Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei

Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Flagellar function in this organism is critical for life cycle progression and pathogenesis, however the regulation of flagellar motility is not well understood. The flagellar axoneme produces a complex beat through the precisely coordinated firing of many proteins, including multiple dynein motors. These motors are found in the inner arm and outer arm complexes. We are studying one of the inner arm dynein motors in the T. brucei flagellum: dynein-f. RNAi knockdown of genes for two components of dynein-f: DNAH10, the a heavy chain, and IC138, an intermediate chain, cause severe motility defects including immotility. To determine if motility defects result from structural disruption of the axoneme, we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy (TEM). Our analysis showed that inner arm dynein size, axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures. These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.

Observations on estrus as monitored by receptivity to mating activity and pattern of vaginal exfoliates in trypanosoma brucei ­ infected WAD goat does synchronized with Estrumate® / Observaciones sobre el estro monitorizado por la receptividad a la actividad de apareamiento y el patrón de exfoliación vaginal en cabras WAD infectadas por trypanosoma brucei sincronizadas con Estrumate®

The effects of experimental Trypanosoma brucei infection on receptivity to mating activity and pattern of vaginal exfoliates were monitored using twenty-one adult WAD goats which were synchronized with double injection, seven days apart of Estrumate®. The twenty-one goats consisted of 3 bucks and 18 does. The does were randomly divided into control group 'A' having 10 does and test group 'B' with 8 does. The goats were fed with Elephant grass in the morning and commercial feed containing 15.23 percent CP at the rate of 0.25kg/head in the afternoons. Freshwater was provided ad libitum. Results showed that while all the control does were observed to stand to be mounted and mated, none of the infected does did. Also, the pattern of the mean percentage vaginal exfoliated cell types encountered between the control and infected doe groups were converse. While parasabal cells changed from 2.90±0.03 percent during proestrus through 3.05 +/- 0.46 percent during estrus to 2.42 +/-0.08 percent at diestrus in the control does, it changed from 22.07 +/- 0.56 percent during expected proestrus through 8.48 +/- 0.05 percent during expected estrus to 28.05 +/-1.09 percent respectively in the infected does. In like manner, intermediate cell changed from 11.10 +/- 0.03 percent during proestrus through 11.10 +/- 0.31 percent during estrus to 1.21 +/- 1.00 percent during diestrus in control does while it changed from 27.27 +/- 0.08 percent during expected proestrus through 42.37 +/- 2.39 percent during expected estrus to 40.24 +/- 1.06 percent during expected diestrus in infected does. Similarly, superficial cells changed from 56.25 +/- 0.75 percent during proestrus through 63.70 +/- 1.05 percent during estrus to 7.37 +/- 0.01 percent during diestrus while it changed from 0.00 percent during expected proestrus through 3.39 +/- 0.02 percent during expected estrus to 63.70 +/- 1.05 percent during estrus to 6.10 +/- 0.01 percent during expected diestrus. In the control does, the ...

Los efectos de la infección experimental por Trypanosoma brucei sobre la receptividad a la actividad de apareamiento y el patrón de exfoliación vaginal fueron monitoreados utilizando 21 cabras WAD adultas sincronizadas con doble inyección, a los siete días de diferencia de Estrumate®. De las 21 cabras utilizadas eran 3 machos y 18 hembras. Las hembras se dividieron al azar en grupo control "A" con 10 sujetos y un grupo de prueba "B" con 8. Las cabras fueron alimentadas con pasto y alimento comercial que contenía 15,23 por ciento de CP en tasa de 0,25kg/por cabeza en las tardes. Agua fresca fue proporcionada ad libitum. Los resultados mostraron que mientras todos las cabras del grupo control pudieron ser montadas y acopladas, ninguna de las infectadas pudo. Además, fue contradictorio el patrón de la media porcentual de los tipos de células vaginales exfoliadas encontradas entre los grupo control e infectadas. Mientras que las células parabasales cambiaron desde un 2,90 +/- 0,03 por ciento durante el proestro, al 3,05 +/- 0,46 por ciento durante el estro y 2,42 +/- 0,08 por ciento al diestro en el grupo control, el grupo infectado cambió desde un 22,07 +/- 0,56 por ciento durante el proestro, al 8,48 +/- 0,05 por ciento durante el estro y 28,05 +/- 1,09 por ciento al diestro. De la misma forma, las célula intermedias cambiaron de un 11,10 +/- 0,03 por ciento durante el proestro, al 11,10 +/- 0,31 por ciento durante el estro y al 1,21 +/- 1,00 por ciento durante el diestro en el grupo control, mientras que en el grupo infectado pasó del 27,27 +/- 0,08 por ciento durante el proestro, al 42,37 +/- 2,39 por ciento durante el estro y al 40,24 +/- 1,06 por ciento durante el diestro. Las células superficiales pasaron desde un 56,25 +/- 0,75 por ciento durante el proestro, 63,70 +/- 1,05 por ciento durante el estro, hasta un 7,37 +/- 0,01 por ciento durante el diestro, mientras en el grupo infectado pasaron de un 0.00 por ciento durante el proestro, al 3,9 +/- 0,02 p...

