Curtis L. Patton

Pentamidine transport in Trypanosoma brucei—Kinetics and specificity

Abstract Pentamidine, an aromatic diamidine used in the treatment and control of African trypanosomiases, is rapidly absorbed by Trypanosoma brucei and is lethal to the parasite both in vitro and in vivo . The pentamidine transport system in T. brucei exhibits saturation kinetics and shows specificity for the aromatic amidine moiety of this compound. The inhibition of uptake by salicyl hydroxamic acid (SHAM) and iodoacetate (IAA), and the retention of intracellular substrate against large chemical gradients indicate a coupling of energy to this system. The system shows high affinity for pentamidine with an average K m value of 2.68 μM. Structural specificity in relationship to varying drug affinities for this transport site and the role it might play in drug resistance are discussed.

African trypanosomes contain calmodulin which is distinct from host calmodulin.

Studies were initiated to determine whether African trypanosomes utilize Ca2+ fluxes to coordinate complex morphological and biochemical life cycle changes. We have identified the ubiquitous intracellular Ca2+ receptor, calmodulin, in two developmental stages of Trypanosoma brucei rhodesiense. The transition from rapidly dividing, slender bloodstream trypomastigotes to slow growing procyclics in axenic culture was accompanied by changes in specific calmodulin content (3 micrograms/mg cell protein to 1 microgram/mg cell protein, respectively) and a shift in intracellular calmodulin distribution, Trypanosome calmodulin is physically and functionally distinct from that of host tissues, including bovine brain and rat erythrocytes. It is similar to but distinct from Tetrahymena calmodulin. Comparisons among these proteins isolated from the four sources were made using the following criteria: (1) mobility on sodium dodecyl sulfate discontinuous polyacrylamide gels; (2) Ca2+-induced conformational changes; (3) CNBr-cleavage fragments; (4) activation of bovine brain cyclic nucleotide phosphodiesterase in both a Ca2+-dependent and calmodulin-dependent manner; (5) activation of human erythrocyte (Ca2+ + Mg2+)-ATPase; and (6) inhibition of calmodulin activity by trifluoperazine and penfluridol. Trifluoperazine but not trifluoperazine sulfoxide was cytotoxic to trypanosomes in vitro. Half maximal effect occurred at 15 microM. We conclude that calmodulin is a functional component of Africal trypanosomes and suggest that it plays an important role in mediating the host-parasite relationship.

Trypanosomatid hydrogen peroxidase metabolism

The rate of whole cell H2O2 metabolism in several salivarian and stercorarian trypanosomes and Leishmania species was measured. These cells metabolized H2O2 at rates between 2.3 and 48.2 nmol/108 cells per min depending upon the species employed. H2O2 metabolism was largely insensitive to NaN3, implying that typical catalase and peroxidase haemoproteins are not important in H2O2 metabolism. The metabolism of H2O2, however, was almost completely inhibited by N‐ethylmaleimide. In representative species, H2O2 metabolism was shown to occur through a trypanothione‐dependent mechanism.

Trypanosoma brucei brucei: Isolation of the major surface coat glycoprotein by lectin affinity chromatography

Abstract We have purified soluble glycoproteins associated with Trypanosoma brucei brucei by lentil lectin affinity chromatography. The major surface coat glycoprotein (SCGp) accounts for 87% of the protein in the preparation with approximately 12 other glycoproteins accounting for the rest. This preparation can be further subdivided by DEAE chromatography. The nonabsorbed protein fraction is 95% SCGp and 1 to 2% each of three other glycoproteins. Elution of the DEAE column with a 0.05-0.5 M NaCl gradient yields a broad peak containing the remaining glycoproteins. The purification of the SCGp is 16- and 17.3-fold at each step out of a theoretical 18-fold possible purification. The preparation after lectin chromatography is stable, showing no degradation after standing 72 hr at 4 C. Additional purification procedures are discussed.

Evaluation of rhodamine 123 as a probe for monitoring mitochondrial function in Trypanosoma brucei spp.

ABSTRACT. Rhodamine 123, a membrane potential‐specific dye, has been evaluated as a probe to monitor the function of the mitochondrion in long slender bloodstream and procyclic trypomastigotes of several Trypanosoma brucei spp. By epifluorescence microscopy, mitochondrial development has been followed in long slender bloodstream and procyclic organisms stained with rhodamine 123. to photograph stained long slender bloodstream forms, it was necessary to develop a method to completely immobilize viable organisms. In both parasite forms, as the cell cycle progressed, the mitochondrion developed from a thread‐like structure to a highly branched organelle. A dramatic reorganization occurred preceding cytokinesis to produce two progeny thread‐like structures which were partitioned into newly formed daughter cells. the organelle within the long slender trypomastigote was found to stain optimally at 0.3 μ/ml of rhodamine 123, while the procyclic form required 3.0 μ/ml. the results suggest that the plasma membrane potential is higher in the long slender parasite than in the procyclic form. the effects of inhibitors that disrupt mitochondrial function were examined in long slender and procyclic parasites, and some of these agents were shown to affect rhodamine 123 accumulation and retention. In long slender trypomastigotes the trypanosome alternative oxidase does not appear to be coupled to proton pumping, whereas in procyclic organisms the effects of inhibitors indicate that this oxidase may be coupled to a pathway that is branched preceding an antimycin A1‐sensitive site.

Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei.

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, (Na+ +K+)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in (3H-mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated Mr = 58000), there are several minor surface glycopeptides (Mr = 76000, 86000 and 92000-100000) which are apparent extrinsic membrane components, and two surface glycopeptides (Mr = 42000 and 130000) which are intrinsic membrane components.

Detection and quantification of variant specific antigen in the plasma of rats and mice infected with Trypanosoma brucei brucei.

Variant specific antigen (VSA), the principal constituent of the surface coat of salivarian trypanosomes, was detected by gel immunoassays in the plasma of rats and mice infected with Trypanosoma brucei brucei. The quantity of VSA in plasma was measured in radial immunodiffusion tests using a monospecific antiserum and purified VSA as a standard. During the first peak of parasitemia, a statistically significant, linear relationship was determined between the number of parasites in the blood (in the range between 4 x 10(8) and 10(9)/ml) and the concentration of VSA in the plasma (28-320 microgram/ml). The VSA from parasites of the first peak was lost within 2 days of remission. Variant antigens of parasites constituting the second peak then began to appear in the plasma of infected rats. All plasma samples had been separated from parasites and blood cells within 15 min of blood collection. The pH of plasma was controlled with a buffered anticoagulant. No soluble parasite antigens, other than VSA, were detected in the plasma of infected hosts. The results of this study extend the observation that salivarian trypanosomes shed surface coat material during the course of infection. Thus, sloughed VSA may be the parasite product that has been hypothesized to cause the nonspecific lymphocyte proliferation, immunosuppression, and/or hypergammaglobulinemia which occur during African trypanosomiasis.

Trypanosoma congolense: surface glycoproteins of two early bloodstream variants. II. Purification and partial chemical characterization.

Abstract Two sequential variant-specific glycoproteins have been purified from two variants of Trypanosoma congolense expressed during a relapsing infection. Isolation of the two glycoproteins, termed VSG-1 and VSG-2, respectively, employed glycerol lysis followed by purification on concanavalin A, Sephadex G-25, and gradient-eluted DE-52 columns. Partially purified VSG proteins were immunologically cross-reactive, but highly purified VSGs showed no cross-reactivity under the conditions employed. Both VSG-1 and VSG-2 consisted of a triplet of polypeptides. Although each member of a triplet subset could be distinguished by isoelectric focusing, all three gave identical N-terminal amino acid sequences and nearly identical tryptic peptide maps. The members of the VSG-1 polypeptide subset differed from those of the VSG-2 subset both with regard to N-terminal amino acid sequence and in tryptic peptide map patterns. Comparison of N-terminal sequences of VSG-1 and VSG-2 did, however, show that the sequences could be aligned to give a modest degree of amino acid homology (27%). This alignment also produced a minimum in the number of two-base changes, suggesting that the observed homology is not a coincidence and that these two proteins may well have arisen by gene duplication followed by retention of multiple point mutations.

Trypanosoma brucei: Nearest neighbor analysis on the major variable surface coat glycoprotein—Crosslinking patterns with intact cells

Abstract Cleavable Crosslinking reagents were used to study interactions among proteins of the surface coat of Trypanosoma brucei . The proteins were resolved by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When intact cells were treated with dithio bis (succinimidylpropionate), we obtained extensive intermolecular Crosslinking of major variable surface coat glycoprotein (VSCG) molecules. This reagent generated no apparent crosslinks between VSCG and other membrane-associated proteins. Complete conversion to oligomers equal to or greater than octamers occurred within 20 min. When purified VSCG in solution was treated with dithio bis (succimidylpropionate), dimers were found. A complex of Cu 2+ and 1,10-phenanthroline was used to catalyze air oxidation of adjacent sulfhydryls to disulfide bonds; however, no crosslinking among VSCG molecules nor between VSCG and other proteins was observed. The results presented indicate that VSCG in solution exists predominately in the form of dimers. Whether VSCG in situ also occurred as dimers could not be determined; however, since we observed trimeric and tetrameric forms of VSCG when untreated cells were analyzed, it is likely that weak interactions occur among the protein molecules. These interactions are less stable than the dimer association observed with purified VSCG. Finally, the analysis indicated that VSCGs of this stock of T. brucei , derived from UGANDA/ 60/TREU/164[ETat3], contained at least one intramolecular disulfide bond. We examined T. brucei stocks 427 and EATRO 110 and obtained similar results. Thus, it appears that intramolecular disulfide bonding is a general feature of T. brucei VSCGs.

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: I. Production of a relapsing infection in rodents

Abstract A strain of Trypanosoma congolense has been cloned, passaged through the tsetse fly, and subsequently recloned. Relapsing infections have been induced in two rats by syringe passage of the cloned trypanosomes. The variant-specific glycoprotein of the initial cloned variant (VSG-1) and those from the two different variants produced in the two relapsing infections (VSG-2 and VSG-3) may be distinguished from each other by their isoelectric-focusing patterns. In this experimental system, cloned T. congolense , like Trypanosoma brucei , undergoes antigenic variation; the conversion of the VSG-1 into the VSG-2 isoelectric-focusing spectrotype was followed. These VSGs may be the products of sequentially expressed genes.