by RNAi caused a cessation of cell growth in the procyclic form and reduced growth rate in the bloodstream form. Depletion of TbTim17 decreased mitochondrial membrane potential more in the procyclic than bloodstream form. However, TbTim17 knockdown reduced the expression level of several nuclear encoded mitochondrial proteins in both the forms. Furthermore, import of presequence containing nuclear encoded mitochondrial proteins was significantly reduced in TbTim17 depleted mitochondria of the procyclic as well as the bloodstream form, confirming that TbTim17 is critical for mitochondrial protein import in both developmental forms. Together, these show that TbTim17 is the translocator of nuclear encoded mitochondrial proteins and its expression is regulated according to mitochondrial activities in T. brucei.     

A potential hexose transporter gene expressed predominantly in the bloodstream form of Trypanosoma brucei.

A cDNA cloned from Trypanosoma brucei brucei codes for a putative membrane protein which is homologous to the erythrocyte glucose transporter and several other sugar transporters from Escherichia coli, yeast, algae and Leishmania. This cDNA hybridizes to a 2.3-kb mRNA that accumulates to a much higher degree in the bloodstream mammalian form than in the procyclic insect form of the parasite. The correlation between the expression of this gene and the hexose metabolism of Leishmania enriettii and T. brucei suggest that these 2 related genes probably encode hexose transporters. The gene encoding this mRNA is a member of a multigene family. The putative hexose transporter gene is highly conserved among Kinetoplastidae, indicating an important role for this protein in the parasite life cycle.     

Esters of 3,4-dihydroxybenzoic acid, highly effective inhibitors of the sn-glycerol-3-phosphate oxidase of Trypanosoma brucei brucei

Alkyl esters of 3,4-dihydroxybenzoic acid are inhibitors of the sn-glycerol-3-phosphate oxidase system of Trypanosoma brucei brucei in vitro and have significant trypanocidal activity in vivo when combined with glycerol. While the parent acid has little inhibitory activity in vitro, the esters are highly active with activity increasing as the chain length of the esterifying alcohol increases. The n-dodecyl ester was more than 400 times as active as salicylhydroxamic acid and 15 times more active than the corresponding p-n-alkyloxybenzhydroxamic acid, one of the most active sn-glycerol-3-phosphate oxidase inhibitors previously reported. When combined with glycerol (to block an alternative pathway of glycolysis) and tested in vitro against intact parasites, this ester was 100 times more effective than salicylhydroxamic acid and 10 times more effective than p-n-dodecyloxybenzhydroxamic acid. It was also active against T. b. brucei in mice when combined with glycerol whereas the latter compound was not. Esters of 3,4,5-trihydroxybenzoic acid (gallic acid) were also highly active while those of 2,3-dihydroxybenzoic acid were much less inhibitory and those of 2,5-dihydroxybenzoic acid were inactive. A related compound, 2',4',5'-trihydroxybutyrophenone, was also active as predicted by its structure but was too toxic to be of interest as a drug candidate.