Myristate exchange in glycolipid A and VSG of African trypanosomes

The variant surface glycoprotein (VSG) of T. brucei is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor which is unique in that its fatty acids are exclusively myristate (a fourteen carbon saturated fatty acid). We showed that the myristate is added to the GPI precursor in a remodeling reaction involving deacylation and reacylation. We now demonstrate that trypanosomes have a second pathway of myristoylation for GPI anchors that we call "myristate exchange" which is distinct from the fatty acid remodeling pathway. We propose that this is an exchange of [3H]myristate into both sn-1 and sn-2 positions of glycolipid A, which already contains myristate, and have demonstrated this using inhibitors and a variety of other methods. We have partially characterized myristate exchange with respect to specificity and susceptibility to some inhibitors. The apparent Km for myristoyl CoA is 7 nM. This myristate-specific process may represent a proof-reading system to ensure that the fatty acids on VSG are exclusively myristate. Although myristate exchange was first discovered for glycolipid A, we now believe that VSG is the true substrate of this reaction. VSG is efficiently labeled by exchange in the presence of cycloheximide, which prevents anchoring of newly synthesized protein. Although its location is not yet know, we have evidence that exchange does not localize to either the endoplasmic reticulum or the plasma membrane. We will present data indicating that surface VSG may be internalized and undergo myristate exchange

The role of glycolipid C in the GPI biosynthetic pathway in Trypanosoma brucei bloodstream forms

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in Trypanosoma brucei bloodstream forms includes the formation of glycolipid C. This molecule is the inositol-acylated form of the GPI anchor precursor, glycolipid A. There is no evidence for the transfer of glycolipid C to protein in vivo and the role of glycolipid C is unclear. In this paper we show that glycolipid C is not synthesised in the presence of phenylmethylsulphonyl fluoride (PMSF) and that glycolipid C is not an obligatory intermediate on the pathway to the formation of glycolipid A. Using pulse-chase experiments we show that glycolipid A and glycolipid C are in a dynamic equilibrium and we suggest that only the forward reaction (glycolipid A conversion to glycolipid C) is inhibited by PMSF

Metabolism of GPIs in mammalian cells

In Trypanosoma brucei, glycosylphosphatidylinositol (GPI) anchors of proteins and free GPIs with identical structures have been characterized. This identity provides strong presumptive evidence that the free GPIs are in fact precursors of the GPI anchors on proteins. In mammalian tissues, however, rather consistent differences in the structures of free GPIs and GPI anchors are observed. The terminal GPIs produced by the mammalian biosynthetic pathway differ from GPI anchors in being almost exclusively fatty acid acylated on the inositol residue, having a greater number of phosphoethanolamine residues, and perhaps in containing a greater percentage of diacylglycerol components. While in principle these differences could be reconciled by remodeling reactions before or after attachment of GPI anchors, it is possible that some of the mammalian free GPIs play cellular roles other than as anchor precursors. We have approached this question by studying the lifetimes of the last three GPIs on the biosynthetic pathway, denoted H6, H7 and H8, in K562 cells and in K562 mutant designated class K that is devoid of GPI-anchored proteins. Pulse-chase metabolic labeling with [3H]-mannose indicated that H6 was a precursor of H7 and H8 and that the H8 lifetime was more than one hour in the parental cells and even longer in the mutant. Preliminary data indicated that the majority of each of the three GPIs was localized in the plasma membrane fraction rather than the endoplasmic reticulum. These observations argue that mammalian GPIs are not utilized exclusively as GPI anchor precursors

Proteins anchored via glycosylphosphatidylinositol and solubilizing phospholipases in Trypanosoma cruzi

The presence of GPI anchors and phospholipases capable of solubilizing them in Trypanosoma cruzi has been investigated in epimastigotes, metacyclic trypomastigotes from axenic cultures and tissue culture trypomastigotes. The GPI anchored proteins in epimastigote forms are scarce when compared to their abundance in the parasite forms which can infect mammals, and GPI-solubilizing phospholipases C have been found in all life cycles stages. In epimastigote and metacyclic forms, the activity is found in the soluble fraction upon cell lysis, whereas in tissue cultured trypomastigotes it is membrane bound and, being mostly sensitive to p-chloromercuriphenylsulfonate, resembles closely the GPI specific phospholipase of Trypanosoma brucei. Sequential immunoprecipitations with monoclonal antibodies and anti-CRD indicated the presence of several sub-populations among the surface proteins of metacyclic trypomastigotes, five of these belonging to the GPI-anchored 90 kD family. Among this family, the epitopes recognized by MAb-1G7 are present in three members, one of them also expressing the 3F6 epitope. There are 2 members recognized only by MAb-3F6 but not by MAb-1G7, one of them being probably galactosylated on the GPI since it can be immunoprecipitated by anti-CRD. Very strangely, the epitope recognized by the MAb-WIC29.26 was always present on the gp72, as originally described, but under certain circumstances appeared cryptic on one of the 90 kD species. During epimastigote transformation into metacyclic trypomastigotes in vitro, the ability of the GPI of the 1G7-antigen to be solubilized by phospholipase C and D varies depending on the age of the culture and presence or absence of fetal calf serum. Different patterns of solubilization were also obtained for 1G7-Ag, depending on whether the test is performed with parasite lysates or with antigen affinity purified from them. Our data indicate that the phospholipase C resistance observed does not arise from acylation on the inositol, as previously described for acetylcholinesterase from human erythrocytes, being rather due to factors which either modify the GPI or affect the action of the phospholipases...