Social Motility in African Trypanosomes

African trypanosomes are devastating human and animal pathogens that cause significant human mortality and limit economic development in sub-Saharan Africa. Studies of trypanosome biology generally consider these protozoan parasites as individual cells in suspension cultures or in animal models of infection. Here we report that the procyclic form of the African trypanosome Trypanosoma brucei engages in social behavior when cultivated on semisolid agarose surfaces. This behavior is characterized by trypanosomes assembling into multicellular communities that engage in polarized migrations across the agarose surface and cooperate to divert their movements in response to external signals. These cooperative movements are flagellum-mediated, since they do not occur in trypanin knockdown parasites that lack normal flagellum motility. We term this behavior social motility based on features shared with social motility and other types of surface-induced social behavior in bacteria. Social motility represents a novel and unexpected aspect of trypanosome biology and offers new paradigms for considering host-parasite interactions.     

Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.     

Social Motility of African Trypanosomes Is a Property of a Distinct Life-Cycle Stage That Occurs Early in Tsetse Fly Transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen—designated as early procyclic forms—express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4–7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.     

Dihydroxyacetone Induced Autophagy in African Trypanosomes

Dihydroxyacetone (DHA) was examined to explore its trypanocidal activity. The compound is easily taken up by trypanosomes via its aquaglyceroporins but is not converted to a glycolytic intermediate due to the lack of a respective kinase. Investigating the DHA-induced cell death it became evident that parasites die by autophagy rather than by necrosis or apoptosis. Since autophagy is not well studied in African trypanosomes our work offers a way to investigate the importance of autophagy for trypanosomes not only for stress coping but also for organelle reconstruction during differentiation.

Antiproliferative Effect of Dihydroxyacetone on Trypanosoma brucei Bloodstream Forms: Cell Cycle Progression, Subcellular Alterations and Cell Death

N.L. Uzcátegui, D. Carmona-Gutiérrez, V. Denninger, C. Schoenfeld, F. Lang, K. Figarella and M. Duszenko

Antimicrob Agents Chemother 2007; In press     

Roles of Adenylate Cyclases in Ciliary Responses of Paramecium to Mechanical Stimulation

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K⁺‐outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K⁺ channels in the membrane. Using gene‐specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus‐induced hyperpolarization‐coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)‐gene and 2 (ac2)‐gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8‐Br‐cAMP and dilution of extracellular [K⁺] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization‐activated inward K⁺ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus‐induced acceleration of ciliary beat in Paramecium.     

Adenylate Cyclases in Vertebrate Retina: Enzymatic Characteristics in Normal and Dystrophic Mouse Retina

Adenylate cyclase activity and the effects of various activators and inhibitors of this enzyme were measured in retinas from normal mice (C57BL/6J) and congenic animals with photoreceptor dystrophy. In normal retina, approximately 250 microM-ATP was required for half-maximal stimulation of the enzyme. Activity was supported by Mg2+ and Mn2+, but Ca2+ was ineffective. The enzyme was inhibited by EGTA and stimulated by 5'-guanylylimidodiphosphate (GPP(NH)P), dopamine, and NaF. The stimulatory effects of GPP(NH)P and dopamine were greater in the presence of EGTA. Examination of microdissected normal retinas revealed that the inner (neural) retina had adenylate cyclase activity four times that of the photoreceptor cell layers, and that EGTA inhibited activity in the inner retina, but had no effect in the outer retina. In dystrophic retinas basal enzyme activity was 60% higher than that in normal retina. The enzyme in this tissue was stimulated by EGTA, GPP(NH)P, and dopamine, and their effects were additive. These results indicate that adenylate cyclase activity in vertebrate retina is under complex regulation by substrate, divalent cations, guanine nucleotides, dopamine, and perhaps calmodulin. In addition, the data demonstrate that adenylate cyclase is not evenly distributed in the retina and that it is regulated differently in the inner and outer retina. Finally, the present results indicate that regulation of this enzyme in dystrophic retina may be qualitatively and quantitatively different from that in normal retina.     

Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention.     

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development.     

Evidence for Recycling of Invariant Surface Transmembrane Domain Proteins in African Trypanosomes

Intracellular trafficking is a vital component of both virulence mechanisms and drug interactions in Trypanosoma brucei, the causative agent of human African trypanosomiasis and n''agana of cattle. Both maintaining the surface proteome composition within a life stage and remodeling the composition when progressing between life stages are important features of immune evasion and development for trypanosomes. Our recent work implicates the abundant transmembrane invariant surface glycoproteins (ISGs) in the uptake of first-line therapeutic suramin, suggesting a potential therapeutic route into the cell. RME-8 is a mediator of recycling pathways in higher eukaryotes and is one of a small cohort of intracellular transport gene products upregulated in mammal-infective trypanosomes, suggesting a role in controlling the copy number of surface proteins in trypanosomes. Here we investigate RME-8 function and its contribution to intracellular trafficking and stability of ISGs. RME-8 is a highly conserved protein and is broadly distributed across multiple endocytic compartments. By knockdown we find that RME-8 is essential and mediates delivery of endocytic probes to late endosomal compartments. Further, we find ISG accumulation within endosomes, but that RME-8 knockdown also increases ISG turnover; combined with previous data, this suggests that it is most probable that ISGs are recycled, and that RME-8 is required to support recycling.     

Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes.     

Purification of African Trypanosomes Can Cause Biochemical Changes in the Parasites1

Bloodstream forms of African trypanosomes are routinely purified from blood components by a combination of centrifugation and chromatography on DEAE cellulose at pH 8.0, Here we report that the nonphysiological conditions used for DEAE chromatography of the parasites result in changes in the ATP levels of the trypanosomes and an enhanced release from the parasites of proteins such as variable surface glycoprotein, peptidase, and phospholipase. Some of these changes can be reduced by the addition of nucleosides to the elution buffer and, after the elution of the parasites, by immediate readjustment of the external pH to the normal physiological level of blood (pH 7.4).     

Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

The variant surface glycoprotein (VSG) of bloodstream form Trypanosoma brucei (Tb) is a critical virulence factor. The VSG glycosylphosphatidylinositol (GPI)-anchor strongly influences passage through the early secretory pathway. Using a dominant-negative mutation of TbSar1, we show that endoplasmic reticulum (ER) exit of secretory cargo in trypanosomes is dependent on the coat protein complex II (COPII) machinery. Trypanosomes have two orthologues each of the Sec23 and Sec24 COPII subunits, which form specific heterodimeric pairs: TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1. RNA interference silencing of each subunit is lethal but has minimal effects on trafficking of soluble and transmembrane proteins. However, silencing of the TbSec23.2/TbSec24.1 pair selectively impairs ER exit of GPI-anchored cargo. All four subunits colocalize to one or two ER exit sites (ERES), in close alignment with the postnuclear flagellar adherence zone (FAZ), and closely juxtaposed to corresponding Golgi clusters. These ERES are nucleated on the FAZ-associated ER. The Golgi matrix protein Tb Golgi reassembly stacking protein defines a region between the ERES and Golgi, suggesting a possible structural role in the ERES:Golgi junction. Our results confirm a selective mechanism for GPI-anchored cargo loading into COPII vesicles and a remarkable degree of streamlining in the early secretory pathway. This unusual architecture probably maximizes efficiency of VSG transport and fidelity in organellar segregation during cytokinesis.